Note: Descriptions are shown in the official language in which they were submitted.
WO 2022/027039
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ANTI-VARIABLE MUC1* ANTIBODIES AND USES THEREOF
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention:
[0002] The present application relates to human, humanized and
non-human anti-MUC1*
antibodies and methods of making and using them. The present application also
relates to
using an immune cell transfected or transduced with a cleavage enzyme for the
treatment of
cancer. The present invention also relates to using an immune cell transfected
or transduced
with a CAR and another protein for the treatment of cancer.
[0003] 2. General Background and State of the Art:
[0004] We previously discovered that a cleaved form of the MUC1
(SEQ ID NO:1)
transmembrane protein is a growth factor receptor that drives the growth of
over 75% of all
human cancers. The cleaved form of MUC1, which we called MUC1* (pronounced muk
1
star), is a powerful growth factor receptor. Cleavage and release of the bulk
of the
extracellular domain of MUC1 unmasks a binding site for activating ligands
dimeric NME1,
NME6, NME7, NME7AB, NME7-X1 or NME8. It is an ideal target for cancer drugs as
it is
aberrantly expressed on over 75% of all cancers and is likely overexpressed on
an even
higher percentage of metastatic cancers (Mahanta et al. (2008) A Minimal
Fragment of
MUC1 Mediates Growth of Cancer Cells. PLoS ONE 3(4): e2054. doi:10.1371/
journal.pone.0002054; Fessler et al. (2009), "MUC1* is a determinant of
trastuzumab
(Herceptin) resistance in breast cancer cells," Breast Cancer Res Treat.
118(1):113-124).
After MUC1 cleavage most of its extracellular domain is shed from the cell
surface. The
remaining portion has a truncated extracellular domain that comprises most or
all of the
primary growth factor receptor sequence called PSMG1-1( (SEQ ID NO:2).
[0005] Antibodies are increasingly used to treat human diseases.
Antibodies generated in
non-human species have historically been used as therapeutics in humans, such
as horse
antibodies. More recently, antibodies are engineered or selected so that they
contain mostly,
or all, human sequences in order to avoid a generalized rejection of the
foreign antibody. The
process of engineering recognition fragments of a non-human antibody into a
human
antibody is generally called 'humanizing'. The amount of non-human sequences
that are used
to replace the human antibody sequences determines whether they are called
chimeric,
humanized or fully human.
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[0006]
Alternative technologies exist that enable generation of humanized or
fully human
antibodies. These strategies involve screening libraries of human antibodies
or antibody
fragments and identifying those that bind to the target antigen, rather than
immunizing an
animal with the antigen. Another approach is to engineer the variable
region(s) of an antibody
into an antibody-like molecule. Another approach involves immunizing a
humanized animal.
The present invention is intended to also encompass these approaches for use
with
recognition fragments of antibodies that the inventors have determined bind to
the
extracellular domain of MUC1*.
[0007]
In addition to treating patients with an antibody, cancer immunotherapies
have
recently been shown to be effective in the treatment of blood cancers. One
cancer
immunotherapy, called CAR T (chimeric antigen receptor T cell) therapy,
engineers a T cell
so that it expresses a chimeric receptor having an extra cellular domain that
recognizes a
tumor antigen, a transmembrane domain and cytoplasmic tail comprising T cell
signaling
and co-stimulatory components (llai H, Wang Y, Lu X, Han W. (2016) Chimeric
Antigen
Receptors Modified T cells for Cancer Therapy. J Natl Cancer Inst. 108(7):
djv439). Such
receptor is composed of a single chain antibody fragment (scFv) that
recognizes a tumor
antigen, linked to a T cell transrnembrane, signaling domain and co-
stimulatory domain or
domains. Upon binding of the receptor to a cancer associated antigen, a signal
is transmitted
resulting in T cell activation, propagation and the targeted killing of the
cancer cells. In
practice, T cells are isolated from a patient or donor and transduced with a
CAR, expanded
and then injected back into the patient. If from a donor, the immune cells may
be mutated or
engineered such that they do not induce graft versus host disease in the
recipient. When the
CAR T cells bind to the antigen on a cancer cell, the CAR T cells attack the
cancer cells and
then expand that population of T cells.
[0008]
Thus far, CAR T therapies have been very successful in the treatment of
blood
cancers but as yet have not shown efficacy against solid tumors in humans.
Because most
blood cancers are B cell malignancies, the CAR T cells can just eliminate all
of the patient's
B cells without causing serious harm to the patient. There is no B cell
equivalent in solid
tumors. Most tumor associated antigens are also expressed on normal tissues;
they are just
expressed at a higher level in cancerous tissues. Thus, the challenge is to
develop an antibody
that recognizes an epitope on a tumor associated antigen that is somehow
different in the
context of the tumor compared to normal tissue. To further minimize the risk
of off-
tumor/on-target killing of normal tissues, the antibody should recognize and
bind to
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cancerous tissues at least two-times more than normal tissues. Antibodies that
are not so
cancer selective may be used therapeutically if they are inducibly expressed
at the tumor site.
[0009] Another cancer therapy that incorporates cancer selective
antibodies is Bispecific
T cell Engagers, also called BiTEs. The BiTE approach attempts to eliminate
the CAR T
associated risk of off-tumor/on-target effects. Unlike CAR T, BiTEs are
bispecific antibodies
that should not pose any greater risk than regular antibody-based therapies.
However, unlike
typical anti-cancer antibodies that bind to and block a cancer antigen, BiTEs
are designed to
bind to an antigen on the tumor cell and simultaneously bind to an antigen on
an immune cell,
such as a T cell. In this way, a BiTE recruits the T cell to the tumor. BiTEs
are engineered
proteins that simultaneously bind to a cancer associated antigen and a T cell
surface protein
such as CD3-epsilon. BiTEs are antibodies made by genetically linking the
scFv's of an
antibody that binds to a T cell antigen, like anti-CD3-epsilon to a scFv of a
therapeutic
monoclonal antibody that binds to a cancer antigen (Patrick A. Baeuerle, and
Carsten
Reinhardt (2009) Bispecific T cell engaging antibodies for cancer therapy.
Cancer Res.
69(12):4941-4944). A drawback of BiTE technology is that, unlike CAR T cells,
they do not
expand in the patient, so have limited persistence.
[0010] Yet another cancer therapy that incorporates cancer
selective antibodies is
antibody drug conjugate, also called ADC, technology. In this case, a toxin,
or a precursor to
a toxin, is linked to a cancer selective antibody. Unlike CAR T cells that use
the CD8 positive
T cell's natural killing to kill cancer cells, ADCs carry a toxic payload to
the tumor.
Drawbacks of ADCs include the potential of delivering the toxic payload to
normal cells and
that most ADCs require binding to a cell surface molecule which then gets
internalized after
binding, with an approximate 10,000 surface molecule required for resultant
cell death.
SUMMARY OF THE INVENTION
[0011] In one aspect, the present invention is directed to a non-
human, human or
humanized anti-MUC1* antibody or antibody fragment or antibody-like protein
that binds to
a region on extracellular domain of MUC1 isoform or cleavage product that is
devoid of the
tandem repeat domains. The non-human, human or humanized anti-MUC1* antibody
or
antibody fragment or antibody-like protein may specifically bind to
[0012] (i) PSMGFR region of MUCl;
[0013] (ii) PSMGFR peptide;
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[0014] (iii) a peptide having amino acid
sequence of
QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10) (SEQ ID NO:3)
[0015] (iv) a peptide having amino acid sequence of
[0016] ASRYNLTISDVSVSDVPFPFSAQSGA (N-19) (SEQ ID NO:4)
[0017] (v) a peptide having amino acid sequence of
[0018] NLTISDVSVSDVPFPFSAQSGA (N-23) (SEQ ID NO:5)
[0019] (vi) a peptide having amino acid sequence of
[0020] ISDVSVSDVPFPFSAQSGA (N-26) (SEQ ID NO:6)
[0021] (vii) a peptide having amino acid sequence of
[0022] SVSDVPFPFSAQSGA (N-30) (SEQ ID NO:7)
[0023] (viii) a peptide having amino acid sequence of
[0024] QFNQYKTEAASRYNLTISDVSVSDVPFPFS (N-10/C-5) (SEQ ID NO:8)
[0025] (ix) a peptide having amino acid sequence of
[0026] ASRYNLTISDVSVSDVPFPFS (N-19/C-5) (SEQ ID NO:9)
[0027] (x) a peptide having amino acid sequence of
[0028] FPFSAQSGA (SEQ ID NO:10)
[0029] The non-human, human or humanized antibody may be IgG1
IgG2, IgG3, IgG4
or IgM. The human or humanized antibody fragment or antibody-like protein may
be scFv or
scFv-Fc.
[0030] The murine, camelid, human or humanized antibody, antibody
fragment or
antibody-like protein as in above may comprise a heavy chain variable region
and light chain
variable region which is derived from mouse monoclonal MNC2, MNE6, 20A10,
3C2B1,
5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11 antibody, and
has at
least 80%, 90% or 95% or 98% sequence identity to the mouse monoclonal MNC2,
MNE6,
20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11
antibody. The heavy chain variable region of CDRI and CDR2 may have at least
90% or
95% or 98% sequence identity to the particularly indicated antibody heavy
chain variable
region sequence set forth in the present application in the sequence listing,
and the light chain
variable region of CDR1 and CDR2 may have at least 90% or 95% or 98% sequence
identity
to the particularly indicated antibody heavy chain variable region sequence
set forth in the
present application in the sequence listing section. The heavy chain variable
region of CDR3
may have at least 80% or 85% or 90% sequence identity to the particularly
indicated antibody
heavy chain variable region sequence set forth in the present application in
the sequence
listing, and the light chain variable region of CDR3 may have at least 80% or
85% or 90%
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sequence identity to the particularly indicated antibody heavy chain variable
region sequence
set forth in the present application in the sequence listing section.
[0031]
The murine, camelid, human or humanized antibody, antibody fragment or
antibody-like protein according to above may include complementarily
determining regions
(CDRs) in the heavy chain variable region and light chain variable region
having at least 90%
or 95% or 98% sequence identity to the particularly indicated antibody heavy
chain CDR1,
CDR2 or CDR3 region and light chain CDR1, CDR2 or CDR3 region sequences set
forth in
the present application in the sequence listing section.
[0032]
In another aspect, the present invention is directed to an anti-MUC1*
extracellular
domain antibody or anti-N-10 antibody, which may be any of the antibodies
described above,
comprised of sequences represented by humanized IgG2 heavy chain, or humanized
IgG1
heavy chain, paired with humanized Kappa light chain, or humanized Lambda
light chain.
The humanized IgG2 heavy chain may be SEQ ID NOS:55, humanized IgG1 heavy
chain
may be SEQ ID NO:58, humanized Kappa light chain may be SEQ ID NO:110, and
humanized Lambda light chain may be SEQ ID NO:114, or a sequence having 90%.
95% or
98% sequence identity thereof.
[0033]
In another aspect, the present invention is directed to an anti-MUC1*
extracellular domain antibody or anti-N-10 antibody, which may be any of the
antibodies
described above, comprised of sequences represented by human IgG2 heavy chain,
or human
IgG1 heavy chain, paired with human Kappa light chain, or human Lambda light
chain. The
human IgG2 heavy chain may be SEQ ID NOS:55, human IgG1 heavy chain may be SEQ
ID
NO:58, human Kappa light chain may be SEQ ID NO:110, and human Lambda light
chain
may be SEQ ID NO:114, or a sequence having 90%, 95% or 98% sequence identity
thereof.
[0034]
In another aspect, the invention is directed to an anti-MUC1*
extracellular domain
antibody or anti-N-10 antibody comprised of sequences of a humanized MNC2
represented
by humanized IgG1 heavy chain, humanized IgG2 heavy chain, paired with
humanized
Lambda light chain, and humanized Kappa light chain.
[0035]
In another aspect, the invention is directed to an anti-MUC1*
extracellular domain
antibody or anti-N-10 antibody comprised of sequences of a humanized MNC2,
MNE6,
20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11
represented by humanized IgG1 heavy chain or humanized IgG2 heavy chain,
paired with
humanized Lambda light chain, or humanized Kappa light chain.
[0036]
In another aspect, the invention is directed to an antibody that is "like"
MNC2,
MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or
H11
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in that they have the same or very similar pattern of binding to subsets of
peptides derived
from the PSMGFR peptide, also do not recognize a linear epitope, competitively
inhibit the
binding of NME1 or NME7 AB to MUC1*, recognize a MUC1 transmembrane cleavage
product produced by cleavage by MMP9 or contain CDR sequences that are at
least 80%
homologous to the MNE6, MNC2, MN18G12, MN20A10, MN25E6, MN28F9, MN5C6F3,
MN3C2B1, and MN1E4 CDR consensus sequences.
[0037]
In another aspect, the invention is directed to an antibody that binds to
the extra
cellular domain of a MUC1 that is devoid of the tandem repeat domain, which
may be a
cleavage product. In one aspect of the invention, the antibody binds to a
peptide having the
sequence of QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10). In one aspect of
the invention, the antibody binds to a peptide having the sequence of
ASRYNLTISDVSVSDVPFPFSAQSGA (N19). In one aspect of the invention, the antibody
binds to a peptide having the sequence of SVSDVPFPFSAQSGA (N-30). In one
aspect of the
invention, the antibody binds to a peptide having the sequence of FPFSAQSGA (N-
36).
Examples of such antibodies include but are not limited to monoclonal
antibodies MNC2,
MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and
H11.
The heavy chain and light chain complementary determining region sequences for
these
antibodies are set forth in the present application in the sequence listing
section.
[0038]
In one aspect of the invention, one or more of these antibodies is
administered to a
patient diagnosed with or at risk of developing a cancer. 'Me antibody may be
human or
humanized. The antibody may be murine or camelid. The antibody may be bivalent
or
monovalent. The antibody may be a fragment, including a single chain fragment,
scFv, of one
of the antibodies. The antibody or antibody fragment may be administered
directly to the
patient or incorporated into a multi-specific antibody-like molecule, a
bispecific antibody, a
bispecific T cell engager , BiTE, or an antibody drug conjugate, ADC. The
antibody or
antibody fragment may be incorporated into a T cell receptor, TCR. The
sequence of the
antibody or antibody fragment may be incorporated into a chimeric antigen
receptor, a
"CAR", or other similar entity, then introduced into an immune cell, ex vivo,
then
administered to a patient diagnosed with or at risk of developing a cancer.
The immune cell,
which may be a T cell or natural killer cell, may be derived from a donor or
from the patient.
In one aspect the immune cell is derived from a stem cell that has been
directed to
differentiate to that immune cell type in vitro. In another aspect the immune
cell is derived
from a stem cell that has been directed to differentiate to that immune cell
type in vitro. In
another aspect, a CAR containing sequences of the antibody are expressed in a
stem cell,
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which then may be differentiated into an immune cell. In one case, the immune
cell is a T
cell. In another case, the immune cell is an NK cell. In one aspect, the
antibody or a CAR
containing sequences of the antibody may be expressed off of an inducible
promoter. In one
case the antibody or the CAR is expressed upon activation of the T cell or
other immune cell.
In one instance, the antibody or the CAR of the invention is expressed off of
an NFAT
response element. In another instance, CAR recognition of a target tumor cell
activates the
immune cell, leading to NFAT inducible expression of a cytokine, such as IL-12
or IL-18, or
expression of a checkpoint inhibitor such as a PD 1 inhibitor or a PDL-1
inhibitor. In yet
another aspect, CAR recognition of a target tumor cell activates the immune
cell, leading to
NFAT inducible expression of a second CAR that contains sequences of a second
antibody.
[0039] In another aspect, the invention is directed to a marine,
camelid, human,
humanized anti-MUC I* antibody or antibody fragment or antibody-like protein
that binds to
the N-10 peptide, according to above, which inhibits the binding of NME
protein to MUCI*.
The NME may be NMEI, NME6, NME7An, NME7-X1, NME7 or NME8.
[0040] In yet another aspect, the invention is directed to a
single chain variable fragment
(scFv) comprising a heavy and light chain variable regions connected via a
linker, further
comprising CDRs of antibodies that bind to MUC1* extracellular domain. The
CDRs may be
derived from MNC2, MNE6, 20A10, 3C2B I, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12,
B2, B7,
B9, 8C7F3, and H11. The scFv may be one that possesses the SEQ ID NOS:233, 235
or 237
(MNE6); SEQ Ill NOS:238-243, 654-655 or 5017-5020 (MNC2); SEQ Ill NOS:1574-
1581
or 5001-5012 (20A10); SEQ ID NOS:1573 or 1813 (3C2B1); SEQ ID NOS:1385 or 1815
(5C6F3); SEQ ID NOS:1599 or 1601 (25E6).
[0041] In still another aspect, the invention is directed to a
chimeric antigen receptor
(CAR) comprising a scFv or a humanized variable region that binds to the
extracellular
domain of a MUCI that is devoid of tandem repeats, a linker molecule, a
transmembrane
domain and a cytoplasmic domain. The single chain antibody fragment may bind
to
[0042] (i) PSMGFR region of MUCl;
[0043] (ii) PSMGFR peptide;
[0044] (iii) a peptide having amino acid
sequence of
QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10) (SEQ ID NO :3)
[0045] (iv) a peptide having amino acid sequence of
[0046] ASRYNLTISDVSVSDVPFPFSAQSGA (N-19) (SEQ ID NO:4)
[0047] (v) a peptide having amino acid sequence of
[0048] NLTISDVSVSDVPFPFSAQSGA (N-23) (SEQ ID NO:5)
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[0049] (vi) a peptide having amino acid sequence of
[0050] ISDVSVSDVPFPFS AQSGA (N-26) (SEQ ID NO:6)
[0051] (vii) a peptide having amino acid sequence of
[0052] SVSDVPFPFSAQSGA (N-30) (SEQ ID NO:7)
[0053] (viii) a peptide having amino acid sequence of
[0054] QFNQYKTEAASRYNLTISDVSVSDVPFPFS (N-10/C-5) (SEQ ID NO:8)
[0055] (ix) a peptide having amino acid sequence of
[0056] ASRYNLTISDVSVSDVPFPFS (N-19/C-5) (SEQ ID NO:9)
[0057] (x) a peptide having amino acid sequence of
[0058] FPFSAQSGA (N-36) (SEQ ID NO:10)
[0059] In the CAR as described above, portions of any of the
variable regions set forth
and described above, or combination thereof may be used in the extracellular
domain of the
CAR. The CAR also comprises a transmembrane region and a cytoplasmic tail that
comprises
sequence motifs that signal immune system activation. The extracellular domain
may be
comprised of murine, camelid, human, non-human, or humanized single chain
antibody
fragments of an MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12,
B2,
B7, B9, 8C7F3, and H11. Additional antibodies from which single chain antibody
fragments
may be made include but are not limited to monoclonal antibodies that are like
MNC2,
MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, and
H11
in that they have the same or very similar pattern of binding to subsets of
peptides derived
from the PSMGFR peptide, may not recognize a linear epitope or competitively
inhibit the
binding of NME1 or NME7AB to MUC1*, or recognize a MUC1 transmembrane cleavage
product produced by cleavage by MMP9 or contain CDR sequences that are at
least 80%
homologous to the MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4,
B12,
B2, B7, B9, 8C7F3, and H11 CDR consensus sequences.
[0060] In the CARs as described above, the extracellular domain
may include a murine,
camelid, human, non-human or humanized single chain antibody fragments of an
MNE6 scFv
set forth as SEQ ID NOS: 233, 235, or 237, MNC2 scFv (SEQ ID NOS:239, 241,243,
655 or
5017-5020), 20A10 scFv as set forth as SEQ ID NOS:1575, 1577, 1579, 1581 or
5001-5012,
3C2B1 scFv as set forth as SEQ ID NOS:1573 or 1813, 5C6F3 scFv as set forth as
SEQ ID
NOS:1385 or 1815, or 25E6 scFv as set forth as SEQ ID NOS:1599 or 1601.
[0061] In the process of humanizing an antibody, one must
annotate the sequence to
identify the different functional regions, such as the complementarity
determining regions
(CDRs), the framework regions and the constant regions. Various computer
programs are
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available that assign certain sequences to the CDRs, framework regions and
constant regions.
Depending on the program used, the exact position where one region ends and
the next
begins may differ by a few amino acids. Typically, humanized single chain
constructs, scFvs,
contain heavy and light chain CDRs supported by intervening framework regions,
wherein
the heavy and light chain are connected through a flexible linker. Depending
on the
annotation program used, the sequence assigned to framework region IV may
extend into the
constant region. In some cases, extension of framework region IV may provide
more stability
to the scFv. Here we provide sequences for humanized scFv's of the invention,
wherein the
length of framework region IV of the light chain may vary. For example, in
some cases the
C-terminus of Framework region IV ends in the amino acids R. T. In other
cases, it ends in R
alone. Still in other cases, the terminal R and T are both omitted. In the
CARs described here,
the extracellular domain may include a murine, camelid, human, non-human or
humanized
single chain antibody fragments with framework region IV having variable
lengths as set
forth as MNE6 scFv (SEQ ID NOS: 5014 or 5016), MNC2 scFv (SEQ ID NOS: 5018 or
5020), or 20A10 scFv (SEQ ID NOS: 5002, 5004, 5006, 5008, 5010 or 5012) or
25E6 scFv
(SEQ ID NOS: 5030 or 5032).
[0062]
In any of the CARs described here, the cytoplasmic tail may be comprised
of one
or more of signaling sequence motifs and co-stimulatory domains, including but
not limited
to CD3-zeta, CD3-zeta-1XX CD27, CD28, 4-1BB, 0X40, CD30, CD40, ICAm-1, LFA-1,
ICOS, CD2, CD5, or CD7. Additionally, the sequence of the intracellular
signaling domain
may contain mutations that dampen the signal to improve persistence or to
improve killing of
low antigen density tumor cells. The cytoplasmic tail may be comprised of one
or more of
signaling sequence motifs and co-stimulatory sequence motifs CD3-zeta, CD27,
CD28, 4-
1BB, 0X40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7. The
transmembrane
and extracellular hinge region of the CAR may or may not be derived from
sequences of the
adjacent co-stimulatory domain. For example, a CAR comprising the 4-1BB co-
stimulatory
domain may have a transmembrane and hinge region derived from CD8 or CD28. In
another
example, a CAR comprising the CD28 co-stimulatory domain may have a
transmembrane
and hinge region derived from CD28. In any of the CARs described above, the
cytoplasmic
tails may include deletions or mutations that dampen signaling. Such deletions
or mutations
in one or more of the three immunoreceptor tyrosine-based activation motifs,
also known as
ITAMs, increase persistence of CAR bearing cells and decrease their
differentiation as
measured by an increase in the CD62L+ CD45RA- population. Such mutations
include but
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are not limited to Tyrosines that are mutated to inhibit phosphorylation and
signaling (Salter
et al, 2018). In another aspect one or two ITAMs are deleted, leaving only one
or two ITAMs
(Feucht et al 2019). In another aspect, the position of the included ITAM or
ITAMs is moved
to a position proximal to the co-stimulatory domain. Suitable ITAM
configurations for
increased persistence of CARs include but are not limited to 1XX, X2X, XX3,
12X and 23X,
wherein the numeral 1, 2 or 3 refers to ITAM1, ITAM2, or ITAM3 and X refers to
the
deletion of that ITAM. In a preferred embodiment ITAM1 is the only functional
ITAM
included in the CAR construct, also known as 1XX. In any of the CARs described
above, the
ITAMs of CD3-zeta may be deleted or mutated to inhibit or dampen signaling. In
any of the
CARs described above, the CD3 of the cytoplasmic tail may comprise deletions
or mutations
in or of the ITAMs including those referred to as 1XX (Feucht et al 2019; SEQ
ID NO:1796-
1797). In any of the CARs described above, the T cell may be engineered to
overexpress c-
Jun as a method to inhibit T cell exhaustion (Lynn et al 2019). The CAR
constructs described
above may be expressed in a T cell, an NK cell, a dendritic cell or other
immune cell, which
may be autologous or allogeneic. Allogeneic cells may be derived from human
stem cells.
[0063]
In any of the CARs described above, the CAR may include a single chain
antibody fragment, scEv comprising a sequence derived from antibody MNE6,
including but
not limited to (SEQ ID NOS:12-13 and 65-66, 56-57, 107-108, 341-342, 391-394),
from
antibody MNC2, including but not limited to (SEQ ID NOS:118-119 and 168-169,
144-145
and 194-195, 654-655, 1788-1789), from antibody 20A10, including but not
limited to (SEQ
ID NOS:988-989 and 1004-1005, 1574-1581, 5001-5012, 1677, 1687), from antibody
3C2B1, including but not limited to (SEQ ID NOS:1820-1823, 1572-1573, 1812-
1813), from
antibody 5C6F3, including but not limited to (SEQ ID NOS:1816-1819, 1384-1385,
1814-
1815), from antibody 25E6, including but not limited to (SEQ ID NOS:1020-1021,
1036-
1037, 1598-1601), wherein the CAR hinge and transmembrane sequences may be
derived
from CD8 (SEQ ID NO:346 and SEQ ID NO:364), or from CD28 (SEQ ID NO:350 and
SEQ
ID NO:368), further comprising a co-stimulatory domain, which may be 41BB (SEQ
ID
NO:659) or CD28 (SEQ ID NO:378) and the CD3-zeta signaling domain may be
derived
from (SEQ ID NO:661) or may contain mutations including those referred to as
1XX (SEQ
ID NO:1796-1797).
[0064]
hi any of the CARs described above, the sequence may be CAR MNE6
CD28/CD3z (SEQ ID NOS:298); CAR MNE6 4-1BB/CD3z (SEQ ID NOS:301); CAR
MNE6 0X40/CD3z (SEQ ID NOS:617); CAR MNE6 CD28/41BB/CD3z (SEQ ID
NOS:304); CAR MNE6 CD28/0X40/CD3z (SEQ ID NOS:619); CAR MNC2 CD3z (SEQ
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ID NOS:607); CAR MNC2 CD8 hinge/transmembrane CD28/CD3z SEQ ID NOS:609);
CAR MNC2 CD8 hinge/transmembrane 4-1BB/CD3z (SEQ ID NOS:611 and SEQ ID NOS:
719); CAR MNC2 CD8 hinge/transmembrane 4-1BB/1XX (SEQ ID NOS:1619 and SEQ ID
NOS: 1621); CAR MNC2 CD8 hinge/transmembrane CD28/1XX (SEQ ID NOS:1623 and
SEQ ID NOS: 1625); CAR MNC2 CD28 hinge/transmembrane CD28/1XX (SEQ ID
NO:5042 and SEQ ID NO: 5044); CAR MNC2 0X40/CD3z (SEQ ID NOS:613); CAR
MNC2 CD28/4-1BB/CD3z (SEQ ID NOS: 307); CAR MNC2 CD28/0X40/CD3z (SEQ ID
NOS:615); CAR 20A10 CD8 hinge/transmembrane 4-1BB/CD3z (SEQ ID NO:1583 and
SEQ ID NO: 1585); CAR 20A10 CD8 hinge/transmembrane CD28/CD3z (SEQ ID NO:1587
and SEQ ID NO: 1589); CAR 20A10 CD8 hinge/transmembrane 4-1BB/1XX (SEQ ID
NO:1591 and SEQ ID NO:1593); CAR 20A10 CD8 hinge/transmembrane CD28/1XX (SEQ
ID NO:1595 and SEQ ID NO:1597); CAR 20A10 CD28 hinge/transmembrane CD28/CD3z
(SEQ ID NO:5022 and SEQ ID NO:5024); CAR 20A10 CD28 hinge/transmembrane
CD28/1XX (SEQ ED NO:5026 and SEQ ID NO:5028); or CAR MNC3 4-1BB/CD3z (SEQ
ID NOS: 601).
[0065]
In another aspect, the invention is directed to a composition that
includes at least
two CARs with different extracellular domain units transfected into the same
cell, which may
be an immune cell, which may be derived from the patient requiring treatment
for a cancer.
The expression of the second CAR may be inducible and driven by the
recognition of a target
by the first CAR. The nucleic acid encoding the second CAR may be linked to an
inducible
promoter. The expression of the second CAR may be induced by an event that
occurs
specifically when the immune cell mounts an immune response to a target tumor
cell. The
antibody fragments of one or both of the CARs may direct the cell to a MUC1*
positive
tumor. The antibody fragments of the first and second CARs may bind to a MUC1*
that is
produced when MUC1 is cleaved by two different cleavage enzymes. Expression of
the
second CAR by the inducible promoter may be induced when the antibody fragment
of the
first CAR engages or binds to a MUC1 or MUC1* on the tumor. One way to do this
is to
induce expression of the second CAR when, or shortly after, an NFAT protein is
expressed or
translocated to the nucleus. For example, a sequence derived from an NFAT
promoter region
is put upstream of the gene for the second CAR. In this way, when the
transcription factors
that bind to the promoter of the NFAT protein are present in sufficient
concentration to bind
to and induce transcription of the NFAT protein, they will also bind to that
same promoter
that is engineered in front of the sequence for transcription of the second
CAR. The NFAT
protein may be NFAT1 also known as NFATc2, NFAT2 also known as NFATc or
NFATcl,
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NFAT3 also known as NFATc4, NFAT4 also known as NFATc3, or NFAT5. In one
aspect
of the invention, the NFAT is NFATcl , NFATc3 or NFATc2. In one aspect of the
invention,
the NFAT is NFAT2 also known as NFATc1. SEQ ID NO:646 shows nucleic acid
sequence
of the upstream transcriptional regulatory region for NFAT2. The recognition
unit of the
second CAR may be an antibody fragment or a peptide, wherein the recognition
units may
bind to NME7, PD-1, PDL-1, or a checkpoint inhibitor.
[0066] The at least two CARs may have one CAR that does not have
a tumor antigen
targeting recognition unit and the other CAR does have a tumor antigen
targeting recognition
unit. In another aspect of the invention, one of the extracellular domain
recognition units may
bind to MUC1* extracellular domain. In another aspect of the invention, one of
the
extracellular domain recognition units may be an antibody fragment and the
other is a
peptide, which may be devoid of transmembrane and signaling motifs; the
peptide may be a
single chain antibody fragment or antibody. In another aspect of the
invention, one of the
recognition units may bind PD-1 or PDL-1. In another aspect of the invention,
one extra
cellular domain recognition unit is an anti-MUCI* antibody, antibody fragment
or scFv
chosen from the group consisting of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6,
18G12,
28F9, 1E4, B12, B2, B7, B9, 8C7F3, and H11. The other recognition unit may be
a CAR or
may be an anti-NME7 antibody.
[0067] In another aspect, the invention is directed to a cell
comprising a CAR with an
extracellular domain that binds to the extra cellular domain of a MUC1
molecule that is
devoid of tandem repeats. In another aspect, the invention is directed to a
cell comprising a
CAR with an extracellular domain that binds to a MUC1* transfected or
transduced cell. The
cell that includes the CAR may be an immune system cell, preferably a T cell,
a natural killer
cell (N K), a dendritic cell or mast cell.
[00681 In another aspect, the invention is directed to an
engineered antibody-like protein.
[0069] In another aspect, the invention is directed to a method
for treating a disease in a
subject comprising administering an antibody according to any claim above, to
a person
suffering from the disease, wherein the subject expresses MUC1 aberrantly. The
disease may
be cancer, such as breast cancer, ovarian cancer, pancreatic cancer, lung
cancer, colon cancer,
gastric cancer or esophageal cancer.
[0070] In another aspect, the invention is directed to an
antibody, antibody fragment or
scFv comprising variable domain fragments derived from an antibody that binds
to an
extracellular domain of MUC1 isoform or cleavage product that is devoid of the
tandem
repeat domains. In a preferred embodiment, the antibody or antibody fragment
binds to the
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N-10 peptide (SEQ ID NO:3), but does not bind to the C-10 peptide (SEQ ID
NO:825). The
variable domain fragments may be derived from mouse monoclonal antibody MNE6
(SEQ
ID NO:13 and 66) or from the humanized MNE6 (SEQ ID NO: 39 and 94), or from
MNE6
scFv (SEQ ID NO: 233, 235 and 237). Or, the variable domain fragments may be
derived
from mouse monoclonal antibody MNC2 (SEQ ID NO: 119 and 169) or from the
humanized
MNC2 (SEQ ID NO: 145 and 195), or from MNC2 scFv (SEQ ID NO: 239, 241 and
243).
Or, the variable domain may be derived from monoclonal antibodies MN18G12,
MN20A10,
MN25E6, MN28F9, MN5C6F3, MN3C2B1, or MN1E4. The heavy chain and light chain
complementary determining region sequences for these antibodies are also set
forth in the
sequence listing herein.
[0071] In another aspect, the invention is directed to a method
for the treatment of a
person diagnosed with, suspected of having or at risk of developing a MUC1 or
MUC1*
positive cancer involving administering to the person an effective amount of
the antibody,
antibody fragment or scFv described above, wherein the species may be murine,
camelid,
human or humanized.
[0072] In another aspect, the invention is directed to a
polypeptide comprising at least
two different scFv sequences, wherein one of the scFv sequences is a sequence
that binds to
extracellular domain of MUC1 isoform or cleavage product that is devoid of the
tandem
repeat domains. The polypeptide may bind to
[0073] (i) PSMGER region of MUC1;
[0074] (ii) PSMGFR peptide;
[0075] (iii) a peptide having amino acid
sequence of
QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10) (SEQ ID NO :3)
[0076] (iv) a peptide having amino acid sequence of
[0077] ASRYNLTISDVSVSDVPFPFSAQSGA (N-19) (SEQ ID NO:4)
[0078] (v) a peptide having amino acid sequence of
[0079] NLTISDVSVSDVPFPFSAQSGA (N-23) (SEQ ID NO:5)
[0080] (vi) a peptide having amino acid sequence of
[0081] ISDVSVSDVPFPFSAQSGA (N-26) (SEQ ID NO:6)
[0082] (vii) a peptide having amino acid sequence of
[0083] SVSDVPFPFSAQSGA (N-30) (SEQ ID NO:7)
[0084] (viii) a peptide having amino acid sequence of
[0085] QFNQYKTEAASRYNLTISDVSVSDVPFPFS (N-10/C-5) (SEQ ID NO:8)
[0086] (ix) a peptide having amino acid sequence of
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[0087] ASRYNLTISDVSVSDVPFPFS (N-19/C-5) (SEQ ID NO:9)
[0088] (x) a peptide having amino acid sequence of
[0089] FPFSAQSGA (N-36) (SEQ ID NO:10)
[0090] In another aspect, the invention is directed to a method
of detecting presence of a
cell that expresses MUC1* aberrantly, comprising contacting a sample of cells
or tissue with
the antibody, antibody fragment or scFv-Fc described above and detecting for
the presence of
the binding of antibody, antibody fragment or scFv-Fc to the cell. The cell
may be cancer
cell.
[0091] In another aspect, the invention is directed to a method
for testing a subject's
cancer for suitability of treatment with a composition comprising antibodies
of the invention,
which may be murine, camelid, human or humanized, or fragments thereof, or
portions of the
variable regions of antibodies MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12,
28F9,
1E4, B12, B2, B7, B9, 8C7F3, or H11, comprising the steps of contacting a
bodily specimen
from the patient, in vitro, ex-vivo, or in vivo, with the antibody and
determining that the
patient exhibits aberrant expression of MUC1* compared to normal tissue or
specimen and
concluding that the patient's cancer will beneficially respond to treatment
with an agent
comprising the antibody or a fragment thereof. The antibody used in these
diagnostics may be
conjugated to an imaging agent.
[0092] In another aspect, the invention is directed to a method
of treating a subject
suffering from a disease comprising, exposing immune cells, which may be '1
cells or INK
cells from the subject, or from a donor, to MUC1* peptides wherein through
various rounds
of maturation, the T cells or NK cells develop MUC1* specific receptors,
creating adapted T
cells or NK cells, and expanding and administering the adapted cells to the
donor patient who
is diagnosed with, suspected of having, or is at risk of developing a MUC1*
positive cancer.
The MUC1* peptide is chosen from among the group:
[0093] (i) PSMGFR region of MUC1;
[0094] (ii) PSMGFR peptide;
[0095] (iii) a peptide having amino acid
sequence of
QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10)
[0096] (iv) a peptide having amino acid sequence of
[0097] ASRYNLTISDVSVSDVPFPFSAQSGA (N-19)
[0098] (v) a peptide having amino acid sequence of
[0099] NLTISDVSVSDVPFPFSAQSGA (N-23)
[00100] (vi) a peptide having amino acid sequence of
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[00101] ISDVSVSDVPFPFSAQSGA (N-26)
[00102] (vii) a peptide having amino acid sequence of
[00103] S VSD VPFPFSAQS GA (N-30)
[00104] (viii) a peptide having amino acid sequence of
[00105] QFNQYKTEAASRYNLTISDVSVSDVPFPFS (N-10/C-5)
[00106] (ix) a peptide having amino acid sequence of
[00107] ASRYNLTISD VS V SD VPFPFS (N -19/C-5)
[00108] (x) a peptide having amino acid sequence of
[00109] FPFSAQS GA (N-36)
[00110] In one aspect of the invention, the antibody that is administered to a
patient for the
treatment or prevention of a MUC1 or MUC1* positive cancer is selected for its
ability to
bind to the N-10 peptide of the PSMGFR. The antibody can be administered
alone, as a
monovalent antibody, as an scFv, or a fragment of the antibody can be
incorporated into a
CAR, a BiTE or an ADC.
[00111] In another aspect of the invention, the antibody that is administered
to a patient for
the treatment or prevention of a MUC1 or MUC1* positive cancer is selected for
its ability to
bind to the QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (N-10) peptide, wherein
the presence of the FPFSAQSGA (N-36) sequence is required for binding. The
antibody can
be administered alone, as a monovalent antibody, as an scFv, or a fragment of
the antibody
can be incorporated into a CAR, a BiTE or an ADC.
[00112] In one aspect of the invention, the antibody that is administered to a
patient for the
treatment or prevention of a MUC1 or MUC1* positive cancer is selected for its
inability to
recognize a linear epitope of MUC1 or MUC1*. The antibody can be administered
alone, as a
monovalent antibody, as an scFv, or a fragment of the antibody can be
incorporated into a
CAR, a BiTE or an ADC.
[00113] In one aspect of the invention, the antibody that is administered to a
patient for the
treatment or prevention of a MUC1 or MUC1* positive cancer is selected for its
ability to
recognize the MUC1 transmembrane cleavage product after it has been cleaved by
MMP9.
The antibody can be administered alone, as an antibody, a monovalent antibody,
as an scFv, a
bispecific antibody, a multi-specific antibody, or a fragment of the antibody
can be
incorporated into a BiTE, an ADC, or a CAR which can be expressed in an immune
cell.
[00114] In one aspect of the invention, the antibody that is administered to a
patient for the
treatment or prevention of a MUCI or MUC I* positive cancer is selected for
its ability to
competitively inhibit the binding of NME7AB or NME7-X1 to the extra cellular
domain of a
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MUC1 that is devoid of tandem repeats. The antibody can be administered alone,
as an
antibody, a monovalent antibody, as an scFv, a bispecific antibody, a multi-
specific antibody,
or a fragment of the antibody can be incorporated into a BiTE, an ADC, or a
CAR which can
be expressed in an immune cell..
[00115] In another aspect, the invention is directed to a method of treating
cancer in a
patient comprising administering to the patient the antibody, antibody
fragment, BiTE, ADC
or CAR expressed in an immune cell of any of the above, in combination with a
checkpoint
inhibitor.
[00116] In the method above, any of the antibodies, or variable regions
thereof, set forth in
the following may be used: MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9,
1E4, B12, B2, 117, B9, 8C7F3, or H11.
[00117] In the method above, any of the variable regions set forth in the
following may be
used:
[00118] (i) an anti-MUC1* extracellular domain antibody or anti-N-10 antibody
comprised of sequences of a humanized MN-E6 represented by humanized IgG2
heavy
chain, or humanized IgG1 heavy chain, paired with humanized Kappa light chain,
or
humanized Lambda light chain;
[00119] (ii) an antibody of (i), wherein the humanized IgG2 heavy chain is SEQ
ID
NOS:53, humanized IgG1 heavy chain is SEQ ID NO:57, humanized Kappa light
chain is
SEQ ID NO:108, and humanized Lambda light chain is SEQ ID NO:112, or a
sequence
having 90%, 95% or 98% sequence identity thereof;
[00120] (iii) an anti-MUC1* extracellular domain antibody or anti-N-10
antibody
comprised of sequences of a humanized MN-C2 represented by humanized IgG1
heavy
chain, humanized IgG2 heavy chain, paired with humanized Lambda light chain,
and
humanized Kappa light chain;
[00121] (iv) an antibody of (iii), wherein the humanized IgG1 heavy chain MN-
C2 (SEQ
ID NOS:159) or IgG2 heavy chain (SEQ ID NOS:164) paired with Lambda light
chain (SEQ
ID NO:219) or Kappa light chain (SEQ ID NO:213), or a sequence having 90%, 95%
or 98%
sequence identity thereof;
[00122] In the method above, in the CAR, the extracellular domain may be
comprised of
humanized single chain antibody fragments of MNC2, MNE6, 20A10, 3C2B1, 5C6F3,
25E6,
18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. The extracellular domain may
be
comprised of humanized single chain antibody fragments of an MN-E6 scFv set
forth as SEQ
ID NOS: 233, 235, or 237), MN-C2 scFv (SEQ ID NOS:239, 241, or 243). In the
CAR, the
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cytoplasmic tail may be comprised of one or more of signaling sequence motifs
and co-
stimulatory domains, including but not limited to CD3-zeta-1XX, CD27, CD28, 4-
1BB,
0X40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7 and CD3-zeta or
variants
1XX, X2X, XX3, 12X, or 23X. Additionally, the sequence of the intracellular
signaling
domain may contain mutations that dampen the signal to improve persistence or
target cell
killing.
[00123] The method above may include two CARs with different extracellular
domain
units transfected into the same cell. One of the extracellular domain
recognition units may
bind to MUCI* extracellular domain. One of the extracellular domain
recognition units may
bind to PD-1. One of the extracellular domain recognition units may be an
antibody fragment
and the other may be a peptide or an anti-MUC1* antibody fragment.
[00124] The method may include an immune cell transfected or transduced with a
plasmid
encoding a CAR and a plasmid encoding a non-CAR species that is expressed from
an
inducible promoter. The non-CAR species may be expressed from an inducible
promoter that
is activated by elements of an activated immune cell. The non-CAR species may
be
expressed from an NFAT inducible promoter. The NFAT may be NFATcl, NFATc3 or
NFATc2. The cleavage enzyme may be MMP2, MMP3, MMP9, MMP13, MMP14, MMP16,
ADAM10, ADAM17, or ADAM28, or a catalytically active fragment thereof. The non-
CAR
species may be a cytokine. The cytokine may be IL-7, IL-12, IL-15 or IL-18.
[00125]
Thc present invention is directed to an antibody, or fragment thereof, for
the diagnosis,
treatment or prevention of cancers wherein the antibody specifically binds to
the PSMGFR peptide
(SEQ ID NO:2) or a fragment thereof of the peptide.
[00126] The antibody binds to the N-10 peptide (SEQ ID NO:3), N-19 peptide
(SEQ ID NO:4),
N-23 peptide (SEQ ID NO:5), N-26 peptide (SEQ ID NO:6), N-30 peptide (SEQ ID
NO:7), N-10/C-5
peptide (SEQ ID NO:8), N-19/C-5 peptide (SEQ ID NO:9), or C-5 peptide (SEQ ID
NO:825).
[00127] The antibody interacts with a peptide comprising conformational
epitope SVSDV (SEQ
ID NO:1751) and FPFSA (SEQ ID NO:1747) within N-26 sequence
ISDVSVSDVPFPFSAQSGA
(SEQ ID NO:6), wherein mutation or deletion of FPFS (SEQ ID NO:1747) destroys
binding of the
antibody or fragment thereof to the N-26 peptide.
[00128] The antibody interacts with a peptide comprising conformational
epitope ASRYNLT
(SEQ ID NO:1745), SVSDV (SEQ ID NO:1751), and FPFSA (SEQ ID NO:1747) within
the N-19
sequence ASRYNLT ISDVSVSDVPFPFSAQSGA (SEQ ID NO:4), wherein mutation or
deletion of
ASRYNLT (SEQ TD NO:1745) destroys binding of the antibody or fragment thereof
to the N-26
peptide.
[00129] The antibody does not bind to the C-10 peptide (SEQ 1D NO:825).
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[00130] The antibody binds to the N-10 peptide (SEQ ID NO:3), but not to the C-
10 peptide (SEQ
ID NO:825).
[00131] The antibody inhibits interaction between NME7AB and MUC1*.
[00132] The antibody inhibits interaction between NME7AH and PSMGFR peptide
(SEQ ID
NO:2).
[00133] The antibody inhibits interaction between NME7AH and N-10 peptide (SEQ
ID NO:3), N-
19 peptide (SEQ ID NO:4), N-23 peptide (SEQ ID NO:5), N-26 peptide (SEQ ID
NO:6), N-30
peptide (SEQ ID NO:7), N-10/C-5 peptide (SEQ ID NO:8), N-19/C-5 peptide (SEQ
ID NO:9), or C-5
peptide (SEQ ID NO:825).
[00134] The antibody recognizes a MUC1 transmembrane enzymatic cleavage
product.
[00135] In the above, the cleavage enzyme is MMP14 or MMP9 or a catalytically
active fragment
thereof of the enzyme.
[00136] The antibody binds to PSMGFR (SEQ ID NO:2) or fragment thereof in
which presence of
an amino acid sequence within PSMGFR (SEQ ID NO:2) induces binding of the
antibody to the
PSMGFR.
[00137] The amino acid sequence of the binding conformationally inducing
peptide is present in
N-10 peptide (SEQ ID NO:3).
[00138] The antibody does not bind to a linear form of the binding
conformationally inducing
peptide sequence wherein the linear form of the peptide is a denatured form.
[00139] The binding conformationally inducing peptide sequence is in the N-26
peptide sequence
ISDVSVSDVPFPFSAQSGA (SEQ ID NO:6), wherein mutation or deletion of FPFS (SEQ
ID
NO:1747) destroys binding of the antibody or fragment thereof to the N-26
peptide.
[00140] The binding conformationally inducing peptide sequence is located
within the N-19
sequence ASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:4), wherein mutation or
deletion of
ASRYNLT (SEQ ID NO:1745) destroys binding of the antibody or fragment thereof
to the N-19
peptide.
[00141] The binding inducing peptide sequence may be located within the N-26
sequence
ISDVSVSDVPFPFSAQSGA (SEQ ID NO:6), wherein mutation or deletion within FPFS
(SEQ ID
NO:1747) destroys binding of the antibody or fragment thereof to PSMGFR.
[00142] The antibodies may have a consensus sequence.
[00143]
heavy chain CDR1 comprises consensus sequence at least 90% identical to
sequence:
F or I at position 1, T at position 2, F at position 3, S at position 4, T, G,
or R at position 5, Y at
position 6, A, G or T at position 7, M at position 8 and S at position 9;
[00144]
heavy Chain CDR2 comprises consensus sequence at least 90% identical to
sequence:
T at position 1, I or S at position 2, I or S at position 3, G or R at
position 5, G or A at position 6, T or
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I at position 9, Y at position 10, Y at position 11, P or S at position12 and
DSVKG for positions 13-
17;
[00145]
heavy chain CDR3 comprises consensus sequence at least 90% identical to
sequence:_ G, L, or N at position 2, G or T at position 4, Y at position 7, D
or E at position 12, A at
position 14, and Y at position 15;
[00146]
light chain CDR1 comprises consensus sequence at least 90% identical to
sequence:
K or R at position 1, A or S at position 2, S at position 3, K or Q at
position 4, S at position 5, L or V
at position 6, L at position 7, T or S at position 10, Y at position 15, and
T, L or M at position 16;
[00147]
light Chain CDR2 comprises consensus sequence at least 90% identical to
sequence:
L or W, or S at position 1, A or T at position 2, S at position 3, N or T at
position 4, L or R at position
5, E or A at position 6, and S at position 7; and
[00148]
light chain CDR3 comprises consensus sequence at least 90% identical to
sequence:
Q at position 1, H or Q at position 2, S, Q or R at position 3, R, S or Y at
position 4, E, L, or S at
position 5, L or S at position 6, P or S at position 7, F or L at position 8
and T at position 9.
[00149] An antibody binding to a conformational epitope within a peptide
having the N-26
sequence ISDVSVSDVPFPFSAQSGA (SEQ ID NO:6), wherein mutation or deletion
within FPFS
(SEQ ID NO:1747), SVSDV (SEQ ID NO:1751), or ASRYNLT (SEQ ID NO:1745) destroys
binding
of the antibody or fragment thereof to PSMGFR.
[00150] The antibody may have a further consensus sequence,
[00151] wherein
[00152]
heavy chain CDR1 comprises consensus sequence at least 90% identical to
sequence:
F or I at position 1, T or A at position 2, F at position 3, S at position 4,
T, G, or R at position 5, Y or
F at position 6, A, G or T at position 7, M at position 8 and S at position 9;
[00153]
heavy Chain CDR2 comprises consensus sequence at least 90% identical to
sequence:
T or A at position 1, I or S at position 2, I or S at position 3, N, S. T or G
at position 4, G or R at
position 5, G or A at position 6, G, T, or D at position 7, Y, K, H or S at
position 8, T or I at position
9, Y or F at position 10, Y at position U, P or S at position12 and D at
position 13, S or T at position
14, V or L at position 15 and KG for positions 16-17;
[00154]
heavy chain CDR3 comprises consensus sequence at least 90% identical to
sequence:
G, L, or N at position 2, G, T, or Y at position 3, G or T at position 4, Y at
position 7, Y, A, or G at
position 10, M, D or F at position 11, D or E at position 12 and AY at
position 14-15;
[00155]
light chain CDR1 comprises consensus sequence at least 90% identical to
sequence:
K or R at position 1, A or S at position 2, S or R at position 3, S, Y, I or V
at position 8, T or S at
position 10, G, S, D, or Q at position 12, V, Y, K or N at position 13, N, S,
or T at position 14, Y or F
at position 15, and I, L or M at position 16;
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[00156] light Chain CDR2 comprises consensus sequence at least
90% identical to sequence:
A, T or V at position 2, S at position 3, N, T, or K at position 4, L or R at
position 5, E, A, F or D at
position 6, and S at position 7; and
[00157] light chain CDR3 comprises consensus sequence at least
90% identical to sequence:
Q, F or W at position 1, H or Q at position 2, R, S, T, Y or N at position 4,
E, L, S or H at position 5,
L, S, V, D or Y at position 6, P or S at position 7, and T at position 9.
[00158] The antibody above which may be MNC2, having
[00159] heavy chain CDR1 comprises consensus sequence
FTFSGYAMS;
[00160] heavy Chain CDR2 comprises consensus sequence
TTSSGGTYTYYPDSVKG;
[00161] heavy chain CDR3 comprises consensus sequence -
LGGDNYYEYFDV--;
[00162] light chain CDR1 comprises consensus sequence RASKS--
VSTSGYSYMH;
[00163] light Chain CDR2 comprises consensus sequence LASNLES;
and
[00164] light chain CDR3 comprises consensus sequence
QHSRELPFT.
[00165] MNE6, having
[00166] heavy chain CDR1 comprises consensus sequence
FTFSRYGMS;
[00167] heavy Chain CDR2 comprises consensus sequence
TISGGGTYIYYPDSVKG;
[00168] heavy chain CDR3 comprises consensus sequence
DNYGRNYDYGMDY--;
[00169] light chain CDR1 comprises consensus sequence
SATSSVSYIH;
[00170] light Chain CDR2 comprises consensus sequence STSNLAS;
and
[00171] light chain CDR3 comprises consensus sequence
QQRSSSPFT.
[00172] B2, having
[00173] heavy chain CDR1 comprises consensus sequence
FAFSTFAMS;
[00174] heavy Chain CDR2 comprises consensus sequence
AISNGGGYTYYPDTLKG;
[00175] heavy chain CDR3 comprises consensus sequence ----
RYYDLYFDL--;
[00176] light chain CDR1 comprises consensus sequence RSSQNIV-
HSNGNTYLE;
[00177] light Chain CDR2 comprises consensus sequence KVSNRFS;
and
[00178] light chain CDR3 comprises consensus sequence
FQDSHVPLT.
[00179] B7, having
[00180] heavy chain CDR1 comprises consensus sequence
FTFSRYGMS;
[00181] heavy Chain CDR2 comprises consensus sequence
TISSGGTYIYYPDSVKG;
[00182] heavy chain CDR3 comprises consensus sequence
DNYGSSYDYAMDY--;
[00183] light chain CDR1 comprises consensus sequence RSSQTIV-
HSNGNTYLE;
[00184] light Chain CDR2 comprises consensus sequence KVSNRFS;
and
[00185] light chain CDR3 comprises consensus sequence
FQDSHVPLT.
[00186] B9, having
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[00187] heavy chain CDR1 comprises consensus sequence
FTFSRYGMS;
[00188] heavy Chain CDR2 comprises consensus sequence
TISSGGTYIYYPDSVKG;
[00189] heavy chain CDR3 comprises consensus sequence
DNYGSSYDYAMDY--;
[00190] light chain CDR1 comprises consensus sequence S
ASSSVSYMH;
[00191] light Chain CDR2 comprises consensus sequence TTSNLAS;
and
[00192] light chain CDR3 comprises consensus sequence QQRSSYPF-
.
[00193] 8C7F3, having
[00194] heavy chain CDR1 comprises consensus sequence
FTFSTYAMS;
[00195] heavy Chain CDR2 comprises consensus sequence
AISNGGGYTYYPDSLKG;
[00196] heavy chain CDR3 comprises consensus sequence RYYDHYFDY-
-;
[00197] light chain CDR1 comprises consensus sequence --
RASESVATYGNNFMQ;
[00198] light Chain CDR2 comprises consensus sequence LASTLDS;
and
[00199] light chain CDR3 comprises consensus sequence
QQNNEDPPT.
[00200] H11, having
[00201] heavy chain CDR1 comprises consensus sequence
FAFSTFAMS;
[00202] heavy Chain CDR2 comprises consensus sequence
AISNGGGYTYYPDTLKG;
[00203] heavy chain CDR3 comprises consensus sequence RYYDLYFDL-
-;
[00204] light chain CDR1 comprises consensus sequence RSSQNIV-
HSNGNTYLE;
[00205] light Chain CDR2 comprises consensus sequence KVSNRFS;
and
[00206] light chain CDR3 comprises consensus sequence
FQDSHVPLT.
[00207] B12, having
[00208] heavy chain CDR1 comprises consensus sequence SYGVH;
[00209] heavy Chain CDR2 comprises consensus sequence
VIWPGGSTNYNSTLMSRM;
[00210] heavy chain CDR3 comprises consensus sequence
DRTPRVGAWFAY; and
[00211] light chain CDR1 comprises consensus sequence
RASESVATYGNNFMQ;
[00212] light Chain CDR2 comprises consensus sequence LASTLDS;
and
[00213] light chain CDR3 comprises consensus sequence
QQNNEDPPT.
[00214] 20A10, having
[00215] heavy chain CDR1 comprises consensus sequence
FTFSTYAMS;
[00216] heavy Chain CDR2 comprises consensus sequence -
SIGRAGSTYYSDSVKG;
[00217] heavy chain CDR3 comprises consensus sequence ---
GPIYNDYDEFAY;
[00218] light chain CDR1 comprises consensus sequence
KSSQSVLYSSNQKNYLA;
[00219] light Chain CDR2 comprises consensus sequence WASTRES;
and
[00220] light chain CDR3 comprises consensus sequence HQYLSSLT.
[00221] 3C2B1, having
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[00222] heavy chain CDR1 comprises consensus sequence
ITFSTYTMS;
[00223] heavy Chain CDR2 comprises consensus sequence
TISTGGDKTYYSDSVKG;
[00224] heavy chain CDR3 comprises consensus sequence -
GTTAMYYYAMDY;
[00225] light chain CDR1 comprises consensus sequence
RASKS¨ISTSDYNYTH ;
[00226] light Chain CDR2 comprises consensus sequence LASNLES;
and
[00227] light chain CDR3 comprises consensus sequence
QHSRELPLT.
[00228]
In another aspect, the invention is directed to an antibody, or fragment
thereof, for the
diagnosis, treatment or prevention of cancers that requires presence of
antibody binding
conformationally inducing peptide ASRYNLT (SEQ ID NO:1745) of PSMGFR (SEQ ID
NO:2). The
antibody may be 25E6, having
[00229] heavy chain CDR1 comprises consensus sequence
FTFSSYGMS;
[00230] heavy Chain CDR2 comprises consensus sequence
TISNGGRHTFYPDSVKG;
[00231] heavy chain CDR3 comprises consensus sequence
QTGTEGWFAY;
[00232] light chain CDR1 comprises consensus sequence
KSSQSLLDSDGKTYLN;
[00233] light Chain CDR2 comprises consensus sequence LVSKLDS ;
and
[00234] light chain CDR3 comprises consensus sequence
WQGTHFPQT.
[00235]
In another aspect, the invention is directed to an antibody, or fragment
thereof, for the
diagnosis, treatment or prevention of cancers that requires presence of
antibody binding
conformationally inducing peptide SVSDV (SEQ ID NO:1761) of PSMGFR (SEQ ID
NO:2). The
antibody may be 5C6F3, having
[00236] heavy chain CDR1 comprises consensus sequence FTFSTYAMS
;
[00237] heavy Chain CDR2 comprises consensus sequence
AISNGGGYTYYPDSLKG;
[00238] heavy chain CDR3 comprises consensus sequence
RYYDHYFDY;
[00239] light chain CDR1 comprises consensus sequence
RSSQTTVHSNGNTYLE;
[00240] light Chain CDR2 comprises consensus sequence KVSNRFS;
and
[00241] light chain CDR3 comprises consensus sequence
FQDSHVPLT.
[00242] The antibody or fragment thereof according all of the above may be
murine,
camelid, human or humanized. The antibody fragment may be scFv or scFv-Fc,
which
variable regions thereof may be murine, camelid, human or humanized.
[00243] In another aspect, the invention is directed to a chimeric antigen
receptor (CAR)
comprising the antibody fragments of above, and may further comprise mutations
in the co-
stimulatory domain or mutations or deletions of one or two of the ITAMs of the
CD3-zeta
signaling domain. Tyrosines may be mutated in CD28 or 4-1BB. CD3-zeta may
contain a
single ITAM such as only ITAM1 also known as 1XX, ITAM2 also known as X2X, or
ITAM3 also known as XX3. In another aspect, CD3-zeta may contain two ITAMs,
wherein
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the positions of the ITAMs may be moved to a more proximal position such as
12X or 23X
(Feucht et al 2019). In yet another aspect, tyrosines of one or two ITAMs may
be mutated to
dampen signaling. In a preferred embodiment, the CD3-zeta domain is 1XX. An
example of
1XX mutations, includes those exemplified in SEQ ID NOS: 1796-1797.
[00244] In another aspect, the invention is directed to an immune cell
comprising the CAR
of above. Immune cell may be T cell, NK cell, dendritic cell, or mast cell. In
one aspect the
immune cell is derived from a stem cell that has been directed to
differentiate to that immune
cell type in vitro. In another aspect, a CAR containing sequences of the
antibody are
expressed in a stem cell, which then may be differentiated into an immune
cell.
[00245] In another aspect, the invention is directed to a cell composition
expressed in a
cell comprising a CARs of above, and second entity having a biological
recognition unit that
has a specificity that is different from that of the CAR. The second entity
may bind PD-1,
PDL-1, or other checkpoint inhibitor, or NME7, or a cytokine such as IL-12 or
IL-18, or c-
Jun.
[00246] In yet another aspect, the invention is directed to an immune cell
engineered to
express a nucleic encoding a CAR of above and a nucleic acid encoding a second
entity as in
any of the claims above wherein the second entity expressed from an inducible
promoter.
The second entity may be expressed from an inducible promoter that is
activated by elements
of an activated immune cell. The second entity may be expressed from an NFAT
inducible
promoter. N1-01'1 may be INFATc 1, NFA1'c3 or NFA1c2. The second entity may be
a
cytokine such as IL-7, IL-15, or IL-18. The nucleic acids encoding the second
entity may be
inserted into a Foxp3 promoter or enhancer region, wherein the cytokine is IL-
18. The
cytokine may be expressed from an NFAT inducible promoter.
[00247] In another aspect, the invention is directed to a BiTE construct
comprising the
antibody fragment of above.
[00248] In yet another aspect, the invention is directed to an antibody drug
conjugate
(ADC) comprising the antibody or antibody fragment of above.
[00249] The present invention is directed to an antibody or fragment thereof
that
specifically:
[00250] (i) binds to PSMGFR (SEQ ID NO:2) and N-10 (SEQ ID NO:3); and
[00251] does not bind to full-length MUCl;
[00252] (ii) does not bind to C-10 (SEQ ID NO:825);
[00253] (iii) competitively inhibits binding of NME1 or N1VIE7An to MUC1*
extra cellular
domain or a PSMGFR peptide;
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[00254] (iv) recognizes a MUC1* generated by cleavage by a cleavage enzyme;
[00255] (v) recognizes a conformational epitope and not a linear epitope; or
[00256] (vi) is cancer selective by immunohistochemistry on tissues.
[00257] Four of the criteria (i) ¨ (vi) may be satisfied. Five of the criteria
(i) ¨ (vi) may be
satisfied. Six of the criteria (i) ¨ (vi) may be satisfied. At least criteria
(vi) may be satisfied.
Cleavage enzyme may be MMP-9.
[00258] In all of the above, the cancer may be breast cancer, pancreatic
cancer, ovarian
cancer, lung cancer, colon cancer, gastric cancer or esophageal cancer.
[00259] The present invention is also directed to a method of diagnosing,
treating or
preventing cancer by administering the antibodies and fragments disclosed
herein to a cancer
patient in need thereof that has been identified as expressing MUC1 aberrantly
and
expressing truncated MUC1, such as MUC1*.
[00260] These and other objects of the invention will be more fully understood
from the
following description of the invention, the referenced drawings attached
hereto and the
claims appended hereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[00261] The patent or application file contains at least one drawing executed
in color.
Copies of this patent or patent application publication with color drawings
will be provided
by the Office upon request and payment of the necessary fee.
[00262] The present invention will become more fully understood from the
detailed
description given herein below, and the accompanying drawings which are given
by way of
illustration only, and thus are not limitative of the present invention, and
wherein;
[00263] Figures 1A-ID show cell growth assay graphs of MUC1* positive cells
treated
with either bivalent `by' anti-MUC1* antibody, monovalent 'my' or Fab, NM23-H1
dimers
or NME7-AB. Bivalent anti-MUC1* antibodies stimulate growth of cancer cells
whereas the
monovalent Fab inhibits growth (Fig. 1A-1B). Classic bell-shaped curve
indicates ligand
induced dimerization stimulates growth. Dimeric NM23-H1, aka NME1, stimulates
growth
of MUC1* positive cancer cells but siRNA to suppress MUC1 expression eliminate
its effect
(Fig. 1C). NME7-AB also stimulates the growth of MUC1* positive cells (Fig.
1D).
[00264] Figures 2A-2I show results of ELISA assays. MUC1* peptides PSMGFR,
PSMGFR minus 10 amino acids from the N-terminus aka N-10, or PSMGFR minus 10
amino
acids from the C-terminus, aka C-10 are immobilized on the plate and the
following are
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assayed for binding: NME7-AB (Fig. 2A), MNC2 monoclonal antibody (Fig. 2B),
MNE6
monoclonal antibody (Fig. 2C), or dimeric NME1 (Fig. 2D). These assays show
that NME1,
NME7-AB and monoclonal antibodies MNC2 and MNE6 all require the first membrane
proximal 10 amino acids of the MUC1* extracellular domain to bind. MUC1*
peptides
PSMGFR minus 10 amino acids from the N-terminus aka N-10, or PSMGFR minus 10
amino
acids from the C-terminus, aka C-10, are immobilized on the plate and the
following are
assayed for binding: MNC3 (Fig. 2E) and MNC8 (Fig. 2F). Fig. 2G shows the
amino acid
sequence of the PSMGFR peptide. Fig. 2H shows the amino acid sequence of the N-
10
peptide. Fig. 21 shows the amino acid sequence of the C-10 peptide.
[00265] Figures 3A-3C show results of competitive ELISA assays. The PSMGFR
MUC1* peptide is immobilized on the plate and dimeric NM23-H1, aka NME1, is
added
either alone or after the MNE6 antibody has been added (Fig. 3A). The same
experiment was
performed wherein NM23-H7, NME7-AB, is added alone or after MNE6 has been
added
(Fig. 3B). Results show that MNE6 competitively inhibits the binding of MUC1*
activating
ligands NME1 and NME7. In a similar experiment (Fig. 3C), PSMGFR or PSMGFR
minus
amino acids from the N-terminus, aka N-10, is immobilized on the plate.
Dimeric NM23-
H1 is then added. Anti-MUC1* antibodies MNE6, MNC2, MNC3 or MNC8 are then
tested
for their ability to compete off the NM23-H1. Results show that although all
three antibodies
bind to the PSMGFR peptides, MNE6 and MNC2 competitively inhibit binding of
the
M1JC1* activating ligands.
[00266] Figures 4A-4F show FACS scans of anti-MUC1* antibody huMNC2scFv
binding
specifically to MUC1* positive cancer cells and MUC1* transfected cells but
not MUC1* or
MUC1 negative cells. ZR-75-1, aka 1500, MUC1* positive breast cancer cells
were stained
with 1:2 or 1:10 dilutions of the 1.5 ug/ml humanized MNC2. After two washes,
cells were
stained with secondary antibody, Anti-Penta-His antibody conjugated to Alexa
488 (Qiagen)
dilutions of 1:200 (Fig. 4A), 1:50 (Fig. 4B), or 1:10 (Fig. 4C) to detect the
6x His tag on the
huMNC2 scFv. Fig. 4A shows huMNC2 binding to ZR-75-1 breast cancer cells where
secondary antibody is added at a 1:200 dilution. Fig. 4B shows huMNC2 binding
to ZR-75-1
breast cancer cells where secondary antibody is added at a 1:50 dilution. Fig.
4C shows
huMNC2 binding to ZR-75-1 breast cancer cells where secondary antibody is
added at a 1:10
dilution. Flow cytometric analysis revealed a concentration-dependent shift of
a subset of
cells, indicating specific binding, which is unseen in the absence of the MNC2
scFv (Fig. 4A-
4C). Fig. 4D shows anti-MUC1* antibody MNE6 staining of MUC1 negative HCT-116
colon cancer cells transfected with the empty vector, single cell clone #8.
Fig. 4E shows anti-
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MUC1* antibody MNE6 staining of HCT-116 colon cancer cells transfected with
MUC1*
single cell clone #10. Fig. 4F shows anti-MUC1* antibody MNE6 staining of ZR-
75-1, aka
1500, MUC1* positive breast cancer cells. As the FACS scans show, both MNC2
and MNE6
only stain MUC1* positive cells and not MUC1 or MUC1* negative cells.
[00267] Figure 5 shows a graph of an ELISA in which surface is coated with
either the
MUC1* PSMGFR peptide or a control peptide. Humanized MNC2 scFv is then
incubated
with the surface, washed and detected according to standard methods. The ELISA
shows that
the huMNC2 scFv binds to the MUC1* peptide with an EC-50 of about 333nM.
[00268] Figures 6A-6B show graphs of cancer cell growth inhibition by MUC1*
antibody
variable region fragment humanized MNC2 scFv hMNC2 scFv potently inhibited the
growth
of ZR-75-1, aka 1500, MUC1* positive breast cancer cells (Fig. 6A) and T47D
MUC1*
positive breast cancer cells (Fig. 6B) with approximately the same EC-50 as
the in vitro
ELISAs.
[00269] Figures 7A-7B show graphs of tumor growth in immune compromised mice
that
have been implanted with human tumors then treated with anti-MUC1* antibody
MNE6 Fab
or mock treatment. Female nu/nu mice implanted with 90-day estrogen pellets
were
implanted with 6 million T47D human breast cancer cells that had been mixed
50/50 with
Matrigel. Mice bearing tumors that were at least 150 mm3 and had three
successive increases
in tumor volume were selected for treatment. Animals were injected sub
cutaneously twice
per week with 80 mg/kg MNE6 Fab and an equal number of mice fitting the same
selection
criteria were injected with vehicle alone (Fig. 7A). Male NOD/SCID mice were
implanted
with 6 million DU-145 human prostate cancer cells that had been mixed 50/50
with Matrigel.
Mice bearing tumors that were at least 150 min3 and had three successive
increases in tumor
volume were selected for treatment. Animals were injected sub-cutaneously
every 48 hours
with 160 mg/kg MNE6 Fab and an equal number of mice fitting the same selection
criteria
were injected with vehicle alone (Fig. 7B). Tumors were measured independently
by two
researchers twice per week and recorded. Statistics were blindly calculated by
independent
statistician, giving a P value of 0.0001 for each. Anti-MUC1* Fab inhibited
breast cancer
growth and prostate cancer growth. Treatment had no effect on weight, bone
marrow cell
type or number.
[00270] Figure 8 shows a graph of an ELISA wherein the surface was immobilized
with
either PSMGFR peptide, PSMGFR minus 10 amino acids from the N-terminus or
minus 10
amino acids from the C-terminus. The huMNE6 scFv-Fc bound to the PSMGFR
peptide and
to the PSMGFR N-10 peptide but not to the PSMGFR C-10 peptide. The parent MNE6
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antibody and the humanized MNE6 require the C-terminal 10 amino acids of
PSMGFR for
binding.
[00271] Figures 9A-9B show graphs of EL1SAs wherein the assay plate surface
was
immobilized with either PSMGFR peptide, PSMGFR minus 10 amino acids from the N-
terminus or minus 10 amino acids from the C-terminus. The MNC3 antibody
variants were
then assayed for binding to the various MUC1* peptides. Fig. 9A shows purified
mouse
monoclonal MNC3 antibody; and Figure 9B shows the humanized MNC3 scFv-Fc.
EL1SAs
show binding to the PSMGFR peptide as well as to certain deletion peptides.
[00272] Figures 10A-10J. Figs. 10A10B are photographs of breast cancer tissue
arrays.
Fig. 10A was stained with VU4H5 which recognizes MUCl-FL (full length); Fig.
10B was
stained with mouse monoclonal antibody MNC2 which recognizes cancerous MUC1*.
Following automated staining (Clarient Diagnostics), the tissue staining was
scored using
Allred scoring method which combines an intensity score and a distribution
score. Figs.
10C1OF are color coded graphs showing the score calculated for MUC1 full-
length staining
for each patient's tissue. Figs. 10G10J are color coded graphs showing the
score calculated
for MUC1* staining for each patient's tissue.
[00273] Figures 11A-11J. Figs. 11A11B are photographs of breast cancer tissue
arrays.
Fig. 11A was stained with VU4H5 which recognizes MUC1-FL (full length); Fig.
11B was
stained with mouse monoclonal antibody MNC2 which recognizes cancerous MUC1*.
Following automated staining (Clarient Diagnostics), the tissue staining was
scored using
Allred scoring method which combines an intensity score and a distribution
score. Figs. 11C-
11F are color coded graphs showing the score calculated for MUC1 full-length
staining for
each patient's tissue. Figs. 11G-11J are color coded graphs showing the score
calculated for
MUC1* staining for each patient's tissue.
[00274] Figures 12A-12H show photographs of normal breast and breast cancer
tissues
stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 2.5
ug/mL,
then stained with a secondary streptavidin HRP antibody. Fig. 12A is a normal
breast tissue.
Figs. 12B-12D are breast cancer tissues from patients as denoted in the
figure. Figs. 12E-12H
are photographs of the corresponding serial sections that were stained with
the secondary
antibody alone.
[00275] Figures 13A-13F show photographs of normal breast and breast cancer
tissues
stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 2.5
ug/mL,
then stained with a secondary streptavidin HRP antibody. Fig. 13A is a normal
breast tissue.
Figs. 13B-13C are breast cancer tissues from patients as denoted in the
figure. Figs. 13D-13F
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are photographs of the corresponding serial sections that were stained with
the secondary
antibody alone.
[00276] Figures 14A-14H show photographs of breast cancer tissues stained with
MNE6
anti-MUC1* antibody at 10 ug/mL, then stained with a rabbit anti mouse
secondary HRP
antibody. Figs. 14A-14D are breast cancer tissues from patient #300. Figs. 14E-
14H are
breast cancer tissues from metastatic patient #291.
[00277] Figures 15A-15F show photographs of normal lung and lung cancer
tissues
stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 2.5
ug/mL,
then stained with a secondary streptavidin HRP antibody. Fig. 15A is a normal
lung tissue.
Figs_ 15B15C are lung cancer tissues from patients as denoted in the figure_
Figs. 15D-15F
are photographs of the corresponding serial sections that were stained with
the secondary
antibody alone.
[00278] Figures 16A-16F show photographs of normal lung and lung cancer
tissues
stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 2.5
ug/mL,
then stained with a secondary streptavidin HRP antibody. Fig. 16A is a normal
lung tissue.
Figs. 16B16C are lung cancer tissues from patients as denoted in the figure.
Figs. 16D-16F
are photographs of the corresponding serial sections that were stained with
the secondary
antibody alone.
[00279] Figures 17A-17F show photographs of normal lung and lung cancer
tissues
stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 25
ug/mL, then
stained with a secondary streptavidin HRP antibody. Fig. 17A is a normal lung
tissue. Figs.
17B-17C are lung cancer tissues from patients as denoted in the figure. Figs.
17D-17F are
photographs of the corresponding serial sections that were stained with the
secondary
antibody alone.
[00280] Figures 18A-18F show photographs of normal lung and lung cancer
tissues
stained with humanized MNE6-scFv-Fc biotinylated anti-MUC1* antibody at 25
ug/mL, then
stained with a secondary streptavidin HRP antibody. Fig. 18A is a normal lung
tissue. Figs.
18B-18C are lung cancer tissues from patients as denoted in the figure. Figs.
18D-18F are
photographs of the corresponding serial sections that were stained with the
secondary
antibody alone.
[00281] Figures 19A-19D show photographs of normal small intestine and
cancerous
small intestine tissues stained with humanized MNE6-scFv-Fc biotinylated anti-
MUC1*
antibody at 5 ug/mL, then stained with a secondary streptavidin HRP antibody.
Fig. 19A is a
normal small intestine tissue. Fig. 19B is small intestine cancer from patient
as denoted in the
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figure. Figs. 19C-19D are photographs of the corresponding serial sections
that were stained
with the secondary antibody alone.
[00282] Figures 20A-20H show photographs of normal small intestine tissues
stained with
humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a
secondary
goat-anti-human HRP antibody. Figs. 20A-20D are normal small intestine tissue.
Figs. 20E-
20H are photographs of the corresponding serial sections that were stained
with the
secondary antibody alone.
[00283] Figures 21A-21H show photographs of cancerous small intestine tissues
stained
with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with
a
secondary goat-anti-human HRP antibody. Figs. 21A-21D are cancerous small
intestine
tissue from a patient as denoted in figure. Figs. 21E-21H are photographs of
the
corresponding serial sections that were stained with the secondary antibody
alone.
[00284] Figures 22A-22H show photographs of cancerous small intestine tissues
stained
with humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with
a
secondary goat-anti-human HRP antibody. Figs. 22A-22D are cancerous small
intestine
tissue from a patient as denoted in figure. Figs. 22E-22H are photographs of
the
corresponding serial sections that were stained with the secondary antibody
alone.
[00285] Figures 23A-23H show photographs of normal colon tissues stained with
humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a
secondary
goat-anti-human HRP antibody. Figs. 23A-23D are normal colon. Figs. 23E-23H
are
photographs of the corresponding serial sections that were stained with the
secondary
antibody alone.
[00286] Figures 24A-24H show photographs of colon cancer tissues stained with
humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a
secondary
goat-anti-human HRP antibody. Figs. 24A-24D are colon cancer tissue from a
metastatic
patient as denoted in figure. Figs. 24E-24H are photographs of the
corresponding serial
sections that were stained with the secondary antibody alone.
[00287] Figures 25A-25H show photographs of colon cancer tissues stained with
humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a
secondary
goat-anti-human HRP antibody. Figs. 25A-25D are colon cancer tissue from a
Grade 2
patient as denoted in figure. Figs. 25E-25H are photographs of the
corresponding serial
sections that were stained with the secondary antibody alone.
[00288] Figures 26A-26H show photographs of colon cancer tissues stained with
humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a
secondary
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goat-anti-human HRP antibody. Figs. 26A-26D are colon cancer tissue from a
metastatic
patient as denoted in figure. Figs. 26E-26H are photographs of the
corresponding serial
sections that were stained with the secondary antibody alone.
[00289] Figures 27A-27H show photographs of prostate cancer tissues stained
with
humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a
secondary
goat-anti-human HRP antibody. Figs. 27A-27D are prostate cancer tissue from a
patient as
denoted in figure. Figs. 27E-27H are photographs of the corresponding serial
sections that
were stained with the secondary antibody alone.
[00290] Figures 28A-28H show photographs of prostate cancer tissues stained
with
humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a
secondary
goat-anti-human HRP antibody. Figs. 28A-28D are prostate cancer tissue from a
patient as
denoted in figure. Figs. 28E-28H are photographs of the corresponding serial
sections that
were stained with the secondary antibody alone.
[00291] Figures 29A-29H show photographs of prostate cancer tissues stained
with
humanized MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a
secondary
goat-anti-human HRP antibody. Figs. 29A-29D are prostate cancer tissue from a
patient as
denoted in figure. Figs. 29E-29H are photographs of the corresponding serial
sections that
were stained with the secondary antibody alone.
[00292]
[00293] Figures 30A-30F show photographs of a triple negative breast cancer
array
stained with anti-MUC1* antibody huMNC2scFv. The first score shown is the
Allred score
and the second is the tumor grade. The percentage of the array that scored
zero, weak,
medium or strong is graphed as a pie chart. Fig. 30A shows the pie chart of
score of anti-
MUC1* antibody staining. Fig. 30B shows a photograph of the array stained with
the
antibody. Figs. 30C-30D show magnified photographs of two of the breast cancer
specimens
from the array. Figs. 30E-30F show more magnified photographs of the portion
of the
specimen that is marked by a box.
[00294] Figures 31A-31F show photographs of an ovarian cancer array stained
with anti-
MUC1* antibody huMNC2scFv. The first score shown is the Allred score and the
second is
the tumor grade. The percentage of the array that scored zero, weak, medium or
strong is
graphed as a pie chart. Fig. 31A shows the pie chart of score of anti-MUC1*
antibody
staining. Fig. 31B shows a photograph of the array stained with the antibody.
Figs. 31C-31D
show magnified photographs of two of the breast cancer specimens from the
array. Figs. 31E-
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31F show more magnified photographs of the portion of the specimen that is
marked by a
box.
[00295] Figures 32A-32F show photographs of a pancreatic cancer array stained
with
anti-MUC1* antibody huMNC2scFv. The first score shown is the Allred score and
the
second is the tumor grade. The percentage of the array that scored zero, weak,
medium or
strong is graphed as a pie chart. Fig. 32A shows the pie chart of score of
anti-MUC1*
antibody staining. Fig. 32B shows a photograph of the array stained with the
antibody. Figs.
32C-32D show magnified photographs of two of the breast cancer specimens from
the array.
Figs. 32E-32F show more magnified photographs of the portion of the specimen
that is
marked by a box.
[00296] Figures 33A-33F show photographs of a lung cancer array stained with
anti-
MUC1* antibody huMNC2scFv. The first score shown is the Allred score and the
second is
the tumor grade. The percentage of the array that scored zero, weak, medium or
strong is
graphed as a pie chart. Fig. 33A shows the pie chart of score of anti-MUC1*
antibody
staining. Fig. 33B shows a photograph of the array stained with the antibody.
Figs. 33C-33D
show magnified photographs of two of the breast cancer specimens from the
array. Figs. 33E-
33F show more magnified photographs of the portion of the specimen that is
marked by a
box.
[00297] Figures 34A-341 show photographs of normal tissues stained with anti-
MUC1*
antibody huMNC2scFv.
[00298] Figures 35A-35D show FACS scans of cells expressing either no MUC1,
MUC1*
or full-length MUC1, wherein the cells were probed with either MNC2 or VU4H5.
Fig. 35A
shows MUC1 negative HCT-116 colon cancer cells probed with antibody MNC2. Fig.
35B
shows HCT cells that have been transfected with MUC1* wherein the extra
cellular domain
is just the sequence of the PSMGFR peptide wherein the cells are probed with
antibody
MNC2. Fig. 35C shows HCT-MUC1-18 cells which are a cleavage resistant single
cell clone
of HCT cells transfected with full-length MUC1, also referred to herein as HCT-
MUC I-
41TR, and cells were probed with antibody MNC2. Fig. 35D shows HCT-MUC1-18
cells
probed with antibody VU4H5 which is an antibody that recognizes the hundreds
of tandem
repeats epitopes in full-length MUC1. As can be seen in the figures, MNC2
recognizes an
ectopic epitope that is not accessible in full-length MUC1.
[00299] Figures 36A-36D show Western blots and corresponding FACs analysis of
HCT-
116 cells which are a MUC1 negative colon cancer cell line, that were then
stably transfected
with either MUC1* or MUC1 full-length. The single cell clones that are shown
are HCT-
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MUC I-41TR, and HCT-MUC1. Fig. 36A shows a Western blot of the parent cell
line HCT-
116, HCT-MUC1-41TR and HCT-MUC1* wherein the gel has been probed with a rabbit
polyclonal antibody, SDIX, that only recognizes cleaved MUCL A visible band
between 25
and 35kDa can be readily seen in Lane 6, loaded with HCT-MUCI*, whereas there
is only a
faint band in Lanes 4 and 5, showing that only a small amount of MUC1 is
cleaved in the
HCT-MUC1-41Tr cells. There is no cleaved MUC1 present in the parent cell line
HCT-116
loaded into Lanes 2 and 3. Fig. 36B is a Western blot that was probed with a
mouse
monoclonal antibody VU4H5 that recognizes the tandem repeats of full-length
MUC1. As
can be seen, only HCT-MUC1-41TR contains full-length MUC1. Fig. 36C shows FACS
scans showing that HCT-MUCI* is 95.7% positive for SDIX which only binds to
MUC1*
and essentially not at all for MUC1 full-length. Fig. 36D shows FACS scans
that show that
HCT-MUC1-41TR cells are 95% positive for full-length MUC1 and only about 11%
positive
for the cleaved form, MUC1*.
[00300] Figure 37A-37C shows western blots and a bar graph of FACS analysis
assessing
the ability of MNC2to recognize a full-length MUC1 after it has been cleaved
by MMP9. Fig.
37A shows a Western blot of HCT-MUC1-18 cells, which are a cleavage resistant
cell line, to
which was added cleavage enzyme MMP9. The cell lysate fraction was run on a
gel and
probed with a polyclonal anti-PSMGFR antibody. The photo shows that in a dose
dependent
manner, MMP9 cleaved MUC1 to MUC1*, the ¨25kDa species. Fig. 37B shows the
Western
blot of the conditioned media from the same experiment. The photo shows that
the addition
of cleavage enzyme MMP9, in a dose dependent manner, increased the release of
the tandem
repeat domain into the conditioned media. Fig. 37C shows FACS analysis of the
experiment.
The graphs show that the addition of MMP9, in a dose dependent manner,
increased
recognition of the cleavage product by anti-MUC1* antibody MNC2 and decreased
the
recognition of the full-length MUC1 which contains the tandem repeat domain.
[00301] Figure 38 shows a photograph of a Western blot in which HCT-MUC1-18
cells,
labeled here as HCT-18, a cleavage resistant single cell clone of HCT cells
transfected with
full-length MUC1, are treated with varying amounts of a catalytically active
ADAM17 or
MMP14. Shed MUC1 tandem repeat domain of full-length MUC1 is
immunoprecipitated
from the conditioned media, and run on a gel that is then probed with VU4H5
that binds to
the tandem repeat epitopes. As can be seen, MMP14 also efficiently cleaves
MUC1 full-
length and sheds the tandem repeat containing extra cellular domain into the
conditioned
media. Cleavage enzyme ADAM17 did not cleave MUC1.
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[00302] Figure 39A-39B shows fluorescence activated cell sorting (FACS)
measurements
of human CD34+ hematopoietic stem cells of human bone marrow stained with anti-
MUC1*
monoclonal antibodies MNC3, MNC2, MNE6 or an isotype control antibody. The
histogram
of the FACS assay and the bar graph showing the data show that the MUC1*
positive cells of
the bone marrow are recognized by one anti-MUC1* antibody, MNC3 but not by
MNE6 or
MNC2. All three antibodies bind to the PSMGFR peptide. The great difference in
the
specificity of these antibodies suggests that MNC3 recognizes a MUC1*-like
form created
when MUC1 is cleaved by an enzyme that is different from MMP9.
[00303] Figure 40A-40G shows the details of FACS analysis of the hematopoietic
stem
cells probed with either MNC3 or MNE6. Fig. 40A shows the FACS scatter plot of
total bone
marrow cells. Fig. 40B shows the FACS scatter plot of the CD34+ cells. Fig.
40C shows the
FACS histogram of the CD34+ cells. Fig. 40D shows the FACS scatter plot of the
earliest
hematopoietic stem cells, which are CD34+/CD38-, stained with either MNC3 or
MNE6. Fig.
40E shows the histogram of the experiment. Fig. 40F shows the histogram
overlay of MNC3
binding to CD34 /CD38- cells versus MNE6. Fig. 40G shows the bar graph of that
FACS
experiment.
[00304]
Figure 41A-42H shows the details of FACS analysis of CD34+/CD3871
hematopoietic stem cells probed with a polyclonal anti-PSMGFR antibody SDIX,
MNE6 or
MNC2. Fig. 41A shows the FACS scatter plot of the CD34+/CD3871 population of
cells. Fig.
41E shows a table of the detailed analysis. Fig. 41B shows the FACS scatter
plot of the
CD34+/CD3871 population of cells probed with the anti-PSMGFR polyclonal
antibody
SDDC. Fig. 41F shows a table of the detailed analysis. Fig. 41C shows the FACS
scatter plot
of the CD34-F/CD38-1 population of cells probed with MNE6. Fig. 41G shows a
table of the
detailed analysis. Fig. 41D shows the FACS scatter plot of the CD34-F/CD38-/1"
population of
cells probed with MNC2. Fig. 41H shows a table of the detailed analysis.
[00305] Figure 42A-42H shows photographs of DU145 prostate cancer cells or
T47D
breast cancer cells that have been treated with either the Fab of anti-MUC1*
antibody MNC2,
MNE6, MNC3 or MNC8. The images show that cancer specific antibodies MNC2 and
MNE6 effectively kill prostate and breast cancer cells while the monoclonal
antibodies
MNC3 and MNC8 do not.
[00306] Figure 43 shows a graph of a PCR experiment comparing expression of a
wide
range of cleavage enzymes expressed in different cells lines, wherein the
values have been
normalized to those expressed in breast cancer cell line T47D. Cell lines that
are compared
are prostate cancer cell line DU145, HCT-MUC1-41TR that is a MUC1 negative
colon
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cancer cell line transfected with a MUC1 whose extracellular domain is
truncated after 41
tandem repeat units and that is not cleaved to the MUC1* form, T47D breast
cancer cell line
and CD34+ bone marrow cells.
[00307] Figure 43 shows a graph of a PCR experiment in which the expression
levels of
various cleavage enzymes are measured in DU145 prostate cancer cells,
HCT116+MUC1FL,
also known as HCT-MUC1-18 a cell line expressing full-length MUCI, T47D breast
cancer
cells, and CD34+ hematopoietic stem cells of the bone marrow. The fold
expression is
relative to the expression of each cleavage enzyme in T47D breast cancer
cells, set as 1.
[00308] Figure 44 shows the graph of the PCR experiment of Figure 43 but with
the Y-
axis maximum set to 5.
[00309] Figures 45A-45P show photographs of a CAR T co-culture assay in which
the
targeting antibody fragment of the CAR is huMNC2scFv wherein CAR44 has a CD8
transmembrane domain, followed by 41BB-3zeta and CAR50 has a CD4 transmembrane
domain, followed by 41BB-3zeta. The target cancer cells are: HCT-FLR which is
HCT-116
cells transfected with MUC1*45 and HCT-MUC1-41TR, which is a stable single
cell clone
HCT-116 cell line that expresses MUC1 with an extracellular domain truncated
after 41
tandem repeats and that does not get cleaved to the MUC1* form on its own. The
HCT-
MUC1-41TR cancer cells were also incubated with conditioned media from cells
transfected
with MMP9 or ADAM17 before co-culture with the CAR T cells. Conditioned media
of the
MMP9 or ADAM17 expressing cells were also incubated with APMA which is an
activator
of those cleavage enzymes. The images shown are an overlay of the 4X bright
field image
and the fluorescent image of the same showing cancer cells dyed with a red
CMTMR
lipophilic dye. Figs. 45A, 45E, 451, 45M show photographs of cells co-cultured
with
untransduced human T cells. Figs. 45B, 45F, 45J, 45N show photographs of cells
co-cultured
with human T cells transduced with anti-MUC1* CAR44 at an MO1 of 10. Figs.
45C, 45G,
45K, 450 show photographs of cells co-cultured with human T cells transduced
with anti-
MUC1* CAR50 at an MO1 of 10. Figs. 45D, 45H, 45L, 4W show photographs of cells
co-
cultured with human T cells transduced with anti-MUC1* CAR44 at an MO1 of 50,
which
increases transduction efficiency. Figs. 45B, 45C, 45D show that both CAR44
and CAR50
transduced T cells recognized MUC1* expressed in these cancer cells, bound to
them,
induced clustering and killed many cancer cells. Figs. 45F, 45G, 45H show that
neither
CAR44 nor CAR50 transduced T cells recognize full-length MUC1 expressed in HCT-
MUC1-41TR cancer cells. There is no T cell induced clustering and the number
of cancer
cells has not decreased. Figs. 45J, 45K, 45L show that activated MMP9 has
cleaved full-
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length MUC1 to a MUC1* form that is recognized by both CAR44 and CAR50
transduced T
cells. There is clearly visible CAR T cell induced clustering and a decrease
in the number of
cancer cells as they are killed. Figs. 45N, 450, 45P show that activated
ADAM17 has either
not cleaved MUC1 or cleaved it at a position not recognized by MNC2. Neither
huMNC2-
CAR44 nor huMNC2-CAR50 transduced T cells recognized these cancer cells.
[00310] Figure 46A-46T shows photographs of a CAR T co-culture assay in which
the
targeting antibody fragment of the CAR is MNC2 scFv wherein CAR44 has a CDS
transmembrane domain, followed by 41BB-3zeta and CAR50 has a CD4 transmembrane
domain, followed by 41BB-3zeta. The target cancer cells are breast cancer T47D
cells that
were also incubated with conditioned media from cells transfected with MMP2,
MMP9 or
ADAM17 before co-culture with the MNC2-CAR T cells. In some cases, the
conditioned
media of the MMP2 and MMP9 expressing cells were also incubated with APMA,
which is
an activator of these cleavage enzymes. The images shown are an overlay of the
4X bright
field image and the fluorescent image of the same showing cancer cells dyed
with a red
CMTMR lipophilic dye. As can be seen, the MNC2-CAR T cells only bind to and
attack the
target cancer cells that express the cleaved form, MUC1*.
[00311] Figures 47A-47I show photographs of cancer cells co-cultured with anti-
MUC1*
CAR T cells, wherein some of the cancer cells were pre-incubated with
activated MMP9
prior to co-culture with the CAR T cells. The cancer cells shown in Figs. 47A-
47C are MUC1
negative colon cancer cell line HC1-116 that have been stably transfected to
express M1JC1*.
The cancer cells shown in Figs. 47D-47F are MUC1 positive breast cancer cell
line T47Ds
that express high levels of both MUC1 full-length and MUC1*. The cancer cells
shown in
Figs. 47G-47I are MUC1 positive breast cancer cell line T47Ds that were pre-
incubated with
activated MMP9. The cells shown in Figs. 47A, 47D and 47G were co-cultured
with
untransduced human T cells and are the controls. The cells shown in Figs. 47B,
47E and 47H
were co-cultured with human T cells that were transduced with huMNC2-CAR44 at
an MOI
of 10, wherein MOI stands for multiplicity of infection and the higher the MOI
the more
CARs are expressed on the T cells. The cells shown in Figs. 47C, 47F and 471
were co-
cultured with human T cells that were transduced with huMNC2-CAR44 at an MOI
of 50. As
can be seen in the photographs, the CAR44 T cells bind to the target MUC1*
positive cancer
cells, surrounding and killing them. Comparing the photograph Fig. 471 with
the others, it can
be seen that the cells that were pre-incubated with MMP9 become much more
susceptible to
CAR T killing when the antibody targeting head of the CAR recognizes MUC1*. It
also
demonstrates that MUC1 cleaved by MMP9 is recognized by huMNC2scFv.
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[00312] Figure 48 shows an xCelligence graph of T47D breast cancer cells in co-
culture
with either untransduced T cells, as a control, or huMNC2-CAR44 T cells over a
45 hour
period. After 18 hours of cancer cell growth, a catalytic sub-unit MMP9 was
added to some
of the cells. At 25 hours, T cells were added. As can be seen, huMNC2-CAR44 T
cell killing
is greatly improved when the T47D cells are pre-incubated with cleavage enzyme
MMP9. In
the xCelligence system, target cancer cells, which are adherent, are plated
onto electrode
array plates. Adherent cells insulate the electrode and increase the
impedance. The number of
adherent cancer cells is directly proportional to impedance. T cells are not
adherent and do
not contribute to impedance. Therefore, increasing impedance reflects growth
of cancer cells
and decreasing impedance reflects killing of cancer cells.
[00313] Figure 49 shows an xCelligence graph of DU145 prostate cancer cells in
co-
culture with either untransduced T cells, as a control, or huMNC2-CAR44 T
cells over a 45
hour period. After 18 hours of cancer cell growth, a catalytic sub-unit MMP9
was added to
some of the cells. At 25 hours, T cells were added. As can be seen, huMNC2-
CAR44 T cell
killing is not affected by pre-incubation with cleavage enzyme MMP9. DU145
cancer cells
express a significantly lower amount of MUC1 which includes the full-length
form as well as
MUC1*. The lower density of MUC1 full-length does not sterically hinder T cell
access to
the membrane proximal MUC1*.
[00314] Figure 50 shows a bar graph of a PCR experiment measuring the amount
of
M1JC1 expressed by a panel of cell lines and primary cells, comprised of
normal cells as well
as cancer cells.
[00315] Figure 51A-51B shows a bar graph of an ELISA assay measuring the
amount of
interferon gamma, IFN-g, secreted by huMNC2-CAR44 human T cells after co-
culture with
the normal cells or the HCT-MUC1* cancer cells for 72 hours. Fig. MA shows the
results of
the experiment where the CAR44 T cell to target cell ratio was 1:1. Fig. 51B
shows the
results of the experiment where the CAR44 T cell to target cell ratio was
0.5:1.
[00316] Figure 52A-52B shows a bar graph of an ELISA assay measuring the
amount of
interleukin-2, IL-2, secreted by huMNC2-CAR44 human T cells after co-culture
with the
normal cells or the HCT-MUC1* cancer cells for 72 hours. Fig. 52A shows the
results of the
experiment where the CAR44 T cell to target cell ratio was 1:1. Fig. 52B shows
the results of
the experiment where the CAR44 T cell to target cell ratio was 0.5:1.
[00317] Figure 53A-53J shows bar graphs of FACS analysis of live versus dead
markers
and photographs of normal cells versus cancer cells after co-culture with
huMNC2-CAR44 T
cells. Fig. 53A.1 shows the bar graph of FACS analysis of live versus dead
cells after HCT-
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MUC1* cancer cells were co-cultured with huMNC2-CAR44 T cells. Fig. 53A.2 and
Fig.
53A.3 show the photographs of the experiment described in Fig. 53A.1. Fig.
53B.1 shows the
bar graph of FACS analysis of live versus dead cells after MCF-12A normal
breast cells were
co-cultured with huMNC2-CAR44 T cells. Fig. 53B.2 and Fig. 53B.3 show the
photographs
of the experiment described in Fig. 53B.1. Fig. 53C.1 shows the bar graph of
FACS analysis
of live versus dead cells after THLE-3 normal liver cells were co-cultured
with huMNC2-
CAR44 T cells. Fig. 53C.2 and Fig. 53C.3 show the photographs of the
experiment described
in Fig. 53C.1. Fig. 53D.1 shows the bar graph of FACS analysis of live versus
dead cells after
T/G HA-HSMC normal heart cells were co-cultured with huMNC2-CAR44 T cells.
Fig.
53D.2 and Fig. 53D.3 show the photographs of the experiment described in Fig.
53D.1_ Fig.
53E.1 shows the bar graph of FACS analysis of live versus dead cells after Hs
1.Tes normal
testes cells were co-cultured with huMNC2-CAR44 T cells. Fig. 53E.2 and Fig.
53E.3 show
the photographs of the experiment described in Fig. 53E.1. Fig. 53F.1 shows
the bar graph of
FACS analysis of live versus dead cells after HEK-293 MUC1 negative cells were
co-
cultured with huMNC2-CAR44 T cells. Fig. 53F.2 and Fig. 53F.3 show the
photographs of
the experiment described in Fig. 53F.1. Fig. 53G.1 shows the bar graph of FACS
analysis of
live versus dead cells after HRCE normal kidney cells were co-cultured with
huMNC2-
CAR44 T cells. Fig. 53G.2 and Fig. 53G.3 show the photographs of the
experiment described
in Fig. 53G.1. Fig. 53H.1 shows the bar graph of FACS analysis of live versus
dead cells
after CCD-18Lu normal lung cells were co-cultured with huMNC2-CAR44 '1 cells.
Fig.
53H.2 and Fig. 53H.3 show the photographs of the experiment described in Fig.
53H.1. Fig.
531.1 shows the bar graph of FACS analysis of live versus dead cells after
HBEC-5i normal
brain cells were co-cultured with huMNC2-CAR44 T cells. Fig. 531.2 and Fig.
531.3 show
the photographs of the experiment described in Fig. 531.1. Fig. 531.1 shows
the bar graph of
FACS analysis of live versus dead cells after Hs.738.St/Int normal stomach and
intestine cells
were co-cultured with huMNC2-CAR44 T cells. Fig. 53J.2 and Fig. 53J.3 show the
photographs of the experiment described in Fig. 531.1.
[00318] Figure 54 shows photographs of a breast cancer tissue array in which
for each
patient there is a specimen from the primary tumor plus a specimen from that
patient's
metastasis. As can be seen in the figure, most often the metastasis expresses
more MUC1*
than the primary tumor.
[00319] Figures 55A-55H show the cytotoxic effect of huMNC2-CAR44 T cells on
MUC1* positive DU145 prostate cancer cells as measured by a variety of assays.
Fig. 55A is
a fluorescent photograph of untransduced T cells co-cultured with the prostate
cancer cells,
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wherein granzyme B is stained with a red fluorophore. Fig. 55B shows merging
of DAPI and
granzyme B. Fig_ 55C is a fluorescent photograph of huMNC2-CAR44 T cells co-
cultured
with the prostate cancer cells, wherein granzyme B is stained with a red
fluorophore. Fig.
55D shows merging of DAPI and granzyme B. Fig. 55E is a FACS scan for
fluorescently
labeled granzyme B for untransduced T cells incubated with the cancer cells.
Fig. 55F is a
FACS scan showing a positive increase in fluorescently labeled granzyme B for
huMNC2-
CAR44 T cells incubated with the cancer cells. Fig. 55G is a graph of the mean
fluorescent
intensity. Fig. 55H is an xCELLigence scan tracking the real-time killing of
DU145 cancer
cells by huMNC2-CAR44 T cells (blue trace) but not by untransduced T cells
(green).
[00320] Figures 56A-56H show the cytotoxic effect of huMNC2-CAR44 T cells on
MUC1* positive CAPAN-2 pancreatic cancer cells as measured by a variety of
assays. Fig.
56A is a fluorescent photograph of untransduced T cells co-cultured with the
pancreatic
cancer cells, wherein granzyme B is stained with a red fluorophore. Fig. 56B
shows merging
of DAPI and granzyme B. Fig. 56C is a fluorescent photograph of huMNC2-CAR44 T
cells
co-cultured with the pancreatic cancer cells, wherein granzyme B is stained
with a red
fluorophore. Fig. 56D shows merging of DAPI and granzyme B. Fig. 56E is a FACS
scan for
fluorescently labeled granzyme B for untransduced T cells incubated with the
cancer cells.
Fig. 56F is a FACS scan showing a positive increase in fluorescently labeled
granzyme B for
huMNC2-CAR44 T cells incubated with the cancer cells. Fig. 56G is a graph of
the mean
fluorescent intensity. Fig. 56H is an xCELLigence scan tracking the real-time
killing of
CAPAN-2 cancer cells by huMNC2-CAR44 T cells (blue trace) but not by
untransduced T
cells (green).
[00321] Figures 57A-57C show xCELLigence scans tracking the real-time killing
of
MUC1* positive cancer cells, but not MUC1* negative cells, by huMNC2-CAR44 T
cells.
Fig. 57A shows that huMNC2-CAR44 T cells effectively kill HCT colon cancer
cells that
have been stably transfected with MUC1*. Fig. 57B shows that huMNC2-CAR44 T
cells
have almost no effect on HCT-MUC1-41TR, which is a MUC1 negative cancer cell
that has
been stably transfected with a MUC1 full-length. In this cell line only about
10% of the cells
have MUC1 cleaved to MUC1*. Fig. 57C shows that huMNC2-CAR44 T cells have no
effect on HCT-116 cells, which is a MUC1 negative colon cancer cell line.
[00322] Figure 58A- 58F shows photographs NOD/SCID/GAMMA mice in an IVIS
instrument measuring photon emission from tumor cells after mice were treated
with nothing,
PBS, untransduced human T cells or huMNC2-CAR44 T cells. Mice had been
injected sub-
cutaneously with HCT-MUC1* tumor cells that had been made Luciferase positive.
Ten (10)
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minutes before the IVIS photographs were taken, the mice were injected into
the
intraperitoneal (ip) space with the Luciferase substrate, Luciferin. Fig. 58A
shows the tumor
bearing mice that had only been treated with phosphate buffered saline, PBS.
Fig. 58B shows
the tumor bearing mice that had only been treated with untransduced T cells.
Fig. 58C shows
the tumor bearing mice that had been treated with a single dose of huMNC2-
CAR44 T cells.
Fig. 58D shows color scale of the images. Fig. 58E shows Kaplan-Meier survival
curves of
the experiment. Fig. 58F shows a table detailing the molecular makeup of the
human T cells
that were isolated from the mouse blood after sacrifice.
[00323] Figure 59A- 59C shows photographs NOD/SCID/GAMMA mice in an IVIS
instrument measuring photon emission from tumor cells after mice were treated
with nothing,
PBS or huMNC2-CAR44 T cells. Mice had been injected sub-cutaneously with T47D-
wt
breast cancer cells or T47D+more MUC1*, which is a mixed population of cells
wherein
95% of the cells were T47D cells that had been stably transfected with even
more MUC1*.
Both T47D-wt and 147D plus more MUC1* cells had been made Luciferase positive.
Ten
(10) minutes before the IVIS photographs were taken, the mice were injected
into the
intraperitoneal (ip) space with the Luciferase substrate, Luciferin. Fig. 59A
shows the tumor
bearing mice that had only been treated with phosphate buffered saline, PBS.
Fig. 59B shows
the T47D-wt tumor bearing mice that had been treated with two (2) doses of
huMNC2-
CAR44 T cells. Fig. T90.1C shows the T47D-MUC1* tumor bearing mice that had
been
treated with two (2) doses of huMNC2-CAR44 T cells.
[00324] Figure 60A- 60C shows photographs NOD/SCID/GAMMA mice in an IVIS
instrument measuring photon emission from tumor cells after mice were treated
with nothing,
PBS, untransduced T cells or huMNC2-CAR44 T cells. Mice had been injected sub-
cutaneously with a mixed population of 70% T47D-wt breast cancer cells and 30%
T47D
cells that had been transfected with even more MUC1*. Both cell types had been
made
Luciferase positive. Ten (10) minutes before the IVIS photographs were taken,
the mice were
injected into the intraperitoneal (ip) space with the Luciferase substrate,
Luciferin. Fig. 60A
shows the tumor bearing mice that had only been treated with phosphate
buffered saline,
PBS. Fig. 60B shows tumor bearing mice that had only been treated with
untransduced T
cells. Fig. 60C shows the tumor bearing mice that had been treated with two
(2) doses of
huMNC2-CAR44 T cells.
[00325] Figures 61A-61J show fluorescent photographs of mice taken on an IVIS
instrument. NSG (NOD/SCID/GAMMA) immune compromised mice that on Day 0 were
sub-cutaneously injected into the flank with 500K human BT-20 cells which are
a MUC1*
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positive triple negative breast cancer cell line. The cancer cells had been
stably transfected
with Luciferase. Tumors were allowed to engraft. On Day 6 after IVIS
measurement, animals
were given a one-time injection of 10 million of either human T cells
transduced with
huMNC2-scFv-CAR44 or untransduced T cells. 5 million T cells were injected
intra-tumor
and 5 million were injected into the tail vein. 10 minutes prior to IVIS
photographs, mice
were IP injected with Luciferin, which fluoresces after cleavage by
Luciferase, thus making
tumor cells fluoresce. Figs. 61A, 61D, 61G show photographs of mice that were
treated with
huMNC2-scFv-CAR44 T cells that had been pre-stimulated by co-culturing for 24
hours with
4ium beads to which was attached a synthetic MUC1*, PSMGFR peptide 24 hours
prior to
administration: Protocol 1. Figs. 61B, 61E, 61H show photographs of mice that
were treated
with huMNC2-scFv-CAR44 T cells that had been pre-stimulated by twice co-
culturing for 24
hours with MUC1* positive cancer cells 24 hours prior to administration:
Protocol 2. Figs.
61C, 61F, 611 show photographs of mice that were treated with untransduced
human T cells.
Fig. 61J is a color scale relating fluorescence in photons/second to color.
[00326] Figures 62A-62M show fluorescent photographs of mice taken on an IVIS
instrument. NSG (NOD/SCID/GAMMA) immune compromised mice that on Day 0 were
injected into the intraperitoneal cavity (IP) with 500K human SKOV-3 cells
which are a
MUC1* positive ovarian cancer cell line. The cancer cells had been stably
transfected with
Luciferase. Tumors were allowed to engraft. On Day 4, animals were injected
into the
intraperitoneal space with 10M either human '1' cells transduced with huMNC2-
scFv-CAR44,
untransduced T cells or PBS. On Day 11, animals were injected again except
that half the
cells were injected into the tail vein and the other half was IP injected.
Animals were imaged
by IVIS on Days 3, 7, 10 and 15. 10 minutes prior to IVIS photographs, mice
were IP
injected with Luciferin, which fluoresces after cleavage by Luciferase, thus
making tumor
cells fluoresce. Figs. 62A, 62D, 62G, and 62J show photographs of mice that
were treated
with huMNC2-scFv-CAR44 T cells that had been pre-stimulated by co-culturing
for 24 hours
with 1ini beads to which was attached a synthetic MUC1*, PSMGFR peptide 24
hours prior
to administration. Figs. 62B, 62E, 6211, and 62K show photographs of mice that
were treated
with untransduced human T cells. Figs. 62C, 62F, 621, and 62L show photographs
of mice
that were treated with PBS. Figs. 62A, 62B and 62C are IVIS images taken Day 3
prior to
CAR T, T cell or PBS administration. Figs. 62D, 62E and 62F show IVIS images
of animals
on Day 7, just four (4) days after treatment. Figs. 62G, 62H, and 621 show
IVIS images of
animals on Day 10. Figs. 62J, 62K, and 62L show IVIS images of animals on Day
15 Fig.
62M is the IVIS color scale relating fluorescence in photons/second to color.
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[00327] Figure 63 shows a graph of an ELISA binding assay in which various
monoclonal
antibodies are tested for their ability to bind to the PSMGFR peptide, the N-
10, C-10,
N+20/C-27, or the N+9/C-9 peptide, wherein the concentration of the antibody
was at
lOug/mL or lug/mL. Note that anti-MUC1* monoclonal antibodies C2 and E6, which
have
been demonstrated to be cancer specific, bind to the PSMGFR peptide, still
bind if the 10 N-
terminal amino acids are missing, but do not bind if the 10 or 9 C-terminal
amino acids are
missing.
[00328] Figure 64A-64B shows a graph of an ELISA binding assay. The antibodies
being
tested were derived from animals immunized with the PSMGFR peptide. The first
selection
criteria was to confirm that the antibodies bound to the immunizing PSMGFR
peptide. Fig.
64A shows a graph of an ELISA of selected antibodies that were further tested
to determine
their ability to bind to the PSMGFR peptide, the N10, the C-10, N+20/C-27, or
N+9/C-9
peptide. All the antibodies except 18B4 were able to bind to the N-10 peptide.
18B4
recognized N+20/C-27 but not the N-10 peptide, implying that its cognate
epitope lies within
the GTINVHDVET sequence. All except 20A10 and C2 showed some binding to the C-
10
and N+9/C-9 peptide, showing that both 20A10 and C2 require the 10 membrane
proximal
amino acids for binding. C2, which requires the 10 membrane proximal amino
acids for
binding has been demonstrated to be cancer specific. Fig. 64B shows the
sequences of the
various peptides. The color of the bars for each antibody in the ELISA graph
are color coded
to match the deductive cognate sequence, or a portion thereof, of that
antibody.
[00329] Figure 65A-65B shows a graph of an ELISA binding assay in which
various
monoclonal antibodies are tested for their ability to bind to the PSMGFR
peptide, the N-10,
the C-10, N+20/C-27, or N+9/C-9 peptide. The antibodies being tested were
derived from
animals immunized with the N+20/C-27 peptide. The first selection criteria was
to confirm
that the antibodies bound to the immunizing N+20/C-27 peptide. Fig. 65A shows
a graph of
ELISA binding assay that tests the ability of each antibody to bind to various
peptides.
Although these antibodies were raised against the N+20/C-27 peptide, all but
one, 45C11,
still bind to the PSMGFR peptide. The binding of 45C11 is weak but deductive
reasoning
shows that the cognate epitope must lie within the SNIKFRPGSVV sequence. 1E4
was able
to bind to the N+20/C-27 peptide, the PSMGFR and the N-10 peptide, consistent
with the
idea that its epitope must lie within the QFNQYKTE sequence. Fig. 65B shows
the sequences
of the various peptides. The color of the bars for each antibody in the ELISA
graph are color
coded to match the deductive cognate sequence, or a portion thereof, of that
antibody.
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[00330] Figure 66A-66B shows a graph of an ELISA binding assay in which
various
monoclonal antibodies are tested for their ability to bind to the PSMGFR
peptide, the N-10,
the C-10, N+20/C-27, or N+9/C-9 peptide. The antibodies being tested were
derived from
animals immunized with the N+9/C-9 peptide. The first selection criteria was
to confirm that
the antibodies bound to the immunizing N+9/C-9 peptide. Fig. 66A shows a graph
of the
ELISA assay. All but one, 39H5, were only able to bind to the immunizing
peptide, N+9/C-9.
39H5 showed very weak binding to the PSMGFR and N-10 peptide, consistent with
the idea
that at least a portion of its cognate epitope must lie within the QFNQYKTE
sequence. Fig.
66B shows the sequences of the various peptides. The color of the bars for
each antibody in
the ELISA graph are color coded to match the deductive cognate sequence, or a
portion
thereof, of that antibody.
[00331] Figure 67A-67D shows results of ELISA assays to further define
antibody
epitopes within the MUCI or MUCI* extra cellular domain. The antibodies shown
in this
figure were all generated by immunizing animals with the PSMGFR peptide.
Binding assays
tested antibodies for their ability to bind to peptides N-19, N-26, N-30, N-
10/C-5, N-19/C-5,
PSMGFR, N-10 and C-10, which are all subsets of the PSMGFR peptide and
numbering
refers back to the PSMGER peptide. Fig. 67A shows the binding of the various
antibodies to
the various peptides. Fig. 67B shows the sequence of the PSMGFR peptide that
has been
extended 20 amino acids at the N-terminus. Fig. 67C shows the sequences of the
PSMGFR-
derived subset peptides. Fig. 6713 shows the sequences that comprise all or
part of the epitope
that is essential for antibody recognition.
[00332] Figure 68A-68D shows results of ELISA assays to further define
antibody
epitopes within the MUCI or MUCI* extra cellular domain. The antibodies shown
in this
figure were all generated by immunizing animals with the N+20/C-27 peptide.
Binding
assays tested antibodies for their ability to bind to peptides N-19, N-26, N-
30, N-10/C-5, N-
19/C-5, PSMGFR, N-10 and C-10, which are all subsets of the PSMGFR peptide and
numbering refers back to the PSMGFR peptide. Fig. 68A shows the binding of the
various
antibodies to the various peptides. Fig. 68B shows the sequence of the PSMGFR
peptide that
has been extended 20 amino acids at the N-terminus. Fig. 68C shows the
sequences of the
PSMGFR-derived subset peptides. Fig. 68D shows the sequences that comprise all
or part of
the epitope that is essential for antibody recognition.
[00333] Figures 69A-69D show results of ELISA assays to further define
antibody
epitopes within the MUC1 or MUC1* extra cellular domain. The antibodies shown
in this
figure were all generated by immunizing animals with the N+9/C-9 peptide.
Binding assays
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tested antibodies for their ability to bind to peptides N-19, N-26, N-30, N-
10/C-5, N-19/C-5,
PSMGFR, N-10 and C-10, which are all subsets of the PSMGFR peptide and
numbering
refers back to the PSMGFR peptide. Fig. 69A shows the binding of the various
antibodies to
the various peptides. Fig. 69B shows the sequence of the PSMGFR peptide that
has been
extended 20 amino acids at the N-terminus. Fig. 69C shows the sequences of the
PSMGFR-
derived subset peptides. Fig. 69D shows the sequences that comprise all or
part of the epitope
that is essential for antibody recognition.
[00334] Figure 70A-70B shows a graph of an ELISA displacement assay. In this
experiment, a multi-well plate was coated with the PSMGFR peptide. In Fig.
70A,
recombinant NME7AB was allowed to bind to the surface-immobilized PSMGFR
peptide.
Various antibodies were added, followed by a wash step. The amount of NME7AB
that
remained attached to the PSMGFR coated plate, after antibody competition, was
measured by
detecting a tag on the NME7AB. As a control, anti-NME7AB antibodies were also
tested for
their ability to displace NME7AB from the PSMGFR. As can be seen in the graph,
antibodies
MNC2, MNE6, 20A10, 3C2B1 and 5C6F3 displace NME7AB from binding to the PSMGFR
peptide, which is an indicator of the antibody being cancer-specific. Fig. 70B
shows that the
epitope within the MUC1* extracellular domain to which these antibodies bind
is the
sequence SVSDVPFPFSAQS GA, wherein binding is destroyed for MNC2, MNE6, 20A10,
3C2B1 if amino acids FPFS are not present or mutated and binding is destroyed
for 5C6F3 if
amino acids S V SDV are not present or mutated.
[00335] Figure 71A-71H shows photographs of Western blots in which antibodies
are
tested for their ability to bind to a linear epitope in full-length MUC1 or
MUC1*. Fig. 71A-
71D shows testing of antibodies for ability to bind to a MUC1 negative cell
line, HCT-116, or
engineered cell lines HCT-MUC1-18, which is a cleavage resistant clone that
expresses full-
length MUC1, or HCT-MUC1*, which is engineered to express only the PSMGFR
sequence
in its extra cellular domain. Fig. 71E-71H shows testing of antibodies for
ability to bind to
breast cancer cell lines T47D or 1500 aka ZR-75-1. Fig. 71A and Fig. 71E show
MNC2, a
monoclonal antibody raised against PSMGFR peptide that binds to N-10 but not C-
10
variants of the PSMGFR peptide. Fig. 71B and Fig. 71F show MNE6, a monoclonal
antibody
raised against PSMGFR peptide that binds to N-10 but not C-10 variants of the
PSMGFR
peptide. Fig. 71C and Fig. 71G show SDIX, a polyclonal antibody raised against
PSMGFR
peptide and which binds to the PSMGFR peptide. Fig. 71D and Fig. 71H show
VU4H5, a
commercially available monoclonal antibody that binds to the tandem repeats of
full-length
MUCL As can be seen, neither MNC2 nor MNE6 bind linear epitopes of MUC1
species.
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[00336] Figure 72A-72P shows photographs of Western blots in which antibodies
are
tested for their ability to bind to a linear epitope in full-length MUC1 or
MUC1*. All these
antibodies were raised against the PSMGFR peptide and bind to the PSMGFR
peptide. Fig.
72A-72H shows testing of antibodies for ability to bind to a MUC1 negative
cell line, HCT-
116, or engineered cell lines HCT-MUC1-18, which is a cleavage resistant clone
that
expresses full-length MUC1, or HCT-MUC1*, which is engineered to express only
the
PSMGFR sequence in its extra cellular domain. Fig. 721-72P shows testing of
antibodies for
ability to bind to breast cancer cell lines T47D or 1500 aka ZR-75-1. Fig. 72A
and Fig. 721
show 20A10. Fig. 72B and Fig. 72J show 25E6. Fig. 72C and Fig. 72K show 18B4.
Fig. 72D
and Fig. 72L show 18G12. Fig. 72E and Fig. 72M show 28F9. Fig. 72F and Fig.
72N show
3C2B1. Fig. 72G and Fig. 720 show 5C6F3. Fig. 72H and Fig. 72P show 5C6F3
wherein the
blot has been exposed for a longer time period to render more visible the
MUC1* specific
bands. As can be seen, antibodies 25E6, 18B4 and to a degree 5C6F3 recognize
linear
epitopes but 20A10, 3C2B1, 18G12 and 28F9 do not.
[00337] Figure 73A-73J shows photographs of Western blots in which antibodies
are
tested for their ability to bind to a linear epitope in full-length MUC1 or
MUC1*. All these
antibodies were raised against the N+20/C-27 variant of the PSMGFR peptide and
bind to the
N+20/C-27 peptide. Fig. 73A-73E shows testing of antibodies for ability to
bind to a MUC1
negative cell line, HCT-116, or engineered cell lines HCT-MUC1-18, which is a
cleavage
resistant clone that expresses full-length MUC1, or HCT-MUCI*, which is
engineered to
express only the PSMGFR sequence in its extra cellular domain. Fig. 73F-73J
shows testing
of antibodies for ability to bind to breast cancer cell lines T47D or 1500 aka
ZR-75-1. Fig.
73A and Fig. 73F show 1E4. Fig. 73B and Fig. 73G show 45C11. Fig. 73C and Fig.
73H
show 31AI. Fig. 73D and Fig. 731 show 32C1. Fig. 73E and Fig. 73J show 29H1.
As can be
seen, antibodies 31A1 and 32C1 recognize linear epitopes.
[00338] Figure 74A-74H shows photographs of Western blots in which antibodies
are
tested for their ability to bind to a linear epitope in full-length MUC1 or
MUCI*. All these
antibodies were raised against the N+9/C-9 variant of the PSMGFR peptide and
bind to the
N+9/C-9 peptide. Fig. 74A-74D shows testing of antibodies for ability to bind
to a MUC1
negative cell line, HCT-I16, or engineered cell lines HCT-MUC1-18, which is a
cleavage
resistant clone that expresses full-length MUC1, or HCT-MUC1*, which is
engineered to
express only the PSMGFR sequence in its extra cellular domain. Fig. 74E-74H
shows testing
of antibodies for ability to bind to breast cancer cell lines T47D or 1500 aka
ZR-75-1. Fig.
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74A and Fig. 74E show 8A9. Fig. 74B and Fig. 74F show 17H6. Fig. 74C and Fig.
74G show
3C5. Fig. 74D and Fig. 74H show 39H5.
[00339] Figure 75A-75P show graphs of FACS analysis. HCT-MUC1-18 cells, which
express full-length MUC1, were incubated with a catalytically active MMP9 or
MMP2 for 24
hours, incubated with an antibody of the invention and then analyzed by FACS
to see if the
antibody bound to the MMP9 or the MMP2 cleaved form of MUC1. Note that the
first bar of
each graph shows that none of the antibodies binds to full-length MUC1 in the
absence of
cleavage. Each bar graph is labeled with both the name of the antibody used in
that assay and
its cognate epitope. The order of the graphs from right to left corresponds to
the distance the
from the cell surface of the antibody's cognate epitope. Fig. 75A shows
antibody 1E4. Fig.
75B shows antibody 28F9. Fig. 75C shows antibody 18G12. Fig. 75D shows
antibody 25E6.
Fig. 75E shows antibody 20A10. Fig. 75F shows antibody 3C5. Fig. 75G shows
antibody
29H1. Fig. 75H shows antibody 32C1. Fig. 751 shows antibody 31A1. Fig. 75J
shows
antibody I8B4. Fig. 75K shows antibody 45C11. Fig. 75L shows antibody 8A9.
Fig. 75M
shows antibody 17H6. Fig. 75N shows antibody 39H5. Fig. 750 shows antibody
3C2B1. Fig.
75P shows antibody 5C6F3.
[00340] Figure 76A-76J show graphs of FACS analyses of reference antibodies
MNC2,
"C2", and VU4H5 binding to either the MUC1-negative cell line HCT-116, HCTs
transfected
with MUC1*, "HCT-MUC1*", a cleavage resistant single cell clone of HCTs
transfected
with M1JC1 full-length, "HC1-M1JC1-18", and MNC2 binding to breast cancer
cells line
T47D or breast cancer cell line 1500 also known as ZR-75-1. MNC2 binds to an
ectopic
binding site on the extra cellular domain of MUC1*, within the membrane
proximal portion
of the PSMGER sequence. The MNC2 binding site is only available after cleavage
and
release of the bulk of the extra cellular domain comprising the tandem repeat
domain.
VU4H5 binds to hundreds of repeating epitopes in the tandem repeat domain.
Fig. 76A-76E
show percent binding and Fig. 76F-76J show Mean Fluorescent Intensity or MFI.
[00341] Figure 77A-77N show graphs of FACS analyses of reference antibody
MNC2,
"C2", binding to a panel of cancer cell lines that are MUC1* positive, with
the exception of
MDA-MB-231, which expresses MUC1 and MUC1* at a level that is so low that it
is often
used as a negative control. MNC2 binds to an ectopic binding site on the extra
cellular
domain of MUC1*, within the membrane proximal portion of the PSMGFR sequence.
The
MNC2 binding site is only available after cleavage and release of the bulk of
the extra
cellular domain comprising the tandem repeat domain. Fig. 77A-77G show percent
binding
and Fig. 77H-77N show Mean Fluorescent Intensity or MFI. Fig. 77A and 77H show
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antibodies binding to lung cancer cell line NCI-H292. Fig. 77B and 771 show
the antibodies
binding to lung cancer cell line NCI-H1975. Fig. 77C and 77J show the
antibodies binding to
ovarian cancer cell line SKOV-3. Fig. 77D and 77K show the antibodies binding
to
pancreatic cancer cell line HPAF-II. Fig. 77E and 77L show the antibodies
binding to
pancreatic cancer cell line Capan-1. Fig. 77F and 77M show the antibodies
binding to
prostate cancer cell line DU145. Fig. 77G and 77N show the antibodies binding
to breast
cancer cell line MDA-MB-231, which is nearly MUC1 and MUC1* negative.
[00342] Figure 78A-78C shows a color coded schematic of the basic PSMGFR
sequence
that has been extended or deleted at both the N- and C-termini. Antibodies of
the invention
were tested against this subset of peptides to further refine the epitopes to
which each
antibody binds or the critical amino acids within the epitope to which each
antibody binds.
Fig. 78A is an aligned schematic of the various subsets of peptides. Fig. 78B
lists the
antibodies that bind to each of the color coded sequences. Fig. 78C lists the
cancer cell lines
that each antibody recognizes.
[00343] Figure 79A-791 shows color coded graphs that resulted from FACS
analyses of
each antibody binding to T47D breast cancer cells and their respective cognate
sequences
within the N-terminally extended PSMGFR sequence. Fig. 79A-79D are FACS graphs
showing the percent cells that were recognized by each antibody. Fig. 79E-79H
are FACS
graphs showing the Mean Fluorescence Intensity, MFI, of each antibody. Fig.
79A and Fig.
79E show the FACS graph of antibodies that were generated by immunizing with
the
PSMGFR peptide. Fig. 79B and Fig. 79F show the FACS graph of antibodies that
were
generated by immunizing with the N+20/C-27 peptide. Fig. 79C and Fig. 79G show
the
FACS graph of antibodies that were generated by immunizing with the N+9/C-9
peptide. Fig.
79D and Fig.79H also show the FACS graph of antibodies that were generated by
immunizing with the PSMGFR peptide. Fig. 791 shows the PSMGFR sequence that is
extended at the N-terminus by 20 amino acids. Fig. 79A, Fig.79E show that
20A10
recognizes MUC1* as it exists on T47D breast cancer cells. Fig. 79D, Fig. 79H
show that
3C2B1 recognizes MUC1* as it exists on T47D breast cancer cells. Fig. 79D,
Fig. 79H show
that 5C6F3 recognizes MUC1* as it exists on T47D breast cancer cells.
[00344] Figure 80A-801 shows color coded graphs that resulted from FACS
analyses of
each antibody binding to 1500, also known as ZR-75-1, breast cancer cells and
their
respective cognate sequences within the N-terminally extended PSMGFR sequence.
Fig.
80A-80C are FACS graphs showing the percent cells that were recognized by each
antibody.
Fig. 80D-80F are FACS graphs showing the Mean Fluorescence Intensity, MFI, of
each
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antibody. Fig. 80A, Fig. 80E Fig. 80D and Fig. 80H show the FACS graph of
antibodies that
were generated by immunizing with the PSMGFR peptide. Fig. 80B and Fig. 80F
show the
FACS graph of antibodies that were generated by immunizing with the N+20/C-27
peptide.
Fig. 80C and Fig. 80G show the FACS graph of antibodies that were generated by
immunizing with the N+9/C-9 peptide. Fig. 801 shows the PSMGFR sequence that
is
extended at the N-terminus by 20 amino acids. Fig. 80A, Fig. 80E show that
antibody 20A10
recognizes MUC1* as it exists on 1500 aka ZR-75-1 breast cancer cells. Fig.
80D, Fig. 80H
show that antibody 3C2B1 recognizes MUC1* as it exists on 1500 aka ZR-75-1
breast cancer
cells. Fig. 80D, Fig. 80H show that antibody 5C6F3 recognizes MUC1* as it
exists on 1500
aka ZR-75-1 breast cancer cells.
[00345] Figure 81A-81G shows color coded graphs that resulted from FACS
analyses of
each antibody binding to NCI-H292 lung cancer cells and their respective
cognate sequences
within the N-terminally extended PSMGFR sequence. Fig. 81A-81C are FACS graphs
showing the percent cells that were recognized by each antibody. Fig. 81D-81F
are FACS
graphs showing the Mean Fluorescence Intensity, MFI, of each antibody. Fig.
81A and Fig.
81D show the FACS graph of antibodies that were generated by immunizing with
the
PSMGFR peptide. Fig. 81B and Fig. 81E show the FACS graph of antibodies that
were
generated by immunizing with the N+20/C-27 peptide. Fig. 81C and Fig. 81F show
the
FACS graph of antibodies that were generated by immunizing with the N+9/C-9
peptide. Fig.
816 shows the PSMCiPR sequence that is extended at the N-terminus by 20 amino
acids. Fig.
81A, Fig. 81D show that antibody 20A10 recognizes MUC1* as it exists on H292
lung
cancer cells.
[00346] Figure 82A-82G shows color coded graphs that resulted from FACS
analyses of
each antibody binding to NCI-H1975 lung cancer cells and their respective
cognate
sequences within the N-terminally extended PSMGFR sequence. Fig. 82A-82C are
FACS
graphs showing the percent cells that were recognized by each antibody. Fig.
82D-82F are
FACS graphs showing the Mean Fluorescence Intensity, MR, of each antibody.
Fig. 82A
and Fig. 82D show the FACS graph of antibodies that were generated by
immunizing with
the PSMGFR peptide. Fig. 82B and Fig. 82E show the FACS graph of antibodies
that were
generated by immunizing with the N+20/C-27 peptide. Fig. 82C and Fig. 82F show
the
FACS graph of antibodies that were generated by immunizing with the N+9/C-9
peptide. Fig.
82G shows the PSMGFR sequence that is extended at the N-terminus by 20 amino
acids. Fig.
82A, Fig. 82D show that antibody 20A10 recognizes MUC1* as it exists on H1975
lung
cancer cells.
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[00347] Figure 83A-83G shows color coded graphs that resulted from FACS
analyses of
each antibody binding to SKOV-3 ovarian cancer cells and their respective
cognate
sequences within the N-terminally extended PSMGFR sequence. Fig. 83A-83C are
FACS
graphs showing the percent cells that were recognized by each antibody. Fig.
83D-83F are
FACS graphs showing the Mean Fluorescence Intensity, MR, of each antibody.
Fig. 83A
and Fig. 83D show the FACS graph of antibodies that were generated by
immunizing with
the PSMGFR peptide. Fig. 83B and Fig. 83E show the FACS graph of antibodies
that were
generated by immunizing with the N+20/C-27 peptide. Fig. 83C and Fig. 83F show
the
FACS graph of antibodies that were generated by immunizing with the N+9/C-9
peptide. Fig.
83G shows the PSMGFR sequence that is extended at the N-terminus by 20 amino
acids. Fig.
83A, Fig. 83D shows that antibody 20A10 recognizes MUC1* as it exists on SKOV-
3
ovarian cancer cells.
[00348] Figure 84A-84G shows color coded graphs that resulted from FACS
analyses of
each antibody binding to DU145 prostate cancer cells and their respective
cognate sequences
within the N-terminally extended PSMGFR sequence. Fig. 84A-84C are FACS graphs
showing the percent cells that were recognized by each antibody. Fig. 84D-84F
are FACS
graphs showing the Mean Fluorescence Intensity, MFI, of each antibody. Fig.
84A and Fig.
84D show the FACS graph of antibodies that were generated by immunizing with
the
PSMGFR peptide. Fig. 84B and Fig. 84E show the FACS graph of antibodies that
were
generated by immunizing with the N+20/C-27 peptide. Fig. 84C and Fig. 84F show
the
FACS graph of antibodies that were generated by immunizing with the N+9/C-9
peptide. Fig.
84G shows the PSMGFR sequence that is extended at the N-terminus by 20 amino
acids. Fig.
84A, Fig. 84E show that antibody 20A10 recognizes MUC1* as it exists on DU145
prostate
cancer cells. Fig. 84D, Fig. 84H show that antibody 3C2B1 recognizes MUC1* as
it exists on
DU145 prostate cancer cells. Fig. 84D, Fig. 84H shows that antibody 5C6F3
recognizes
MUC1* as it exists on DU145 prostate cancer cells.
[00349] Figure 85A-85G shows color coded graphs that resulted from FACS
analyses of
each antibody binding to HPAF-II pancreatic cancer cells and their respective
cognate
sequences within the N-terminally extended PSMGER sequence. Fig. 85A-85C are
FACS
graphs showing the percent cells that were recognized by each antibody. Fig.
85D-85F are
FACS graphs showing the Mean Fluorescence Intensity, MM, of each antibody.
Fig. 85A
and Fig. 85D show the FACS graph of antibodies that were generated by
immunizing with
the PSMGFR peptide. Fig. 85B and Fig. 85E show the FACS graph of antibodies
that were
generated by immunizing with the N+20/C-27 peptide. Fig. 85C and Fig. 85F show
the
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FACS graph of antibodies that were generated by immunizing with the N+9/C-9
peptide. Fig.
85G shows the PSMGFR sequence that is extended at the N-terminus by 20 amino
acids. Fig.
85A, Fig. 85D show that antibody 20A10 recognizes MUC1* as it exists on HPAF
11
pancreatic cancer cells.
[00350] Figure 86A-86G shows color coded graphs that resulted from FACS
analyses of
each antibody binding to Capan-1 pancreatic cancer cells and their respective
cognate
sequences within the N-terminally extended PSMGFR sequence. Fig. 86A-86C are
FACS
graphs showing the percent cells that were recognized by each antibody. Fig.
86D-86F are
FACS graphs showing the Mean Fluorescence Intensity, MM, of each antibody.
Fig. 86A
and Fig. 86D show the FACS graph of antibodies that were generated by
immunizing with
the PSMGFR peptide. Fig. 86B and Fig. 86E show the FACS graph of antibodies
that were
generated by immunizing with the N+20/C-27 peptide. Fig. 86C and Fig. 86F show
the
FACS graph of antibodies that were generated by immunizing with the N+9/C-9
peptide. Fig.
86G shows the PSMGFR sequence that is extended at the N-terminus by 20 amino
acids.
[00351] Figure 87A-87G shows color coded graphs that resulted from FACS
analyses of
each antibody binding to MDA-MB-231 breast cancer cells, which are nearly MUC1
negative, and their respective cognate sequences within the N-terminally
extended PSMGFR
sequence. Fig. 87A-87C are FACS graphs showing the percent cells that were
recognized by
each antibody. Fig. 87D-87F are FACS graphs showing the Mean Fluorescence
Intensity,
MN, of each antibody. Fig. 87A and Pig. 87D show the FACS graph of antibodies
that were
generated by immunizing with the PSMGFR peptide. Fig. 87B and Fig. 87E show
the FACS
graph of antibodies that were generated by immunizing with the N+20/C-27
peptide. Fig.
87C and Fig. 87F show the FACS graph of antibodies that were generated by
immunizing
with the N+9/C-9 peptide. Fig. 87G shows the PSMGFR sequence that is extended
at the N-
terminus by 20 amino acids.
[00352] Figure 88A-88L show photographs of normal liver tissue specimens, each
from
the same donor but stained with a different antibody of the invention. Fig.
88A-88F show the
entire tissue core. Fig. 88G-88L show the 40X magnification of a particular
area of the tissue.
The tissues are ordered from right to left with antibodies that bind to the
most membrane
proximal, that is to say most C-terminal portion of the PSMGFR peptide, on the
right and
antibodies that bind to the most N-terminal portions of the MUC1 extra
cellular domain, even
beyond the PSMGFR region, on the left. As can be seen in the figure, the most
cancer-
specific antibodies are those that bind to the more membrane proximal portions
of the
PSMGFR sequence and antibodies that bind to the most distal, N-terminal
portions lose
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cancer specificity, with those antibodies that bind to epitopes outside of the
PSMGFR having
lost all cancer specificity. As can be seen, Fig. 88F and 88L show that
antibody 3C2B1,
which binds the portion of MUC1* extracellular domain that comprises all or
part of the
sequence FPFS or PFPFSAQSGA, does not bind to normal liver.
[00353] Figure 89A-89H show photographs of normal heart tissue specimens,
stained
with different antibodies of the invention. Fig. 89A-89D show the entire
tissue core. Fig.
89E-89HL show the 40X magnification of a particular area of the tissue. Fig.
89A and Fig.
89E show staining with MNC2-scFv. Fig. 89B and Fig. 89F show staining with
MNE6. Fig.
89C and Fig. 89G show staining with 20A10. Fig. 89D and Fig. 89H show staining
with
3C2B1. These antibodies bind to an epitope that comprises all or part of the
sequence FPFS
or PFPFSAQSGA. All these antibodies are all able to bind to the PSMGFR
peptide, bind to
the N-10 peptide but do not bind to the C-10 peptide. In addition, these
antibodies disrupt the
binding of NME7An to the MUC1* extra cellular domain as exemplified by the
PSMGFR
peptide. Further, these antibodies recognize a MUC1 cleavage product when the
cleavage
enzyme is MMP9. As can be seen in the figure, these antibodies show no binding
to normal
heart tissue. Fig. 89A, Fig. 89E show that reference antibody MNC2 does not
bind to normal
heart tissue. Fig. 89B, Fig. 89F show that reference antibody MNE6 does not
bind to normal
heart tissue. Fig. 89C, Fig. 89G show that antibody 20A10 does not bind to
normal heart
tissue. Fig. 89D, Fig. 89H shows that antibody 3C2B1 does not bind to normal
heart tissue.
[00354] Figure 90A-90D show photographs of normal heart tissue specimens,
stained
with different antibodies of the invention. Fig. 90A-90B show the entire
tissue core. Fig.
90C-90D show the 40X magnification of a particular area of the tissue. Fig.
90A and Fig.
90C show staining with MNC3. Fig. 90B and Fig. 90D show staining with 25E6.
These
antibodies bind to an epitope that comprises all or part of the sequence
ASRYNLT. These
antibodies are all able to bind to the PSMGFR peptide, bind to the N-10
peptide but also bind
to the C-10 peptide. As can be seen, these antibodies are not as cancer-
specific and show
sonic binding to normal heart tissue.
[00355] Figure 91A-91B show photographs of normal heart tissue specimens,
stained with
an antibody of the invention 1E4. Fig. 91A show the entire tissue core. Fig.
91B show the
40X magnification of a particular area of the tissue. Antibody 1E4 binds to an
epitope that
comprises all or part of the sequence QFNQYKTEA. Antibody 1E4 can bind to the
N-10
peptide but also binds to the C-10 peptide. As can be seen in the figure, 1E4
binds to normal
heart tissue. As can be seen, these antibodies are not as cancer-specific and
show some
binding to normal heart tissue.
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[00356] Figure 92A-92H show photographs of normal heart tissue specimens,
stained
with different antibodies of the invention. Fig. 92A-92D show the entire
tissue core. Fig.
92E-92HL show the 40X magnification of a particular area of the tissue. Fig.
92A and Fig.
92E show staining with 18B4. Fig. 92B and Fig. 92F show staining with 31A1.
Fig. 92C and
Fig. 92G show staining with 32C1. Fig. 92D and Fig. 92H show staining with
29H1. These
antibodies bind to an epitope that comprises all or part of the sequence
GTINVHDVET,
which is the most N-terminal part of the PSMGFR peptide. None of these
antibodies are able
to bind to the N-10 peptide. As can be seen in the figure, all of these
antibodies except 18B4
show binding to normal heart tissue.
[00357] Figure 93A-93D show photographs of normal heart tissue specimens,
stained
with antibodies of the invention. Fig. 93A-93B show the entire tissue core.
Fig. 93C-93D
show the 40X magnification of a particular area of the tissue. Fig. 93A and
Fig. 93C show
staining with antibody 8A9. Fig. 93B and Fig 93D show staining with antibody
17H6. Both
antibodies bind to an epitope that that is outside of the PSMGFR region and
comprises all or
part of the sequence VQLTLAFRE. As can be seen in the figure, both antibodies
show strong
binding to normal heart tissue.
[00358] Figure 94A-94B show photographs of normal heart tissue specimens,
stained with
an antibody of the invention 45C11. Fig. 94A show the entire tissue core. Fig.
94B show the
40X magnification of a particular area of the tissue. Antibody 45C11 binds to
an epitope that
is outside of the PSMGFR region and comprises all or part of the sequence
SNIKFRPGSVV. Antibody 45C11 cannot bind to the N-10 peptide. As can be seen in
the
figure, 45C11 binds strongly to normal heart tissue.
[00359] Figure 95A-95H show photographs of normal liver tissue specimens,
stained with
different antibodies of the invention. Fig. 95A-95D show the entire tissue
core. Fig. 95E-
95HL show the 40X magnification of a particular area of the tissue. Fig. 95A
and Fig. 95E
show staining with reference antibody MNC2-scFv. Fig. 95B and Fig. 95F show
staining
with reference antibody MNE6. Fig. 95C and Fig. 95G show staining with 20A10.
Fig. 95D
and Fig. 95H show staining with 3C2B1. These antibodies bind to an epitope
that comprises
all or part of the sequence FPFS or PFPFSAQSGA. All these antibodies are all
able to bind
to the PSMGFR peptide, bind to the N-10 peptide but do not bind to the C-10
peptide. In
addition, these antibodies disrupt the binding of NME7An to the MUC1* extra
cellular
domain as exemplified by the PSMGFR peptide. Further, these antibodies
recognize a MUC1
cleavage product when the cleavage enzyme is MMP9. As can be seen in the
figure, these
antibodies show no binding to normal liver tissue.
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[00360] Figure 96A-96D show photographs of normal liver tissue specimens,
stained with
different antibodies of the invention. Fig. 96A-96B show the entire tissue
core. Fig. 96C-96D
show the 40X magnification of a particular area of the tissue. Fig. 96A and
Fig. 96C show
staining with MNC3. Fig. 96B and Fig. 96D show staining with 25E6. These
antibodies bind
to an epitope that comprises all or part of the sequence ASRYNLT. These
antibodies are all
able to bind to the PSMGER peptide, bind to the N-10 peptide but also bind to
the C-10
peptide. As can be seen, these antibodies are not as cancer-specific and show
some binding to
normal liver tissue.
[00361] Figure 97A-97B show photographs of normal liver tissue specimens,
stained with
an antibody of the invention 1E4. Fig. 97A show the entire tissue core. Fig.
97B show the
40X magnification of a particular area of the tissue. Antibody 1E4 binds to an
epitope that
comprises all or part of the sequence QFNQYKTEA. Antibody 1E4 can bind to the
N-10
peptide but also binds to the C-10 peptide. As can be seen in the figure, 1E4
binds to normal
liver tissue.
[00362] Figure 98A-98H show photographs of normal liver tissue specimens,
stained with
different antibodies of the invention. Fig. 98A-98D show the entire tissue
core. Fig. 98E-98H
show the 40X magnification of a particular area of the tissue. Fig. 98A and
Fig. 98E show
staining with 18B4. Fig. 98B and Fig. 98F show staining with 31A1. Fig. 98C
and Fig. 98G
show staining with 32C1. Fig. 98D and Fig. 98H show staining with 29H1. These
antibodies
bind to an epitope that comprises all or part of the sequence GT1N VHDVE'1',
which is the
most N-terminal part of the PSMGER peptide. None of these antibodies are able
to bind to
the N-10 peptide. As can be seen in the figure, 32C1 shows some binding to
normal liver and
29H1 shows extremely strong binding to normal liver tissue.
[00363] Figure 99A-99D show photographs of normal liver tissue specimens,
stained with
antibodies of the invention. Fig. 99A-99B show the entire tissue core. Fig.
99C-99D show the
40X magnification of a particular area of the tissue. Fig. 99A and Fig. 99C
show staining
with antibody 8A9. Fig. 99B and Fig. 99D show staining with antibody 17H6.
Both
antibodies bind to an epitope that that is outside of the PSMGFR region and
comprises all or
part of the sequence VQLTLAFRE. As can be seen in the figure, 8A9 shows strong
binding
to normal liver tissue. 17H6 is a weal( antibody and it is possible that it
was not used at a high
enough concentration in this study.
[00364] Figure 100A-100B show photographs of normal liver tissue specimens,
stained
with an antibody of the invention 45C11. Fig. 100A show the entire tissue
core. Fig. 100B
show the 40X magnification of a particular area of the tissue. Antibody 45C11
binds to an
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epitope that is outside of the PSMGFR region and comprises all or part of the
sequence
SNIKFRPGSVV. Antibody 45C11 cannot bind to the N-10 peptide. As can be seen in
the
figure, 45C11 binds strongly to normal liver tissue.
[00365] Figure 101A-101H show photographs of normal lung tissue specimens,
stained
with different antibodies of the invention. Fig. 101A-101D show the entire
tissue core. Fig.
101E-101H show the 40X magnification of a particular area of the tissue. Fig.
101A and Fig.
101E show staining with MNC2-scFv. Fig. 101B and Fig. 101F show staining with
MNE6.
Fig. 101C and Fig. 101G show staining with 20A10. Fig. 101D and Fig. 101H show
staining
with 3C2B1. These antibodies bind to an epitope that comprises all or part of
the sequence
FPFS or PFPFSAQSGA. All these antibodies are all able to bind to the PSMGFR
peptide,
bind to the N-10 peptide but do not bind to the C-10 peptide. In addition,
these antibodies
disrupt the binding of NME7AB to the MUC1* extra cellular domain as
exemplified by the
PSMGFR peptide. Further, these antibodies recognize a MUC1 cleavage product
when the
cleavage enzyme is MMP9. As can be seen in the figure, these antibodies show
no binding to
normal lung tissue.
[00366] Figure 102A-102D show photographs of normal lung tissue specimens,
stained
with different antibodies of the invention. Fig. 102A-102B show the entire
tissue core. Fig.
102C-102D show the 40X magnification of a particular area of the tissue. Fig.
102A and Fig.
102C show staining with MNC3. Fig. 102B and Fig. 102D show staining with 25E6.
These
antibodies bind to an epitope that comprises all or part of the sequence ASK
YNL'1'. 'Mese
antibodies are all able to bind to the PSMGFR peptide, bind to the N-10
peptide but also bind
to the C-10 peptide. As can be seen, these antibodies are not as cancer-
specific and show
some binding to normal lung tissue.
[00367] Figure 103A-103B show photographs of normal lung tissue specimens,
stained
with an antibody of the invention 1E4. Fig. 103A show the entire tissue core.
Fig. 103B show
the 40X magnification of a particular area of the tissue. Antibody 1E4 binds
to an epitope that
comprises all or part of the sequence QFNQYKTEA. Antibody 1E4 can bind to the
N-10
peptide but also binds to the C-10 peptide.
[00368] Figure 104A-104H show photographs of normal lung tissue specimens,
stained
with different antibodies of the invention. Fig. 104A-104D show the entire
tissue core. Fig.
104E-104H show the 40X magnification of a particular area of the tissue. Fig.
104A and Fig.
104E show staining with 18B4. Fig. 104B and Fig. 104F show staining with 31A1.
Fig. 104C
and Fig. 104G show staining with 32C1. Fig. 104D and Fig. 104H show staining
with 29H1.
These antibodies bind to an epitope that comprises all or part of the sequence
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GTINVHDVET, which is the most N-terminal part of the PSMGFR peptide. None of
these
antibodies are able to bind to the N-10 peptide. As can he seen in the figure,
all these
antibodies show strong binding to normal lung tissue.
[00369] Figure 105A-105D show photographs of normal lung tissue specimens,
stained
with antibodies of the invention. Fig. 105A-105B show the entire tissue core.
Fig. 105C-
105D show the 40X magnification of a particular area of the tissue. Fig. 105A
and Fig. 105C
show staining with antibody 8A9. Fig. 105B and Fig. 105D show staining with
antibody
17H6. Both antibodies bind to an epitope that that is outside of the PSMGFR
region and
comprises all or part of the sequence VQLTLAFRE. As can be seen in the figure,
8A9 shows
strong binding to normal lung tissue. 17H6 is a weak antibody and it is
possible that it was
not used at a high enough concentration in this study.
[00370] Figure 106A-106B show photographs of normal lung tissue specimens,
stained
with an antibody of the invention 45C11. Fig. 106A show the entire tissue
core. Fig. 106B
show the 40X magnification of a particular area of the tissue. Antibody 45C11
binds to an
epitope that is outside of the PSMGFR region and comprises all or part of the
sequence
SNIKFRPGSVV. Antibody 45C11 cannot bind to the N-10 peptide. As can be seen in
the
figure, 45C11 binds to normal lung tissue.
[00371] Figure 107A-107H show photographs of normal bone marrow tissue
specimens,
stained with different antibodies of the invention. Fig. 107A-107D show the
entire tissue
core. Hg. 107B-107H show the 40X magnification of a particular area of the
tissue. Fig.
107A and Fig. 107E show staining with MNC2-scFv. Fig. 107B and Fig. 107F show
staining
with MNE6. Fig. 107C and Fig. 107G show staining with 20A10. Fig. 107D and
Fig. 107H
show staining with 3C2B1. These antibodies bind to an epitope that comprises
all or part of
the sequence FPFS or PFPFSAQSGA. All these antibodies are all able to bind to
the
PSMGFR peptide, bind to the N-10 peptide but do not bind to the C-10 peptide.
In addition,
these antibodies disrupt the binding of NME7An to the MUC1* extra cellular
domain as
exemplified by the PSMGFR peptide. Further, these antibodies recognize a MUC 1
cleavage
product when the cleavage enzyme is MMP9. As can be seen in the figure, these
antibodies
show no binding to normal bone marrow tissue.
[00372] Figure 108A-108D show photographs of normal bone marrow tissue
specimens,
stained with different antibodies of the invention. Fig. 108A-108B show the
entire tissue
core. Fig. 108C-108D show the 40X magnification of a particular area of the
tissue. Fig.
108A and Fig. 108C show staining with MNC3. Fig. 108B and Fig. 108D show
staining with
25E6. These antibodies bind to an epitope that comprises all or part of the
sequence
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ASRYNLT. These antibodies are all able to bind to the PSMGER peptide, bind to
the N-10
peptide but also bind to the C-10 peptide.
[00373] Figure 109A-109B show photographs of normal bone marrow tissue
specimens,
stained with an antibody of the invention 1E4. Fig. 109A show the entire
tissue core. Fig.
109B show the 40X magnification of a particular area of the tissue. Antibody
1E4 binds to an
epitope that comprises all or part of the sequence QFNQYKTEA. Antibody 1E4 can
bind to
the N-10 peptide but also binds to the C-10 peptide. 1E4 binds to normal bone
marrow.
[00374] Figure 110A-110H show photographs of normal bone marrow tissue
specimens,
stained with different antibodies of the invention. Fig. 110A-110D show the
entire tissue
core. Fig. 110E-110H show the 40X magnification of a particular area of the
tissue. Fig.
110A and Fig. 110E show staining with 18B4. Fig. 110B and Fig. 110F show
staining with
31A1. Fig. 110C and Fig. 110G show staining with 32C1. Fig. 110D and Fig. 110H
show
staining with 29H1. These antibodies bind to an epitope that comprises all or
part of the
sequence GTINVHDVET, which is the most N-terminal part of the PSMGER peptide.
None
of these antibodies are able to bind to the N-10 peptide. As can be seen in
the figure, all these
antibodies show strong binding to normal bone marrow tissue.
[00375] Figure 111A-111D show photographs of normal bone marrow tissue
specimens,
stained with antibodies of the invention. Fig. 111A-111B show the entire
tissue core. Fig.
111C-111D show the 40X magnification of a particular area of the tissue. Fig.
111A and Fig.
111C show staining with antibody 8A9. Fig. 111B and Fig. 111D show staining
with
antibody 17H6. Both antibodies bind to an epitope that that is outside of the
PSMGER region
and comprises all or part of the sequence VQLTLAFRE. As can be seen in the
figure, 8A9
shows strong binding to normal bone marrow tissue. 17H6 is a weak antibody and
it is
possible that it was not used at a high enough concentration in this study.
[00376] Figure 112A-112B show photographs of normal bone marrow tissue
specimens,
stained with an antibody of the invention 45C11. Fig. 112A show the entire
tissue core. Fig.
112B show the 40X magnification of a particular area of the tissue. Antibody
45C11 binds to
an epitope that is outside of the PSMGFR region and comprises all or part of
the sequence
SNIKFRPGSVV. Antibody 45C11 cannot bind to the N-10 peptide. As can be seen in
the
figure, 45C11 binds to normal bone marrow tissue.
[00377] Figure 113A-113C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the anti-PSMGER antibody 20A10 at
0.25ug/mL.
Fig. 113A shows photographs of the tissue micro array. Fig. 113B shows map of
the array
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with abbreviated tissue descriptors. Fig. 113C detailed description of the
tissue micro array
with non-identifying donor data.
[00378] Figure 114A-114X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the anti-PSMGFR antibody 20A10 at 0.25ug/mL,
magnified to 6X and 20X. Fig. 114A and Fig. 114E are adrenal gland. Fig. 114B
and Fig.
114F are breast. Fig. 114C and Fig. 114G are fallopian tube. Fig. 114D and
Fig. 114H are
kidney. Fig. 1141 and Fig. 114M are heart muscle. Fig. 114J and Fig. 114N are
liver. Fig.
114K and Fig. 1140 are lung. Fig. 114L and Fig. 114P are ureter. Fig. 114Q and
Fig. 114U
are eye. Fig. 114R and Fig. 114V are cerebral cortex. Fig. 114S and Fig. 114W
are bone
marrow. Fig. 114T and Fig. 114X are skeletal muscle.
[00379] Figure 115A-115C shows photographs, array map and description of
breast
cancer tissue array BR1141 stained with the anti-PSMGFR antibody 20A10 at
0.25ug/mL.
Fig. 115A shows photographs of the tissue micro array. Fig. 115B shows map of
the array
with abbreviated tissue descriptors. Fig. 115C detailed description of the
tissue micro array
with non-identifying donor data.
[00380] Figure 116A-116F shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the anti-PSMGFR antibody 20A10 at 0.25ug/mL,
magnified to 6X and 20X. Fig. 116A and Fig. 116D are photographs of a Grade 2
invasive
ductal carcinoma. Fig. 116B and Fig. 116E are photographs of a Grade 2
invasive ductal
carcinoma. Fig. 116C and Fig. 116F are photographs of a Grade 2 invasive
ductal carcinoma.
[00381] Figure 117A-117C shows photographs, array map and description of
pancreatic
cancer tissue array PA805c stained with the anti-PSMGFR antibody 20A10 at
0.25ug/mL.
Fig. 117A shows photographs of the tissue micro array. Fig. 117B shows map of
the array
with abbreviated tissue descriptors. Fig. 117C detailed description of the
tissue micro array
with non-identifying donor data.
[00382] Figure 118A-118F shows photographs of specific tissues from pancreatic
cancer
tissue array PA805c stained with the anti-PSMGFR antibody 20A10 at 0.25tig/mL,
magnified
to 6X and 20X. Fig. 118A and Fig. 118D are photographs of a Grade 2 papillary
adenocarcinoma. Fig. 118B and Fig. 118E are photographs of a Grade 2-3 ductal
carcinoma.
Fig. 118C and Fig. 118F are photographs of a Grade 3 invasive adenocarcinoma.
[00383] Figure 119A-119C shows photographs, array map and description of
esophageal
cancer tissue array BC001113 stained with the anti-PSMGFR antibody 20A10 at
0.25ug/mL.
Fig. 119A shows photographs of the tissue micro array. Fig. 119B shows map of
the array
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with abbreviated tissue descriptors. Fig. 119C detailed description of the
tissue micro array
with non-identifying donor data.
[00384] Figure 120A-120F shows photographs of specific tissues from esophageal
cancer
tissue array BC001113 stained with the anti-PSMGFR antibody 20A10 at
0.25ug/mL,
magnified to 6X and 20X. Fig. 120A and Fig. 120D are photographs of the
specimen at
position Al. Fig. 120B and Fig. 120E are photographs of the specimen at
position A7. Fig.
120C and Fig. 120F are photographs of the specimen at position AS.
[00385] Figure 121A-121C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the anti-PSMGFR antibody 3C2B1 at
20ug/mL.
Fig. 121A shows photographs of the tissue micro array. Fig. 121B shows map of
the array
with abbreviated tissue descriptors. Fig. 121C detailed description of the
tissue micro array
with non-identifying donor data.
[00386] Figure 122A-122X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the anti-PSMGFR antibody 3C2B1 at 20ug/mL,
magnified to 6X and 20X. Fig. 122A and Fig. 122E are adrenal gland. Fig. 122B
and Fig.
122F are breast. Fig. 122C and Fig. 122G are fallopian tube. Fig. 122D and
Fig. 122H are
kidney. Fig. 1221 and Fig. 122M are heart muscle. Fig. 122J and Fig. 122N are
liver. Fig.
122K and Fig. 1220 are lung. Fig. 122L and Fig. 122P are ureter. Fig. 122Q and
Fig. 122U
are eye. Fig. 122R and Fig. 122V are cerebral cortex. Fig. 122S and Fig. 122W
are bone
marrow. Fig. 1221 and Fig. 122X are skeletal muscle.
[00387] Figure 123A-123C shows photographs, array map and description of
pancreatic
cancer tissue array PA1003 stained with the anti-PSMGFR antibody 3C2B1 at
20ug/mL. Fig.
123A shows photographs of the tissue micro array. Fig. 123B shows map of the
array with
abbreviated tissue descriptors. Fig. 123C detailed description of the tissue
micro array with
non-identifying donor data.
[00388] Figure 124A-124F shows photographs of specific tissues from pancreatic
cancer
tissue array PA1003 stained with the anti-PSMGFR antibody 3C2B1 at 2Oug/mL,
magnified
to 6X and 20X. Fig. 124A and Fig. 124D are photographs of a Grade 2
adenocarcinoma. Fig.
124B and Fig. 124E are photographs of a Grade 2 adenocarcinoma. Fig. 124C and
Fig. 124F
are photographs of a Grade 2 adenocarcinoma.
[00389] Figure 125A-125C shows photographs, array map and description of
breast
cancer tissue array BR1141 stained with the anti-PSMGFR antibody 3C2B1 at
20ug/mL. Fig.
125A shows photographs of the tissue micro array. Fig. 125B shows map of the
array with
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abbreviated tissue descriptors. Fig. 125C detailed description of the tissue
micro array with
non-identifying donor data.
[00390] Figure 126A-126F shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the anti-PSMGFR antibody 3C2B1 at 20ug/mL,
magnified
to 6X and 20X. Fig. 126A and Fig. 126D are photographs of a Grade 2 invasive
ductal
carcinoma. Fig. 126B and Fig. 126E are photographs of a Grade 2 invasive
ductal carcinoma.
Fig. 126C and Fig. 126F are photographs of a Grade 2 invasive carcinoma.
[00391] Figure 127A-127C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the anti-PSMGFR antibody 5C6F3 at
lug/mL.
Fig. 127A shows photographs of the tissue micro array. Fig. 127B shows map of
the array
with abbreviated tissue descriptors. Fig. 127C detailed description of the
tissue micro array
with non-identifying donor data.
[00392] Figure 128A-128X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the anti-PSMGFR antibody 5C6F3 at lug/mL,
magnified
to 6X and 20X. Fig. 128A and Fig. 128E are adrenal gland. Fig. 128B and Fig.
128F are
breast. Fig. 128C and Fig. 128G are fallopian tube. Fig. 128D and Fig. 128H
are kidney.
Fig. 1281 and Fig. 128M are heart muscle. Fig. 128J and Fig. 128N are liver.
Fig. 128K and
Fig. 1280 are lung. Fig. 128L and Fig. 128P are ureter. Fig. 128Q and Fig.
128U are eye.
Fig. 128R and Fig. 128V are cerebral cortex. Fig. 128S and Fig. 128W are bone
marrow. Fig.
128'1' and Fig. 128X are skeletal muscle.
[00393] Figure 129A-129C shows photographs, array map and description of
pancreatic
cancer tissue array PA1003 stained with the anti-PSMGFR antibody 5C6F3 at 1-
2Oug/mL.
Fig. 129A shows photographs of the tissue micro array. Fig. 129B shows map of
the array
with abbreviated tissue descriptors. Fig. 129C detailed description of the
tissue micro array
with non-identifying donor data.
[00394] Figure 130A-130F shows photographs of specific tissues from pancreatic
cancer
tissue array PA1003 stained with the anti-PSMGFR antibody 5C6F3 at lug/mL,
magnified to
6X and 20X. Fig. 130A and Fig. 130D are photographs of a Grade 2
adenocarcinoma. Fig.
130B and Fig. 130E are photographs of a Grade 2 adenocarcinoma. Fig. 130C and
Fig. 130F
are photographs of a Grade 2 adenocarcinoma.
[00395] Figure 131A-131C shows photographs, array map and description of
breast
cancer tissue array BR1141 stained with the anti-PSMGFR antibody 5C6F3 at
lug/mL. Fig.
131A shows photographs of the tissue micro array. Fig. 131B shows map of the
array with
58
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abbreviated tissue descriptors. Fig. 131C detailed description of the tissue
micro array with
non-identifying donor data.
[00396] Figure 132A-132F shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the anti-PSMGFR antibody 5C6F3 at lug/mL,
magnified to
6X and 20X. Fig. 132A and Fig. 132D are photographs of a Grade 2 invasive
ductal
carcinoma. Fig. 132B and Fig. 132E are photographs of a Grade 2 invasive
ductal carcinoma.
Fig. 132C and Fig. 132F are photographs of a Grade 2 invasive carcinoma.
[00397] Figure 133A-133C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the anti-PSMGFR antibody 18B4 at
lOug/mL.
Fig. 133A shows photographs of the tissue micro array. Fig. 133B shows map of
the array
with abbreviated tissue descriptors. Fig. 133C detailed description of the
tissue micro array
with non-identifying donor data.
[00398] Figure 134A-134X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the anti-PSMGFR antibody 18B4 at lOug/mL,
magnified
to 6X and 20X. Fig. 134A and Fig. 134E are adrenal gland. Fig. 134B and Fig.
134F are
breast. Fig. 134C and Fig. 134G are fallopian tube. Fig. 134D and Fig. 134H
are kidney.
Fig. 1341 and Fig. 134M are heart muscle. Fig. 134J and Fig. 134N are liver.
Fig. 134K and
Fig. 1340 are lung. Fig. 134L and Fig. 134P are ureter. Fig. 134Q and Fig.
134U are eye.
Fig. 134R and Fig. 134V are cerebral cortex. Fig. 134S and Fig. 134W are bone
marrow. Fig.
1341 and Fig. 134X are skeletal muscle.
[00399] Figure 135A-135C shows photographs, array map and description of
breast
cancer tissue array BR1141 stained with the anti-PSMGFR antibody 18B4 at
lOug/mL. Fig.
135A shows photographs of the tissue micro array. Fig. 135B shows map of the
array with
abbreviated tissue descriptors. Fig. 135C detailed description of the tissue
micro array with
non-identifying donor data.
[00400] Figure 136A-136F shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the anti-PSMGFR antibody 18B4 at lOug/mL,
magnified to
6X and 20X. Fig. 136A and Fig. 136D are photographs of a Grade 2 invasive
ductal
carcinoma. Fig. 136B and Fig. 136E are photographs of a Grade 2 invasive
ductal carcinoma.
Fig. 136C and Fig. 136F are photographs of a Grade 2 invasive ductal
carcinoma.
[00401] Figure 137A-137C shows photographs, array map and description of
esophageal
cancer tissue array BC001113 stained with the anti-PSMGFR antibody 18B4 at
lOug/mL.
Fig. 137A shows photographs of the tissue micro array. Fig. 137B shows map of
the array
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with abbreviated tissue descriptors. Fig. 137C detailed description of the
tissue micro array
with non-identifying donor data.
[00402] Figure 138A-138F shows photographs of specific tissues from esophageal
cancer
tissue array BC001113 stained with the anti-PSMGFR antibody 18B4 at lOug/mL,
magnified
to 6X and 20X. Fig. 138A and Fig. 138D are photographs of the specimen at
position Al.
Fig. 138B and Fig. 138E are photographs of the specimen at position A7. Fig.
138C and Fig.
138F are photographs of the specimen at position A8.
[00403] Figure 139A-139C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the anti-PSMGFR antibody 18G12 at
lOug/mL.
Fig. 139A shows photographs of the tissue micro array. Fig. 139B shows map of
the array
with abbreviated tissue descriptors. Fig. 139C detailed description of the
tissue micro array
with non-identifying donor data.
[00404] Figure 140A-140X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the anti-PSMGFR antibody 18G12 at lOug/mL,
magnified to 6X and 20X. Fig. 140A and Fig. 140E are adrenal gland. Fig. 140B
and Fig.
140F are breast. Fig. 140C and Fig. 140G are fallopian tube. Fig. 140D and
Fig. 140H are
kidney. Fig. 1401 and Fig. 140M are heart muscle. Fig. 140J and Fig. 140N are
liver. Fig.
140K and Fig. 1400 are lung. Fig. 140L and Fig. 140P are ureter. Fig. 140Q and
Fig. 140U
are eye. Fig. 140R and Fig. 140V are cerebral cortex. Fig. 140S and Fig. 140W
are bone
marrow. Fig. 140T and Fig. 140X are skeletal muscle.
[00405] Figure 141A-141C shows photographs, array map and description of
breast
cancer tissue array BR1141 stained with the anti-PSMGFR antibody 18G12 at
15ug/mL. Fig.
141A shows photographs of the tissue micro array. Fig. 141B shows map of the
array with
abbreviated tissue descriptors. Fig. 141C detailed description of the tissue
micro array with
non-identifying donor data.
[00406] Figure 142A-142F shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the anti-PSMGFR antibody 18G12 at 15tig/mL,
magnified
to 6X and 20X. Fig. 142A and Fig. 142D are photographs of a Grade 2 invasive
ductal
carcinoma. Fig. 142B and Fig. 142E are photographs of a Grade 2 invasive
ductal carcinoma.
Fig. 142C and Fig. 142F are photographs of a Grade 2 invasive ductal
carcinoma.
[00407] Figure 143A-143C shows photographs, array map and description of
pancreatic
cancer tissue array PA1003 stained with the anti-PSMGFR antibody 18G12 at
15ug/mL. Fig.
143A shows photographs of the tissue micro array. Fig. 143B shows map of the
array with
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abbreviated tissue descriptors. Fig. 143C detailed description of the tissue
micro array with
non-identifying donor data.
[00408] Figure 144A-144F shows photographs of specific tissues from pancreatic
cancer
tissue array PA1003 stained with the anti-PSMGFR antibody 18G12 at 15ug/mL,
magnified
to 6X and 20X. Fig. 144A and Fig. 144D are photographs of a Grade 2
adenocarcinoma. Fig.
144B and Fig. 144E are photographs of a Grade 2 adenocarcinoma. Fig. 144C and
Fig. 144F
are photographs of a Grade 2-3 adenocarcinoma with lymph node involvement.
[00409] Figure 145A-145C shows photographs, array map and description of
esophageal
cancer tissue array BC001113 stained with the anti-PSMGFR antibody 18G12 at
30ug/mL.
Fig. 145A shows photographs of the tissue micro array. Fig. 145B shows map of
the array
with abbreviated tissue descriptors. Fig. 145C detailed description of the
tissue micro array
with non-identifying donor data.
[00410] Figure 146A-146F shows photographs of specific tissues from esophageal
cancer
tissue array BC001113 stained with the anti-PSMGFR antibody 18G12 at 30ug/mL,
magnified to 6X and 20X. Fig. 146A and Fig. 146D are photographs of the
specimen at
position Al. Fig. 146B and Fig. 146E are photographs of the specimen at
position A7. Fig.
146C and Fig. 146F are photographs of the specimen at position A8.
[00411] Figure 147A-147C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the anti-PSMGFR antibody 25E6 at
5.0ug/mL.
Pig. 147A shows photographs of the tissue micro array. Fig. 147B shows map of
the array
with abbreviated tissue descriptors. Fig. 147C detailed description of the
tissue micro array
with non-identifying donor data.
[00412] Figure 148A-148X shows photographs of specific tissues from FDA normal
tissue array 1021 stained with the anti-PSMGFR antibody 25E6 at 5.0ug/mL,
magnified to
6X and 20X. Fig. 148A and Fig. 148E are adrenal gland. Fig. 148B and Fig. 148F
are breast.
Fig. 148C and Fig. 148G are fallopian tube. Fig. 148D and Fig. 148H are
kidney. Fig. 1481
and Fig. 148M are heart muscle. Fig. 148J and Fig. 148N are liver. Fig. 148K
and Fig.
1480 are lung. Fig. 148L and Fig. 148P are ureter. Fig. 148Q and Fig. 148U are
eye. Fig.
148R and Fig. 148V are cerebral cortex. Fig. 148S and Fig. 148W are bone
marrow. Fig.
148T and Fig. 148X are skeletal muscle.
[00413] Figure 149A-149C shows photographs, array map and description of
breast
cancer tissue array BR1141 stained with the anti-PSMGFR antibody 25E6 at
5.0ug/mL. Fig.
149A shows photographs of the tissue micro array. Fig. 149B shows map of the
array with
61
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abbreviated tissue descriptors. Fig. 149C detailed description of the tissue
micro array with
non-identifying donor data.
[00414] Figure 150A-150F shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the anti-PSMGFR antibody 25E6 at 5.0ug/mL,
magnified to
6X and 20X. Fig. 150A and Fig. 150D are photographs of a Grade 2 invasive
ductal
carcinoma. Fig. 150B and Fig. 150E are photographs of a Grade 2 invasive
ductal carcinoma.
Fig. 150C and Fig. 150F are photographs of a Grade 2 invasive ductal
carcinoma.
[00415] Figure 151A-151C shows photographs, array map and description of
pancreatic
cancer tissue array PA1003 stained with the anti-PSMGFR antibody 25E6 at
5.0ug/mL. Fig.
151A shows photographs of the tissue micro array. Fig. 151B shows map of the
array with
abbreviated tissue descriptors. Fig. 151C detailed description of the tissue
micro array with
non-identifying donor data.
[00416] Figure 152A-152F shows photographs of specific tissues from pancreatic
cancer
tissue array PA1003 stained with the anti-PSMGFR antibody 25E6 at 5.0ug/mL,
magnified to
6X and 20X. Fig. 152A and Fig. 152D are photographs of a Grade 2
adenocarcinoma. Fig.
152B and Fig. 152E are photographs of a Grade 1 adenocarcinoma. Fig. 152C and
Fig. 152F
are photographs of a Grade 1 adenocarcinoma.
[00417] Figure 153A-153C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the anti-PSMGFR antibody 28F9 at
15.0ug/mL.
Pig. 153A shows photographs of the tissue micro array. Fig. 153B shows map of
the array
with abbreviated tissue descriptors. Fig. 153C detailed description of the
tissue micro array
with non-identifying donor data.
[00418] Figure 154A-154X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the anti-PSMGFR antibody 28F9 at 15.0ug/mL,
magnified to 6X and 20X. Fig. 154A and Fig. 154E are adrenal gland. Fig. 154B
and Fig.
154F are breast. Fig. 154C and Fig. 154G are fallopian tube. Fig. 154D and
Fig. 154H are
kidney. Fig. 1541 and Fig. 154M are heart muscle. Fig. 154J and Fig. 154N are
liver. Fig.
154K and Fig. 1540 are lung. Fig. 154L and Fig. 154P are ureter. Fig. 154Q and
Fig. 154U
are eye. Fig. 154R and Fig. 154V are cerebral cortex. Fig. 154S and Fig. 154W
are bone
marrow. Fig. 154T and Fig. 154X are skeletal muscle.
[00419] Figure 155A-155C shows photographs, array map and description of
breast
cancer tissue array BR1141 stained with the anti-PSMGFR antibody 28F9 at
15.0ug/mL. Fig.
155A shows photographs of the tissue micro array. Fig. 155B shows map of the
array with
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abbreviated tissue descriptors. Fig. 155C detailed description of the tissue
micro array with
non-identifying donor data.
[00420] Figure 156A-156F shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the anti-PSMGFR antibody 28F9 at 15.0ug/mL,
magnified
to 6X and 20X. Fig. 156A and Fig. 156D are photographs of a Grade 2 invasive
ductal
carcinoma. Fig. 156B and Fig. 156E are photographs of a Grade 2 invasive
ductal carcinoma.
Fig. 156C and Fig. 156F are photographs of a Grade 2 invasive ductal
carcinoma.
[00421] Figure 157A-157C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the N+20/C-27 antibody 1E4 at
7.5ug/mL. Fig.
157A shows photographs of the tissue micro array. Fig. 157B shows map of the
array with
abbreviated tissue descriptors. Fig. 157C detailed description of the tissue
micro array with
non-identifying donor data.
[00422] Figure 158A-158X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the N+20/C-27 antibody 1E4 at 7.5ug/mL,
magnified to
6X and 20X. Fig. 158A and Fig. 158E are adrenal gland. Fig. 158B and Fig. 158F
are breast.
Fig. 158C and Fig. 158G are fallopian tube. Fig. 158D and Fig. 158H are
kidney. Fig. 1581
and Fig. 158M are heart muscle. Fig. 158J and Fig. 158N are liver. Fig. 158K
and Fig.
1580 are lung. Fig. 158L and Fig. 158P are ureter. Fig. 158Q and Fig. 158U are
eye. Fig.
158R and Fig. 158V are cerebral cortex. Fig. 158S and Fig. 158W are bone
marrow. Fig.
158'1' and Fig. 158X are skeletal muscle.
[00423] Figure 159A-159C shows photographs, array map and description of
breast
cancer tissue array BR1007 stained with the N+20/C-27 antibody 1E4 at
10.0ug/mL. Fig.
159A shows photographs of the tissue micro array. Fig. 159B shows map of the
array with
abbreviated tissue descriptors. Fig. 159C detailed description of the tissue
micro array with
non-identifying donor data.
[00424] Figure 160A-160F shows photographs of specific tissues from breast
cancer
tissue array BR1007 stained with the N+20/C-27 antibody 1E4 at 10.0ug/mL,
magnified to
6X and 20X. Fig. 160A and Fig. 160D are photographs of a Grade 2 invasive
ductal
carcinoma with positive lymph nodes. Fig. 160B and Fig. 160E are photographs
of a Grade 2
invasive ductal carcinoma. Fig. 160C and Fig. 160F are photographs of a Grade
2 invasive
ductal carcinoma.
[00425] Figure 161A-161C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the N+20/C-27 antibody 29H1 at
0.5ug/mL. Fig.
161A shows photographs of the tissue micro array. Fig. 161B shows map of the
array with
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abbreviated tissue descriptors. Fig. 161C detailed description of the tissue
micro array with
non-identifying donor data.
[00426] Figure 162A-162X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the N+20/C-27 antibody 29H1 at 0.5ug/mL,
magnified to
6X and 20X. Fig. 162A and Fig. 162E are adrenal gland. Fig. 162B and Fig. 162F
are breast.
Fig. 162C and Fig. 162G are fallopian tube. Fig. 162D and Fig. 162H are
kidney. Fig. 1621
and Fig. 162M are heart muscle. Fig. 162J and Fig. 162N are liver. Fig. 162K
and Fig.
1620 are lung. Fig. 162L and Fig. 162P are ureter. Fig. 162Q and Fig. 162U are
eye. Fig.
162R and Fig. 162V are cerebral cortex. Fig. 162S and Fig. 162W are bone
marrow. Fig.
162T and Fig. 162X are skeletal muscle.
[00427] Figure 163A-163C shows photographs, array map and description of
breast
cancer tissue array BR1141 stained with the N+20/C-27 antibody 29H1 at
0.5ug/mL. Fig.
163A shows photographs of the tissue micro array. Fig. 163B shows map of the
array with
abbreviated tissue descriptors. Fig. 163C detailed description of the tissue
micro array with
non-identifying donor data.
[00428] Figure 164A-164F shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the N+20/C-27 antibody 29H1 at 0.5ug/mL,
magnified to
6X and 20X. Fig. 164A and Fig. 164D are photographs of a Grade 2 invasive
ductal
carcinoma. Fig. 164B and Fig. 164E are photographs of a Grade 2 invasive
ductal carcinoma.
Pig. 164C and Fig. 164F are photographs of a Grade 2 invasive ductal
carcinoma.
[00429] Figure 165A-165C shows photographs, array map and description of
pancreatic
cancer tissue array PA1003 stained with the N+20/C-27 antibody 29H1 at
0.5ug/mL. Fig.
165A shows photographs of the tissue micro array. Fig. 165B shows map of the
array with
abbreviated tissue descriptors. Fig. 165C detailed description of the tissue
micro array with
non-identifying donor data.
[00430] Figure 166A-166F shows photographs of specific tissues from pancreatic
cancer
tissue array PA1003 stained with the N+20/C-27 antibody 29H1 at 0.5ug/mL,
magnified to
6X and 20X. Fig. 166A and Fig. 166D are photographs of a Grade 2
adenocarcinoma. Fig.
166B and Fig. 166E are photographs of a Grade 2 adenocarcinoma. Fig. 166C and
Fig. 166F
are photographs of a Grade 3 adenocarcinoma.
[00431] Figure 167A-167C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the N+20/C-27 antibody 31A1 at
0.5ug/mL. Fig.
167A shows photographs of the tissue micro array. Fig. 167B shows map of the
array with
64
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abbreviated tissue descriptors. Fig. 167C detailed description of the tissue
micro array with
non-identifying donor data.
[00432] Figure 168A-168X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the N+20/C-27 antibody 31A1 at 0.5ug/mL,
magnified to
6X and 20X. Fig. 168A and Fig. 168E are adrenal gland. Fig. 168B and Fig. 168F
are breast.
Fig. 168C and Fig. 168G are fallopian tube. Fig. 168D and Fig. 168H are
kidney. Fig. 1681
and Fig. 168M are heart muscle. Fig. 168J and Fig. 168N are liver. Fig. 168K
and Fig.
1680 are lung. Fig. 168L and Fig. 168P are ureter. Fig. 168Q and Fig. 168U are
eye. Fig.
168R and Fig. 168V are cerebral cortex. Fig. 168S and Fig. 168W are bone
marrow. Fig.
168T and Fig. 168X are skeletal muscle.
[00433] Figure 169A-169C shows photographs, array map and description of
breast
cancer tissue array BR1141 stained with the N+20/C-27 antibody 31A1 at
0.5ug/mL. Fig.
169A shows photographs of the tissue micro array. Fig. 169B shows map of the
array with
abbreviated tissue descriptors. Fig. 169C detailed description of the tissue
micro array with
non-identifying donor data.
[00434] Figure 170A-170F shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the N+20/C-27 antibody 31A1 at 0.5ug/mL,
magnified to
6X and 20X. Fig. 170A and Fig. 170D are photographs of a Grade 2 invasive
ductal
carcinoma. Fig. 170B and Fig. 170E are photographs of a Grade 2 invasive
ductal carcinoma.
Pig. 170C and Fig. 170F are photographs of a Grade 2 invasive ductal
carcinoma.
[00435] Figure 171A-171C shows photographs, array map and description of
pancreatic
cancer tissue array PA1003 stained with the N+20/C-27 antibody 31A1 at
0.5ug/mL. Fig.
171A shows photographs of the tissue micro array. Fig. 171B shows map of the
array with
abbreviated tissue descriptors. Fig. 171C detailed description of the tissue
micro array with
non-identifying donor data.
[00436] Figure 172A-172F shows photographs of specific tissues from pancreatic
cancer
tissue array PA1003 stained with the N+20/C-27 antibody 31A1 at 0.5ug/mL,
magnified to
6X and 20X. Fig. 172A and Fig. 172D are photographs of a Grade 1
adenocarcinoma. Fig.
172B and Fig. 172E are photographs of a Grade 2 adenocarcinoma. Fig. 172C and
Fig. 172F
are photographs of a Grade 3 adenocarcinoma.
[00437] Figure 173A-173C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the N+20/C-27 antibody 32C1 at
0.25ug/mL. Fig.
173A shows photographs of the tissue micro array. Fig. 173B shows map of the
array with
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abbreviated tissue descriptors. Fig. 173C detailed description of the tissue
micro array with
non-identifying donor data.
[00438] Figure 174A-174X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the N+20/C-27 antibody 32C1 at 0.25ug/mL,
magnified
to 6X and 20X. Fig. 174A and Fig. 174E are adrenal gland. Fig. 174B and Fig.
174F are
breast. Fig. 174C and Fig. 174G are fallopian tube. Fig. 174D and Fig. 174H
are kidney.
Fig. 1741 and Fig. 174M are heart muscle. Fig. 174J and Fig. 174N are liver.
Fig. 174K and
Fig. 1740 are lung. Fig. 174L and Fig. 174P are ureter. Fig. 174Q and Fig.
174U are eye.
Fig. 174R and Fig. 174V are cerebral cortex. Fig. 174S and Fig. 174W are bone
marrow. Fig.
174T and Fig. 174X are skeletal muscle.
[00439] Figure 175A-175C shows photographs, array map and description of
breast
cancer tissue array BR1141 stained with the N+20/C-27 antibody 32C1 at
5.0ug/mL. Fig.
175A shows photographs of the tissue micro array. Fig. 175B shows map of the
array with
abbreviated tissue descriptors. Fig. 175C detailed description of the tissue
micro array with
non-identifying donor data.
[00440] Figure 176A-176F shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the N+20/C-27 antibody 32C1 at 5.0ug/mL,
magnified to
6X and 20X. Fig. 176A and Fig. 176D are photographs of a Grade 2 invasive
ductal
carcinoma. Fig. 176B and Fig. 176E are photographs of a Grade 2 invasive
ductal carcinoma.
Pig. 176C and Fig. 176F are photographs of a Grade 2 invasive ductal
carcinoma.
[00441] Figure 177A-177C shows photographs, array map and description of
esophageal
cancer tissue array ES1001 stained with the N+20/C-27 antibody 32C1 at
1.0ug/mL. Fig.
177A shows photographs of the tissue micro array. Fig. 177B shows map of the
array with
abbreviated tissue descriptors. Fig. 177C detailed description of the tissue
micro array with
non-identifying donor data.
[00442] Figure 178A-178F shows photographs of specific tissues from esophageal
cancer
tissue array BC001113 stained with the N+20/C-27 antibody 32C1 at 1.0ug/mL,
magnified to
6X and 20X. Fig. 178A and Fig. 178D are photographs of a squamous cell
carcinoma. Fig.
178B and Fig. 178E are photographs of an adenocarcinoma. Fig. 178C and Fig.
178F are
photographs of a squamous cell carcinoma.
[00443] Figure 179A-179C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the N+20/C-27 antibody 45C11 at
12.5ug/mL.
Fig. 179A shows photographs of the tissue micro array. Fig. 179B shows map of
the array
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with abbreviated tissue descriptors. Fig. 179C detailed description of the
tissue micro array
with non-identifying donor data.
[00444] Figure 180A-180X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the N+20/C-27 antibody 45C11 at 12.5ug/mL,
magnified
to 6X and 20X. Fig. 180A and Fig. 180E are adrenal gland. Fig. 180B and Fig.
180F are
breast. Fig. 180C and Fig. 180G are fallopian tube. Fig. 180D and Fig. 180H
are kidney.
Fig. 1801 and Fig. 180M are heart muscle. Fig. 180J and Fig. 180N are liver.
Fig. 180K and
Fig. 1800 are lung. Fig. 180L and Fig. 180P are ureter. Fig. 180Q and Fig.
180U are eye.
Fig. 180R and Fig. 180V are cerebral cortex. Fig. 180S and Fig. 180W are bone
marrow. Fig.
180T and Fig. 180X are skeletal muscle.
[00445] Figure 181A-181C shows photographs, array map and description of
breast
cancer tissue array BR1007 stained with the N+20/C-27 antibody 45C11 at
10.0ug/mL. Fig.
181A shows photographs of the tissue micro array. Fig. 181B shows map of the
array with
abbreviated tissue descriptors. Fig. 181C detailed description of the tissue
micro array with
non-identifying donor data.
[00446] Figure 182A-182F shows photographs of specific tissues from breast
cancer
tissue array BR1007 stained with the N+20/C-27 antibody 45C11 at 10.0ug/mL,
magnified to
6X and 20X. Fig. 182A and Fig. 182D are photographs of a Grade 2 invasive
ductal
carcinoma with positive lymph nodes. Fig. 182B and Fig. 182E are photographs
of a Grade 2
invasive ductal carcinoma. Fig. 182C and Fig. 182F are photographs of a Grade
2 invasive
ductal carcinoma.
[00447] Figure 183A-183C shows photographs, array map and description of
pancreatic
cancer tissue array PA805c stained with the N+20/C-27 antibody 45C11 at
12.5ug/mL. Fig.
183A shows photographs of the tissue micro array. Fig. 183B shows map of the
array with
abbreviated tissue descriptors. Fig. 183C detailed description of the tissue
micro array with
non-identifying donor data.
[00448] Figure 184A-184F shows photographs of specific tissues from pancreatic
cancer
tissue array PA805c stained with the N+20/C-27 antibody 45C11 at 12.5ug/mL,
magnified to
6X and 20X. Fig. 184A and Fig. 184D are photographs of a Grade 2 papillary
adenocarcinoma. Fig. 184B and Fig. 184E are photographs of a Grade 2-3 ductal
carcinoma.
Fig. 184C and Fig. 184F are photographs of a Grade 3 invasive adenocarcinoma.
[00449] Figure 185A-185C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the N+9/C-9 antibody 3C5 at
10.0ug/mL. Fig.
185A shows photographs of the tissue micro array. Fig. 185B shows map of the
array with
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abbreviated tissue descriptors. Fig. 185C detailed description of the tissue
micro array with
non-identifying donor data.
[00450] Figure 186A-186X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the N+9/C-9 antibody 3C5 at 10.0ug/mL,
magnified to
6X and 20X. Fig. 186A and Fig. 186E are adrenal gland. Fig. 186B and Fig. 186F
are breast.
Fig. 186C and Fig. 186G are fallopian tube. Fig. 186D and Fig. 186H are
kidney. Fig. 1861
and Fig. 186M are heart muscle. Fig. 186J and Fig. 186N are liver. Fig. 186K
and Fig.
1860 are lung. Fig. 186L and Fig. 186P are ureter. Fig. 186Q and Fig. 186U are
eye. Fig.
186R and Fig. 186V are cerebral cortex. Fig. 186S and Fig. 186W are bone
marrow. Fig.
186T and Fig. 186X are skeletal muscle.
[00451] Figure 187A-187C shows photographs, array map and description of
pancreatic
cancer tissue array PA1003 stained with the N+9/C-9 antibody 3C5 at 10.0ug/mL.
Fig. 187A
shows photographs of the tissue micro array. Fig. 187B shows map of the array
with
abbreviated tissue descriptors. Fig. 187C detailed description of the tissue
micro array with
non-identifying donor data.
[00452] Figure 188A-188F shows photographs of specific tissues from pancreatic
cancer
tissue array PA1003 stained with the N+9/C-9 antibody 3C5 at 10.0ug/mL,
magnified to 6X
and 20X. Fig. 188A and Fig. 188D are photographs of a Grade 2 adenocarcinoma.
Fig. 188B
and Fig. 188E are photographs of a Grade 2 adenocarcinoma. Fig. 188C and Fig.
188F are
photographs of a Grade 2-3 adenocarcinoma with lymph node involvement.
[00453] Figure 189A-189C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the N+9/C-9 antibody 8A9 at
15.0ug/mL. Fig.
189A shows photographs of the tissue micro array. Fig. 189B shows map of the
array with
abbreviated tissue descriptors. Fig. 189C detailed description of the tissue
micro array with
non-identifying donor data.
[00454] Figure 190A-190X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the N+9/C-9 antibody 8A9 at 15.0ug/mL,
magnified to
6X and 20X. Fig. 190A and Fig. 190E are adrenal gland. Fig. 190B and Fig. 190F
are breast.
Fig. 190C and Fig. 190G are fallopian tube. Fig. 190D and Fig. 190H are
kidney. Fig. 1901
and Fig. 190M are heart muscle. Fig. 190J and Fig. 190N are liver. Fig. 190K
and Fig.
1900 are lung. Fig. 190L and Fig. 190P are ureter. Fig. 190Q and Fig. 190U are
eye. Fig.
190R and Fig. 190V are cerebral cortex. Fig. 190S and Fig. 190W are bone
marrow. Fig.
190T and Fig. 190X are skeletal muscle.
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[00455] Figure 191A-191C shows photographs, array map and description of
pancreatic
cancer tissue array PA1003 stained with the N+9/C-9 antibody 8A9 at 15.0ug/mL.
Fig. 191A
shows photographs of the tissue micro array. Fig. 191B shows map of the array
with
abbreviated tissue descriptors. Fig. 191C detailed description of the tissue
micro array with
non-identifying donor data.
[00456] Figure 192A-192F shows photographs of specific tissues from pancreatic
cancer
tissue array PA1003 stained with the N+9/C-9 antibody 8A9 at 15.0ug/mL,
magnified to 6X
and 20X. Fig. 192A and Fig. 192D are photographs of a Grade 2 adenocarcinoma.
Fig. 192B
and Fig. 192E are photographs of a Grade 2 adenocarcinoma. Fig. 192C and Fig.
192F are
photographs of a Grade 2 adenocarcinoma.
[00457] Figure 193A-193C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the N+9/C-9 antibody 17H6 at
30.0ug/mL. Fig.
193A shows photographs of the tissue micro array. Fig. 193B shows map of the
array with
abbreviated tissue descriptors. Fig. 193C detailed description of the tissue
micro array with
non-identifying donor data.
[00458] Figure 194A-194X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the N+9/C-9 antibody 17H6 at 30.0ug/mL,
magnified to
6X and 20X. Fig. 194A and Fig. 194E are adrenal gland. Fig. 194B and Fig. 194F
are breast.
Fig. 194C and Fig. 194G are fallopian tube. Fig. 194D and Fig. 194H are
kidney. Fig. 1941
and Pig. 194M are heart muscle. Fig. 194J and Pig. 1941N are liver. Pig. 194K
and Pig.
1940 are lung. Fig. 194L and Fig. 194P are ureter. Fig. 194Q and Fig. 194U are
eye. Fig.
194R and Fig. 194V are cerebral cortex. Fig. 194S and Fig. 194W are bone
marrow. Fig.
194T and Fig. 194X are skeletal muscle.
[00459] Figure 195A-195C shows photographs, array map and description of
pancreatic
cancer tissue array PA805c stained with the N+9/C-9 antibody 17H6 at
30.0ug/mL. Fig.
195A shows photographs of the tissue micro array. Fig. 195B shows map of the
array with
abbreviated tissue descriptors. Fig. 195C detailed description of the tissue
micro array with
non-identifying donor data.
[00460] Figure 196A-196F shows photographs of specific tissues from pancreatic
cancer
tissue array PA805c stained with the N+9/C-9 antibody 17H6 at 30.0ug/mL,
magnified to 6X
and 20X. Fig. 196A and Fig. 196D are photographs of a Grade 2 papillary
adenocarcinoma.
Fig. 196B and Fig. 196E are photographs of a Grade 2-3 ductal carcinoma with
lymph node
involvement. Fig. 196C and Fig. 196F are photographs of a Grade 3 invasive
adenocarcinoma.
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[00461] Figure 197A-197C shows photographs, array map and description of FDA
normal tissue array MN01021 stained with the N+9/C-9 antibody 39H5 at
5.0ug/mL. Fig.
197A shows photographs of the tissue micro array. Fig. 197B shows map of the
array with
abbreviated tissue descriptors. Fig. 197C detailed description of the tissue
micro array with
non-identifying donor data.
[00462] Figure 198A-198X shows photographs of specific tissues from FDA normal
tissue array MN01021 stained with the N+9/C-9 antibody 39H5 at 5.0ug/mL,
magnified to
6X and 20X. Fig. 198A and Fig. 198E are adrenal gland. Fig. 198B and Fig. 198F
are breast.
Fig. 198C and Fig. 198G are fallopian tube. Fig. 198D and Fig. 198H are
kidney. Fig. 1981
and Fig. 198M are heart muscle. Fig. 198J and Fig. 198N are liver. Fig. 198K
and Fig.
1980 are lung. Fig. 198L and Fig. 198P are ureter. Fig. 198Q and Fig. 198U are
eye. Fig.
198R and Fig. 198V are cerebral cortex. Fig. 198S and Fig. 198W are bone
marrow. Fig.
198T and Fig. 198X are skeletal muscle.
[00463] Figure 199A-199C shows photographs, array map and description of
pancreatic
cancer tissue array PA1003 stained with the N+9/C-9 antibody 39H5 at 5.0ug/mL.
Fig. 199A
shows photographs of the tissue micro array. Fig. 199B shows map of the array
with
abbreviated tissue descriptors. Fig. 199C detailed description of the tissue
micro array with
non-identifying donor data.
[00464] Figure 200A-200F shows photographs of specific tissues from pancreatic
cancer
tissue array PA1003 stained with the N+9/C-9 antibody 39H5 at 5.0ug/mL,
magnified to 6X
and 20X. Fig. 200A and Fig. 200D are photographs of a Grade 2 adenocarcinoma.
Fig. 200B
and Fig. 200E are photographs of a Grade 2 adenocarcinoma. Fig. 200C and Fig.
200F are
photographs of a Grade 2 adenocarcinoma.
[00465] Figure 201A-201C show graphs of EL1SA assays to determine the binding
of
another set of antibodies generated by immunizing animals with the PSMGFR
peptide. Fig.
201A shows binding to the PSMGFR peptide. Fig. 201B shows binding to the N-10
peptide.
Fig. 201C shows binding to the C-10 peptide. As can be seen, none of the
antibodies bound to
the C-10 peptide. F3, B12, B2, B7, B9, 8C7F3 and H11 all bound to the PSMGFR
peptide
and to the N-10 peptide.
[00466] Figure 202A-202C shows photographs of pancreatic cancer tissue array
PA1003
that has been stained with monoclonal antibody 1E4, monoclonal antibody 18B4
or the
polyclonal anti-PSMGFR antibody SDIX. 18B4 binds to the GTINVHDVET epitope at
the
most N-terminal portion of the PSMGFR peptide, while the 1E4 antibody binds to
the
QFNQYKTEA epitope that is immediately adjacent and C-terminal to the 18B4
epitope.
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[00467] Figure 203A-203F shows magnified images of the tissue specimen at
position A2
of the pancreatic cancer array PA1003. Fig. 203A and Fig. 203B show the
specimen stained
with antibody 1E4. Fig. 203C and Fig. 203D show the specimen stained with
antibody 18B4.
Fig. 203E and Fig. 203F show the specimen stained with polyclonal antibody
SDIX.
[00468] Figure 204A-204D shows magnified images of the tissue specimen at
position D4
of the pancreatic array PA1003. Fig. 204A and Fig. 204B show the specimen
stained with
antibody 18B4. Fig. 204C and Fig. 204D show the specimen stained with
polyclonal antibody
SDIX.
[00469] Figure 205A-205D shows magnified images of the tissue specimen at
position El
of the pancreatic cancer array PA1003. Fig. 205A and Fig. 205B show the
specimen stained
with antibody 18B4. Fig. 205C and Fig. 205D show the specimen stained with
polyclonal
antibody SDIX.
[00470] Figure 206A-206D shows magnified images of the tissue specimen at
position C3
of the pancreatic cancer array PA1003. Fig. 206A and Fig. 206B show the
specimen stained
with antibody 1E4. Fig. 206C and Fig. 206D show the specimen stained with
polyclonal
antibody SDIX.
[00471] Figure 207A-207D shows magnified images of the tissue specimen at
position D1
of the pancreatic cancer array PA1003. Fig. 207A and Fig. 207B show the
specimen stained
with antibody 1E4. Fig. 207C and Fig. 207D show the specimen stained with
polyclonal
antibody SDIX.
[00472] Figure 208A-208C shows photographs of the pancreatic cancer array
PA1003.
Fig. 208A shows the specimen stained with polyclonal antibody SDIX. Fig. 208B
shows the
specimen stained with antibody 20A10. Fig. 208C shows the specimen stained
with antibody
29H1.
[00473] Figure 209A-209D shows photographs of the esophageal cancer array
ES1001
stained with various antibodies. Fig. 209A shows the array stained with
polyclonal antibody
SDIX. Fig. 209B shows the array stained with antibody 20A10. Fig. 209C shows
the array
stained with antibody 29H1. Fig. 209D shows the array stained with antibody
31A1.
[00474] Figure 210A-210C shows photographs of the pancreatic cancer array
PA1003
stained with various antibodies. Fig. 210A shows the array stained with
polyclonal antibody
SDIX. Fig. 210B shows the array stained with antibody 20A10. Fig. 210C shows
the array
stained with antibody 29H1.
[00475] Figure 211A- 211C show graphs of an ELISA experiment measuring the
amount
of IL-18 secreted into the condition media of MUC1* positive cancer cells co-
cultured with
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huMNC2-CAR44 T cells wherein the cells also bear an NFAT inducible IL-18. Fig.
211A
shows the graph of IL-18 secreted into the supernatant of T47D breast cancer
cells co-
cultured with untransduced human T cells. Fig. 211B shows the graph of 1L-18
secreted into
the supernatant of T47D breast cancer cells co-cultured with huMNC2-CAR44 T
cells that
also bore an NFAT inducible IL-18 gene inserted into a portion of the Foxp3
enhancer. Fig.
211C shows the graph of IL-18 secreted into the supernatant of T47D breast
cancer cells co-
cultured with huMNC2-CAR44 T cells that also bore an NFAT inducible 1L-18 gene
inserted
into a portion of the IL-2 enhancer.
[00476] Figure 212A- 212X shows photographs of T47D breast cancer cells (red)
doped
with varying percentages of T47D cells engineered to express more MUC1*
(green). The
target cancer cells have been co-cultured with huMNC2-CAR44 T cells with NFAT
inducible
IL-18 wherein the IL-18 gene has been inserted into either the Foxp3
enhancer/promoter or
the IL-2 enhancer/promoter. Fig. 212A-212C, 212I-212K, and 212Q-212S show the
cancer
cells co-cultured with untranduced T cells. Fig. 212D-212F, 212L-212N, and
212T-212V
show the cancer cells co-cultured with hiMNC2-CAR44 T cells with the NFAT
inducible IL-
18 gene inserted into the Foxp3 enhancer/promoter. Fig. 212G-212H, 2120-212P,
and
212W-212X show the cancer cells co-cultured with hiMNC2-CAR44 T cells with the
NFAT
inducible IL-18 gene inserted into the IL-2 enhancer/promoter.
[00477] Figure 213A- 213B shows graphs of ELISA experiments in which levels of
IL-18
secreted into the conditioned media are measured for huMNC1-CAR44 T cells with
NFAT
inducible IL-18 gene, inserted into the Foxp3 enhancer or promoter, co-
cultured with either
MUC1* positive cancer cells or MUC1 negative non-cancerous cells. Fig. 213A
shows IL-18
secretion from huMNC2-CAR44 T cells with NFAT inducible IL-18 in co-culture
with T47D
breast cancer cells where the population has been doped with 5%, 10% or 30%
T47D cells
that had been transfected with even more MUC1*. Fig. 213B shows IL-18
secretion from
huMNC2-CAR44 T cells with NFAT inducible IL-18 in co-culture with non-
cancerous,
MUC1 negaive HEK293 cells where the cell population has been doped with 5%,
10% or
30% T47D cells that had been transfected with more MUC1*.
[00478] Figure 214A-214X shows photographs of T47D breast cancer cells (red)
or non-
cancerous HEK293 cells (also red), where both cell types have been doped with
varying
percentages of T47D cells engineered to express more MUC1* (green). These
target cancer
cells have been co-cultured with huMNC2-CAR44 T cells with NFAT inducible IL-
18
wherein the IL-18 gene has been inserted into the Foxp3 enhancer/promoter.
Fig. 214A-214F
shows either T47D cells or HEK293 cells that have not been doped with T47D
cells
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engineered to express high MUC1* density. Fig. 214G-214L shows either T47D
cells or
HEK293 cells that have been doped with 5% T47D cells engineered to express
high MUC1*
density. Fig. 214M-214R shows either T47D cells or HEK293 cells that have been
doped
with 10% T47D cells engineered to express high MUC1* density. Fig. 214S-214X
shows
either T47D cells or HEK293 cells that have been doped with 30% T47D cells
engineered to
express high MUC1* density. Fig. 214A-B, G-H, M-N, and S-T show T47D breast
cancer
cells. Fig. 214C-F, 1-L, O-R, and U-X show HEK293 cells. As can be seen in the
figures, the
induced secretion of IL-18 resulted in low MUC1* density T47D cells being
killed but did
not induce non-specific killing of the MUC1* negative HEK293 cells.
[00479] Figure 215A-215C shows the consensus sequences of the heavy chain CDRs
wherein the consensus sequences were generated for each group of antibodies
that bound to
the same epitope in the PSMGFR and N-terminally extended PSMGFR peptide. Fig.
215A
shows consensus sequences for heavy chain CDR1. Fig. 215B shows consensus
sequences
for heavy chain CDR2. Fig. 215C shows consensus sequences for heavy chain
CDR3.
[00480] Figure 216A-216C shows the consensus sequences of the light chain CDRs
wherein the consensus sequences were generated for each group of antibodies
that bound to
the same epitope in the PSMGFR and N-terminally extended PSMGFR peptide. Fig.
216A
shows consensus sequences for light chain CDRL Fig. 216B shows consensus
sequences for
light chain CDR2. Fig. 216C shows consensus sequences for light chain CDR3.
[00481] Figure 217 shows alternative formats for bispecific antibodies and
other
bispecific immunotherapeutics subdivided into five major classes: BsIgG,
appended IgG,
BsAb fragments, bispecific fusion proteins and BsAb conjugates. Heavy chains
are shown in
dark blue, dark pink and dark green and corresponding light chains are in
lighter shades of
the same colors. Connecting peptide linkers are shown by thin black lines and
engineered
disulfide bonds by thin green lines. Approximate molecular weights are shown
assuming
¨12.5 kDa per immunoglobulin domain. BsAb formats that have advanced into
clinical
testing are highlighted (*). For interpretation of the references to color in
this figure
description, the reader is referred to the web version of the article, Spiess
et al. Molecular
Immunology 67, 95-106 (2015), the contents of which are incorporated by
reference in their
entirety, in particular with respect to the description of Figure 1 of Spiess
et al., as well as
other descriptions of various methods of making and using bispecific antibody
fragments).
[00482] Figure 218 shows graphs of tumor volume measured by an IVIS instrument
wherein the tumor cells have been genetically modified to express Luciferase.
The substrate
Luciferin was injected 10 minutes before the photo emissions were measured in
the sedated
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animal. On Day 1 of the experiment, animals were injected sub-cutaneously with
250,000
human breast tumor cells. Tumors were made heterogeneous, comprised of two
different
tumor cell types. A first tumor cell population was T47D-wt, a breast cancer
cell line that
expresses both full-length MUC1 and the growth factor receptor form MUC1*,
which we
engineered to express mCherry fluorescence. The second tumor cell population
was the same
T47D breast cancer cells, except that they had been stably transduced to
express even more
MUC1* and OFF fluorescence, referred to here as T47D-MUC1*. In this
experiment,
animals were implanted with T47D-wt plus T47D-MUC1*, wherein the population of
T47D-
MUC1* made up 30%, 15% or 7.5% of the tumor population. Animals were then
administered a one-time injection of either PBS, huMNC2-41BB-3z CAR T cells (4-
1BB),
huMNC2-CD28-3z CAR T cells (CD28) or huMNC2-CD28-1XX CAR T cells (CD28-1XX
or 1XX). The CAR T cells were injected into the tail vein at an Effector to
Target ratio (E:T)
of 10:1, 5:1, or 1:1.
[00483] Figure 219A-219B shows IVIS photographs and graphs of IVIS tumor
volume
measurement. Fig. 219A shows photographs of the mice that had been implanted
with tumors
in which 30% of the cancer cell population was T47D-MUC1*, referred to here as
high
antigen expressing cells. The various CAR T cells were administered at a CAR T
to tumor
cell ratio of 10:1 wherein 250,000 tumor cells were implanted and the animals
were injected
days later with 2,500,000 CAR T cells. Fig. 219B shows a graph of the tumor
volume by
IVIS measurement by day. As can be seen, animals injected with the huMNC2-
C1328-1XX
had much smaller tumors than the animals treated huMNC2-4-1BB-3z or huMNC2-
CD28-3z,
which is the same CAR T except without the 1XX mutations in the CD3-zeta
domain.
[00484] Figure 220A-220T shows the IVIS graphs for 30% tumors treated at a CAR
T to
Tumor ratio of 10:1. Here graphs are shown for each individual animal rather
than the
average of the treatment group.
[00485] Figure 221A-221B shows IVIS photographs and graphs of IVIS tumor
volume
measurement. Fig. 221A shows photographs of the mice that had been implanted
with tumors
in which 30% of the cancer cell population was T47D-MUC1*, referred to here as
high
antigen expressing cells. The various CAR T cells were administered at a CAR T
to tumor
cell ratio of 1:1 wherein 250,000 tumor cells were implanted and the animals
were injected 5
days later with 250,000 CAR T cells. Fig. 221B shows a graph of the tumor
volume by IVIS
measurement by day. As can be seen, animals injected with the huMNC2-CD28-1XX
had
much smaller tumors than the animals treated huMNC2-4-1BB-3z or huMNC2-CD28-
3z,
which is the same CAR T except without the 1XX mutations in the CD3-zeta
domain.
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However, with the lower dose of CAR T cells, even tumors in the huMNC2-CD28-
1XX
treated group begin to grow again.
[00486] Figure 222A-222T shows the IVIS graphs for 30% tumors treated at a CAR
T to
Tumor ratio of 1:1. Here graphs are shown for each individual animal rather
than the average
of the treatment group.
[00487] Figure 223A-223B shows IVIS photographs and graphs of IVIS tumor
volume
measurement. Fig. 223A shows photographs of the mice that had been implanted
with tumors
in which 7.5% of the cancer cell population was T47D-MUC1*, referred to here
as high
antigen expressing cells. The various CAR T cells were administered at a CAR T
to tumor
cell ratio of 10:1 wherein 250,000 tumor cells were implanted and the animals
were injected
days later with 2,500,000 CAR T cells. Fig. 223B shows a graph of the tumor
volume by
IVIS measurement by day. As can be seen, animals injected with the huMNC2-CD28-
1XX
had much smaller tumors than the animals treated huMNC2-4-1BB-3z or huMNC2-
CD28-3z,
which is the same CAR T except without the 1XX mutations in the CD3-zeta
domain.
However, even tumors in the huMNC2-CD28-1XX treated group begin to grow again,
which
is consistent with the idea that a small percentage of high antigen expressing
tumor cells
impedes the killing of the rest of the tumor.
[00488] Figure 224A-224T shows the IVIS graphs for 7.5% tumors treated at a
CAR T to
Tumor ratio of 10:1. Here graphs are shown for each individual animal rather
than the
average of the treatment group.
[00489] Figure 225A-225B shows IVIS photographs and graphs of IVIS tumor
volume
measurement. Fig. 225A shows photographs of the mice that had been implanted
with tumors
in which 7.5% of the cancer cell population was T47D-MUC1*, referred to here
as high
antigen expressing cells. The various CAR T cells were administered at a CAR T
to tumor
cell ratio of 1:1 wherein 250,000 tumor cells were implanted and the animals
were injected 5
days later with 250,000 CAR T cells. Fig. 225B shows a graph of the tumor
volume by IVIS
measurement by day. As can be seen, animals injected with the huMNC2-CD28-1XX
had
smaller tumors than the animals treated huMNC2-4-1BB-3z or huMNC2-CD28-3z,
which is
the same CAR T except without the 1XX mutations in the CD3-zeta domain.
However, at
low antigen density combined with very low dose of CAR T cells, even tumors in
the
huMNC2-CD28-1XX treated group begin to grow again.
[00490] Figure 226A-226T shows the IVIS graphs for 7.5% tumors treated at a
CAR T to
Tumor ratio of 1:1. Here graphs are shown for each individual animal rather
than the average
of the treatment group.
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[00491] Figure 227 shows the tabulation of CD3 positive human T cells that
were
harvested from the spleens of the test animals post sacrifice. In this table,
cells isolated from
mice implanted with tumors comprised of 30% T47D-MUC1* and treated with CAR T
cells
at a 10:1 ratio. As can be seen, the huMNC2-CD28-1XX treated mice, that had
smaller
tumors, have the greater numbers of CAR T cells and CD8 positive killer T
cells. TIM3,
LAG3 and PD-1 are molecular markers of T cell exhaustion. The table shows that
the
huMNC2-CD28-1XX CAR T cells harvested from the animals express lower levels of
exhaustion markers, consistent with the idea that the 1XX mutations in CD3-
zeta increase
CAR T cell persistence in vivo.
[00492] Figure 228 shows the tabulation of CD3 positive human T cells that
were
harvested from the spleens of the test animals post sacrifice. In this table,
cells isolated from
mice implanted with tumors comprised of 30% T47D-MUC1* and treated with CAR T
cells
at a 1:1 ratio. As can be seen, the huMNC2-CD28-1XX treated mice, that had
smaller tumors,
have the greater numbers of CAR T cells and CD8 positive killer T cells. TIM3,
LAG3 and
PD-1 are molecular markers of T cell exhaustion. The table shows that the
huMNC2-CD28-
1XX CAR T cells harvested from the animals express lower levels of exhaustion
markers,
consistent with the idea that the 1XX mutations in CD3-zeta increase CAR T
cell persistence
in vivo.
[00493] Figure 229 shows the tabulation of CD3 positive human T cells that
were
harvested from the blood of the test animals post sacrifice. In this table,
cells isolated from
mice implanted with tumors comprised of 30% T47D-MUC1* and treated with CAR T
cells
at a 1:1 ratio. As can be seen, the huMNC2-CD28-1XX treated mice, that had
smaller tumors,
have the greater numbers of CAR T cells and CD8 positive killer T cells and
have lower
levels of T cell exhaustion markers.
[00494] Figure 230 shows the tabulation of CD3 positive human T cells that
were
harvested from the spleens of the test animals post sacrifice. In this table,
cells isolated from
mice implanted with tumors comprised of 7.5% T47D-MUC1* and treated with CAR T
cells
at a 10:1 ratio. As can be seen, the huMNC2-CD28-1XX treated mice, that had
smaller
tumors, have the greater numbers of CAR T cells and CD8 positive killer T
cells. TIM3,
LAG3 and PD-1 are molecular markers of T cell exhaustion. The table shows that
the
huMNC2-CD28-1XX CAR T cells harvested from the animals express lower levels of
exhaustion markers, consistent with the idea that the 1XX mutations in CD3-
zeta increase
CAR T cell persistence in vivo.
76
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[00495] Figure 231 shows the tabulation of CD3 positive human T cells that
were
harvested from the blood of the test animals post sacrifice. In this table,
cells isolated from
mice implanted with tumors comprised of 7.5% T47D-MUC1* and treated with CAR T
cells
at a 10:1 ratio. As can be seen, the huMNC2-CD28-1XX treated mice, that had
smaller
tumors, have the greater numbers of CAR T cells and CD8 positive killer T
cells. TIM3,
LAG3 and PD-1 are molecular markers of T cell exhaustion. The table shows that
the
huMNC2-CD28-1XX CAR T cells harvested from the animals express lower levels of
exhaustion markers, consistent with the idea that the 1XX mutations in CD3-
zeta increase
CAR T cell persistence in vivo.
[00496] Figure 232 shows the tabulation of CD3 positive human T cells that
were
harvested from the spleens of the test animals post sacrifice. In this table,
cells isolated from
mice implanted with tumors comprised of 7.5% T47D-MUC1* and treated with CAR T
cells
at a 1:1 ratio. As can be seen, the huMNC2-CD28-1XX treated mice, that had
smaller tumors,
have the greater numbers of CAR T cells and CD8 positive killer T cells. TIM3,
LAG3 and
PD-1 are molecular markers of T cell exhaustion. The table shows that the
huMNC2-CD28-
1XX CAR T cells harvested from the animals express lower levels of exhaustion
markers,
consistent with the idea that the 1XX mutations in CD3-zeta increase CAR T
cell persistence
in vivo.
[00497] Figure 233 shows the tabulation of CD3 positive human T cells that
were
harvested from the blood of the test animals post sacrifice. In this table,
cells isolated from
mice implanted with tumors comprised of 7.5% T47D-MUC1* and treated with CAR T
cells
at a 1:1 ratio. As can be seen, the huMNC2-CD28-1XX treated mice, that had
smaller tumors,
have the greater numbers of CAR T cells and CD8 positive killer T cells. TIM3,
LAG3 and
PD-1 are molecular markers of T cell exhaustion. The table shows that the
huMNC2-CD28-
1XX CAR T cells harvested from the animals express lower levels of exhaustion
markers,
consistent with the idea that the 1XX mutations in CD3-zeta increase CAR T
cell persistence
in vivo.
[00498] Figure 234A-234U shows photographs of the tumors excised from test
animals
and shows their weight in grams. Tumors were excised from animals implanted
with tumors
made up of 30% T47D-MUC1* high antigen density cells and 70% T47D-wt low
antigen
density cells. Animals were treated with CAR T cells at an effector to target
ratio of 10:1.
[00499] Figure 235A-235N2 shows magnified photographs of dissociated tumors
excised
from animals implanted with tumors made up of 30% T47D-MUC1* high antigen
density
cells and 70% T47D-wt low antigen density cells. Animals were treated with CAR
T cells at
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an effector to target ratio of 10:1. Shown are overlays of bright field images
and fluorescent
images, wherein the red fluorescence, mCherry, shows the low antigen density
cells and the
green fluorescence, GFP, shows the low antigen density cells. Fig. 235A-235J
show tumor
cells excised from the control animals treated only with PBS. Fig. 235K-235T
show tumor
cells excised from the animals treated with huMNC2-41BB-3z CAR T cells. Fig.
235U-
235D2 show tumor cells excised from the animals treated with huMNC2-CD28-1XX
CAR T
cells. Fig. 235E2-235N2 show tumor cells excised from the animals treated with
huMNC2-
CD28-3z CAR T cells.
[00500] Figure 236A-236U shows photographs of the tumors excised from test
animals
and shows their weight in grams. Tumors were excised from animals implanted
with tumors
made up of 30% T47D-MUC1* high antigen density cells and 70% T47D-wt low
antigen
density cells. Animals were treated with CAR T cells at an effector to target
ratio of 1:1. Fig.
236A-236E show tumors excised from animals mock treated with PBS. Fig. 236F-
236J show
tumors excised from animals treated with huMNC2-41BB-3z. Fig. 236K-2360 show
tumors
excised from animals treated with huMNC2-CD28-1XX. Fig. 236P-236T show tumors
excised from animals treated with huMNC2-CD28-3z. Fig. 236U shows a bar graph
of the
weights of tumors excised from the test animals.
[00501] Figure 237A-237D2 shows magnified photographs of dissociated tumors
excised
from animals implanted with tumors made up of 30% T47D-MUC1* high antigen
density
cells and 70% '1'47D-wt low antigen density cells. Animals were treated with
CAR '1 cells at
an effector to target ratio of 1:1. Shown are overlays of bright field images
and fluorescent
images, wherein the red fluorescence, mCherry, shows the low antigen density
cells and the
green fluorescence, GFP, shows the low antigen density cells. Fig. 237A-237J
show tumor
cells excised from the control animals treated only with PBS. Fig. 237K-237T
show tumor
cells excised from the animals treated with huMNC2-41BB-3z CAR T cells. Fig.
237U-
237D2 show tumor cells excised from the animals treated with huMNC2-CD28-1XX
CAR T
cells. Fig. 237E2-237N2 show tumor cells excised from the animals treated with
huMNC2-
CD28-3z CAR T cells.
[00502] Figure 238A-238T shows photographs of the tumors excised from test
animals
and shows their weight in grams. Tumors were excised from animals implanted
with tumors
made up of 7.5% T47D-MUC1* high antigen density cells and 92.5% T47D-wt low
antigen
density cells. Animals were treated with CAR T cells at an effector to target
ratio of 10:1.
[00503] Figure 239A-239M2 shows magnified photographs of dissociated tumors
excised
from animals implanted with tumors made up of 7.5% T47D-MUC1* high antigen
density
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cells and 92.5% T47D-wt low antigen density cells. Animals were treated with
CAR T cells
at an effector to target ratio of 10:1. Shown are overlays of bright field
images and
fluorescent images, wherein the red fluorescence, mCherry, shows the low
antigen density
cells and the green fluorescence, GFP, shows the low antigen density cells.
Fig. 239A-239J
show tumor cells excised from animals mock treated with PBS. Fig. 239K-239T
show tumor
cells excised from the animals treated with huMNC2-41BB-3z CAR T cells. Fig.
239U-
239C2 show tumor cells excised from the animals treated with huMNC2-CD28-1XX
CAR T
cells. Fig. 239D2-239M2 show tumor cells excised from the animals treated with
huMNC2-
CD28-3z CAR T cells.
[00504] Figure 240A-2400 shows photographs of the tumors excised from test
animals
and shows their weight in grams. Tumors were excised from animals implanted
with tumors
made up of 7.5% T47D-MUC1* high antigen density cells and 92.5% T47D-wt low
antigen
density cells. Animals were treated with CAR T cells at an effector to target
ratio of 1:1.
[00505] Figure 241A-241D2 shows magnified photographs of dissociated tumors
excised
from animals implanted with tumors made up of 7.5% T47D-MUC1* high antigen
density
cells and 92.5% T47D-wt low antigen density cells. Animals were treated with
CAR T cells
at an effector to target ratio of 1:1. Shown are overlays of bright field
images and fluorescent
images, wherein the red fluorescence, mCherry, shows the low antigen density
cells and the
green fluorescence, GFP, shows the low antigen density cells. Fig. 241A-241J
show tumor
cells excised from control animals treated only with PBS. Fig. 241K-2411 show
tumor cells
excised from the animals treated with huMNC2-41BB-3z CAR T cells. Fig. 241U-
241D2
show tumor cells excised from the animals treated with huMNC2-CD28-1XX CAR T
cells.
Fig. 241E2-241N2 show tumor cells excised from the animals treated with huMNC2-
CD28-
3z CAR T cells.
[00506] Figure 242A-242R shows photographs of live animals, where IVIS
measures
tumor volume, mCherry detects the low antigen cells within the tumor and GFP
detects the
high antigen cells within the tumor. Post sacrifice photographs are shown of
the excised
tumors and a graph of tumor weights. A graph of the GFP positive high antigen
tumor cells is
also shown. Both the live GFP photographs and the graph of FACS measurement of
high
antigen cells show that huMNC2-CD28-1XX has killed all the high antigen cells
and most of
the low antigen cells even with the lowest levels of high antigen cells in the
tumor and at very
low dose of CAR T cells.
[00507] Figure 243A-243F shows photographs taken at two different timepoints.
Here,
IVIS photographs measure tumor volume, mCherry fluorescent photographs measure
low
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antigen cells and GFP fluorescent photographs measure high antigen cells. In
this case, the
animals were implanted with tumors made up of 30% high antigen density cells
(GFP+) and
70% low antigen density cells (mCherry+). Animals had been given a single dose
of CAR T
cells at a 10:1 or a 1:1 CART to tumor cell ratio.
[00508] Figure 244 shows graphs of IVIS tumor volume measurements over time.
Arrows
indicate timepoints when fluorescent photographs, mCherry and GFP, of live
animals were
taken. In this case, the animals were implanted with tumors made up of 30%
high antigen
density cells (GFP+) and 70% low antigen density cells (mCherry+). Animals had
been given
a single dose of CAR T cells at a 10:1 or a 1:1 CAR T to tumor cell ratio.
[00509] Figure 245 shows graphs of IVIS measurements of tumor volume, mCherry
measurements of the growth rate of low antigen cells and GFP measurements of
the growth
rate of high antigen cells, between two timepoints. As can be seen, at high
CAR T dose, there
is no increase in the number of high antigen density cells (GFP) over time in
mice treated
with either huMNC2-41BB-3z or huMNC2-CD28-1XX. However, at low CAR T dose
there
is some of the mice treated with huMNC2-41BB-3z do show an increase in the
growth of
high antigen density cells, whereas mice treated with huMNC2-CD28-1XX do not.
More
importantly, at high or low CAR T dose, mice treated with huMNC2-41BB-3z show
increases in the growth of low antigen density cells, whereas in mice treated
with huMNC2-
CD28-1XX the growth of low antigen density cells is more controlled.
[00510] Figure 246A-246B shows IVIS photographs and graphs of IVIS tumor
volume
measurement. Fig. 246A shows photographs of the mice that had been implanted
with tumors
in which 15% of the cancer cell population was T47D-MUC1*, referred to here as
high
antigen expressing cells. The various CAR T cells were administered at a CAR T
to tumor
cell ratio of 10:1 wherein 250,000 tumor cells were implanted and the animals
were injected
days later with 2,500,000 CAR T cells. Fig. 246B shows a graph of the tumor
volume by
IVIS measurement by day. In this experiment, on Day 52, animals outlined in
red were
implanted with 250,000 more 100% high antigen density tumor cells. The animals
outlined in
green received an additional dose of 2,500,000 CAR T cells.
[00511] Figure 247A-247T shows graphs of the IVIS measured growth of the
tumors in
each individual animal. The red arrows indicate injection of more tumor cells
and the green
arrows indicate injection of 2,500,000 additional CAR T cells. As can be seen,
the injection
of additional tumor cells does not increase the tumor growth in animals
treated with
huMNC2-CD281XX CAR T cells. However, the injection of additional tumor cells
does
increase tumor growth in animals treated with huMNC2-CD28-3z or huMNC2-41BB-3z
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CAR T cells. This result is consistent with CAR T cells with 1XX mutations in
CD3z prolong
CAR T cell persistence in vivo. It can also be seen that the injection of
additional CAR T
cells suppressed tumor cells in some of the animals in all groups. This is
consistent with the
idea that tumor recurrence was not due to tumor escape because the fresh CAR T
cells still
recognized the tumor cells and killed them.
[00512] Figure 248A-247D shows a cartoon of the experimental strategy wherein
animals
were implanted with heterologous tumors comprised of two cell types that carry
two different
florescent labels. Animals were implanted with both T47D breast cancer cells
that bear
mCherry and fluoresce red and also implanted with T47D cells that have been
engineered to
express even more MUC1* and bear GFP making them fluoresce green_ Fig. 248A
shows
cartoon of animals implanted with tumors in which 30% of the tumors express
high levels of
MUC1* and those tumor cells fluoresce green. Fig. 248B shows cartoon of
animals
implanted with tumors in which 15% of the tumors express high levels of MUC1*
and those
tumor cells fluoresce green. Fig. 248C shows cartoon of animals implanted with
tumors in
which 7.5% of the tumors express high levels of MUC1* and those tumor cells
fluoresce
green. Fig. 248D lists the variables used in these experiments.
[00513] Figure 249A-247F shows cartoons of the experimental strategy and data.
Fig.
249A shows cartoons of MUC1 full-length as it appears on normal epithelial
cells. Fig. 249B
shows four tissue specimens stained with huMNC2-scFv-Fc. Fig. 249C shows
cartoons
depicting heterogeneous tumors expressing either high (30%-left) or low (7.5%-
right)
percentages of high MUC1* expressing tumor cells. Fig. 249D shows flow
cytometry
verifying the percentages of the heterogeneous tumors before their
implantation. Fig. 249E
shows bar graphs of bioluminescence measured on an IVIS instrument for animals
implanted
with 30% high MUC1* cells and treated with the various CAR T cells at an
effector to target
ratio of 10:1 (top) or 1:1 (bottom). Fig. 249F shows bar graphs of
bioluminescence measured
on an IVIS instrument for animals implanted with 7.5% high MUC1* cells and
treated with
the various CAR T cells at an effector to target ratio of 10:1 (top) or 1:1
(bottom).
[00514] Figure 250A-250F shows bar graphs of bioluminescence of the tumors
measured
on an IVIS instrument for animals implanted with 30% high MUC1* cells or 7.5%
high
MUC1* tumors and treated with the various CAR T cells at various effector to
target ratios.
Fig. 250A shows IVIS graph of animals implanted with tumors in which 30%
expressed high
levels of MUCI* and where animals were treated with CAR T cells at an effector
to target
ratio of 10:1. Fig. 250B shows IVIS graph where effector to target ratio was
5:1. Fig. 250C
shows IVIS graph where effector to target ratio was 1:1. Fig. 250D shows IVIS
graph of
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animals implanted with tumors in which 7.5% expressed high levels of MUC1* and
where
animals were treated with CAR T cells at an effector to target ratio of 10:1.
Fig. 250E shows
IVIS graph where effector to target ratio was 5:1. Fig. 250F shows IVIS graph
where effector
to target ratio was 1:1.
[00515] Figure 251A-251D shows photographs of bioluminescence of the tumors
measured on an IVIS instrument for animals implanted with 30% high MUC1* cells
or 7.5%
high MUC1* tumors and treated with the various CAR T cells at effector to
target ratios of
10:1 or 1:1. Fig. 251A shows IVIS photographs for animals implanted with
tumors in which
30% expressed high levels of MUC1* and where animals were treated with of CAR
T cells at
an effector to target ratio of 10:1. Fig. 250B shows IVIS photographs where
effector to target
ratio was 1:1. Fig. 251C shows IVIS photographs for animals implanted with
tumors in which
7.5% expressed high levels of MUC1* and where animals were treated with of CAR
T cells
at an effector to target ratio of 10:1. Fig. 250D shows IVIS photographs where
effector to
target ratio was 1:1.
[00516] Figure 252A-252D shows magnified fluorescent photographs of
dissociated
tumors excised from animals implanted with tumors made up of either 30% or
7.5% T47D-
MUC1* high antigen density cells and the remainder are low antigen density
cells. Animals
were treated with various CART cells at effector to target ratios of either
10:1 or 1:1. Shown
are overlays of bright field images and fluorescent images, wherein the red
fluorescence,
mCherry, shows the low antigen density cells and the green fluorescence. CiPP,
shows the
low antigen density cells. Fig. 252A shows tumor cells excised from animals
implanted with
30% high antigen density tumors and treated with various CAR T cells at
effector to target
ratio of 10:1. Fig. 252B shows tumor cells excised from animals implanted with
30% high
antigen density tumors and treated with various CAR T cells at effector to
target ratio of 1:1.
Fig. 252C shows tumor cells excised from animals implanted with 7.5% high
antigen density
tumors and treated with various CAR T cells at effector to target ratio of
10:1. Fig. 252D
shows tumor cells excised from animals implanted with 7.5% high antigen
density tumors
and treated with various CAR T cells at effector to target ratio of 1:1.
[00517] Figure 253A-253H shows cartoons of the experimental strategy and data.
Fig.
253A shows cartoons of MUC1 full-length as it appears on normal epithelial
cells. Fig. 253B
shows four tissue specimens stained with huMNC2-scFv-Fc. Fig. 253C shows
cartoons
depicting heterogeneous tumors expressing either high or low percentages of
high MUC1*
expressing tumor cells. Fig. 253D shows flow cytometry verifying 15% percent
of the tumor
cells expressed high levels of MUC1* before their implantation. Fig. 253E
shows bar graphs
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of bioluminescence measured on an IVIS instrument for animals implanted with
15% high
MUC1* cells and treated with the various CAR T cells at an effector to target
ratio of 10:1.
Fig. 253F shows immunofluorescent photographs of the excised tumors for
animals
implanted with 15% high antigen density tumors and treated with the various
CAR Ts at an
effector to target ratio of 10:1. Fig. 253G shows bar graphs of
bioluminescence measured on
an IVIS instrument for animals implanted with 15% high MUC1* cells and treated
with the
various CAR T cells at an effector to target ratio of 1:1. Fig. 253H shows
immunofluorescent
photographs of the excised tumors for animals implanted with 15% high antigen
density
tumors and treated with the various CAR Ts at an effector to target ratio of
1:1.
[00518] Figure 254A-254B shows bar graphs of bioluminescence measured on an
IVIS
instrument for animals implanted with 15% high MUC1* cells and treated with
the various
CAR T cells at an effector to target ratio of 10:1. Fig. 254A shows the graph
of animals
treated at an effector to target ratio of 10:1. Fig. 254B shows the graph of
animals treated at
an effector to target ratio of 1:1.
[00519] Figure 255A-255B shows photographs of bioluminescence of tumors
measured
on an IVIS instrument for animals implanted with 15% high MUC1* cells and
treated with
the various CAR T cells at an effector to target ratio of 10:1. Fig. 255A
shows the
photographs of animals treated at an effector to target ratio of 10:1. Fig.
255B shows the
photographs of animals treated at an effector to target ratio of 1:1.
[00520] Figure 256A-256B shows fluorescent photographs of the excised tumors
showing
the mCherry positive, low antigen density cells in red and the GFP positive,
high antigen
density cells in green. Animals were all implanted with a mixture of tumor
cells that before
implantation were 15% high MUC1* expressing cells. Fig. 256A shows the
photographs of
animals treated at an effector to target ratio of 10:1. Fig. 256B shows the
photographs of
animals treated at an effector to target ratio of 1:1.
[00521] Figure 257 shows a table of results of flow cytometry in which cells
of the
excised tumors were assayed for the presence of human T cells and CAR T cells,
which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
30% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 10:1. When the number of CAR T cells
detected was less
than 25, that number is shown in red and the further analysis of those few
cells is called into
question.
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[00522] Figure 258 shows a table of results of flow cytometry in which cells
of the
excised tumors were assayed for the presence of human T cells and CAR T cells,
which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
15% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 10:1. When the number of CAR T cells
detected was less
than 25, that number is shown in red and the further analysis of those few
cells is called into
question.
[00523] Figure 259 shows a table of results of flow cytometry in which cells
of the
excised tumors were assayed for the presence of human T cells and CAR T cells,
which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
7.5% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 10:1. When the number of CAR T cells
detected was less
than 25, that number is shown in red and the further analysis of those few
cells is called into
question.
[00524] Figure 260 shows a table of results of flow cytometry in which cells
of the
excised tumors were assayed for the presence of human T cells and CAR T cells,
which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
30% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 5:1. When the number of CAR T cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00525] Figure 261 shows a table of results of flow cytometry in which cells
of the
excised tumors were assayed for the presence of human T cells and CAR T cells,
which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
7.5% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 5:1. When the number of CAR T cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00526] Figure 262 shows a table of results of flow cytometry in which cells
of the
excised tumors were assayed for the presence of human T cells and CAR T cells,
which were
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then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
30% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 1:1. When the number of CAR T cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00527] Figure 263 shows a table of results of flow cytometry in which cells
of the
excised tumors were assayed for the presence of human T cells and CAR T cells,
which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
15% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 1:1. When the number of CAR T cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00528] Figure 264 shows a table of results of flow cytometry in which cells
of the
excised tumors were assayed for the presence of human T cells and CAR T cells,
which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
7.5% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 1:1. When the number of CAR '1 cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00529] Figure 265 shows a table of results of flow cytometry in which cells
of the
excised spleens of the treated animals were assayed for the presence of human
T cells and
CAR T cells, which were then enumerated and analyzed for the presence of
markers of T cell
exhaustion. Shown here is the analysis of tumors excised from animals that had
been
implanted with tumors that were 30% high antigen expressing cells, wherein
animals were
treated with various CAR T cells and an effector to target ratio of 10:1. When
the number of
CAR T cells detected was less than 25, that number is shown in red and the
further analysis
of those few cells is called into question.
[00530] Figure 266 shows a table of results of flow cytometry in which cells
of the
excised spleens of the treated animals were assayed for the presence of human
T cells and
CAR T cells, which were then enumerated and analyzed for the presence of
markers of T cell
exhaustion. Shown here is the analysis of tumors excised from animals that had
been
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implanted with tumors that were 15% high antigen expressing cells, wherein
animals were
treated with various CAR T cells and an effector to target ratio of 10:1. When
the number of
CAR T cells detected was less than 25, that number is shown in red and the
further analysis
of those few cells is called into question.
[00531] Figure 267 shows a table of results of flow cytometry in which cells
of the
excised spleens of the treated animals were assayed for the presence of human
T cells and
CAR T cells, which were then enumerated and analyzed for the presence of
markers of T cell
exhaustion. Shown here is the analysis of tumors excised from animals that had
been
implanted with tumors that were 7.5% high antigen expressing cells, wherein
animals were
treated with various CAR T cells and an effector to target ratio of 10:1. When
the number of
CAR T cells detected was less than 25, that number is shown in red and the
further analysis
of those few cells is called into question.
[00532] Figure 268 shows a table of results of flow cytometry in which cells
of the
excised spleens of the treated animals were assayed for the presence of human
T cells and
CAR T cells, which were then enumerated and analyzed for the presence of
markers of T cell
exhaustion. Shown here is the analysis of tumors excised from animals that had
been
implanted with tumors that were 30% high antigen expressing cells, wherein
animals were
treated with various CAR T cells and an effector to target ratio of 5:1. When
the number of
CAR T cells detected was less than 25, that number is shown in red and the
further analysis
of those few cells is called into question.
[00533] Figure 269 shows a table of results of flow cytometry in which cells
of the
excised spleens of the treated animals were assayed for the presence of human
T cells and
CAR T cells, which were then enumerated and analyzed for the presence of
markers of T cell
exhaustion. Shown here is the analysis of tumors excised from animals that had
been
implanted with tumors that were 15% high antigen expressing cells, wherein
animals were
treated with various CAR T cells and an effector to target ratio of 5:1. When
the number of
CAR T cells detected was less than 25, that number is shown in red and the
further analysis
of those few cells is called into question.
[00534] Figure 270 shows a table of results of flow cytometry in which cells
of the
excised spleens of the treated animals were assayed for the presence of human
T cells and
CAR T cells, which were then enumerated and analyzed for the presence of
markers of T cell
exhaustion. Shown here is the analysis of tumors excised from animals that had
been
implanted with tumors that were 7.5% high antigen expressing cells, wherein
animals were
treated with various CAR T cells and an effector to target ratio of 5:1. When
the number of
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CAR T cells detected was less than 25, that number is shown in red and the
further analysis
of those few cells is called into question.
[00535] Figure 271 shows a table of results of flow cytometry in which cells
of the
excised spleens of the treated animals were assayed for the presence of human
T cells and
CAR T cells, which were then enumerated and analyzed for the presence of
markers of T cell
exhaustion. Shown here is the analysis of tumors excised from animals that had
been
implanted with tumors that were 30% high antigen expressing cells, wherein
animals were
treated with various CAR T cells and an effector to target ratio of 1:1. When
the number of
CAR T cells detected was less than 25, that number is shown in red and the
further analysis
of those few cells is called into question.
[00536] Figure 272 shows a table of results of flow cytometry in which cells
of the
excised spleens of the treated animals were assayed for the presence of human
T cells and
CAR T cells, which were then enumerated and analyzed for the presence of
markers of T cell
exhaustion. Shown here is the analysis of tumors excised from animals that had
been
implanted with tumors that were 15% high antigen expressing cells, wherein
animals were
treated with various CAR T cells and an effector to target ratio of 1:1. When
the number of
CAR T cells detected was less than 25, that number is shown in red and the
further analysis
of those few cells is called into question.
[00537] Figure 273 shows a table of results of flow cytometry in which cells
of the
excised spleens of the treated animals were assayed for the presence of human
'1 cells and
CAR T cells, which were then enumerated and analyzed for the presence of
markers of T cell
exhaustion. Shown here is the analysis of tumors excised from animals that had
been
implanted with tumors that were 7.5% high antigen expressing cells, wherein
animals were
treated with various CAR T cells and an effector to target ratio of 1:1. When
the number of
CAR T cells detected was less than 25, that number is shown in red and the
further analysis
of those few cells is called into question.
[00538] Figure 274 shows a table of results of flow cytometry in which blood
from the
treated animals were assayed for the presence of human T cells and CAR T
cells, which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
30% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 10:1. When the number of CAR T cells
detected was less
than 25, that number is shown in red and the further analysis of those few
cells is called into
question.
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[00539] Figure 275 shows a table of results of flow cytometry in which blood
from the
treated animals were assayed for the presence of human T cells and CAR T
cells, which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
15% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 10:1. When the number of CAR T cells
detected was less
than 25, that number is shown in red and the further analysis of those few
cells is called into
question.
[00540] Figure 276 shows a table of results of flow cytometry in which blood
from the
treated animals were assayed for the presence of human T cells and CAR T
cells, which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
7.5% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 10:1. When the number of CAR T cells
detected was less
than 25, that number is shown in red and the further analysis of those few
cells is called into
question.
[00541] Figure 277 shows a table of results of flow cytometry in which blood
from the
treated animals were assayed for the presence of human T cells and CAR T
cells, which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
30% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 5:1. When the number of CAR T cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00542] Figure 278 shows a table of results of flow cytometry in which blood
from the
treated animals were assayed for the presence of human T cells and CAR T
cells, which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
15% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 5:1. When the number of CAR T cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00543] Figure 279 shows a table of results of flow cytometry in which blood
from the
treated animals were assayed for the presence of human T cells and CAR T
cells, which were
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then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
7.5% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 5:1. When the number of CAR T cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00544] Figure 280 shows a table of results of flow cytometry in which blood
from the
treated animals were assayed for the presence of human T cells and CAR T
cells, which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
30% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 1:1. When the number of CAR T cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00545] Figure 281 shows a table of results of flow cytometry in which blood
from the
treated animals were assayed for the presence of human T cells and CAR T
cells, which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
15% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 1:1. When the number of CAR '1 cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00546] Figure 282 shows a table of results of flow cytometry in which blood
from the
treated animals were assayed for the presence of human T cells and CAR T
cells, which were
then enumerated and analyzed for the presence of markers of T cell exhaustion.
Shown here
is the analysis of tumors excised from animals that had been implanted with
tumors that were
7.5% high antigen expressing cells, wherein animals were treated with various
CAR T cells
and an effector to target ratio of 1:1. When the number of CAR T cells
detected was less than
25, that number is shown in red and the further analysis of those few cells is
called into
question.
[00547] Figure 283A-283L shows photographs of MUC1* positive breast cancer
cells,
T47D, in culture with human T cells to which have been added various
concentrations of
bispecific antibody 20A10-0KT3-BiTE. 20A10 is a humanized anti-MUC1* antibody
and
OKT3 is an antibody that binds to CD3 that is present on human T cells. As can
be seen in
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the figure, the addition of the bispecific antibody mediates the joining
together of T cells and
cancer cells, seen here as cell clustering. In Fig_ 283A the concentration of
the bispecific
antibody is 1,000 ng/mL. In Fig. 283B concentration is 333 ng/mL. In Fig. 283C
concentration is 111 ng/mL. In Fig. 283D concentration is 37 ng/mL. In Fig.
283E
concentration is 12.3 ng/mL. In Fig. 283F concentration is 4.1 ng/mL. In Fig.
283G
concentration is 1.3 ng/mL. In Fig. 283H concentration is 0.4 ng/mL. In Fig.
2831
concentration is 0.15 ng/mL. In Fig. 283J concentration is 0.05 ng/mL. Fig.
283K is a control
well in which both T cells and cancer cells are present, but no bispecific
antibody has been
added. Fig. 283L is a control well in which bispecific antibody has been added
to cancer
cells, but no T cells are present.
[00548] Figure 284A-284L shows photographs of MUC1* positive breast cancer
cells,
T47D, in culture with human T cells to which have been added various
concentrations of
bispecific antibody 20A10-12F6-BiTE. 20A10 is a humanized anti-MUC1* antibody
and
12F6 is an antibody that binds to CD3 that is present on human T cells. As can
be seen in the
figure, the addition of the bispecific antibody mediates the joining together
of T cells and
cancer cells, seen here as cell clustering. In Fig. 284A the concentration of
the bispecific
antibody is 1,000 ng/mL. In Fig. 284B concentration is 333 ng/mL. In Fig. 284C
concentration is 111 ng/mL. In Fig. 284D concentration is 37 ng/mL. In Fig.
284E
concentration is 12.3 ng/mL. In Fig. 284F concentration is 4.1 ng/mL. In Fig.
284G
concentration is 1.3 ng/mL. In Fig. 284H concentration is 0.4 ng/mL. In Hg.
2841
concentration is 0.15 ng/mL. In Fig. 284J concentration is 0.05 ng/mL. Fig.
284K is a control
well in which both T cells and cancer cells are present, but no bispecific
antibody has been
added. Fig. 284L is a control well in which bispecific antibody has been added
to cancer
cells, but no T cells are present.
[00549] Figure 285A-285L shows photographs of HCT-MUC1*-transduced cancer
cells
in culture with human T cells to which have been added various concentrations
of bispecific
antibody 20A10-0KT3-BiTE. 20A10 is a humanized anti-MUC1* antibody and OKT3 is
an
antibody that binds to CD3 that is present on human T cells. As can be seen in
the figure, the
addition of the bispecific antibody mediates the joining together of T cells
and cancer cells,
seen here as cell clustering. In Fig. 285A the concentration of the bispecific
antibody is 1,000
ng/mL. In Fig. 285B concentration is 333 ng/mL. In Fig. 285C concentration is
111 ng/mL.
In Fig. 285D concentration is 37 ng/mL. In Fig. 285E concentration is 12.3
ng/mL. In Fig.
285F concentration is 4.1 ng/mL. In Fig. 285G concentration is L3 ng/mL. In
Fig. 285H
concentration is 0.4 ng/mL. In Fig. 2851 concentration is 0.15 ng/mL. In Fig.
285J
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concentration is 0.05 ng/mL. Fig. 285K is a control well in which both T cells
and cancer
cells are present, but no bispecific antibody has been added. Fig. 285L is a
control well in
which bispecific antibody has been added to cancer cells, but no T cells are
present.
[00550] Figure 286A-286L shows photographs of HCT-MUC1*-transduced cancer
cells
in culture with human T cells to which have been added various concentrations
of bispecific
antibody 20A10-12F6-BiTE. 20A10 is a humanized anti-MUC1* antibody and 12F6 is
an
antibody that binds to CD3 that is present on human T cells. As can be seen in
the figure, the
addition of the bispecific antibody mediates the joining together of T cells
and cancer cells,
seen here as cell clustering. In Fig. 286A the concentration of the bispecific
antibody is 1,000
ng/mL. In Fig. 286B concentration is 333 ng/mL. In Fig. 286C concentration is
111 ng/mL.
In Fig. 286D concentration is 37 ng/mL. In Fig. 286E concentration is 12.3
ng/mL. In Fig.
286F concentration is 4.1 ng/mL. In Fig. 286G concentration is 1.3 ng/mL. In
Fig. 286H
concentration is 0.4 ng/mL. In Fig. 2861 concentration is 0.15 ng/mL. In Fig.
286J
concentration is 0.05 ng/mL. Fig. 286K is a control well in which both T cells
and cancer
cells are present, but no bispecific antibody has been added. Fig. 286L is a
control well in
which bispecific antibody has been added to cancer cells, but no T cells are
present.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[00551] In the present application, "a" and "an" are used to refer to both
single and a
plurality of objects.
[00552] As used herein, occasionally, in short hand, a polypeptide is
indicated as being
"transduced or transfected" into a cell. In these occurrences, it is
understood that the nucleic
acid encoding the polypeptide sequence is transduced or transfected into the
cell, as it is an
impossibility that a polypeptide could be transduced or transfected into a
cell.
[00553] As used herein, occasionally when referring to number of cells
injected into an
animal or otherwise contextually wherein the number of cells is referred to,
"M" refers to
millions, and "K" refers to thousands.
[00554] As used herein, interchangeable designations for various monoclonal
antibodies
are used, such as, "MNC2", which is interchangeable with "C2", "Min-C2" and
"MNC2";
"MNE6", which is interchangeable with "E6", "Min-E6" and "MNE6"; "MNC3", which
is
interchangeable with "C3", "Min-C3" and "MNC3"; and "MNC8", which is
interchangeable
with "C8", "Min-C8" and "MNC8". The monoclonal antibodies provided herein
follow the
same convention.
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[00555] As used herein, "h" or "hu" placed before an antibody construct is
short-hand for
humanized.
[00556] As used herein, the term "antibody-like" means a molecule that may be
engineered such that it contains portions of antibodies but is not an antibody
that would
naturally occur in nature. Examples include but are not limited to CAR
(chimeric antigen
receptor) T cell technology and the Ylanthia technology. The CAR technology
uses an
antibody epitope fused to a portion of a T cell so that the body's immune
system is directed to
attack a specific target protein or cell. The Ylanthia technology consists of
an "antibody-
like" library that is a collection of synthetic human Fabs that are then
screened for binding to
peptide epitopes from target proteins. The selected Fab regions can then be
engineered into a
scaffold or framework so that they resemble antibodies.
[00557] As used herein, "PSMGFR" is abbreviation for Primary Sequence of the
MUC1
Growth Factor Receptor which is identified by SEQ ID NO:2, and thus is not to
be confused
with a six amino acid sequence. "PSMGFR peptide" or "PSMGFR region" refers to
a peptide
or region that incorporates the Primary Sequence of the MUC1 Growth Factor
Receptor
(SEQ ID NO:2).
[00558] As used herein, the "MUC1*" extra cellular domain is defined primarily
by the
PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA
(SEQ ID NO:2)). Because the exact site of MUC1 cleavage depends on the enzyme
that clips
it, and that the cleavage enzyme varies depending on cell type, tissue type or
the time in the
evolution of the cell, the exact sequence of the MUC1* extra cellular domain
may vary at the
N-terminus.
[00559] Other clipped amino acid sequences may include
SNIKFRPGS V V V QLTLAFREGTIN V HD VETQFN QYKTEAASRY (SEQ ID N 0:620); or
SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:621).
[00560] As used herein, the term "PSMGFR" is an acronym for Primary Sequence
of
MI JC1 Growth Factor Receptor as set forth
as
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:2). In
this regard, the "N-number" as in "N-10 PSMGFR" or simply "N-10", "N-15
PSMGFR" or
simply "N-15", or "N-20 PSMGFR" or simply "N-20" refers to the number of amino
acid
residues that have been deleted at the N-terminal end of PSMGFR. Likewise "C-
number" as
in "C-10 PSMGFR" or simply "C-10", "C-15 PSMGFR" or simply "C-15", or "C-20
PSMGFR" or simply "C-20" refers to the number of amino acid residues that have
been
deleted at the C-terminal end of PSMGFR. A mixture of deletions and additions
is also
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possible. For instance, N+20/C-27 refers to a peptide fragment of wild-type
MUC1 in which
20 amino acids are added to the PSMGFR at the N-terminus and 27 amino acids
are deleted
from the C-terminus.
[00561] As used herein, the "extracellular domain of MUC1*" refers to the
extracellular
portion of a MUC1 protein that is devoid of the tandem repeat domain. In most
cases,
MUC1* is a cleavage product wherein the MUC1* portion consists of a short
extracellular
domain devoid of tandem repeats, a transmembrane domain and a cytoplasmic
tail. The
precise location of cleavage of MUC1 is not known perhaps because it appears
that it can be
cleaved by more than one enzyme. The extracellular domain of MUC1* will
include most of
the PSMGFR sequence but may have an additional 10-20 N-terminal amino acids.
[00562] As used herein "sequence identity" means homology in sequence of a
particular
polypeptide or nucleic acid to a reference sequence of nucleic acid or amino
acid such that
the function of the homologous peptide is the same as the reference peptide or
nucleic acid.
Such homology can be so close with the reference peptide such that at times
the two
sequences may be 90%, 95% or 98% identical yet possess the same function in
binding or
other biological activities.
[00563] As used herein, "MUC1 positive" cell refers to a cell that expresses a
gene for
MUC1, MUC1-Y or MUC1-Z or other MUC1 variant.
[00564] As used herein, "MUC1 negative" cell refers to a cell that does not
express a gene
for MUC1.
[00565] As used herein. "MUC1* positive- cell refers to a cell that expresses
a gene for
MUC1, wherein that gene's expressed protein is a transmembrane protein that is
devoid of
tandem repeats, which may be a consequence of post-translational modification,
cleavage,
alternative splicing, or transfecting or transducing a cell with a MUC1
protein that is devoid
of tandem repeats.
[00566] As used herein, "MUC1* negative" cell refers to a cell that may or may
not
express a gene for MUC1 but does not express a MUC1 transmembrane protein that
is devoid
of tandem repeats.
[00567] As used herein, "MUC1 positive" cancer cell refers to a cancer cell
that
overexpresses the gene for MUC1, expresses MUC1 in an aberrant pattern,
wherein its
expression is not restricted to the apical border and/or expresses a MUC1 that
is devoid of
tandem repeats.
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[00568] As used herein, "MUC1 negative" cancer cell refers to a cancer cell
that may or
may not express a gene for MUC1 but does not overexpress MUC1 or does not
overexpress a
MUC1 transmembrane protein that is devoid of tandem repeats.
[00569] As used herein, "MUC1* positive" cancer cell refers to a cancer cell
that
overexpresses a MUC1 transmembrane protein that is devoid of tandem repeats.
[00570] As used herein, "MUC1* negative" cancer cell refers to a cancer cell
that may or
may not express a gene for MUC1 but does not overexpress a MUC1 transmembrane
protein
that is devoid of tandem repeats.
[00571] As used herein "conformational epitope" refers to a peptide sequence
that is
required to be present in a specific three-dimensional structure or
conformation for an
antibody to bind. However the antibody binds when the peptide sequence is in
the three-
dimensional structure or conformation and is not bound when linear. A common
technique
for determining whether an antibody binds to a linear stretch or a
conformational epitope is to
use the antibody to probe a denaturing Western blot. Traveling through a
denaturing gel
linearizes proteins and peptides. Antibodies that do not work in a denaturing
Western but do
recognize the native target, for example expressed on an intact cell, are
determined to
recognize a conformational epitope. As used herein, the antibody may or may
not actually
bind to the "conformational epitope", however the presence of the
"conformational epitope"
sequence is required to render a three dimensional structure so that the MUC1*
region on
cancer cells is able to be bound by the antibody that is specific for cancer
treatment. Thus, the
conformational epitope is an amino acid sequence that induces the binding of
the antibody to
the MUC1* region on cancer cells. Thus, a term "conformational inducing
peptide sequence"
may be used, which indicates that a peptide sequence is present within a
larger peptide not as
a binding site but that induces binding of an antibody to the larger peptide
by causing a three-
dimensional structure to form that facilitates the binding of the antibody to
the larger peptide.
[00572] MUC1* antibodies (anti-PSMGFR) for treatment or prevention of cancers
[00573] We discovered that a cleaved form of the WW1 (SEQ fE) NO:1)
transmembrane
protein is a growth factor receptor that drives the growth of over 75% of all
human solid
tumor cancers. The cleaved form of MUC1, which we called MUC1* (pronounced muk
1
star), is a powerful growth factor receptor. Enzymatic cleavage releases the
bulk of the
MUC1 extracellular domain. It is the remaining portion comprising a truncated
extracellular
domain, transmembrane domain and cytoplasmic tail that is called MUC1*.
Cleavage and
release of the bulk of the extracellular domain of MUC1 unmasks a binding site
for activating
ligands dimeric NME1, NME6, NME8, NME7AB, NME7-X1 or NME7. Cell growth assays
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show that it is ligand-induced dimerization of the MUC1* extracellular domain
that promotes
growth (Fig. 1A-1D). MUC1* positive cells treated with either bi val en t 'by
' anti -MUC1*
antibody, monovalent 'my' or Fab, NM23-H1 dimers or NME7-AB. Bivalent anti-
MUC1*
antibodies stimulate growth of cancer cells whereas the monovalent Fab
inhibits growth.
Classic bell-shaped curve indicates ligand induced dimerization stimulates
growth. Dimeric
NM23-H1, aka NME1, stimulates growth of MUC1* positive cancer cells but siRNA
to
suppress MUC1 expression eliminate its effect (Fig. 1C). NME7-AB also
stimulates the
growth of MUC1* positive cells (Fig. 1D).
[00574] MUC1* is an excellent target for cancer drugs as it is aberrantly
expressed on over
75% of all cancers and is likely overexpressed on an even higher percentage of
metastatic
cancers. After MUC1 cleavage, most of its extracellular domain is shed from
the cell surface.
The remaining portion has a truncated extracellular domain that at least
comprises the
primary growth factor receptor sequence, PSMGFR (SEQ ID NO:2). Antibodies that
bind to
the PSMGFR sequence and especially those that competitively inhibit the
binding of
activating ligands such as NME proteins, including NME1, NME6, NME8, NME7An,
NME7-
X1 and NME7, are ideal therapeutics and can be used to treat or prevent MUC1
positive or
MUC1* positive cancers, as stand-alone antibodies, antibody fragments or
variable region
fragments thereof incorporated into multi-specific antibody-like molecules,
bispecific
antibodies, antibody-drug conjugates or chimeric antigen receptors also called
CARs, which
are then transfected or transduced into immune cells, then administered to a
patient.
Therapeutic anti-MUC1* antibodies can be monoclonal, polyclonal, antibody
mimics,
engineered antibody-like molecules, full antibodies or antibody fragments.
Examples of
antibody fragments include but are not limited to Fabs, scFv, and scFv-Fc.
Human or
humanized antibodies are preferred for use in the treatment or prevention of
cancers. In any
of these antibody-like molecules, mutations can be introduced to prevent or
minimize dimer
formation. Anti-MUC1* antibodies that are monovalent or bispecific are
preferred because
MUC1* function is activated by ligand induced dimerization. Typical binding
assays show
that NME1 and NME7An bind to the PSMGFR peptide portion of MUC1* (Fig. 2A,
2D).
Further, they show that these activating growth factors bind to the membrane
proximal
portion of MUC1*, as they do not bind to the PSMGFR peptide if the 10 C-
terminal amino
acids are missing. Similarly, anti-MUC1* antibodies MNC2 and MNE6 bind to the
PSMGFR
peptide if an only if the 10 C-terminal amino acids are present (Fig. 2B, 2C).
Antibodies
MNC3 and MNC8 bind to epitopes that are different from MNC2 and MNE6, as they
do not
depend on the presence of the 10 C-terminal amino acids of the PSMGFR peptide
(Fig. 2E,
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2F). Antibodies MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12,
B2,
B7, B9, 8C7F3, and Hl 1 antibody and other antibodies of the invention or
fragments derived
from them, can be administered to a patient for the treatment or prevention of
cancers, as
stand-alone antibodies or incorporated into a BiTE, an ADC, a multi-specific
antibody-like
molecule, bispecific antibodies, with or without an FC region or a portion of
an Fc region, a
bi-scFv, a di-scFv, a tandem di-scEv, a diabody, triabody, tribody, tetrabody
and other
antibody-like molecules that are multi-valent and multi-specific. The antibody
or antibody
fragment may be murine, human, humanized, camelid, rabbit or other non-human
species.
[00575]
BiTEs or chimeric antigen receptors also called CARs that have been
transduced
into immune cells. MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4,
B12,
B2, B7, B9, 8C7F3, and H11 antibody and other anti-MUC1* antibodies that
competitively
inhibit the binding of NME1 and NME7AB are preferred. The antibody or antibody
fragment
may be murine, human, humanized, camelid, rabbit or other non-human species.
[00576] Therapeutic anti-MUC1* antibodies for use as a stand-alone antibody
therapeutic
or for integration into a BiTE, a CAR, an ADC, or any of the multi-specific
antibody-like
molecules can be selected based on specific criteria. The parent antibody can
be generated
using typical methods for generating monoclonal antibodies in animals.
Alternatively, they
can be selected by screening antibody or antibody fragment libraries,
including but not
limited to strategies described in Beckman U.S. Patent No. 9.944,719B2, which
is
incorporated by reference herein for description of methods of screening
antibodies.
Antibodies suitable for therapeutic use are chosen based on their ability to
bind to a MUC1*
peptide, which can be:
[00577] (i) PSMGFR region of MUCl;
[00578] (ii) PSMGFR peptide;
[00579] (iii) a peptide having amino acid sequence of
QFNQYKTEAAS RYNLTISDVS VSDVPFPFS AQS GA (N-10)
[00580] (iv) a peptide having amino acid sequence of
[00581] ASRYNLTISDVSVSDVPFPFSAQSGA (N-19)
[00582] (v) a peptide having amino acid sequence of
[00583] NLTISDVSVSDVPFPFSAQSGA (N-23)
[00584] (vi) a peptide having amino acid sequence of
[00585] IS DVS VS DVPFPFS AQS GA (N-26)
[00586] (vii) a peptide having amino acid sequence of
[00587] SVSDVPFPFSAQSGA (N-30)
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[00588] (viii) a peptide having amino acid sequence of
[00589] QFNQYKTEA A SRYNLTISDVSVS DVPFPFS (N-10/C-5)
[00590] (ix) a peptide having amino acid sequence of
[00591] ASRYNLTIS D VS VS DVPFPFS (N- 19/C-5) or
[00592] (x) a peptide having amino acid sequence of
[00593] FPFSAQS GA (N-36).
[00594] Resultant antibodies or antibody fragments generated or selected in
this way can
then be further selected by passing additional screens. For example,
antibodies or antibody
fragments become more preferred based on their ability to bind to MUC1*
positive cancer
cells or tissues but not to MUC1 negative cancer cells or to normal tissues.
Further, anti-
MUC1* antibodies or antibody fragments may be de-selected as anti-cancer
therapeutics if
they bind to stem or progenitor cells. Anti-MUC1* antibodies or antibody
fragments become
more preferred if they have the ability to competitively inhibit the binding
of activating
ligands, such as NME7AB or NME7-XI, to MUC1*. Figs. 3A-3C shows that MNE6 and
MNC2 competitively inhibit the binding of activating ligands NME1 and NME7 to
MUC1*.
[00595] A process for selecting anti-MUCI* antibodies for use in treating a
patient
diagnosed with a MUC1 positive cancer, at risk of developing a MUC1 positive
cancer or
suspected of having a MUC1 positive cancer comprises one or more of the
following steps of
selecting antibodies or antibody fragments that 1) bind to the PSMGFR peptide;
2) bind to
the N-10 PSMGFR peptide; 3) selectively bind to cancer cells; 4) do not bind
to C-10
PSMGFR peptide; and 5) competitively inhibited the binding of dimeric NME1 or
NME7-
AB to the PSMGFR peptide. For example, Figs. 3A-3C show that monoclonals MNE6
and
MNC2 satisfy all five criteria, while monoclonals MNC3 and MNC8 do not
competitively
inhibit the binding of activating ligands NME1 and NME7 (Fig. 3C). Recall that
the MUC1*
growth factor receptor is activated by ligand-induced dimerization of its
extracellular domain.
Therefore, the ideal antibody therapeutic, if used as a straight stand-alone
antibody
therapeutic, should not dimerize the MUCI* extracellular domain. For this
therapeutic
format , suitable antibodies in this regard include monovalent antibodies such
as those
generated in lamas and camels, Fabs, scFv's, single domain antibodies (sdAb),
scFv-Fc as
long as the Fc portion is constructed such that it does not homo-dimerize.
[00596] FACS scans show that anti-MUC1* antibodies MNC2 and MNE6 specifically
bind to MUC1* positive solid tumor cancer cells and MUC1* transfected cells
but not
MUC1* negative or MUC1 negative cells. In one example, a humanized MNC2 scFv
is
shown to bind to ZR-75-1, aka 1500, MUC1* positive breast cancer cells (Fig.
4A-4C).
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MNE6 was shown to bind to MUC1 negative HCT-116 colon cancer cells if an only
if they
were transfected with MUC1*. MNE6 also bound to MUC1* positive cancer cells
such as
ZR-75-1, aka 1500, MUC1* positive breast cancer cells (Fig. 4D-4F). Binding
assays such as
ELISAs, immunofluorescence, and the like all confirm that MNC2 and MNE6 bind
to the
PSMGFR peptide and to live MUC1 positive cancer cells. Humanized anti-MUC1*
antibodies are selected based on their ability to also bind to the PSMGFR
peptide or to
MUC1 positive cancer cells. Figure 5 shows that humanized MNC2 scFv binds with
high
affinity to the MUC1* peptide PSMGFR with an EC-50 of about 333nM. Humanized
MNC2
scFv, like Fabs, potently inhibits the growth of MUC1* positive cancer cells
as is shown in
one example in Figs. 6A, 6B. Like the parent antibodies, humanized scFv's show
the same
binding pattern. huMNE6-scFv binds to the PSMGFR peptide, binds to the N-10
peptide but
does not bind to the C-10 peptide (SEQ ID NO:825) (Fig. 8). However, murine or
humanized
MNC3-scFv, which is less suitable for the treatment of cancers, binds to the,
PSMGFR
peptide, binds to the N-10 peptide and also binds to the C-10 peptide (Fig.
9), which we know
is the epitope to which the activating ligand NME7An binds.
[00597] The Fabs of MNE6 and MNC2 or the comparable single chain variable
regions
derived from them potently inhibit the growth of MUC1* positive cancers in
vitro and in
vivo. In several examples, the Fabs of Anti-MUC1* antibodies inhibited the
growth of human
MUC1* positive cancers in vivo. In one case, immune-compromised mice were
implanted
with human breast tumors then treated with MN E6 Fab after tumor engraftment.
Fig. 7A
shows that MNE6 Fab potently inhibited the growth of MUC1* positive breast
cancers.
Female nu/nu mice implanted with 90-day estrogen pellets were implanted with 6
million
T47D human breast cancer cells that had been mixed 50/50 with Matrigel. Mice
bearing
tumors that were at least 150 min and had three successive increases in tumor
volume were
selected for treatment. Animals were injected sub-cutaneously twice per week
with 80 mg/kg
MNE6 Fab and an equal number of mice fitting the same selection criteria were
injected with
vehicle alone (Fig. 7A).
[00598] In another aspect, MNE6 was shown to halt the growth of prostate
cancer. Fig. 7B
shows that MNE6 Fab potently inhibited the growth of MUC1* positive prostate
cancers.
Male NOD/SCID mice were implanted with 6 million DU-145 human prostate cancer
cells
that had been mixed 50/50 with Matrigel. Mice bearing tumors that were at
least 150 mmA3
and had three successive increases in tumor volume were selected for
treatment. Animals
were injected sub-cutaneously every 48 hours with 160 mg/kg MNE6 Fab and an
equal
number of mice fitting the same selection criteria were injected with vehicle
alone (Fig. 7B).
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Tumors were measured independently by two researchers twice per week and
recorded.
Statistics were blindly calculated by independent statistician, giving a P
value of 0.0001 for
each. Anti-MUC1* Fab inhibited breast cancer growth and prostate cancer
growth. Treatment
had no effect on weight, bone marrow cell type or number. The MNE6 Fab
effectively
inhibited the growth of the tumors, while the control group's tumors continued
to grow until
sacrifice. No adverse effects of treatment were observed or detected.
[00599] Recombinant forms of MNE6 and MNC2 were constructed that like the Fab
are
monomeric. In this case, MNE6 was humanized and MNC2 was humanized. There are
a
number of methods known to those skilled in the art for humanizing antibodies.
In addition to
humanizing, libraries of human antibodies or antibody fragments can be
screened to identify
other fully human antibodies that bind to the PSMGFR.
[00600] A single chain of the humanized MNE6 variable region, called an scFv,
was
genetically engineered such that it was connected to the Fc portion of the
antibody (SEQ ID
NO:256 and 257). Fc regions impart certain benefits to antibody fragments for
use as
therapeutics. The Fc portion of an antibody recruits complement, which in
general means it
can recruit other aspects of the immune system and thus amplify the anti-tumor
response
beyond just inhibiting the target. The addition of the Fc portion also
increases the half-life of
the antibody fragment (Czajkowsky DM, Hu J, Shao Z and Pleass RJ. (2012) Fc-
fusion
proteins: new developments and future perspectives. EMBO Mol Med. 4(10):1015-
1028).
However, the Fc portion of an antibody homo-dimerizes, which in the case of
anti-M1JC1*
antibody based therapeutics is not optimal since hg and-induced dimerization
of the MUC1*
receptor stimulates growth. Therefore, mutations in the Fc region that resist
dimer formation
are preferred for anti-MUC1* anti-cancer therapeutics. Deletion of the hinge
region and other
mutations in the Fc region that make the Fc-mutant resistant to dimerization
were made and
could be used as therapeutics.
[00601] A human or humanized MNE6 antibody or antibody fragment, Fab, MNE6
scFv
or hu MNE6 scFv-Fcmut are effective anti-cancer agents that can be
administered to a person
diagnosed with a MUC1 or MUC1* positive cancer, suspected of having a MUC1 or
MUC1*
positive cancer or is at risk of developing a MUC1 or MUC1* positive cancer.
[00602] Humanizing
[00603] Humanized antibodies or antibody fragments or fully human antibodies
that bind
to the extracellular domain of -MUC1* are preferred for therapeutic use. The
techniques
described herein for humanizing antibodies are but a few of a variety of
methods known to
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those skilled in the art. The invention is not meant to be limited by the
technique used to
humanize the antibody.
[00604] Humanization is the process of replacing the non-human regions of a
therapeutic
antibody (usually mouse monoclonal antibody) by human one without changing its
binding
specificity and affinity. The main goal of humanization is to reduce
immunogenicity of the
therapeutic monoclonal antibody when administered to human. Three distinct
types of
humanization are possible. First, a chimeric antibody is made by replacing the
non-human
constant region of the antibody by the human constant region. Such antibody
will contain the
mouse Fab region and will contain about 80-90% of human sequence. Second, a
humanized
antibody is made by grafting of the mouse CDR regions (responsible of the
binding
specificity) onto the variable region of a human antibody, replacing the human
CDR (CDR-
grafting method). Such antibody will contain about 90-95% of human sequence.
Third and
last, a full human antibody (100% human sequence) can be created by phage
display, where a
library of human antibodies, antibody-like molecules or antibody fragments is
screened to
select antigen specific human antibody or by immunizing transgenic mice
expressing human
antibody.
[00605] A general technique for humanizing an antibody is practiced
approximately as
follows. Monoclonal antibodies are generated in a host animal, typically in
mice. Monoclonal
antibodies are then screened for affinity and specificity of binding to the
target. Once a
monoclonal antibody that has the desired effect and desired characteristics is
identified, it is
sequenced. The sequence of the animal-generated antibody is then aligned with
the sequences
of many human antibodies in order to find human antibodies with sequences that
are the most
homologous to the animal antibody. Biochemistry techniques are employed to
paste together
the human antibody sequences and the animal antibody sequences. Typically, the
non-human
CDRs are grafted into the human antibodies that have the highest homology to
the non-
human antibody. This process can generate many candidate humanized antibodies
that need
to be tested to identify which antibody or antibodies has the desired affinity
and specificity.
[00606] Once a human antibody or a humanized antibody has been generated it
can be
further modified for use as an Fab fragment, as a full antibody, or as an
antibody-like entity
such as a single chain molecule containing the variable regions, such as scFv
or an scFv-Fc.
In some cases it is desirable to have Fc region of the antibody or antibody-
like molecule
mutated such that it does not dimerize.
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[00607] In addition to methods that introduce human sequences into antibodies
generated
in non-human species, fully human antibodies can be obtained by a variety of
methods
known to those skilled in the art, including screening human antibody
libraries with a peptide
fragment of an antigen. A fully human antibody that functions like MNE6 or
MNC2, 20A10
or other antibodies of the invention can be generated by screening a human
antibody library
or library of antibody fragments with a peptide having the sequence of the
PSMGFR N-10
peptide. In another method, human antibodies are generated in genetically
modified mice.
Humanized anti-MUC1* antibodies or antibody fragments were generated based on
the
sequences of the mouse monoclonal antibodies MNE6, MNC2, 20A10, 3C2B1, 5C6F3
and
25E6. In one aspect of the invention, a patient diagnosed with a MUC1*
positive cancer is
treated with an effective amount of a murine or camelid antibody or antibody
fragment
comprising sequences from MNC2 (SEQ ID NO:118-119 and 168-169), MNE6 (SEQ ID
NOS: 12-13 and 65-66), 20A10 (SEQ ID NOS:988-989 and 1004-1005), 3C2B1 (SEQ ID
NOS:1820-1821 and 1822-1823), 5C6F3 (SEQ ID NO:1816-1817 and 1818-1819), 25E6
(SEQ ID NO:1020-1021 and 1036-1037 ), 18G12, 28F9, 1E4, B12, B2, B7, B9,
8C7F3, or
H11. In another aspect of the invention, a patient diagnosed with a MUC1*
positive cancer is
treated with an effective amount of human or humanized antibody or antibody
fragment
comprising sequences from MNE6 (SEQ ID NOS:56-57 and 107-108, or 341-342, or
391-
392, or 393-394) or MNC2 (SEQ ID NO:144-145 and 194-195. or 654-655, or 239-
249, or
5017-5020), 20A10 (SEQ ID NOS:1576-1581 or 5001-5012), 3C2B1 (SEQ Ill NOS:1820-
1823
or 1812-1813), 5C6F3 (SEQ ID NOS:1816-1819, or 1814-1815), 25E6 (SEQ ID
NOS:1020-
1021 and 1036-1037, or 1600-1601), 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3,
or H11.
In a preferred embodiment, a patient diagnosed with a MUC1* positive cancer is
treated with
an effective amount of humanized antibody or antibody fragment comprising
sequences of
MNC2 (SEQ ID NO:654-655), MNE6 (SEQ ID NO:341-342), 20A10 (SEQ ID NO:1580-
1581), 3C2B1 (SEQ ID NO:1812-1813), 5C6F3 (SEQ ID NO:1814-1815), 25E6 (SEQ ID
NO:1600-1601). In another aspect of the invention, a patient diagnosed with a
MUC1*
positive cancer is treated with an effective amount of humanized monovalent
form of the
antibodies such as MNC2 (SEQ ID NOS:239, 241, 243,396 or 5018-5020), MNE6 (SEQ
ID
NO:), 20A10 (SEQ ID NOS:1574-1581 or SEQ ID NOS:5001-5012) , 3C2B1 (SEQ ID
NO:1813), 5C6F3 (SEQ ID NO:1815), 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9,
8C7F3, or
H11, wherein monovalent means the corresponding Fab fragment, the
corresponding scFv or
the corresponding scFv-Fc fusion. In a preferred embodiment, a patient
diagnosed with a
MUC1* positive cancer is treated with an effective amount of a humanized scFv
or
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monomeric humanized scFv-Fc of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12,
28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. Since the MUC1* growth factor
receptor is
activated by ligand induced dimerization of its extracellular domain, and
because the Fc
portion of an antibody homo-dimerizes, it is preferable that a construct that
includes an Fc
portion uses a mutated Fc region that prevents or minimizes dimerization.
[00608]
Antibodies that bind to PSMGFR (SEQ ID NO:2) peptide, and more
specifically
to the N-10 peptide, of the extracellular domain of the MUC1* receptor are
potent anti-cancer
therapeutics that are effective for the treatment or prevention of MUC1*
positive cancers.
They have been shown to inhibit the binding of activating ligands dimeric NME1
(SEQ ID
NO:1781) and NME7AB (SEQ ID NOS:827) to the extracellular domain of MUC1*.
Anti-
MUC1* antibodies that bind to the PSMGFR sequence inhibit the growth of MUC1*-
positive
cancer cells, specifically if they inhibit ligand-induced receptor
dimerization. Fabs of anti-
MUC1* antibodies have been demonstrated to block tumor growth in animals.
Thus,
antibodies or antibody fragments that bind to the extracellular domain of
MUC1* would be
beneficial for the treatment of cancers wherein the cancerous tissues express
MUC1*.
[00609] Antibodies that bind to PSMGFR region of MUC1* or bind to a synthetic
PSMGFR peptide are preferred. Especially preferred are antibodies that bind to
the N-10
peptide but not to the C-10 peptide. Still more preferred are antibodies that
bind to the N-26
peptide wherein mutation or deletion of the PFPFS sequence (SEQ ID NO:1747)
destroys binding
of the antibody or fragment thereof to the N-26 peptide. We have identified
several monoclonal
antibodies that bind to the extracellular domain of MUC1*. Among this group
are mouse
monoclonal antibodies MNC2 (SEQ ID NOS:118-131, 144-158, 163-164, 168-181, 194-
209), MNE6 (SEQ ID NOS:12-25, 39-59, 65-78, 93-114), 20A10 (SEQ ID NOS:988-
1019,
1574-1597, 1659-1666); 3C2B1 (SEQ ID NOS:1386-1413, 1572-1573), 5C6F3 (SEQ ID
NOS:1356-1385), 25E6 (SEQ ID NOS:1020-1051, 1598-1617, 1667-1674), 18G12 (SEQ
ID
NOS:956-987), 28F9 (SEQ ID NOS:1052-1083), 1E4 (SEQ ID NOS:1116-1227), B12
(SEQ
ID NOS:1414-1431, 1733-1742), B2 (SEQ ID NOS:1432-1459), B7 (SEQ ID NOS:1460-
1487), B9 SEQ ID NOS:1544-1571), 8C7F3 (SEQ ID NOS:1488-1515), or H11 (SEQ ID
NOS:1516-1543), the variable regions of which were sequenced and are given as
for MNE6
SEQ ID NOS: 12-13 and 65-66, for MNC2 SEQ ID NOS: 118-119 and 168-169. The
CDRs
of these antibodies make up the recognition units of the antibodies and are
the most important
parts of the mouse antibody that should be retained when grafting into a human
antibody. The
sequences of the CDRs for each mouse monoclonal are as follows, heavy chain
sequence
followed by light chain: MNE6 CDR1 (SEQ ID NO:16-17 and 69-70) CDR2 (SEQ ID
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NO:20-21 and 73-74) CDR3 (SEQ ID NO: 24-25 and 77-78), MNC2 CDR1 (SEQ ID
NO:122-123 and 172-173) CDR2 (SEQ ID NO:126-127 and 176-177) CDR3 (SEQ ID
NO:130-131 and 180-181), 20A10 CDR1 (SEQ ID NO:991-992 and 1008-1009) CDR2
(SEQ ID NO:996-997 and 1012-1013) CDR3 (SEQ ID NO:1000-1001 and 1016-1017),
3C2B1 CDR1 (SEQ ID NO:1388-1389 and 1402-1403) CDR2 (SEQ ID NO:1392-1393 and
1406-1407) CDR3 (SEQ ID NO:1396-1397 and 1410-1411), 5C6F3 CDR1 (SEQ ID
NO:1358-1359 and 1372-1373) CDR2 (SEQ ID NO:1362-1363 and 1376-1377) CDR3 (SEQ
ID NO:1366-1367 and 1380-1381), and 25E6 CDR1 (SEQ ID NO:1024-1025 and 1040-
1041) CDR2 (SEQ ID NO:1028-1029 and 1044-1045) CDR3 (SEQ ID NO:1032-1033 and
1048-1049). In some cases, portions of the framework regions that by modeling
are thought
to be important for the 3-dimensional structure of the CDRs, are also imported
from the
mouse sequence.
[00610] Monoclonal antibodies MNE6, MNC2, 20A10, 3C2B1 and 25E6 have greater
affinity for MUC1* as it appears on cancer cells. Monoclonal antibodies MNC3
and MNC8
have greater affinity for MUC1* as it appears on stem cells.
[00611] All seven antibodies have been humanized, which process has resulted
in several
humanized forms of each antibody. CDRs derived from the variable regions of
the mouse
antibodies were biochemically grafted into a homologous human antibody
variable region
sequence. Humanized variable regions of MNE6 (SEQ ID NOS: 38-39 and 93-94),
MNC2
(SEQ Ill NOS: 144-145 and 194-195), 20A10 (SEQ Ill NOS:1576-1581 and 5001-
5012),
3C2B1 (SEQ ID NOS:1812-1813), 5C6F3 (SEQ ID NOS: 1814-1815). 25E6 (SEQ ID
NOS:1600-1601). MNC3 (SEQ ID NOS: 439-440 and 486-487) and MNC8 (SEQ ID NOS:
525-526 and 543-544) were generated by grafting the mouse CDRs into the
variable region of
a homologous human antibody. The humanized heavy chain variable constructs
were then
fused into constant regions of either human IgG1 heavy chain constant region
(SEQ ID
NOS:58-59) or human IgG2 heavy chain constant region (SEQ ID NO:54-55), which
are then
paired with either humanized light chain variable constructs fused to a human
kappa chain
(SEQ ID NO: 109-110) or human lambda chain (SEQ ID NO: 113-114) constant
region.
Other IgG isotypes could be used as constant region including IgG3 or IgG4.
[00612] Examples of humanized MNE6 variable region into an IgG2 heavy chain
(SEQ ID
NOS:52-53) and into an IgG1 heavy chain (SEQ ID NOS:56-57), humanized MNC2
variable
into an IgG1 heavy chain (SEQ ID NOS: 157-158) or into an IgG2 heavy chain
(SEQ ID
NOS: 163-164) paired with either Lambda light chain (SEQ ID NO: 1 1 1-112 and
216-219) or
Kappa chain (SEQ ID NO:107-108 and 210-213) and , humanized MNC3 (SEQ ID NOS:
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455-456, 453-454 and 500-501, 502-503) and MNC8 (SEQ ID NOS: 541-542, 539-540
and
579-580, 581-582) antibodies were generated. Which IgG constant region is
fused to the
humanized variable region depends on the desired effect since each isotype has
its own
characteristic activity. The isotype of the human constant region is selected
on the basis of
things such as whether antibody dependent cell cytotoxicity (ADCC) or
complement
dependent cytotoxicity (CDC) is desired but can also depend on the yield of
antibody that is
generated in cell-based protein expression systems. In a preferred embodiment,
humanized
anti-MUC1* antibodies or antibody fragments are administered to a person
diagnosed with or
at risk of developing a MUC1-positive cancer.
[00613] One method for testing and selecting the humanized anti-MUC1*
antibodies that
would be most useful for the treatment of persons with cancer or at risk of
developing cancers
is to test them for their ability to inhibit the binding of activating ligands
to the MUC1*
extracellular domain. Dimeric NME1 can bind to and dimerize the MUC1*
extracellular
domain and in so doing stimulates cancer cell growth. Antibodies and antibody
fragments
that compete with NME1 for binding to the MUC1* extracellular domain are
therefore anti-
cancer agents. NME7AB is another activating ligand of MUC1*. In some cases, it
is
preferable to identify antibodies that block the binding of NME7, or an NME7AB
truncation
or cleavage product of NME7-X1, to the MUC1* extracellular domain. Antibodies
and
antibody fragments that compete with NME7 and NME7 variants for binding to the
MUC1*
extracellular domain are effective as anti-cancer therapeutics. These
antibodies include but
are not limited to MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4,
B12, B2,
B7, B9, 8C7F3, or H11 as well as single chain versions, such as scFv, of these
antibodies
and humanized version thereof. Other NME proteins also bind to MUC1 or MUC1*
including NME1, NME6 and NME8. Antibodies that compete with these proteins for
binding
to MUC1* may also be useful as therapeutics. In a preferred embodiment,
murine, camelid,
human or humanized anti-MUC1* antibodies or antibody fragments are
administered to a
person diagnosed with or at risk of developing a MUC1-positive cancer. In a
more preferred
embodiment, single chain antibody fragments, or monomeric scFv-Fc fusions,
derived from
humanized sequences of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9,
1E4,
B12, B2, B7, B9, 8C7F3, or H11 are administered to a person diagnosed with or
at risk of
developing a MUC1-positive cancer.
[00614] Single chain variable fragments, scFv, or other forms that result in a
monovalent
antibody or antibody-like protein are also useful. In some cases it is desired
to prevent
dimerization of the MUC1* extracellular domain. Single chain variable
fragments, Fabs and
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other monovalent antibody-like proteins have been shown to be effective in
binding to the
extracellular domain of MUC1* and blocking MUC1* dimerization. These single
chain
variable fragments, Fabs and other monovalent antibody-like molecules
effectively blocked
cancer growth in vitro and in animals xenografted with human MUCl-positive
cancer cells.
Thus, humanized single chain variable fragments or monovalent anti-MUC1*
antibodies or
antibody-like molecules would be very effective as an anti-cancer therapeutic.
Such
humanized single chain antibodies, Fabs and other monovalent antibody-like
molecules that
bind to the MUC1* extracellular domain or to a PSMGER peptide are therefore
useful as
anti-cancer therapeutics. Anti-MUC1* single chain variable fragments are
generated by
grafting non-human CDRs of antibodies, which bind to extracellular domain of
MUC1* or
bind to PSMGER peptide, into a framework of a homologous variable region human
antibody. The resultant humanized heavy and light chain variable regions are
then connected
to each other via a suitable linker, wherein the linker should be flexible and
of length that it
allows heavy chain binding to light chain but discourages heavy chain of one
molecule
binding to the light chain of another. For example a linker of about 10-15
residues.
Preferably, the linker includes RGlycine)4 (Serine)113 (SEQ ID NOS: 401-402),
but is not
limited to this sequence as other sequences are possible.
[00615] In one aspect, the humanized variable regions of MNE6 (SEQ ID NOS: 38-
39 and
93-94), MNC2 (SEQ ID NOS: 144-145 and 194-195), or other antibodies of the
invention are
biochemically grafted into a construct that connects heavy and light chains
via a linker.
Examples of humanized single chain anti-MUC1* antibodies comprising humanized
sequences from the variable regions of MNE6 and MNC2, were generated. Several
humanized MNE6 single chain proteins were generated (SEQ ID NOS: 232-237, 397-
398).
Several humanized MNC2 single chain proteins were generated (SEQ ID NOS: 238-
243,
395-396, 654-655, 5017-5018, 5019-5020). Several humanized 20A10 single chain
proteins
were generated (SEQ ID NOS:1576-1581 and 5001-5012). Several humanized 3C2B1
single
chain proteins were generated (SEQ ID NOS:1812-1813). Several humanized 5C6F3
single
chain proteins were generated (SEQ ID NOS:1814-1815). Several humanized 25E6
single
chain proteins were generated (SEQ ID NOS:1600-1601). In a preferred
embodiment,
humanized anti-MUC1* antibody fragments, including variable fragments, scFv
antibody
fragments derived from MNE6 scFv, MNC2 scFv, 20A10, 3C2B1, 5C6F3, 25E6, 18G12,
28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 scFv, which may be incorporated into
different
therapeutic formats including CARs, Bispecific antibodies, BiTEs, antibody
drug conjugates,
are administered to a person diagnosed with or at risk of developing a MUC1-
positive cancer.
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[00616] One aspect of the invention is a method for treating a patient
diagnosed with,
suspected of having, or at risk of developing a MUC1 positive or MUC1*
positive cancer,
wherein the patient is administered an effective amount of an agent containing
a monomeric
form of MNE6, MNC2, or 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2,
B7,
B9, 8C7F3, or H11, wherein the antibody variable fragment portions are human
or have been
humanized and wherein the Fc portion of the antibody-like protein, if present,
has been
mutated such that it resists dimer formation.
[00617] CAR T and cancer immunotherapy techniques
[00618] In another aspect of the invention, some or all of the single chain
portions of anti-
MUC1* antibody fragments are biochemically fused onto immune system molecules,
using
several different chimeric antigen receptor, 'CAR' strategies. The idea is to
fuse the
recognition portion of an antibody, typically as a single chain variable
fragment, to an
immune system molecule that has a transmembrane domain and a cytoplasmic tail
that is able
to transmit signals that activate the immune system for example activating the
immune cell to
kill the cell that is recognized by the recognition unit. The recognition unit
can be an antibody
fragment, a single chain variable fragment, scFv, or a peptide. In one aspect,
the recognition
portion of the extracellular domain of the CAR is comprised of sequences from
the human,
humanized or non-human variable regions of MNE6 (SEQ ID NOS:12-13 and 65-66,
56-57
and 107-108, 38-39 and 93-94, 341-342, 391-394), MNC2 (SEQ ID NOS:118-119 and
168-
169, or 144-145 and 194-195, 654-655, 239-243, or 5017-5020), 20A10 (SEQ ID
NOS:988-
989 and 1004-1005, or 1574-1581, 1677, 1687 or 5001-5012), 3C2B1 (SEQ ID
NOS:1386-
1413, or 1820-1823, 1572-1573, or 1812-1813), 5C6F3 (SEQ ID NOS:1816-1819, or
1384-
1385, or 1814-1815), 25E6 (SEQ ID NOS:1020-1021, or 1036-1037, or 1598-1601),
18G12,
28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. These are examples of murine or
humanized
antibodies of the invention, or their single chain fragments, scFv' s, which
can be incorporated
into CARs, BiTEs or ADCs. In another aspect, the recognition unit is comprised
of
sequences from a single chain variable fragment. Examples of single chain
constructs are
given. Several humanized MNE6 single chain proteins, scFv, were generated (SEQ
ID NOS:
232-237). Several humanized MNC2 single chain proteins, scFv, were generated
(SEQ ID
NOS: 238-243, 654-655, or 5017-5020). Several humanized 20A10 single chain
proteins,
scFv, were generated (SEQ ID NOS:1576-1581, 1677, 1687 and 5001-5012).
Humanized
single chain proteins were also derived from 3C2B1 (SEQ ID NOS:1812-1813),
5C6F3 (SEQ
ID NOS:18141815) and 25E6 (SEQ ID NOS:1600-1601). The extracellular hinge of
the
CAR can be derived from a variety of proteins, including CD8 (SEQ ID NOS:345-
346), CD4
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(SEQ ID NOS:347-348) or CD28 (SEQ ID NOS:349-350). The transmembrane region of
the
CAR can also be derived from CD3-zeta (SEQ ID NOS:361-362), CD8 (SEQ ID
NOS:363-
364), CD4 (SEQ ID NOS:365-366), CD28 (SEQ ID NOS:367-368), 4-1BB (SEQ ID
NOS:369-370), 0X40 (SEQ ID NOS:371-372), antibody domains or other
transmembrane
region, including the transmembrane region of the proximal cytoplasmic co-
stimulatory
domain, such as CD28, 4-1BB or other. The cytoplasmic tail of the CAR can be
comprised
of one or more motifs that signal immune system activation. A group of
cytoplasmic
signaling motifs, sometimes referred to as co-stimulatory domains, includes
but is not limited
to CD27, CD28 (SEQ ID NOS:377-378), 4-1BB (SEQ ID NOS:379-380), 0X40, CD30,
CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, CD7 and Fc receptor gamma domain. The
signaling domain can be CD3-zeta (SEQ ID NOS:373-374, or 375-376) or a
modified CD3-
zeta called 1XX (SEQ ID NOS:1796-1797). A minimal CAR may have the CD3-zeta or
an
Fc receptor gamma domain then one or two of the above domains in tandem on the
cytoplasmic tail. In one aspect, the cytoplasmic tail comprises CD3-zeta, or a
mutant thereof
such as 1XX, plus a co-stimulatory domain such as CD28, 4-1BB and/or 0X40. In
another
aspect, one or two ITAMs of CD3-zeta are deleted or mutated to slow signaling
which
increases persistence and decreases differentiation of the immune cell.
[00619] The extracellular domain recognition unit of a MUC1* targeting CAR can
comprise variable regions of any non-human, humanized or human antibody that
is able to
bind to at least 12 contiguous amino acids of the PSMGFR peptide (SEQ Ill
NO:2) or more
preferably the N-10 peptide (SEQ ID NO:3), still more preferably, is able to
bind to the N-10
peptide (SEQ ID NO:3), but is not able to bind to the C-10 peptide (SEQ ID
NO:825). In one
aspect, the MUC1* targeting portion of the CAR comprises variable regions from
non-
human, humanized or human MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9,
1E4, B12, B2, B7, B9, 8C7F3, or H11. In a preferred embodiment, the MUC1*
targeting
portion of the CAR comprises variable regions from non-human, humanized or
human
MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6. Examples of a few antibodies of the
invention,
incorporated into CARs as either murine or humanized are given as MNE6 (SEQ ID
NOS:297-298, 300-301, 303-304, 1626-1633 and 5045-5048), MNC2 (SEQ ID NOS:306-
307, 608-611, 718-719, 1618-1625, 5041-5044, and 1784-1785), 20A10 (SEQ ID
NOS:1582-
1597, 5021-5028, 1798-1799, 1692,1699, and 1706), and 25E6 (SEQ ID NOS:1602-
1617,
5033-5040). Similarly, single chain antibodies derived from 3C2B1 (SEQ ID
NOS:1572-
1573 or 1812-1813) or 5C6F3 (SEQ ID NOS:1384-1385 or 1814-1815) can be
substituted for
the single chain antibody fragment in any of the CARs listed above. In the
humanization
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process, the antibody CDRs can be inserted into a number of different
framework regions; as
a demonstration we generated three versions of a humanized 20A10 which differ
only in the
framework regions. These have been incorporated into CARs (SEQ ID NOS:1675,
1678,
1685) that when transduced into human T cells are able to recognize target
MUC1*
expressing cells and kill them. In one aspect, the extracellular domain
recognition unit of a
CAR is comprised essentially of a humanized MNC2, MNE6, 20A10, 3C2B1, 5C6F3,
25E6,
18612, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 single chain variable
fragment scFv.
The recognition domain, which is typically an antibody fragment, can be fused
to an
extracellular region, often referred to as the hinge. The hinge can be derived
from a variety of
extracellular regions or peptides, including but not limited to the hinge
region of CD8 (SEQ
ID NOS:345-346), CD4 (SEQ ID NOS:347-348) or CD28 (SEQ ID NOS:349-350). The
transmembrane region of the CAR can be derived from a number of protein
transmembrane
domains, including but not limited to CD8 (SEQ ID NOS:363-364), or can be the
transmembrane domain of CD3-zeta (SEQ ID NOS:361-362), CD4 (SEQ ID NOS:365-
366),
CD28 (SEQ ID NOS:367-368), 41BB (SEQ ID NOS:369-370), 0X40 (SEQ ID NOS:371-
372) or other transmembrane region. The cytoplasmic domain of a CAR with
antibody
fragment targeting MUC1* extracellular domain can be comprised of one or more
selected
from the group comprising an immune system co-stimulatory cytoplasmic domain
and a
cytoplasmic signaling domain. The group of immune system co-stimulatory
domains includes
but is not limited to CD27. CD28, 4-1BB, 0X40, CD30, CD40, ICAm-1,
ICOS,
CD2, CD5, CD7 and Fc receptor gamma domain (SEQ ID NOS:373-382). The group of
immune system signaling domains includes but is not limited to CD3-zeta (SEQ
ID
NOS:373-376) and CD3-zeta-1XX (SEQ ID NOS:1796-1797). The CD3-zeta signaling
domain may be wild type or may contain deletions or mutations of one or two of
the three
ITAMs. In one aspect, the CD3-zeta domain contains only one functional ITAM.
In a
preferred embodiment that ITAM is ITAM1 also known as the 1XX variation of CD3-
zeta.
[00620] The CARs described can be transfected or transduced into a cell of the
immune
system. In a preferred embodiment, a MUC1* targeting CAR is transfected or
transduced into
a T cell or an NK cell. The immune cell can be autologous or allogeneic.. In
one aspect, the T
cell is a CD3+ T cell, which may be CD8 or CD4 positive. In another case it is
a dendritic
cell. In another case it is a B cell. In another case it is a mast cell. In
yet another case it is a
Natural Killer, NK, cell. In another aspect, the immune cell is derived from a
stem cell that
has been directed to differentiate to that immune cell type in vitro. In
another aspect, a CAR
containing sequences of the antibody are expressed in a stem cell, which then
may be
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differentiated into an immune cell. In one case, the immune cell is a T cell.
In another case,
the immune cell is an NK cell. The cell can be from a patient or from a donor.
If from a
donor, it can be engineered to remove molecules that would trigger rejection.
Cells
transfected or transduced with a CAR of the invention can be expanded ex vivo
or in vitro
then administered to a patient. Administrative routes are chosen from a group
containing but
not limited to bone marrow transplant, intravenous injection, in situ
injection or transplant. In
a preferred embodiment, the MUC1* targeting CAR is administered to a person
diagnosed
with or at risk of developing a MUCl-positive cancer.
There are many possible anti-MUC1* CAR constructs that can be transduced into
T cells or
other immune cells for the treatment or prevention of MUC1* positive cancers.
CARs are
made up of modules and the identity of some of the modules is relatively
unimportant, while
the identity of other modules is critically important. We and others have
shown that
intracellular signaling modules, such as CD3-zeta (SEQ ID NOS: 373-376), CD28
(SEQ ID
NOS: 377-378) and 41BB (SEQ ID NOS: 379-380), alone or in combinations
stimulate
immune cell expansion, cytokine secretion and immune cell mediated killing of
the targeted
tumor cells (Pule MA, Straathof KC, Dotti G, Heslop HE, Rooney CM and Brenner
MK
(2005) A chimeric T cell antigen receptor that augments cytokine release and
supports clonal
expansion of primary human T cells. Mol Ther. 12(5):933-941; Hombach AA,
Heiders J,
Foppe M, Chmielewski M and Abken H. (2012) 0X40 costimulation by a chimeric
antigen
receptor abrogates CD28 and 1L-2 induced 1L-10 secretion by redirected CD4(+)
'1 cells.
Oncoimmunology. 1(4):458-466; Kowolik CM, Topp MS, Gonzalez S, Pfeiffer T,
Olivares S,
Gonzalez N, Smith DD, Forman SJ, Jensen MC and Cooper U. (2006) CD28
costimulation
provided through a CD19-specific chimeric antigen receptor enhances in vivo
persistence and
antitumor efficacy of adoptively transferred T cells. Cancer Res. 66(22):10995-
11004;
Loskog A, Giandomenico V, Rossig C, Pule M, Dotti G and Brenner MK. (2006)
Addition of
the CD28 signaling domain to chimeric T cell receptors enhances chimeric T
cell resistance
to T regulatory cells. Leukemia. 20(10):1819-1828; Milone MC, Fish JD,
Carpenito C,
Carroll RG, Binder GK, Teachey D, Samanta M, Lakhal M, Gloss B, Danet-
Desnoyers G,
Campana D, Riley JL, Grupp SA and June CH. (2009) Chimeric receptors
containing CD137
signal transduction domains mediate enhanced survival of T cells and increased
antileukemic
efficacy in vivo. Mol Ther. 17(8):1453-1464; Song DG, Ye Q, Carpenito C,
Poussin M,
Wang LP, Ji C, Figini M, June CH, Coukos G, Powell DJ Jr. (2011) In vivo
persistence,
tumor localization, and antitumor activity of CAR-engineered T cells is
enhanced by
costimulatory signaling through CD137 (4-1BB). Cancer Res. 71(13):4617-4627).
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Antibodies of the invention including but not limited to fragments of MNC2,
MNE6, 20A10,
3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11 can also
be
incorporated into CARs that have mutated cytoplasmic tails, such as mutated or
deleted
tyrosines of one or more of the ITAMs. In any of the CARs described above, the
cytoplasmic
tails may include mutations or deletions that dampen signaling, which
increases persistence
and decreases host cell differentiation. Such mutations include but are not
limited to
Tyrosines that are mutated to inhibit phosphorylation and signaling (Salter et
al, 2018; ). In
any of the CARs described above, the ITAMs of CD3-zeta may be mutated to
inhibit or
dampen signaling (Feucht et al 2019). In any of the CARs described above, the
CD3-zeta of
the cytoplasmic tail may comprise mutations or deletions in the ITAMs
including those
referred to as 1XX (SEQ ID NOS:1796-1797). In another aspect one or two ITAMs
are
deleted, leaving only one or two ITAMs (Feucht et al 2019). In another aspect,
the position of
the included ITAM or ITAMs is moved to a position proximal to the co-
stimulatory domain.
Suitable ITAM configurations for increased persistence of CARs include but are
not limited
to 1XX, X2X, )0(3, 12X and 23X, wherein the numeral 1, 2 or 3 refers to ITAM1,
ITAM2,
or ITAM3 and X refers to the deletion of that ITAM. In a preferred embodiment
ITAM 1 is
the only functional ITAM included in the CAR construct, also known as 1XX.
Examples of
antibodies of the invention incorporated into CARs with 1XX mutations in ITAMs
of CD3-
zeta are given in the following sequences: MNC2 (SEQ ID NOS: 1618-1625, 5041-
5044 and
17841785), MNE6 (SEQ ID N05:16261633 and 5045-5048 ), 20A10 (SEQ ID NOS:1590-
1597, 5021-5028, and 1798-1799), 25E6 (SEQ ID NOS:1610-1617 and 5037-5040).
The
transmembrane and extracellular hinge region of the CAR may or may not be
derived from
sequences of the adjacent co-stimulatory domain. For example, a CAR comprising
the 4-1BB
co-stimulatory domain may have a transmembrane and hinge region derived from
CD8 or
CD28. In another example, a CAR comprising the CD28 co-stimulatory domain may
have a
transmembrane and hinge region derived from CD28. Examples of antibodies of
the
invention incorporated into CARs with 1XX mutations in ITAMs of CD3-zeta,
which have a
CD28 co-stimulatory domain as well as transmembrane and hinge region derived
from CD28,
are given in the following sequences: MNC2 (SEQ ID NOS:5041-5044 and 1784-
1785)
MNE6 (SEQ ID NOS:5045-5048), 20A10 (SEQ ID NOS:5025-5028, 1798-1799,
1692,1699,
and 1706); 25E6 (SEQ ID NOS:5037-5040). In any of the CARs described here, the
cytoplasmic region may be comprised of one or more of signaling sequence
motifs and co-
stimulatory domains, including but not limited to CD3-zeta, CD3-zeta-1XX,
CD27, CD28, 4-
1BB, 0X40, CD30, CD40, ICAm-1, LFA-1, ICOS, CD2, CD5, or CD7. Additionally,
the
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sequence of the intracellular signaling domain may contain mutations, such as
CD3-zeta-1XX
(SEQ ID NOS:1796-1797) that dampen the signal to improve persistence or target
cell killing.
Signaling domain CD3-zeta may be wild type or may contain mutations or
deletions of one or
two ITAMs. In a preferred embodiment, ITAMs 2 and 3 are deleted or
inactivated, leaving a
single ITAM, which is ITAM1 also known as the 1XX construct.
[00621] In one aspect of the invention, the hinge and transmembrane regions of
CAR are
derived from CD8 (SEQ ID NO: 301, 719, 1675 or 1605). In another aspect of the
invention,
the hinge and transmembrane regions of CAR are derived from CD28 (SEQ ID
NO:5048,
5044, 5024 or 5036). In one aspect of the invention, the co-stimulatory domain
is CD28 (SEQ
ID NO: 298, 609, 1589, 1609). In another aspect of the invention, the co-
stimulatory domain
is 4-1BB (SEQ ID NO: 301, 719, 1585 or 1605). In a preferred embodiment, the
antibody
fragment that is the targeting head of the CAR, binds to the extracellular
domain of a MUC1
that is devoid of the tandem repeat domain. In a more preferred embodiment,
the antibody
fragment that is the targeting head of the CAR, binds to a region of the MUC1*
extracellular
domain that contains the 35 most membrane proximal amino acids, also referred
to here as N-
(SEQ ID NO:3). In a still more preferred embodiment, the antibody fragment
that is the
targeting head of the CAR, binds to N-10 (SEQ ID NO:3) but does not bind to C-
10 (SEQ ID
NO:825). In a yet more preferred embodiment, the antibody fragment that is the
targeting
head of the CAR, binds to N-10 (SEQ ID NO:3), does not bind to C-10 (SEQ ID
NO:825)
and either does not bind to a linear epitope, that is to say doesn't work in a
standard Western
blot, or competes with NME7AB for binding to the N-10 peptide (SEQ ID NO:3).
In the
CARs described here, the extracellular domain may include a murine, camelid,
human, non-
human or humanized single chain antibody fragment with framework region IV of
the light
chain having variable lengths as set forth as MNE6 scFv (SEQ ID NOS:5014 or
5016),
MNC2 scFv (SEQ ID NOS:5018 or 5020) or 20A10 scFv (SEQ ID NOS 5002, 5004,
5006,
5008, 5010 or 5012), 25E6 scFv (SEQ ID NOS: 5030 or 5032). In any of the CARs
described
above, the Framework region TV of the light chain of the single chain antibody
fragment may
have the terminal amino acids R and T deleted or just T deleted. We note that
the CDRs of
antibodies can be inserted into a background of a number of different
framework regions. As
an example, 20A10 CDRs were inserted into three different sets of framework
regions (SEQ
ID NOS: 1692, 1699 and 1706) and all were able to function when transduced
into T cells. In
any of the CARs described above, the T cell may be engineered to overexpress c-
Jun as a
method to inhibit T cell exhaustion (Lynn et al 2019). A variety of promoters
can be used
upstream of the genes for CARs and other compositions of the invention,
including insertion
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into a naturally occurring promoter in the cell, such as the TRAC locus, using
CRISPR,
Sleeping Beauty or similar technology for site directed insertion of a gene.
Among the
promoters commonly used are the CMV promoter, or a mini CMV (SEQ ID NO: 1634),
a
minimal IL-2 promoter (SEQ ID NO: 1635), or Minimal Promoter minip (SEQ ID NO:
1636).
[00622] Single chain antibody fragments that included the variable domain of
the
monoclonal anti-MUC1* antibodies called MNE6 or MNC2 were engineered into a
panel of
CARs. The MUC1* targeting CARs were then transduced, separately or in
combinations,
into immune cells. When challenged with surfaces presenting a MUC1* peptide,
an antigen
presenting cell transfected with MUC1*, or MUC1* positive cancer cells, the
immune cells
that were transduced with MUC1* targeting CARs elicited immune responses,
including
cytokine release, killing of the targeted cells and expansion of the immune
cells.
[00623] For example, the gene encoding the CARs and activated T cell induced
genes
described herein can be virally transduced into an immune cell using viruses,
or inserted into
a region downstream of one of the cell's promoters or enhancers, such as the
TRAC (T cell
receptor alpha chain) locus. Virus delivery systems and viral vectors
including but not limited
to retroviruses, including gamma-retroviruses, lentivirus, adenoviruses, adeno-
associated
viruses, baculoviruses, poxvirus, herpes simplex viruses, oncolytic viruses,
HF10, T-Vec and
the like can be used. In addition to viral transduction, CARs and activated T
cell induced
genes described herein can be directly spliced into the genome of the
recipient cell using
methods such as CRISPR technology, CRISPR-Cas9 and -CPF1, TALEN, Sleeping
Beauty
transposon system, and SB 100X.
[00624] Similarly, the identity of molecules that make up the non-targeting
portions of the
CAR, except for the CD3-zeta identity, such as the extracellular domain,
transmembrane
domain and membrane proximal portion of the cytoplasmic domain, are not
essential to the
function of a MUC1*-targeting CAR. For example, the extracellular domain,
transmembrane
domain and membrane proximal portion of the cytoplasmic domain can be
comprised of
portions of CD8, CD4, CD28, or generic antibody domains such as Fc, CH2CH3, or
CH3.
Further, the non-targeting portions of a CAR can be a composite of portions of
one or more
of these molecules or other family members. However, the identity of CD3-zeta
is critical, as
mutations, such as those referred to as 1XX or CD3-zeta-1XX, greatly affect in
vivo
persistence of CAR T cells. CAR T cells that express CARs whose cytoplasmic
tail includes
CD3-zeta-1XX have prolonged activity in vivo because they do not get exhausted
as quickly
as cells containing wild-type CD3-zeta. In addition, we have discovered that
CARs with the
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1XX signaling domain are more effective against cells characterized by low
antigen density.
Cancer cells with low antigen density may comprise a sub-population of a
heterogeneous
tumor. Cancer cells with low antigen density may be characteristic of early
cancer cells that
can lead to cancer recurrence. Additionally, tumors at the time of treatment
may be
comprised of cancer cells that express low levels of a particular cancer
antigen.
[00625] Thus, in one embodiment, patients diagnosed with a cancer or at risk
of
developing a cancer or a cancer recurrence are treated with immune cells that
express a CAR
comprising a 1XX signaling domain. In one aspect, the patient is diagnosed
with or at risk of
developing a MUC1* cancer. In a preferred embodiment the recognition unit of
the CAR
comprises an antibody fragment that binds to the N-10 peptide (SEQ ID NO:3)
but does not
bind to the C-10 peptide (SEQ ID NO:825). In a more preferred embodiment, the
antibody
fragment is derived from MNC2, MNE6, 20A10, 3C2B1, 5C6F3, or 25E6.
[00626] In another embodiment, a patient diagnosed with a cancer comprised of
tumor
cells that express low levels of a targeted antigen, or diagnosed with an
early cancer, or a
patient who has been treated but still has residual tumor cells and is at risk
of a cancer
recurrence is treated with immune cells that express a CAR comprising a 1XX
signaling
domain, which enables the CAR T cells to kill both high and low antigen
density cancer cells.
In one aspect, the patient is diagnosed with or at risk of developing a MUC1*
cancer. In a
preferred embodiment the recognition unit of the CAR comprises an antibody
fragment that
binds to the N-10 peptide (SEQ Ill NO:3) but does not bind to the C-10 peptide
(SEQ ID
NO:825). In a more preferred embodiment, the antibody fragment is derived from
MNC2,
MNE6, 20A10, 3C2B1, 5C6F3, or 25E6.
[00627] In yet another aspect of the invention, a patient diagnosed with a
cancer or at risk
of developing a cancer or a cancer recurrence is treated with an immune cell
expressing a
CAR with a wild-type CD3-zeta and is also treated with an immune cell
expressing a CAR
with a mutated CD3-zeta, such as CD3-zeta-1XX. In this way, the tumor is
attacked by an
immune cell expressing a CAR with full CD3-zeta signaling that efficiently
kills off the high
antigen expressing cells, but which are prematurely exhausted, while the cells
expressing a
CAR with a mutated CD3-zeta, such as CD3-zeta-1XX, persist longer in the
patient and kill
of the low antigen expressing cells that likely give rise to tumor recurrence.
In one aspect, the
patient is diagnosed with or at risk of developing a MUC1* cancer. In a
preferred
embodiment the recognition unit of the CAR comprises an antibody fragment that
binds to
the N-10 peptide (SEQ ID NO:3) but does not bind to the C10 peptide (SEQ ID
NO:825). In
a more preferred embodiment, the antibody fragment is derived from MNC2, MNE6,
20A10,
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3C2B1, 5C6F3, or 25E6. In one aspect, a patient is treated with a CAR T cell
in which the
CAR has a wild type CD3-zeta signaling domain, wherein the CAR is chosen from
among
the group comprising MNC2 CARs (SEQ ID NO:306-307, 608-611, 718-719), MNE6
CARs
(SEQ ID NO:297-298, 300-301, 303-304), 20A10 CARs (SEQ ID NO:1582-1589, 5021-
5024), 25E6 CARs (1602-1609, 5033-5036), a CAR comprising an antibody fragment
derived from 3C2B1 wherein the signaling domain is wild type CD3-zeta, and a
CAR
comprising an antibody fragment derived from 5C6F3 wherein the signaling
domain is wild
type CD3-zeta. In another aspect, a patient is treated with a CAR T cell in
which the CAR has
a CD3-zeta-1XX signaling domain, wherein the CAR is chosen from among the
group
comprising MNC2 CARs (SEQ ID NO:1618-1625, 5041-5044, 1784-1785), MNE6 CARs
(SEQ ID NO:1626-1633, 5045-5048), 20A10 CARs (SEQ ID NO:1590-1597, 5025-5028,
1798-1799), 25E6 CARs (SEQ ID NOS:1610-1617, 5037-5040), a CAR comprising an
antibody fragment derived from 3C2B1 wherein the signaling domain is CD3-zeta-
1XX, and
a CAR comprising an antibody fragment derived from 5C6F3 wherein the signaling
domain
is CD3-zeta-1XX. In yet another aspect, a patient is treated with immune cells
that express
both a CAR having a wild type CD3-zeta signaling domain and a CAR having a 1XX
signaling domain.
[00628] We have shown that CAR T cells bearing the 1XX mutations in the CD3-
zeta are
more effective than CAR T cells with wild type CD3-zeta at preventing tumor
recurrence.
Figure 218 shows graphs of tumor volume measured by an IVIS instrument wherein
the
tumor cells have been genetically modified to express Luciferase. The
substrate Luciferin
was injected 10 minutes before the photo emissions were measured in the
sedated animal. On
Day 1 of the experiment, animals were injected sub-cutaneously with 250,000
human breast
tumor cells. Tumors were made heterogeneous, comprised of two different tumor
cell types.
A first tumor cell population was T47D-wt, a breast cancer cell line that
expresses both full-
length MUC1 and the growth factor receptor form MUC1*, which we engineered to
express
mCherry fluorescence. The second tumor cell population was the same T47D
breast cancer
cells, except that they had been stably transduced to express even more MUC1*
and GFP
fluorescence, referred to here as T47D-MUC1*. In this experiment, animals were
implanted
with T47D-w t plus T47D-MUC1*, wherein the population of T47D-MUC1* made up
30%,
15% or 7.5% of the tumor population. Animals were then administered a one-time
injection
of either PBS, huMNC2-41BB-3z CAR T cells (4-1BB), huMNC2-CD28-3z CAR T cells
(CD28) or huMNC2-CD28-1XX CAR T cells (CD28-1XX or 1XX). The CAR T cells were
injected into the tail vein at an Effector to Target ratio (E:T) of 10:1, 5:1,
or 1:1. As can be
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seen in these graphs, huMNC2-CD28-1XX out-performs huMNC2-CD28-3z and huMNC2-
41BB-3z and the difference is more pronounced in tumors with low antigen
density and when
treatment is at a low CAR T cell dose. In follow-on experiments described
here, it is clear
that 1)0C CAR T cells not only persist longer in vivo and stave off T cell
exhaustion, but they
also are much more effective at killing low antigen density tumor cells, which
is a recognized
problem in the industry and will lead to cancer recurrence. Figure 219A-219B
shows IVIS
photographs and graphs of IVIS tumor volume measurement. Fig. 219A shows
photographs
of the mice that had been implanted with tumors in which 30% of the cancer
cell population
was T47D-MUC1*, referred to here as high antigen expressing cells. The various
CAR T
cells were administered at a CAR T to tumor cell ratio of 10:1 wherein 250,000
tumor cells
were implanted and the animals were injected 5 days later with 2,500,000 CAR T
cells. Fig.
219B shows a graph of the tumor volume by IVIS measurement by day. As can be
seen,
animals injected with the huMNC2-CD28-1XX had much smaller tumors than the
animals
treated huMNC2-4-1BB-3z or huMNC2-CD28-3z, which is the same CAR T except
without
the DO( mutations in the CD3-zeta domain. Figure 220A-220T shows the IVIS
graphs for
30% tumors treated at a CAR T to Tumor ratio of 10:1. Here graphs are shown
for each
individual animal rather than the average of the treatment group. Figure 221A-
221B and
Figure 222A-222T show essentially the same experiment except that the CAR T
cell dose is
lower with only 250,000 CAR T cells administered in a single injection. As can
be seen,
animals injected with the huMNC2-CD28-1XX had much smaller tumors than the
animals
treated huMNC2-4-1BB-3z or huMNC2-CD28-3z, which is the same CAR T except
without
the 1,0C mutations in the CD3-zeta domain. However, with the lower dose of CAR
T cells,
even tumors in the huMNC2-CD28-1XX treated group begin to grow again, albeit
more
slowly.
[00629] In these next experiments (Fig. 223 ¨ Fig. 226) we show that in
animals implanted
with tumors in which only 7.5% of the tumor expresses high antigen density, it
is even harder
for standard CAR T cells to kill of the tumor. However, the same CAR T cells
with the 1XX
mutations in CD3-zeta are still quite effective at killing the tumor and
inhibiting tumor
recurrence. At a moderate CAR T cell dose having an effector to target ratio
of 10:1,
huMNC2-41BB-3z and huMNC2-CD28-3z show some long-term killing, which is
exceeded
by huMNC2-CD28-1XX. However, with low antigen density, which is a mimic of
early
cancer cells and residual, resistant cancer cells, and a low CAR T cell dose,
having an
effector to target ratio of 1:1, huMNC2-41BB-3z and huMNC2-CD28-3z did no
better than
the control PBS, whereas huMNC2-CD28-1XX continued to kill the tumor.
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[00630] Animals were sacrificed between Day 69 and Day 90. Figures 227-233
show the
tabulation of CD3 positive human T cells that were harvested from the spleens
and from the
blood of the test animals post sacrifice. As can be seen, the huMNC2-CD28-1XX
treated
mice, that had smaller tumors, have a greater number of live, persisting CAR T
cells and CD8
positive killer T cells than the animals treated with the CAR T cells with
wild type CD3-zeta.
It is also seen that the molecular markers of T cell exhaustion, TIM3, LAG3
and PD-1, are
greatly reduced or absent in the cells retrieved from the animals treated with
huMNC2-CD28-
1XX CAR T cells. This result is consistent with the idea that the 1XX
mutations in CD3-zeta
increase CAR T cell persistence in vivo.
[00631] After sacrifice, the tumors were excised and analyzed as well. Excised
tumors
were photographed and weighed (Fig. 234, Fig. 236, Fig. 238, Fig. 240) weights
were
recorded. Tumors were then enzymatically dissociated and fluorescent
photographs were
taken to characterize and quantify the cells that caused the tumor recurrence.
Recall that
tumors were a heterogeneous population of high antigen expressing cells that
were GFP
positive and low antigen density cells that were mCherry positive. Red and
green fluorescent
photographs of the residual tumors show that when tumors have a high
percentage of high
antigen density cells and a high CAR T cell dose, the all the CAR T cells were
able to kill the
tumor cells that expressed high levels of the target antigen. However, tumor
recurrence was
mainly due to the growth of the low antigen density cells that standard CAR T
cells do not
kill as readily. When tumors were made up of a low percentage of high antigen
cells and the
CAR T cell dose was low, standard CAR T cells did no better than the control
injection of
PBS. However, the CAR T cells with the 1XX mutations in the CD3-zeta domain
killed the
low antigen density cells considerably better which greatly inhibited tumor
recurrence (Fig.
235, Fig. 237, Fig. 239, Fig. 241).
[00632] Figure 242 shows photographs of live animals fluorescently imaged.
Fluorescent
photographs taken at two different timepoints showed that for animals
implanted with a
moderate percentage of high antigen cells and treated with a CAR with wild
type CD3-zeta or
a CAR with DO( mutations, the growth rate of low antigen density cells was
faster in the
animals treated with the CAR with wild type CD3-zeta compared to animals
treated with the
CAR with the CD3-1XX. Al.low CAR T cell dose, the standard CARs showed a
faster
growth rate o both low and high antigen density tumor cells. This is
consistent with the idea
that a CAR with a wild type CD3-zeta is not as efficient as CARs with 1XX at
killing low
antigen density cells and that with a lower number of CAR T cells, the
standard CARs get
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exhausted faster than CARs with the 1XX mutations in CD3-zeta (Fig. 242, Fig.
243, Fig.
244, Fig. 245.
[00633] In another experiment, we injected mice at Day 52 with either 250,000
high
antigen density tumor cells or 2,500,000 more CAR T cells. As can be seen in
Figure 246 and
Figure 247, the injection of additional tumor cells does not increase the
tumor growth in
animals treated with huMNC2-CD28-1XX CAR T cells. However, the injection of
additional
tumor cells does increase tumor growth in animals treated with huMNC2-CD28-3z
or
huMNC2-41BB-3z CAR T cells. This result is consistent with CAR T cells with
1XX
mutations in CD3z prolong CAR T cell persistence in vivo. It can also be seen
that the
injection of additional CAR T cells suppressed tumor cells in some of the
animals in all
groups. This is consistent with the idea that tumor recurrence was not due to
tumor escape
because the fresh CAR T cells still recognized the tumor cells and killed
them.
[00634] A significant problem for the treatment of cancers are the tumor cells
that express
low levels of tumor-associated antigen, especially with regard to cellular
therapies where, to
date, the killing of tumor cells has been dependent on the antigen density of
the tumor cells.
Tumor cells expressing low levels of the target antigen escape CAR T cells as
well as
engineered CAR NK cells. Essentially all solid tumors are heterogeneous and
comprised of
cells that express different levels of the target antigen. To further study
this problem and to
develop therapeutics that are able to detect and kill tumor cells expressing
low levels of the
target antigen, we devised a strategy for making heterogeneous tumors made up
of both high
and low antigen expressing cells, wherein the two different cell types bear
fluorescent labels
that can be detected in vivo as well as ex-vivo after sacrifice of the test
animals (See Fig. 248
- Fig. 256). As can be seen in the IVIS graphs of Fig. 249 and Fig. 250, CARs
with standard
CD3z signaling domains or with 1XX mutations kill tumor cells expressing high
levels of
antigen when treated with high, 10:1, dose of CAR T cells. However, for CARs
with standard
CD3-z domains, the difference between treated and untreated wanes for animals
implanted
with tumor cells expressing low percentage of high antigen expressing cells,
even when the
dose of CAR T cells is high. This result argues that CARs with mutated CD3-z
signaling
domain, like 1XX, have enhanced ability to detect tumor cells that express low
percentages of
high antigen expressing cells. Fig. 251 shows that in animals that were
implanted with
250,000 tumor cells, then treated once with either 2.5M CAR T cells or 250,000
CAR T cells,
tumors recur and the timing and degree of recurrence is greater in animals
treated with CARs
bearing standard CD3z than in animals bearing CD3z with DOC mutations. The
excised
tumors were fluorescently photographed at mCherry wavelength and at GFP
wavelength. As
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can be seen in Fig. 252 tumor recurrence is minimal for the tumors of animals
treated with
huMNC2-CD28-1XX. Further, the figure shows that tumor recurrence is dominated
by the
low antigen expressing cells that are missed by the standard CAR T cells. As
can be seen in
Fig. 253 and Fig. 256, when tumors expressing low percentage of high antigen
expressing
cells are coupled with treating with low dose CAR T cells, a combination of
CAR T cell
exhaustion plus escape of low antigen cells drives tumor resistance. We note
that for the
tumors labeled 15% high antigen density, flow cytometry showed that a
population of tumor
cell clusters was present, which has been reported to result in a greater rate
of tumor growth
in vivo.
[00635] Anti-MUC1* CAR T cells persist longer and stave off T cell exhaustion
when the
CD3-zeta signaling domain is mutated to slow signaling as in mutating some of
the
Tyrosines, for example as we have done with the huMNC2-CD28-1XX, see Tables in
Fig.
257 ¨ Fig. 282. Excised tumors from the test animals were analyzed by flow
cytometry for
the presence of human CAR T cells and their expression of exhaustion markers.
It is notable
that even in a high antigen density tumor, such as 30% of the cells express
high levels of the
antigen, in this case MUC1*, wherein animals were treated with a high effector
to target ratio
of 10:1 CAR T cells to tumor cells, there is a significantly higher number of
CAR T cells in
the tumors of mice treated with the anti-MUC1* CAR T with CD3z mutations, in
this case
DOC. As can be seen in the Table of Fig. 257, the mice treated with huMNC2-
CD28-1XX
had an average of 1,516 CAR '1 cells in the excised tumor, compared to only
196 CAR '1'
cells for the huMNC2-41BB-3z and 395 CAR T cells for the huMNC2-CD28-3z
treated
mice. In the huMNC2-CD28-1XX treated mice, the CAR T cells expressed the
lowest
percentages of exhaustion markers. That means that for tumors with a
significant amount of
high antigen expressing cells and at high dose of CAR T cells, the mutated
CD3z, as in 1XX
mutations, gives the CAR T cells an advantage in terms of being able to
recognize tumor
cells and also being able to stave off exhaustion. Comparison of CAR T cell
persistence
among CARs, with or without mutated CD3z, is assessed by looking at these same
30% high
antigen expressing tumors, but wherein animals were treated with a low dose of
CAR T cells:
250,000 CAR T cells and 250,000 tumor cells, i.e. 1:1 ratio. The Table of Fig.
262 shows that
CARs with a standard CD3z signaling domain, administered to animals at low
dose have
almost no measurable CAR T cells in their tumors after about 70-90 days after
CAR T cell
injection. These data argue that at low CAR T dose, the CAR T cells need to
work harder and
get exhausted faster. In contrast, animals treated with low dose of 250,000
huMNC2-CD28-
1XX CAR T cells to 250,000 implanted tumor cells had on average 1,444 CAR T
cells in
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their tumors at day of sacrifice, which is about the same as when the animals
were treated
with high dose of huMNC2-CD28-1XX CAR T cells. This result shows that the
mutated
CD3z signaling domain resists T cell exhaustion.
[00636] However, looking at the excised tumors from animals implanted with
tumors that
only have 7.5% expressing high levels of antigen but treated with high dose of
CAR T cells -
2.5M CAR T cells to 250,000 implanted tumor cells ¨ shows that mutations that
slow the
signaling of CD3-z also do something else. It follows that with low antigen
density but high
dose of CAR T cells, the CAR T cells would signal less due to lesser numbers
of antigen
molecules and thus would not get exhausted. Unexpectedly, as can be seen in
the Table of
Fig. 259, 7.5% high antigen expressing tumors excised from animals treated
with high, 10:1
dose of CAR T cells still do not have significant numbers of CAR T cells in
their tumors if
treated with CARs having wild type CD3z. Tumors from animals treated with
huMNC2-
CD28-1XX had on average 1,555 CAR T cells in the excised tumor, compared with
only 167
in tumors from huMNC2-41BB-3z treated mice and 275 in tumors from huMNC2-CD28-
3z
treated mice. This result strongly argues that, unexpectedly, CAR T cells with
a 1XX mutated
CD3z, or similar mutated signaling domain, have enhanced ability to recognize
and kill
tumor cells that express low levels of the target antigen.
[00637] Similarly, standard CAR T cells could not recognize nor kill tumors
that
expressed a low percentage, 7.5%, of high antigen expressing cells when
animals were also
treated with a low dose of CAR '1 cells. The "[able of Fig. 264 shows that of
the mice
implanted with tumors that were only 7.5% high antigen expressing cells that
were treated
with low dose CAR T cells (250,000 CAR T cells and 250,000 tumor cells, 1:1
ratio) only the
animals treated with huMNC2-CD28-1XX had measurable CAR T cells in their
tumors, 841
on average, and those CAR T cells showed almost no signs of exhaustion.
[00638] Similar results were obtained when cells from the spleens of the
animals were
analyzed (Fig. 265 ¨ Fig. 273). One striking example is shown in Fig. 273.
Analysis of
spleens excised from animals bearing tumors that were 7.5% high antigen
expressing cells
when implanted, showed that only spleens from animals treated with huMNC2-CD28-
1XX
had detectable CAR T cells, wherein they had on average 1,413 CAR T cells in
their tumors.
[00639] The trend was also observed when the blood from the treated animals
was
analyzed by flow cytometry as shown in Fig. 274 ¨ Fig. 282.
[00640] These experiments demonstrate that anti-MUC1* CAR T cells kill MUC1*
positive tumor cells and that the killing is enhanced when the CAR
incorporates a mutated
CD3z domain such as one with the DOC mutations. The experiments also show an
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unexpected result that CAR T cells with the 1XX CD3z domain have enhanced
ability to
recognize and kill tumor cells that express low levels of the target antigen.
Thus, CAR T cells
bearing 1XX, including but not limited to CARs that recognize MUC1*, have
improved
killing of solid tumor cancers, where tumors are more heterogeneous than blood
cancers.
Also, CAR T cells bearing DOC, including but not limited to CARs that
recognize MUC1*,
are far better than standard CAR T cells at preventing tumor recurrence.
[00641] One aspect of the invention is a method for treating a patient
diagnosed with,
suspected of having, or at risk of developing a MUC1 positive or MUC1*
positive cancer,
wherein the patient is administered an effective amount of immune cells that
have been
transduced with a MUC1* targeting CAR. In another aspect of the invention, the
immune
cells are T cells isolated from a patient, which are then transduced with CARs
wherein the
targeting head of the CAR binds to MUC1*, and after expansion of transduced T
cells, the
CAR T cells are administered in an effective amount to the patient. In yet
another aspect of
the invention, the immune cells are T cells isolated from a patient, which are
then transduced
with CARs wherein the targeting head of the CAR comprises portions of MNC2,
MNE6,
20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11,
and
after optional expansion of transduced T cells, the CAR T cells are
administered in an
effective amount to the patient. In another aspect of the invention, the
antibody fragment of
MNC2, MNE6. 20A10, 3C2B1, 5C6F3, or 25E6 is incorporated into a chimeric
antigen
receptor of a natural killer cell, or INK cell.
[00642] Specificity of anti-MUC1* targeting antibodies
[00643] As these experiments demonstrate, the critical portion of a CAR is the
antibody
fragment that directs the immune cell to the tumor cell. As we will show in
the following
section, MNE6, MNC2, 20A10, 3C2B1 and 5C6F3 are highly specific for the form
of
MUC1* that is expressed on tumor cells. The next most important part of a CAR
is the
cytoplasmic tail bearing immune system co-stimulatory domains and the
signaling domain
CD3-zeta or variations thereof. The identity of these domains modulates the
degree of
immune response but does not affect the specificity. As shown, the identity of
the
transmembrane portion of a CAR is the least important. It appears that as long
as the
transmembrane portion has some flexibility and is long enough to allow the
antibody
fragment to reach its cognate receptor on the tumor cell, it will suffice.
CARs comprising the
MNE6 targeting antibody fragment, and intracellular co-stimulatory domains
41BB and
CD3-zeta but having a variety of different extracellular, transmembrane and
short
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cytoplasmic tail all worked in that they specifically killed the targeted
cells while stimulating
the expansion of the host T cells.
[00644] The most accurate way of demonstrating antibody specificity is testing
the
antibody on normal human tissue specimens compared to cancerous tissue
specimens. MNC2
and MNE6 were previously shown to specifically bind to MUC1 or MUC1* positive
cancer
cells. Several breast tumor arrays were assayed using several anti-MUC1 or
MUC1*
antibodies. Essentially the studies involving serial sections of breast cancer
tissue specimens
from over 1,200 different breast cancer patients showed that very little full-
length MUC1
remains on breast cancer tissues. The vast majority of the MUC1 expressed is
MUC1* and is
stained by MNC2. The analysis was performed by Clarient Diagnostics and tissue
staining
was scored using the Allred method. For example, Fig. 10 shows serial sections
of breast
cancer tissue arrays that were stained with either VU4H5, a commercially
available anti-
MUC1 antibody that binds to the tandem repeats, or MNC2 that binds to MUC1*.
Figs. 10
and 11 are photographs of breast cancer tissue arrays stained with either
VU4H5 which
recognizes MUCl-FL (full length) or MNC2 which recognizes cancerous MUC1*.
Tissue
staining was scored using Allred scoring method which combines an intensity
score and a
distribution score. Below the photographs of the tissue arrays are color-coded
graphs
displaying the results. As can be seen, the arrays stained with VU4H5 are very
light and
many tissues do not stain at all despite the published reports that MUC1 is
aberrantly
expressed on over 96% of all breast cancers as evidenced by nucleic acid based
diagnostics.
In contrast, the arrays stained with MNC2 are very dark (red versus yellow or
white in
graph). Additionally, many tissues did not stain at all with anti-full-length
MUC1 but stained
very dark with MNC2, (see green boxes in graph). Similarly, we stained normal
or cancerous
breast tissues with humanized MNE6 scFv-Fc. The antibody fragment was
biotinylated so it
could be visualized by a secondary streptavidin based secondary. As can be
seen in Fig. 12,
hMNE6 scFv-Fc does not stain normal breast tissue but stains cancerous breast
tissue.
Further, the intensity and homogeneity of staining increases with tumor grade
and/or
metastatic grade of the patient (Fig. 12-13). Similarly, hMNE6 scFv-Fc did not
stain normal
lung tissue but did stain lung cancer tissue (Fig. 14-18) and the intensity
and distribution of
staining increased as tumor grade or metastatic grade increased. Fig. 19 shows
photographs
of normal small intestine and cancerous small intestine tissues stained with
humanized
MNE6-scFv-Fc biotinylated anti-MUCI* antibody at 5 ug/mL, then stained with a
secondary
streptavidin HRP antibody. A) is a normal small intestine tissue. B) is small
intestine cancer
from patient as denoted in the figure. C,D are photographs of the
corresponding serial
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sections that were stained with the secondary antibody alone. Fig. 20 shows
photographs of
normal small intestine tissues stained with humanized MNE6-scFv-Fc anti-MUC1*
antibody
at 50 ug/mL, then stained with a secondary goat-anti-human HRP antibody. A-D
are normal
small intestine tissue. E-H are photographs of the corresponding serial
sections that were
stained with the secondary antibody alone. Fig. 21 shows photographs of
cancerous small
intestine tissues stained with humanized MNE6-scFv-Fc anti-MUC1* antibody at
50 ug/mL,
then stained with a secondary goat-anti-human HRP antibody. A-D are cancerous
small
intestine tissue from a patient as denoted in figure. E-H are photographs of
the corresponding
serial sections that were stained with the secondary antibody alone. Fig. 22
shows
photographs of cancerous small intestine tissues stained with humanized MNE6-
scFv-Fc anti-
MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP
antibody.
A-D are cancerous small intestine tissue from a patient as denoted in figure.
E-H are
photographs of the corresponding serial sections that were stained with the
secondary
antibody alone. Fig. 23 shows photographs of normal colon tissues stained with
humanized
MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary
goat-anti-
human HRP antibody. A-D are normal colon. E-H are photographs of the
corresponding
serial sections that were stained with the secondary antibody alone. Fig. 24
shows
photographs of colon cancer tissues stained with humanized MNE6-scFv-Fc anti-
MUC1*
antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP
antibody. A-D are
colon cancer tissue from a metastatic patient as denoted in figure. E-H are
photographs of the
corresponding serial sections that were stained with the secondary antibody
alone. Fig. 25
shows photographs of colon cancer tissues stained with humanized MNE6-scFv-Fc
anti-
MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP
antibody.
A-D are colon cancer tissue from a Grade 2 patient as denoted in figure. E-H
are photographs
of the corresponding serial sections that were stained with the secondary
antibody alone. Fig.
26 shows photographs of colon cancer tissues stained with humanized MNE6-scFv-
Fc anti-
MUC1* antibody at 50 ug/mL, then stained with a secondary goat-anti-human HRP
antibody.
A-D are colon cancer tissue from a metastatic patient as denoted in figure. E-
H are
photographs of the corresponding serial sections that were stained with the
secondary
antibody alone. Fig. 27 shows photographs of prostate cancer tissues stained
with humanized
MNE6-scFv-Fc anti-MUC1* antibody at 50 ug/mL, then stained with a secondary
goat-anti-
human HRP antibody. A-D are prostate cancer tissue from a patient as denoted
in figure. E-H
are photographs of the corresponding serial sections that were stained with
the secondary
antibody alone. Fig. 28 shows photographs of prostate cancer tissues stained
with humanized
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MNE6-scFv-Fc anti-MUC I* antibody at 50 ug/mL, then stained with a secondary
goat-anti-
human HRP antibody. A-D are prostate cancer tissue from a patient as denoted
in figure. E-H
are photographs of the corresponding serial sections that were stained with
the secondary
antibody alone. Fig. 29 shows photographs of prostate cancer tissues stained
with humanized
MNE6-scFv-Fc anti-MUC I* antibody at 50 ug/mL, then stained with a secondary
goat-anti-
human HRP antibody. A-D are prostate cancer tissue from a patient as denoted
in figure. E-H
are photographs of the corresponding serial sections that were stained with
the secondary
antibody alone.
[00645] One aspect of the invention is a method for treating a patient
diagnosed with,
suspected of having, or at risk of developing a MUC1 positive or MUC1*
positive cancer,
wherein a specimen is obtained from the patient's cancer and is tested for
reactivity with an
antibody that binds to PSMGFR SEQ ID NO:2, or more specifically to the N-10
peptide
(SEQ ID NO:3), or yet more specifically binds to N-10 peptide (SEQ ID NO:3),
but does not
bind to C-10 peptide (SEQ ID NO:825). The patient is then treated with an
scFv, scFv-Fc or
CAR T that comprises antibody variable fragments from the antibody that
reacted with their
cancer specimen or can be chosen from among MNC2, MNE6, 20A10, 3C2B1, 5C6F3,
25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. Another aspect of the
invention
is a method for treating a patient diagnosed with, suspected of having, or at
risk of developing
a MUC1 positive or MUC1* positive cancer, wherein a specimen is obtained from
the
patient's cancer and is tested for reactivity with MNC2, MNE6, 20A10, 3C2B1,
5C6F3,
25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11; the patient is then
treated with
the antibody, antibody fragment, scFv, scFv-Fc-mut, BiTE or CAR T that
comprises portions
of the antibody that reacted with their cancer specimen.
00646] As we previously reported, it is MUC1*, the transmembrane cleavage
product, not
full-length MUC1, the is a growth factor receptor that drives tumor growth.
The growth
factors that activate MUC1* bind to ectopic sites that are only exposed after
cleavage and
release of the tandem repeat portion of MUC1. Antibodies of the invention,
like the activating
growth factors, cannot bind to full-length MUCl. FACS analysis clearly shows
that anti-
MUC1* antibody MNC2 is unable to bind to HCT-116, MUC1 negative cells (Fig.
35A),
binds robustly to those cells if they are transfected with MUC1* (Fig. 35B),
but will not bind
to HCT cells transfected with full-length MUC1 (Fig. 35C). A commercially
available anti-
tandem repeat antibody VU4H5 clearly recognizes full-length MUC1 (Fig. 35D).
[00647] We discovered that MUC I can be cleaved to MUC1* by more than one
cleavage
enzyme and that the site of cleavage affects its fold and consequently affects
which
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monoclonal antibody is able to recognize that form of MUC1*. Different cancer
cells or
cancerous tissues express different cleavage enzymes. We tested various
cleavage enzyme
inhibitors on different cancer cell lines and found that an inhibitor that
inhibits cleavage of
MUC1 in one cancer cell line did not inhibit its cleavage in another cancer
cell line.
Similarly, PCR experiments showed that cleavage enzymes are expressed at
different levels
in different cells or cell lines. For example, hematopoietic stem cells of the
bone marrow
express a MUC1* that is recognized by monoclonal antibody MNC3 but not MNE6 or
MNC2 (Fig. 39). The growth of DU145 prostate cancer cells and T47D breast
cancer cells is
inhibited by the Fabs of MNC2 and MNE6 but not by the Fabs of MNC3 or MNC8,
indicating that the cancer cell lines express a MUC1* that is recognized by
MNE6 and
MNC2 but not by MNC3 or MNC8 (Fig. 42). PCR experiments show that CD34
positive
cells of the bone marrow express about 2,500-times more MMP2 and about 350-
times more
ADAM28 than T47D breast cancer cells, while DU145 prostate cancer cells
express about
2,000-times more ADAM TS16, about 400-times more MMP14 and about 100-times
more
MMP1 than T47D breast cancer cells (Fig. 43 and Fig. 44). Conversely, T47D
breast cancer
cells express about 80-times more MMP9 than the bone marrow cells and about
twice as
much as DU145 prostate cancer cells. Various cleavage enzyme inhibitors were
tested for
their ability to inhibit cleavage in different kinds of cancer cells.
[00648] General strategy for using antibodies, antibody fragments and CARs
that
target the extracellular domain of MUC1*
[00649] In one aspect of the invention, a second factor, which may be a
cleavage enzyme,
an antibody, a cytokine, or a second CAR, and a CAR are transduced into the
same T cell. In
another aspect of the invention, the second factor is on an inducible promoter
such that its
expression is activated when the CAR engages the targeted cancer cells. In
some cases, the
expression of the second factor is controlled by an inducible promoter. In one
aspect of the
invention, expression of the second factor is induced when the immune cell is
activated, for
example when it recognizes or engages its target. In one example, a T cell is
transfected or
transduced with a second factor whose expression is induced when the T cell
recognizes a
target cancer cell. One way to do this is to induce expression of the second
factor when, or
shortly after, an NFAT protein is expressed or translocated to the nucleus.
For example, a
sequence derived from an NFAT promoter region is put upstream of the gene for
the second
factor. In this way, when the transcription factors that bind to the promoter
of the NFAT
protein are present in sufficient concentration to bind to and induce
transcription of the NFAT
protein, they will also bind to that same promoter that is engineered in front
of the sequence
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for transcription of the second factor. The NFAT protein may be NFAT1 also
known as
NFATc2, NFAT2 also known as NFATc or NFATc 1 , NFAT3 also known as NFATc4,
NFAT4 also known as NFATc3, or NFAT5. In one aspect of the invention, the NFAT
is
NFATcl, NFATc3 or NFATc2. In one aspect of the invention, the NFAT is NFAT2
also
known as NFATcl. SEQ ID NO:646 shows nucleic acid sequence of the upstream
transcriptional regulatory region for NFAT2. The promoter sequence for NFAT
gene may
include the nucleic acid sequence of SEQ ID NO:781-783 or SEQ ID NO:815 as
examples,
but it can be seen that the optimal sequence or minimal sequence for
expression of the second
factor may be obtained by making fragments, extensions or mutations of the
promoter and
testing for the strength of the promoter with respect to expression of the
second factor. In one
aspect of the invention, the transcriptional regulatory region for NFAT2 is
engineered
upstream of the gene encoding the second factor, which if for cleavage enzyme
MMP9 (SEQ
ID NO:647) or the catalytic sub-unit of MMP9 (SEQ ID NO:648). In one aspect of
the
invention, the NFAT is NFATc3 and the promoter sequence of NFATc3 includes
nucleic acid
sequences from SEQ ID NO:816. In one aspect of the invention, the
transcriptional
regulatory region for NFATc3 is engineered upstream of the gene encoding the
second factor,
here as an example is MMP9. In another aspect of the invention, the NFAT is
NFATc2. SEQ
ID NO:817-818 shows nucleic acid sequence of the upstream transcriptional
regulatory
region for NFATc2. In one aspect of the invention, the transcriptional
regulatory region for
NFATc2 is engineered upstream of the gene encoding the second factor, which
may be
cleavage enzyme MMP9 (SEQ ID NO:647) or the catalytic sub-unit of MMP9 (SEQ ID
NO: 648).
[00650] Another method for having the expression of the second factor induced
when the
T cell or CAR T cell is activated is to have the gene for the second factor on
an inducible
promoter where the NFAT protein itself binds to and induces transcription of
the second
factor. In this case, an NFAT response element (NFAT RE) may be positioned
upstream of
the gene for the second factor or fragment of the second factor. The NFAT may
bind to its
responsive element upstream of the second factor alone or as part of a
complex. The NFAT
protein may be NFATcl, NFATc2, NFATc3, NFATc4, or NFAT5. In a preferred
embodiment, the NFAT protein is NFAT2 aka NFATcl, aka NFATc. The gene of the
second
factor or fragment thereof is cloned downstream of an NFAT-response element
(SEQ ID
NO:649), which may be repeats of the response element (SEQ ID NO:650) and CMV
minimal promoter (mCMV) (SEQ ID NO:651) to induce expression of second factor
by
NFAT protein. The NFAT response element may include nucleic acid sequence of
NFAT
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consensus sequence (SEQ ID NO:804). The NFAT response element may include the
nucleic
acid sequence of SEQ ID NOS:805-814 as examples, hut it can be seen that the
optimal
sequence or minimal sequence for expression of the second factor may be
obtained by
making fragments, extensions or mutations of the responsive element nucleic
acid and testing
for the strength of the responsive element with respect to expression of the
second factor. The
enhancer region of Foxp3 also contains NFAT response elements within the 120-
bp from
2079 to 2098 (SEQ ID NO:821). The NFAT response element may include nucleic
acid
NFAT consensus sequence of (5' -cattttttccat-3') (SEQ ID NO:819) or (5' -
tttttcca-3' ) (SEQ
ID NO:820), which NFATcl specifically binds to (Xu et al., Closely related T-
memory stem
cells correlate with in vivo expansion of CAR. CD19-T cells and are preserved
by IL-7 and
IL-15, Blood 2014 123:3750-3759), or repeats thereof. The NFAT response
elements may
also be separated by nucleic acid spacer sequences. Other NFAT responsive
elements may
exist and may further be discovered, and a skilled artisan in the art when
directed to
determine NFAT responsive element may do so by carrying out molecular
biological assays
to obtain it given the guidance of at least the responsive elements as set
forth as SEQ ID
NOS: 804-814 albeit as only mere examples. In one aspect of the invention, the
cleavage
enzyme that is downstream of the NFAT-response element and CMV minimal
promoter is
MMP9 (SEQ ID NO:652). In another aspect of the invention, the cleavage enzyme
is a
catalytic sub-unit of MMP9 (SEQ ID NO:653).
[00651] Because NFATs 1-4 are regulated by the calcineurin pathway, potential
toxicities
that may arise in a patient can be stopped by treatment with an
immunosuppressive agent
such as FK506, Cyclosporin, Cyclosporin A, or Tacrolimus that block
calcineurin activity
and inhibit NFAT translocation to the nucleus. The T cell transduced or
transfected with a
cleavage enzyme on an inducible promoter may also be transfected or transduced
with a CAR
that recognizes a protein or molecule on the cancer cell. In a specific
example, the cleavage
enzyme is one that is able to cleave MUC1 full-length and the CAR bears an
antibody
fragment that directs it to MUC1* on the surface of cancer cells.
[00652] To determine which cleavage enzymes cleave MUC1 on cancer cells, we
tested a
series of MMP and ADAM enzyme inhibitors. These experiments pointed to MMP9 as
being
an important cleavage enzyme in cancer cells. To confirm that MMP9 cleaves
MUC1 on
cancer cells, we transfected HCT-116 MUC1 negative colon cancer cells with a
mimic of
full-length MUC1 having 41 tandem repeat domains: HCT-MUC1-41TR. Through
single cell
cloning we were able to establish this cell line wherein MUC1 only minimally
gets cleaved to
MUC1*. Figs. 36A-36D show Western blots and FACS analysis showing that HCT-
MUC1-
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41TR is 95% positive for full-length MUC1 and only 5-10% positive for the
cleaved form,
MUC1*. HCT-MUC1-41TR cells were incubated with MMP9 at varying concentrations
and
then assayed by immunofluorescence to measure binding of MNC2 monoclonal
antibody to
the resultant cells. As can be seen in Figs. 37A-37C binding of MNC2 increased
as the
concentration of MMP9 added to the cells increased. These experiments show
that MMP9
cleaves MUC1 to a form that is recognized by MNC2. The human cancer tissue
array studies
we performed (Fig. 30A-30F, Fig. 31A-31F, Fig. 32A-32F, Fig. 33A-33F) show
that MNC2
recognizes the form of cleaved MUC1 that is present on cancerous tissue but
not on healthy
cells or tissues (Fig. 34A-34I). Importantly, MNC2 does not recognize the form
of cleaved
MUC1 that is expressed on healthy hematopoietic stem cells of the bone marrow
(Figs. 39-
41).
[00653] In one aspect of the invention, an immune cell is transduced with both
a CAR to
target the immune cell to the tumor, and a cleavage enzyme. The CAR and the
cleavage
enzyme can be encoded on the same plasmid or on two different plasmids. In one
aspect, the
cleavage enzyme is on an inducible promoter. In another aspect, expression of
the cleavage
enzyme is induced by a protein that is expressed when the immune cell is
activated. In one
case, expression of the cleavage enzyme is induced by an NFAT protein. In
another aspect,
expression of the cleavage enzyme is induced by NFATcl. In another aspect,
expression of
the cleavage enzyme is induced when one of the NFAT proteins binds to an NFAT
response
element that is inserted upstream of the gene for the cleavage enzyme or a
catalytically active
fragment thereof. In one aspect, the cleavage enzyme is MMP9 or a fragment of
MMP9 that
is catalytically active.
[00654]
In one aspect of the invention, the cleavage enzyme is MMP9 (SEQ ID
NO:643).
Some cleavage enzymes are naturally expressed as pro-enzymes that need to be
activated.
This can be accomplished by biochemical means, by expressing a co-enzyme that
activates a
cleavage enzyme or by engineering the enzyme in an activated form. The
invention
anticipates overcoming this problem by co-expressing the cleavage enzyme with
its activator.
In one aspect of the invention, the cleavage enzyme is MMP9 and the co-
activator is MMP3.
In another aspect of the invention, the cleavage enzyme is expressed in a form
that is already
active, for example by expressing a fragment of the cleavage enzyme that still
has catalytic
function. In one case, the cleavage enzyme is an MMP9 fragment that is
catalytically active.
One example of an MMP9 catalytic fragment is given as SEQ ID NO:645.
[00655] MMP9, which must be activated by MMP3, is overexpressed in a large
percentage
of solid tumors. Further, it is known that MNC2 anti-MUC1* monoclonal antibody
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recognizes MUC1 after it is cleaved by MMP9. The various breast, ovarian,
pancreatic and
lung cancer tissue arrays that were shown in Figs. 30-33 were probed with MNC2-
scFv,
further indicating that MUC1 in these cancers is being cleaved by MMP9. To see
if cleavage
of tumors by MMP9 would increase T cell access to the tumor, we did a series
of experiments
using a cell line that expresses full-length MUC1, HCT-MUC1-41TR, a breast
cancer cell
line that is a high expresser of both full-length MUC1 and MUC1* and a MUC1
negative cell
line that we transfect with MUC1*4. We transfected cells with MMP9 and MMP3,
which
activates MMP9. We took the supernatant of those cells, which contained
activated MMP9,
and added it to the various cells, which were then co-cultured with T cells
transduced with an
anti-MUC I* CAR: huMNC2-CAR44. The result was greatly increased CAR T cell
killing of
the targeted MUCl/MUC1* positive cancer cells, compared to the control cells
that were not
incubated with a MUC1 cleavage enzyme.
[00656] APMA is a biochemical that activates MMPs. We used APMA along with the
conditioned media of cells that we transfected with either MMP9 or ADAM17 to
see if any of
these cleavage enzymes would cleave MUC1 on the HCT-MUC1-41TR cell line that
only
expresses full-length MUC1. As controls, we also tested the enzymes on HCT-
MUCI* cells.
The MUC1 and MUC1* expressing cells were stained with a red dye, CMTMR. Human
T
cells that were transduced with an anti-MUCI* CARs, CAR44 or CAR50 were co-
cultured
with the cancer cells. Untransduced T cells were used as a control (Fig. 45A-
45P). As can be
seen in Fig. 45B, Fig. 45C, and Fig. 45D, the anti-MUCI* CAR '1 cells
effectively
recognized and clustered the HCT-MUC1* cancer cells, which is a sign of T cell
activation
and killing. However, no CAR T cell induced clustering is visible in the wells
containing
HCT-MUC1-41TR, the full-length MUC1 expressing cells (Fig. 45F, Fig. 45G, and
Fig.
45H). However, the cells that were incubated with activated MMP9 show dramatic
increase
in CAR T cell induced clustering (Fig. 45J, Fig. 45K, and Fig. 45L),
indicating that MMP9
cleaved the full-length MUC1 to a form of MUC1* that is recognized by MNC2
monoclonal
antibody and more specifically by huMNC2-scFv. ADAM17 had no apparent effect.
ADAM17 either did not cleave MUC1 or cleaved it at a position that is not
recognized by
MNC2, which is more likely (Fig. 45N-45P).
[00657] We performed the same experiment, this time using T47D breast cancer
cells that
were hard to kill using anti-MUC1* CAR T cells presumably because they express
high
levels of full-length MUC1 as well as MUC1* (Fig. 46A-46T). As can be seen in
Figs. 46B,
46C, and 46D, anti-MUC I* CAR44 and CAR50 have little effect on the T47D
cancer cells.
Only in Fig. 46D, which is CAR44 at the highest level of CAR expression in the
T cells, do
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we see a small amount of CAR T cell induced clustering. However, the presence
of activated
MMP2 (Fig. 46J, 46K, 46L) or activated MMP9 (Fig. 46R, 46S, 46T) shows a
dramatic
increase in CAR T cell recognition, clustering and killing, showing that
cleavage of full-
length MUC1 increases T cell access to the cancer cells. To ensure that the
addition of the
APMA was not inducing cleavage or anti-MUC I* CAR T recognition by some other
mechanism, we made a catalytically active form of MMP9 and added it to T47D
cells that
were then co-cultured with MNC2-CAR44 T cells (Fig. 47A-471). As can be seen
in the
figure, MNC2-CAR T cells recognize and cluster cells transfected with MUC1*
(Fig. 47B-
47C), poorly cluster T47D breast cancer cells that express both full-length
MUC1 and
MUC1* (Fig. 47E-47F), but robustly bind to and cluster the T47D cells after
the addition of
the catalytically active MMP9 (Fig. 47H-47I). This results supports the claim
that MNC2
does not recognize full-length MUC1 but does recognize the growth factor
receptor MUC1*.
Note that the full-length MUC1 expressed on this cell line may sterically
hinder the binding
of CAR T cells near the cell membrane.
[00658] In another example, T47D MUC1 positive tumor cells were incubated with
a
recombinant catalytic domain of MMP9 (Enzo Life Sciences, Inc., Farmingdale,
NY) at
either 100ng/mL or 500ng/mL. Western blot analysis showed that the MUC1/MUC1*
positive cancer cells underwent extensive cleavage of MUC1 to MUC1*. In
another example,
T47D breast cancer cells were pre-incubated with a human recombinant MMP9
catalytic
domain protein then co-cultured with anti-M1JC1* CAR44 '1 cells. "lhe specific
killing of the
T47D cells by CAR44 T cells was monitored in real-time on an xCelligence
instrument that
measures impedance as a function of time. This analysis uses electrode arrays
upon which
cancer cells are plated. The adherent cancer cells insulate the electrode and
cause an increase
in impedance as they grow. Conversely, T cells are not adherent and remain in
suspension so
do not increase or decrease impedance. However, if the T cells or CAR T cells
kill the cancer
cells on the electrode plate, the cancer cells ball up and float as they die,
which causes the
impedance to decrease. The addition of MMP9 catalytic domain dramatically
increased the
killing of T47D cancer cells. Fig. 48 shows an xCelligence graph of T47D
breast cancer cells
in co-culture with either untransduced T cells, as a control, or huMNC2-CAR44
T cells over
a 45 hour period. After 18 hours of cancer cell growth, a catalytic sub-unit
MMP9 was added
to some of the cells. At 25 hours, T cells were added. As can be seen, huMNC2-
CAR44 T
cell killing is greatly improved when the T47D cells are pre-incubated with
cleavage enzyme
MMP9. In the xCelligence system, target cancer cells, which are adherent, are
plated onto
electrode array plates. Adherent cells insulate the electrode and increase the
impedance. The
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number of adherent cancer cells is directly proportional to impedance. T cells
are not
adherent and do not contribute to impedance. Therefore, increasing impedance
reflects
growth of cancer cells and decreasing impedance reflects killing of cancer
cells. Prostate
cancer cell line DU145 expresses both MUC1 and MUC1* but at a much lower level
of
expression than T47D cells. DU145 cells are efficiently killed by anti-MUCI*
CAR T cells
in the presence or absence of a cleavage enzyme.
[00659] Fig. 49 shows an xCelligence graph of DU145 prostate cancer cells in
co-culture
with either untransduced T cells, as a control, or huMNC2-CAR44 T cells over a
45 hour
period. After 18 hours of cancer cell growth, a catalytic sub-unit MMP9 was
added to some
of the cells. At 25 hours, T cells were added. As can be seen, huMNC2-CAR44 T
cell killing
of low density MUC1/MUC1* positive cancer cells is not affected by pre-
incubation with
cleavage enzyme MMP9. DU145 cancer cells express a significantly lower amount
of MUC1
which includes the full-length form as well as MUC1*. The lower density of
full-length
MUC1 does not sterically hinder T cell access to the membrane proximal MUC1*.
DU145
cells represent an early stage cancer that expresses both full length and
cleaved MUC1 but at
lower levels so that T cell access is not sterically hindered. T47D cells
represent mid-stage
cancers that express high levels of both MUC1 and MUC1*, wherein the density
of MUC1
full-length sterically hinders access of T cells to the tumor. HCT-MUCI* cells
are a MUC1
negative cell line that has been stably transfected with MUC1*45, and they
represent late
stage cancer cells. It is significant that MUC1 cleaved to MUC1* by MMP9 is
recognized by
the anti-MUC1* antibody MNC2, which is the targeting head of the CAR. Immune
cell
access to tumor antigens on the cancer cell surface can be sterically hindered
by the presence
of bulky extra cellular domain proteins or other obstructing elements also
known as the tumor
micro-environment. The aforementioned serve as an example that can be extended
to
improve the efficacy of CAR T therapies that target other tumor antigens. In
one aspect of the
invention, an immune cell is transfected or transduced with both a CAR
comprising an
antibody fragment that targets a tumor antigen and a cleavage enzyme. In
another aspect of
the invention, an immune cell is transfected or transduced with both a CAR
comprising an
antibody fragment that targets a tumor antigen and a cleavage enzyme that
cleaves a tumor
antigen to a form recognized by the antibody fragment of the CAR. In one
aspect, an immune
cell is transfected or transduced with both a CAR comprising an antibody
fragment that
targets a tumor antigen and a cleavage enzyme that cleaves a tumor antigen to
a form
recognized by the antibody fragment of the CAR, wherein the antibody fragment
of the CAR
recognizes MUC1* extra cellular domain and the cleavage enzyme cleaves MUC1 to
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MUC1*. In one aspect, an immune cell, which may be a T cell or an NK cell, is
transfected
or transduced with a CAR comprising an antibody fragment derived from MNC2,
MNE6,
MNC3 or MNC8 and a cleavage enzyme chosen from the group comprising MMPI,
MMP2,
MMP3, MMP7, MMP8, MMP9, MMP11, MMP12, MMP13, MMP14, MMP16, ADAM9,
ADAM10, ADAM17, ADAM 19, ADAMTS16, ADAM28 or a catalytically active fragment
thereof. In one aspect of the invention, the immune cell is derived from a
stem cell that has
been directed to differentiate into an immune cell type in vitro. In another
aspect, a CAR
containing sequences of the antibody are expressed in a stem cell, which then
may be
differentiated into an immune cell. In one case, the immune cell is a T cell.
In another case,
the immune cell is an NK cell.
[00660] In one aspect of the invention, a person diagnosed with cancer or at
risk of
developing cancer is administered a sufficient amount of an immune cell
transduced with
both a CAR and a cleavage enzyme. In another aspect of the invention, a person
diagnosed
with cancer or at risk of developing cancer is administered a sufficient
amount of an immune
cell transduced with both a CAR and a cleavage enzyme, wherein the cleavage
enzyme is on
an inducible promoter that is activated by proteins that are expressed when
the immune cell
becomes activated. In another aspect of the invention, a person diagnosed with
cancer or at
risk of developing cancer is administered a sufficient amount of an immune
cell transduced
with both a CAR and a cleavage enzyme, wherein the cleavage enzyme is on an
inducible
promoter that is activated by one or more NEAT. In one case the NEAT is NFATc
1. In
another aspect, the NFAT is NFATc3. In another aspect, the NFAT is NFATc2. In
any of the
instances above, the extra cellular domain of the CAR comprises a fragment of
an anti-
MUC1* antibody. In one aspect, the anti-MUC1* antibody is MNC2scFv or a
humanized
form of MNC2scFv. In another aspect, the anti-MUCI* antibody is MNE6scFv or a
humanized form of MNE6scFv. In any of the instances above, the immune cell can
be a T
cell, an NK cell, a mast cell, or a dendritic cell. In one aspect the immune
cell is derived from
a stem cell that has been directed to differentiate to that immune cell type
in vitro. In another
aspect, a CAR containing sequences of the antibody are expressed in a stem
cell, which then
may be differentiated into an immune cell. In one case, the immune cell is a T
cell. In another
case, the immune cell is an NK cell.
[00661] It is not intended that the present invention be limited to one or two
specific
methods of having expression of a cleavage enzyme induced by an activated T
cell. We have
demonstrated specific expression of a cleavage enzyme only upon T cell
activation by
constructing a plasmid with the cleavage enzyme gene downstream of an NFAT
promoter
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sequence or downstream of one or more repeats of NFAT response elements. In
another
aspect of the invention, expression of the cleavage enzyme is induced by
constructing a
plasmid where the cleavage enzyme gene is inserted downstream of an 1L-2
promoter
sequence or downstream of an IL-2 response element, then inserting the plasmid
into an
immune cell. In another aspect of the invention, expression of the cleavage
enzyme is
induced by constructing a plasmid where the cleavage enzyme gene is inserted
downstream
of a Calcineurin promoter sequence or downstream of a Calcineurin response
element, then
inserting the plasmid into an immune cell and then administering to a patient
for the
treatment or prevention of cancers. There are also drug-inducible plasmids
that can be used to
induce expression of the cleavage enzyme or used to stop expression induced by
an element
of an activated T cell. These drug inducible systems may include tetracycline-
inducible
systems, Tet-on, Tet-off, tetracycline response elements, doxycycline,
tamoxifen inducible
systems, ecdysone inducible systems and the like.
[00662] It is not intended that the present invention be limited to one or two
specific
promoters used in the plasmids encoding the CARs or inducible cleavage
enzymes. As is
known by those skilled in the art, many promoters can be interchanged
including SV40,
PGK1, Ubc, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GAL1, GAL10, TEF1, GDS,
ADH1, CaMV35S, Ubi, H1 and U6.Another solution to the problem of steric
hindrance of
CAR T cell access, caused by bulky cell surface proteins such as MUCl-FL, is
to increase the
length of the linker region of the CAR that is expressed by the 'I cell. In
standard design
CARs, the length of the extracellular linker region between the transmembrane
portion and
the antibody fragment is about 45-50 amino acids in length. We made long-arm
CARs where
the length of the extracellular linker is extended from about 50 amino acids
to 217 ¨ 290
amino acids. Co-culture assays show that CARs with longer extracellular
linkers have
improved access to the tumor-associated antigen on the target cancer cells.
[00663] BiTEs, bispecific antibodies and antibody-like molecules with multi-
specificity
[00664] Antibodies of the invention can be incorporated into a molecule
comprising at
least two binding arms wherein at least two bind to different antigens.
Several examples of
multi-specific antibody-like molecules have been described (Brinkmann and
Kontermann,
MABS, Vol. 9, No. 2, 182-212 (2017); Spiess et al. Molecular Immunology 67, 95-
106
(2015)), which references are incorporated by reference herein in their
entirety, but in
particular with respect to disclosures of making of bispecific antibodies and
fragments of
antibodies that are bispecific. It is also understood that various
terminologies such as
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"CrossMab", "DutaMab" found in Figure 217 of the present application and
Figure 1 of
Spiess et al. cited herein are terms of art and should be interpreted within
the context of the
description provided in Spiess et al. or Brinkmann and Kondermann cited
herein.
[00665] The variable regions of antibodies of the invention, or fragments
thereof, can be
incorporated into molecular formats wherein one molecule is able to
simultaneously bind to
at least two different antigens. The stability of these various antibody-like
molecules can be
increased by the introduction of Cysteines for the formation of disulfide
bonds. The stability
of antibodies of the invention as well as of various antibody-like molecules,
which may be
multi-specific can be increased by the introduction of mutations as more fully
described in
Beckman, US9708388B2, which reference is incorporated by reference herein in
its entirety,
but in particular with respect to disclosures of making of bispecific
antibodies and fragments
of antibodies that are bispecific or multispecific. The invention contemplates
incorporating
variable regions, or fragments thereof, of antibodies of the invention in
combination with
other antibodies having other binding specificities, or fragments thereof,
into a variety of
antibody-like formats including but not limited to the following. Among the
possible
antibody-like formats are bispecific antibodies, which may or may not include
an Fc region.
Bispecific antibodies that contain at least one Fc region will have a higher
molecular weight
and an extended half-life in the body. In a variation of a bispecific Fc-
containing antibody,
variable regions, or fragments thereof, derived from antibodies with yet other
binding
specificities are appended to the N- or C-terminus of the IgC1 scaffold to
yield a tetravalent,
multi-specificity antibody-like molecule. Combinations of variable regions, or
their
fragments, of antibodies with diverse binding specificities, which may be
incorporated into
scFvs, can be appended to an IgG scaffold or other scaffold to yield multi-
valent, multi-
specificity antibody-like molecules. Antibodies of the invention can be
incorporated into
mini-antibodies wherein scFvs are fused to the C-terminus of the CH3 portion
or hinge region
of an antibody Fc region. Divalent ,or bivalent) single-chain variable
fragments (di-seFvs,
bi-
scFvs) can be engineered by linking two scFvs. This can be done by producing a
single
peptide chain with two VII and two VI. regions, yielding tandem scFvs. Another
possibility is
the creation of scFvs with linker peptides that are too short for the two
variable regions to
fold together (about five amino acids), forcing scFvs to dimerize. This type
is known
as diabodies. Diabodies have been shown to have dissociation constants up to
40-fold lower
than corresponding scFvs, meaning that they have a much higher affinity to
their target.
Consequently, di.abody drugs could be dosed much lower than other therapeutic
antibodies
and are capable of highly specific targeting of tumors in vivo. Still shorter
linkers (one or two
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amino acids), and optional addition of disulfide bonds, lead to the formation
of turners, so-
called triabodies or tribodies. Tetrabodies and higher order inultimers have
also been
produced. They exhibit an even higher affinity to their targets than
diabodies.
[00666] Other formats of a bispecific antibody-like molecules are bispecific
tandem di-
sc.Fvs and bispecific T cell engagers, known as BiTEs (Bi
________________________ Ile antibody constructs). BiTEs are
fusion proteins consisting of two scFvs of different antibodies, on a single
peptide chain, of
about 55 kilodaltons. Typically, one of the binding arms binds to a molecule
on a T cell, such
as the CD3 receptor, and the other binding arm binds to a tumor cell via a
tumor specific
molecule, such as aberrantly expressed MUC1*. All of these lo.rmats can be
composed from
one or more variable fragments of antibodies of the invention, with
specificity fOr at least two
different antigens, to generate multi-valent and multi-specific antibody-like
molecules. In one
aspect, a variable domain fragment derived from a first antibody binds to a
first antigen on a
first surface and a variable domain fragment derived from a second antibody
binds to a
second antigen on a second surface, wherein at least one of the surfaces may
be the surface of
a cell. in another aspect of the invention, at least one of the variable
domain fragments
comprising a multi-valent, multi-specific antibody-like molecule binds to an
antigen that is
not associated with a surface. In one aspect, the antigen that is not
associated with a surface is
a cytokine.
[00667] As an example of how antibodies of the invention can be incorporated
into
bispecific antibodies, we constructed a bispecific antibody using a knob-in-
hole, also known
as Kai (Spiess et al. Molecular Immunology 67, 95-106 (2015)), format. In this
example, a
first arm of the antibody is the humanized and-MU-CT* antibody 20A10, also
known as
hu20A1.0, with a 14616 framework region; the second arm of the antibody is
either the anti-
CD3 antibody OKT3 or 12.F6, which both bind to the same epitope on human T
cells. The
resultant bispecific antibodies are referred to here as 20A10-0KT3-BiTE and
20A 10-12146-
BiTE. In a demonstration of function, the bispecific antibodies are added at
various
concentrations to cells in culture wherein both human T cells and -NII.JC1.*
positive cancer
cells are present. In one case the cancer cells are T47D breast cancer cells
and in the other
case a MUC1.* negative line LICT-1.16 colon cancer cells have been
tra.n.sduced to express
MUC1*, called HCT-MUC1*. As can be seen in the photographs shown in Fig. 283A-
283L,
Fig. 284A-284L, Fig. 285A-285L and Fig. 286A-286L, the addition of either
bispecific
antibody mediated the joining together of the T cells and the MUCI* positive
cancer cells as
evidenced by a bispecific dose--dependent cell clustering. Two control
experiments were
performed. in one control, no bispecific antibody is added, but both T cells
and MUC1*
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cancer cells are present. No clustering is observed. -In another control,
bispecific antibody is
added to MUC I* positive cancer cells, hut no T cells are present. These data
demonstrate that
an ti- MUC1* antibodies of the invention can be readily incorporated into a
bispecific format.
In these examples, the second arm of the bispecific antibody was an antibody
that recognizes
CD3 on T cells. However, it is not intended that the invention be limited to
bispecific
antibodies where one arm binds to MIJCI* and the other binds a T cell. A
person skilled in
the art could readily substitute the second arm of the .bispecific antibody
with an antibody that
binds to an NK cell, or another receptor on a cancer cell for increased
affinity. As MNC2
binds to the same epitope as 20.AI 0, it is evident that a person skilled in
the art could readily
substitute MNC2 or humanized MNC2 for the 20A10_ frt one aspect of the.
invention a person
diagnosed with a MUC1* positive cancer, or at risk of developing a MUC I*
positive cancer,
is treated with an effective amount of a bispecific antibody that
simultaneously binds to a
MUC1* positive cancer cell and a human T cell. In another aspect of the
invention, the
bispecific antibody is hu20A10-0KT3-BiTE. In another aspect of the invention,
the
bispecific antibody is hu20A10-12F6-BiTE, In another aspect of the invention,
the bispecific
antibody is huMNC2-0KT3-BiTE. hi another aspect of the invention, the
bispecific antibody
is h uMNC2-12F6-BiTE.
[00668] Another aspect of the invention is a method for treating a patient
diagnosed with,
suspected of having, or at risk of developing a MUC1 positive or MUC1*
positive cancer,
wherein the patient is administered an effective amount of one of the multi-
valent, multi-
specific antibody-like molecules described here wherein at least one of the
variable domains,
or fragment thereof, is derived from an antibody that binds to PSMGFR (SEQ ID
NO:2), or
more specifically to N-10 peptide (SEQ ID NO:3), or more specifically to N-19
peptide (SEQ
ID NO:4), or N-23 peptide (SEQ ID NO:5), or N-26 peptide (SEQ ID NO:6), or N-
30 peptide
(SEQ ID NO:7) or still more specifically that binds to a fragment of the
PSMGFR peptide
wherein said binding depends on the presence of the amino acids FPFSAQSGA (SEQ
ID
NO:10). In one case, the antibody variable fragment that binds to MUC1*
comprises portions
of MNC2, MNE6, 20A10, 3C2111, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9,
8C7F3, or H11. In one case, at least one of the antibody variable fragments
comprising a
multi-valent, multi-specific antibody-like molecule that binds to MUC1*
comprises portions
of MNC2, MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9,
8C7F3, or H11 and at least one other antibody variable fragment binds to an
antigen on an
immune cell, which may be a T cell or NK cell.
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[00669]
Another aspect of the invention is a method for treating a patient
diagnosed with,
suspected of having, or at risk of developing a MUC1 positive or MUC1*
positive cancer,
wherein the patient is administered an effective amount of a BiTE wherein one
antibody
variable fragment of the BiTE binds to a T cell surface antigen and the other
antibody
variable fragment of the BiTE binds to PSMGFR (SEQ ID NO:2), or more
specifically to N-
peptide (SEQ ID NO:3), or more specifically to N-19 peptide (SEQ ID NO:4), or
N-23
peptide (SEQ ID NO:5), or N-26 peptide (SEQ ID NO:6), or N-30 peptide (SEQ ID
NO:7) or
still more specifically that binds to a fragment of the PSMGFR peptide wherein
said binding
depends on the presence of the amino acids FPFSAQSGA (SEQ ID NO:10). In one
case, the
antibody variable fragment of the BiTE that binds to MUC1* comprises portions
of MNC2,
MNE6, 20A10, 3C2B1, 5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or
H11.
[00670] In another aspect of the invention, MUC1* peptides including PSMGFR
(SEQ ID
NO:2), or most or all of N-10 peptide are used in adoptive T cell approaches.
In this case, a
patient's T cells are exposed to the MUC1* peptides and through various rounds
of
maturation, the T cells develop MUC1* specific receptors. The adapted T cells
are then
expanded and administered to the donor patient who is diagnosed with,
suspected of having,
or is at risk of developing a MUC1* positive cancer.
[00671] A series of CARs were also made that had MNC2 and humanized MNC2 as
the
extra cellular, targeting head of the CAR. The constructs for these CARs were
inserted into a
plasmid that was then inserted into a Lenti viral vector. Human '1 cells were
then transduced
with the lenti viral vector carrying the MNC2 CARs and huMNC2 CARs. MNC2-scFv-
CARs
that were mouse sequence or humanized were generated. In one aspect of the
invention, the
CAR comprised huMNC2-scFv-short hinge region-transmembrane domain derived from
CD8-short intracellular piece-4-1BB-3zeta. In another aspect, the
transmembrane domain
was derived from CD4 transmembrane sequence. In another aspect, the
intracellular co-
stimulatory domain was CD28-3zeta. In yet another aspect, the intracellular co-
stimulatory
domain was CD28-4-1BB-3zeta.
[00672] There are a variety of methods for assessing whether or not T cells
recognize a
target cell and are in the process of mounting an immune response. T cells
cluster when they
recognize a target or foreign cell. This can be readily seen with the naked
eye or at low
magnification. The appearance of CAR T cell clustering when co-cultured with
target cancer
cells is one measure of: a) whether or not they recognize the cells as target
cells; and b)
whether or not they are getting activated to attack the targeted cells, which
in this case are
cancer cells. Figures 45-47 show photographs of MUC1* positive T47D breast
cancer cells
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that were either stably transfected with niCherry or dyed with CMTMR, so are
red, which
were co-cultured with either human T cells without a CAR or human T cells
transduced with
huMNC2-scFv-CAR44, or with huMNC2-scFv-CAR50. The CAR T cells are clear. As
can
be seen, there is no T cell induced clustering of the cancer cells when the T
cell does not
carry a CAR. However, when T cells carrying a MUC1* targeting CAR, there is
dramatic
clustering of the MUC1* positive cancer cells.
[00673] After T cells recognize and cluster target cells, they overexpress
perforM and
granzyme B. Together these two molecules activate a cell death pathway in the
targeted cell.
It is thought that the perforin makes a hole in the target cell into which the
T cell injects
granzyme B which then activates apoptotic proteases, causing the target cell
to lyse. Figs. 55
and Fig. 56 show huMNC2-scFV-CAR44 T cells binding to target MUC1* positive
prostate
cancer and pancreatic cancer cells and injecting granzyme B.
[00674] Another measure of whether or not a T cell has recognized a target
cell and is
activated to kill that cell, is the upregulation and secretion of cytokines,
interferon gamma
(IFN-g) and interleukin-2 (IL-2), by the T cell. Activation of CAR T cells, as
evidenced by
IFN-g and IL-2 secretion, can be readily measured in vitro. CAR T cells are co-
cultured with
target cells and after an incubation period, the conditioned media is assayed
by ELISA to
detect secreted IFN-g and IL-2. In order to determine the cancer-specificity
of CAR T cells
wherein the targeting head of the CAR was either huMNC2 or huMNE6, these
experiments
were performed with huMNC2-CAR44 '1 cells and huMNE6-CAR44 '1' cells in co-
culture
with MUC1* positive cancer cells and normal cells. Table 1 details the MUC1
positive
normal or primary cells that were tested.
[00675] Table 1: Normal Cell Lines and Primary Cells
[00676]
ATCC
ttll Line Dsigriation Tissut Oi igin. .
Hep.G2 Liver
The THLE-2 (ATCC CRL-10149 and the
THLE-3 (ATCC CRL-11233) cell lines
were derived from primary normal liver
cells by infection with SV40 large T
antigen. THLE-2 and THLE-3 cells
THLE-3 CRL-11233 Liver express phenotypic characteristics
of
normal adult liver epithelial cells. They are
non tumorigenic when injected into
athymic nude mice, have near-diploid
karyotypes, and do not express alpha-
fetoprotein.
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ATCC
Male, Caucasian
2.0 months old
Induction Fold CYP1A2 (a) 14.0
Induction Fold CYP2B6 (b) 13.0
Induction Fold CYP3A4 (c) 44.0
Basal Activity CYP1A2 2.6
Basal Activity CYP2B6 0.7
Lonza Basal Activity CYP3A4 14.0
Primary HUM181141 Liver Additional Information:
Hepatocytes Inducer / Marker Metabolite
(a) 0.05 mM Omeprazole / Acetaminophen
(b) 1 mIVI Phenobarbital /
Hydroxybupropion
(c) 0.01 mlVI Rifampicin / 6-Beta-
Hydroxytestosterone
Basal activity is expressed as:
pmol/million cells/minute
T/G HA- CRL - 1999 Aortic Smooth 11 months
VSMC Muscle Female, Caucasian
This fibroblast-like cell line was derived
from the lung tissue of a 2 month, 17-day-
old Black female.
CCD-18Lu CCL-205 Lung The donor had cerebral
anoxia, cardiac
anomaly, sepsis, endocardial cushion
defect and fetal alcoholic syndrome.
Female, Black
2.5 months
B rain Derived from small fragments
of human
HBEC-5i CRL-3245 cerebral cortex obtained from
patients who
endothelium
had died of various causes.
18 weeks gestation fetus
Male, Caucasian
Part of the NBL Cell Line Collection. This
Hs cell line is neither produced nor fully
CRL-7869 Stomach/Intestine
738. Stunt characterized by ATCC. We do
not
guarantee that it will maintain a specific
morphology, purity, or any other property
upon passage.
The MCF-12A cell line is a non-
tumorigenic epithelial cell line established
from tissue taken at reduction
mamrnoplasty from a nulliparous patient
MCF-12A CRL-10782 Breast with fibrocystic breast
disease that
contained focal areas of intraductal
hyperplasia. The line was produced by
long term culture in serum free medium
with low Ca++ concentration. MCF-12A
was derived from adherent cells in the
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ATCC
Designation.... Origiun
population.
Male, Caucasian
second trimester
Part of the NBL Cell Line Collection. This
cell line is neither produced nor fully
Hs 1.Tes CRL-7002 Testis
characterized by ATCC. We do not
guarantee that it will maintain a specific
morphology, purity, or any other property
upon passage.
Lonza:
catalogue # Human Renal Cortical Cells
(HRCE) are
CC-2554 Lot from proximal and distal
tubules.
HRCE Kidney
Donor info: 49 year old female, passage 2,
0000542104 95% viability, doubling time
(hours) 24 hrs
[00677]
[00678] Figure 50 is a graph of PCR measurement of the various cell lines
tested, wherein
mRNA levels of MUC1 are measured. The cancer cell lines that were tested in
these assays
were HCT-MUC1* and T47D breast cancer cells. These cells were co-cultured with
huMNC2-CAR44 human T cells. Co-culture of huMNC2-CAR44 T cells with the cancer
cells induced the CAR T cells to secrete large amounts of IFN-g and IL-2 into
the
surrounding media, yet co-culture with the MUCI positive normal cells induced
no secretion
of the cytokines (Fig. 51 and Fig. 52). In addition to testing for IFN-g and
IL-2 secretion by
the CAR T cells, the normal cells were assayed for signs of cell death, which
could have been
induced by the CAR T cells if the antibody targeting head were not extremely
cancer-
specific. After co-culture with huMNC2-CAR44 T cells, the cells were incubated
with a cell
death marker, then assayed by FACS. huMNC2-CAR44 T cells induced no cell death
in the
normal cells (Fig. 53A-53J).
[00679] In addition to FACS analysis, many researchers now use an xCELLigence
instrument to measure CAR T killing of cancer cells. FACS is not the best
method for
tracking T cell induced cell killing because the T cells lyse the target cell.
By FACS it is
difficult to measure dead cells because they are excluded as cell debris, so
one must infer an
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amount of cell killing and by various methods determine if the missing cells
are T cells or
cancer cells.
[00680] The xCELLigence instrument uses electrode arrays upon which cancer
cells are
plated. The adherent cancer cells insulate the electrode and so cause an
increase in impedance
as they grow. Conversely, T cells are not adherent and remain in suspension so
do not
contribute to insulation of the electrode which would increase impedance.
However, if the T
cells or CAR T cells kill the cancer cells on the electrode plate, the cancer
cells ball up and
float off as they die, which causes the impedance to decrease. The xCELLigence
instrument
measures impedance as a function of time, which is correlated to cancer cell
killing. In
addition, the electrode plates also have a viewing window. When CAR T cells
effectively kill
the adsorbed target cancer cells, there is a decrease in impedance but also
one can see that
there are no cancer cells left on the plate surface.
[00681] Figs. 55A-55H show the cytotoxic effect of huMNC2-CAR44 T cells on
MUC1*
positive DU145 prostate cancer cells as measured by a variety of assays. Fig.
55A is a
fluorescent photograph of untransduced T cells co-cultured with the prostate
cancer cells,
wherein granzyme B is stained with a red fluorophore. Fig. 55C is a
fluorescent photograph
of huMNC2-CAR44 T cells co-cultured with the prostate cancer cells, wherein
granzyme B is
stained with a red fluorophore. Fig. 55D is the DAPI and granzyme B merge.
Fig. 55E is a
FACS scan for fluorescently labeled granzyme B for untransduced T cells
incubated with the
cancer cells. Fig. 55F is a FACS scan showing a positive increase in
fluorescently labeled
granzyme B for huMNC2-CAR44 T cells incubated with the cancer cells. Fig. 55G
is a graph
of the mean fluorescent intensity. Fig. 55H is an xCELLigence scan tracking
the real-time
killing of DU145 cancer cells by huMNC2-CAR44 T cells (blue trace) but not by
untransduced T cells (green). Figs. 56A-56H show the cytotoxic effect of
huMNC2-CAR44
T cells on MUC1* positive CAPAN-2 pancreatic cancer cells as measured by a
variety of
assays. Fig. 56A is a fluorescent photograph of untransduced T cells co-
cultured with the
pancreatic cancer cells, wherein granzyme B is stained with a red fluorophore.
Fig. 56B is the
DAPI and granzyme B merge. Fig. 56C is a fluorescent photograph of huMNC2-
CAR44 T
cells co-cultured with the pancreatic cancer cells, wherein granzyme B is
stained with a red
fluorophore. Fig. 56D is the DAPI and granzyme B merge. Fig. 56E is a FACS
scan for
fluorescently labeled granzyme B for untransduced T cells incubated with the
cancer cells.
Fig. 56F is a FACS scan showing a positive increase in fluorescently labeled
granzyme B for
huMNC2-CAR44 T cells incubated with the cancer cells. Fig. 56G is a graph of
the mean
fluorescent intensity. Fig. 56H is an xCELLigence scan tracking the real-time
killing of
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CAPAN-2 cancer cells by huMNC2-CAR44 T cells (blue trace) but not by
untransduced T
cells (green). Figs. 57A-57C show xCELLigence scans tracking the real-time
killing of
MUC1* positive cancer cells, but not MUC1* negative cells, by huMNC2-CAR44 T
cells.
Fig. 57A shows that huMNC2-CAR44 T cells effectively kill HCT colon cancer
cells that
have been stably transfected with MUC1*. Fig. 57B shows that huMNC2-CAR44 T
cells
have almost no effect on HCT-MUC1-41TR, which is a MUC1 negative cancer cell
that has
been stably transfected with a MUC1 full-length. In this cell line only about
10% of the cell
have MUC1 cleaved to MUC1*. Fig. 57C shows that huMNC2-CAR44 T cells have no
effect on HCT-116 cells, which is a MUC1 negative colon cancer cell line.
[00682] These data demonstrate that T cells transduced with a CAR wherein the
antibody
fragment targeting head is MNC2, effectively kill MUC1* positive cancer cells.
These data
specifically show that huMNC2-scFV-CAR44 transduced into human T cells
effectively kill
MUC1* positive cancer cells. Because we and others have now demonstrated that
the most
important aspect of CAR T function is the targeting antibody fragment, it
follows that an
immune cell or a T cell transduced with any CAR having the antibody fragment
MNC2-scFV
or huMNC2-scFV would have similar efficacy against MUC1 or MUC1* positive
tumors.
For example, the hinge region that connects the scFv to the transmembrane
portion could be
any flexible linker. The intracellular co-stimulatory domains could be CD28-
3zeta, CD28-4-
1BB-3zeta or any combination of immune cell co-stimulatory domains.
[00683] Figure 61 shows an experiment in which huMNC2-scFv-CAR44 transduced
human T cell that were bead stimulated (Protocol 1) or cancer cell stimulated
(Protocol 2)
were tested for their ability to inhibit tumor growth in animals. Human cancer
cells that had
been stably transfected with Luciferase were injected into female
NOD/SCID/GAMMA
(NSG) mice between 11 and 15 weeks of age. 500,000 BT-20 breast cancer cells
were
injected sub-cutaneously into a rear flank. Tumor engraftment was verified by
injecting the
animals with Luciferin and then imaging the fluorescent cancer cells using an
IVIS
instrument. IVIS images taken Day 5 post implantation showed the presence of
tumor cells.
On Day 6 after IVIS measurement, animals were given a one-time injection of 10
million of
either human T cells transduced with huMNC2-scFv-CAR44 or untransduced T
cells. 5
million T cells were injected intra-tumor and 5 million were injected into the
tail vein. 10
minutes prior to IVIS photographs, mice were IP injected with Luciferin, which
fluoresces
after cleavage by Luciferase, thus making tumor cells fluoresce. Figs. 61A,
61D, 61G show
photographs of mice that were treated with huMNC2-scFv-CAR44 T cells that had
been pre-
stimulated by co-culturing for 24 hours with 41im beads to which was attached
a synthetic
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MUC1*, PSMGFR peptide 24 hours prior to administration, "Protocol 1". Figs.
61B, 61E,
61H show photographs of mice that were treated with huMNC2-scFv-CAR44 T cells
that had
been pre-stimulated by twice co-culturing for 24 hours with MUC1* positive
cancer cells 24
hours prior to administration, "Protocol 2". As can be seen in Figure 61,
huMNC2-CAR44
T cells that were peptide-bead stimulated inhibited tumor growth better than
cells pre-
stimulated by incubation with live cancer cells, which likely contaminated the
target cells and
increased the tumor volume.
[00684] huMNC2-scFv-CAR44 transduced human T cell that were bead stimulated
(Protocol 1) or cancer cell stimulated (Protocol 2) were also tested for their
ability to inhibit
tumor growth in animals. Human cancer cells that had been stably transfected
with Luciferase
were injected into female NOD/SCID/GAMMA (NSG) mice between 11 and 15 weeks of
age. In another experiment, 500,000 BT-20 MUC1* positive triple negative
breast cancer
cells were injected sub-cutaneously into a rear flank. Tumor engraftment was
verified by
injecting the animals with Luciferin and then imaging the fluorescent cancer
cells using an
IVIS instrument. IVIS images taken Day 6 post implantation showed the presence
of tumor
cells. On Day 6, after IVIS imaging, 10M huMNC2-scFv-CAR44 T cells were
administered
to the animals. 5M of the CAR T cells were administered by intratumor
injection and the
other 5M were administered by tail vein injection. Control group was injected
by same
administration routes with the same number of untransduced T cells. IVIS
measurements of
tumor burden were taken on Days 6, 8, and 12. As can be seen in Figs. 61A-
61,1, both groups
of mice treated with huMNC2-CAR44 T cells showed a decrease in tumor burden
compared
to the control group.
[00685] huMNC2-scFv-CAR44 transduced human T cell that were bead stimulated
(Protocol 1) were also tested for their ability to inhibit ovarian cancer
growth in animals.
Human SKOV-3 MUC1* positive ovarian cancer cells that had been stably
transfected with
Luciferase were injected into female NOD/SCID/GAMMA (NSG) mice between 11 and
15
weeks of age. In one experiment, 500,000 SKOV-3 cancer cells were injected
into the
intraperitoneal cavity to mimic metastatic ovarian cancer in humans. Tumor
engraftment was
verified by injecting the animals with Luciferin and then imaging the
fluorescent cancer cells
using an IVIS instrument. IVIS images taken Day 3 post implantation showed the
presence of
tumor cells. On Day 4 and Day 11, post tumor implantation, 10M huMNC2-scFv-
CAR44 T
cells were IP administered to the animals. On Day 4, CAR T cells were IP
injected. On Day
11 half the CAR T cells were injected into the intraperitoneal space and the
other half was
injected into the tail vein. Control groups were injected by same
administration routes with
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either the same number of untransduced T cells or same volume of PBS.
Subsequent IVIS
measurements of tumor burden were taken on Day 7, Day 10 and Day 15. As can be
seen in
Figs. 62A-62L, control mice have tumors that are growing at a much faster rate
than the
huMNC2-CAR44 T cell treated mice. Fig. 62M shows the IVIS color bar
correlating
photons/second to color.
[00686] One aspect of the invention is a method for treating a patient
diagnosed with,
suspected of having, or at risk of developing a MUC1 positive or MUC1*
positive cancer,
wherein the patient is administered an effective amount of an antibody,
antibody fragment
which may be incorporated into a BiTE, a bispecific antibody, a multi-specific
antibody, an
ADC or a CAR expressed in an immune cell wherein the antibody or fragment
thereof is
derived from an antibody of the invention. In a preferred embodiment, the
antibody or
fragment thereof is derived from MNC2, MNE6, 20A10, 3C2B1, 5C6F3 or 25E6.
[00687] One aspect of the invention is a method for treating a patient
diagnosed with,
suspected of having, or at risk of developing a MUC1 positive or MUC1*
positive cancer,
wherein the patient is administered an effective amount of immune cells that
have been
transduced with a MUC1* targeting CAR, wherein the CAR is chosen from among
the group
consisting of MNE6-CD8-CD28-3z (SEQ ID NOS:297-298); MNE6-CD4-CD28-3z (SEQ ID
NOS:748-749); MNE6-CD8-41BB-3z (SEQ ID NOS:300-301); MNE6-CD4-41BB-3z (SEQ
ID NOS:750-751); MNE6-CD8-CD28-41BB-3z (SEQ ID NOS:303-304); MNE6-CD4-
CD28-41BB-3z (SEQ ID NOS:754-755); MNE6scFv-Fc-8-41BB-CD3z (SEQ ID NOS:310-
311); MNE6scFv-IgD-Fc-8-41BB-CD3z (SEQ ID NOS :770-771); MNE6scFv-FcH-8-41BB-
CD3z (SEQ ID NOS:315-316); MNE6scFv-IgD-FcH-8-41BB-CD3z (SEQ ID NOS:772-
773); MNE6scFv-Fc-4-41BB-CD3z (SEQ ID NOS :318-319); MNE6scFv-FcH-4-41BB-
CD3z (SEQ ID NOS:321-322); MNE6scFv-IgD-8-41BB-CD3z (SEQ ID NOS:323-324);
MNE6scFv-IgD-4-41BB-CD3z (SEQ ID NOS :327-328); MNE6scFv-X4-8-41BB-CD3z
(SEQ ID NOS:330-331); MNE6scFv-X4-4-41BB-CD3z (SEQ ID NOS:333-334);
MNE6scFv-8-4-41BB-CD3z (SEQ ID NOS:336-337), or any of the aforementioned CARs
wherein the MNE6 is replaced by fragment derived from MNC2, MNE6, 20A10,
3C2B1,
5C6F3, 25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. Another aspect
of the
invention is a method for treating a patient diagnosed with, suspected of
having, or at risk of
developing a cancer, wherein the patient is administered an effective amount
of immune cells
that have been transduced with one of the aforementioned CARs wherein the MNE6
is
replaced by a peptide comprising antibody variable domain fragments that are
specific for a
cancer antigen. In any of the above methods, the immune cell may be a T cell
and may
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further be isolated from the patient to be treated. Alternatively, the immune
cell, which may
be a T cell is isolated from a donor. In yet another aspect, the immune cell
is derived from a
stem cell that has been directed to differentiate to that immune cell type in
vitro. In another
aspect, a CAR containing sequences of the antibody are expressed in a stem
cell, which then
may be differentiated into an immune cell. In one case, the immune cell is a T
cell. In another
case, the immune cell is an NK cell.
[00688] Another aspect of the invention is a method for treating a patient
diagnosed with,
suspected of having, or at risk of developing a MUC1 positive or MUC1*
positive cancer,
wherein the patient is administered an effective amount of immune cells that
have been
transduced with a MUC1* targeting CAR. In a preferred embodiment, the CAR may
include
a single chain antibody fragment, scFv, comprising a sequence derived from
antibody MNC2,
such as MNC2-scFv (SEQ ID NO:239, 241, 243, or 654-655), from antibody 20A10,
such as
SEQ ID NO:1574-1581 or 5001-5012, from antibody 3C2B1, such as SEQ ID NO:1572-
1573, which may be humanized for example as in SEQ ID NO:1812-1813, or from
antibody
5C6F3, such as SEQ ID NO:1384-1385), which may be humanized for example as in
SEQ ID
NO:1814-1815, or from antibody 25E6 such as SEQ ID NO:1598-1599 or 1600-1601,
wherein the hinge and transmembrane sequences may be derived from CD8 (SEQ ID
NO:346
and SEQ ID NO:364), or from CD28 (SEQ ID NO:350 and SEQ ID NO:368), further
comprising a co-stimulatory domain, which may be 41BB (SEQ ID NO:659) or CD28
(SEQ
Ill NO:378) and the CD3-zeta signaling domain may be derived from (SEQ ID
NO:661) or
may contain mutations including those referred to as 1XX (SEQ ID NO:1796-
1797).
[00689] Other MUC1 cleavage sites
[00690] It is known that MUC1 is cleaved to the growth factor receptor form,
MUC1*, on
some healthy cells in addition to cancer cells. For example, MUC1 is cleaved
to MUC1* on
healthy stem and progenitor cells. A large percentage of bone marrow cells are
MUC1*
positive. Portions of the intestine are MUC1* positive.
[00691] The inventors have discovered that MUC1 can be cleaved at different
positions
that are relatively close to each other but the location of cleavage changes
the fold of the
remaining portion of the extracellular domain. As a result, monoclonal
antibodies can be
identified that bind to MUC1* cleaved at a first position but do not bind to
MUC1* that has
been cleaved at a second position. This discovery is disclosed in
W02014/028668, filed
August 14, 2013, the contents of which are incorporated by reference herein
its entirety. We
identified a set of anti-MUC1* monoclonal antibodies that bind to MUC1* as it
appears on
cancer cells but do not bind to MUC1* as it appears on stem and progenitor
cells.
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Conversely, we identified a second set of monoclonal antibodies that bind to
stem and
progenitor cells but do not bind to cancer cells. One method used to identify
stem specific
antibodies is as follows: supernatants from monoclonal hybridomas were
separately adsorbed
onto 2 multi-well plates. Stem cells, which are non-adherent cells, were put
into one plate and
cancer cells which are adherent were put into an identical plate. After an
incubation period,
the plates were rinsed and inverted. If the non-adherent stem cells stuck to
the plate, then the
monoclonal antibody in that particular well recognizes stem cells and will not
recognize
cancer cells. Antibodies that did not capture stem cells or antibodies that
captured cancer
cells were identified as cancer specific antibodies. FACS analysis has
confirmed this method
works.
[00692] Antibodies MNE6 and MNC2 are examples of cancer-specific antibodies.
Antibodies MNC3 and MNC8 are examples of stem-specific antibodies. Although
both sets
of antibodies are able to bind to a peptide having the PSMGFR sequence, FACS
analysis
shows that the anti-MUC1* polyclonal antibody and MNC3 bind to MUC1* positive
bone
marrow cells but MNE6 does not. The MUC1* polyclonal antibody was generated by
immunizing a rabbit with the PSMGFR peptide. Similarly, MNC3 binds to stem
cells of the
intestinal crypts but MNE6 does not. Conversely, MNE6 antibody binds to
cancerous tissue
while the stem-specific MNC3 does not. Competition ELISA experiments indicate
that the C-
terminal 10 amino acids of the PSMGFR peptide are required for MNE6 and MNC2
binding,
but not for MNC3 and MNC8. Therefore, another method for identifying
antibodies that are
cancer specific is to immunize with a peptide having the sequence of the
PSMGFR peptide
minus the 10 N-terminal amino acids or use that peptide to screen for
antibodies or antibody
fragments that will be cancer specific. Antibodies that bind to a peptide with
a sequence of
PSMGFR peptide minus the N-terminal 10 amino acids, referred to herein as N-10
peptide,
but do not bind to a peptide with a sequence of PSMGFR peptide minus the C-
terminal 10
amino acids, C-10 peptide, are cancer specific antibodies for use in the
treatment or
prevention of cancers.
[00693] The extracellular domain of MUC1 is also cleaved on stem cells and
some
progenitor cells, where activation of cleaved MUC1 by ligands NME1 in dimer
form or
NME7 promotes growth and pluripotency and inhibits differentiation. The
transmembrane
portion of MUC1 that remains after cleavage is called MUC1* and the
extracellular domain
is comprised essentially of the Primary Sequence of MUC1 Growth Factor
Receptor
(PSMGFR) sequence. However, the exact site of cleavage can vary depending on
cell type,
tissue type, or which cleavage enzyme a particular person expresses or
overexpresses. In
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addition to the cleavage site that we previously identified which leaves the
transmembrane
portion of MUC1* comprising most or all of the PSMGFR (SEQ ID NO:2), other
cleavage
sites could possibly result in an extended MUC1* comprised of most or all of
SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:620); or
[00694] SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:621).
[00695] To test this hypothesis, and to determine if antibodies to an N-
terminally extended
PSMGFR, would generated more cancer-specific antibodies than antibodies that
bind to the
PSMGFR, we generated monoclonal antibodies by immunization with peptides:
[00696] (PSMGFR)
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO :2),
[00697] (N+20/C-27) SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTE (SEQ
ID NO:822), or
[00698] (N+9/C-9)
VQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVP (SEQ ID NO:824)
[00699] Monoclonal antibodies generated from immunization with the same
peptide can
also show differences in reactivity to the same cancerous tissue specimen.
These results
indicate that the monoclonal antibodies recognize different conformations of
the truncated
MUC1 extra cellular domain produced by immunizing with different length
peptides,
mimicking different cleavage sites, or from cleavage at different sites in the
host animal.
Antibodies that recognize different cleavage site conformations may be cancer
sub-type
specific or patient specific, depending on which cleavage enzyme their tumor
expresses. In
one aspect of the invention, a patient diagnosed with a certain type of cancer
is treated with
an antibody of the invention that recognizes a cleaved MUC1 wherein the
antibody is specific
for cleavage by a specific enzyme that is known to be typically expressed by
that sub-type of
cancer. In another aspect, a patient tumor is analyzed to determine which
enzyme his or her
tumor expresses and an antibody that recognizes a MUC1 cleaved by that enzyme
is then
administered to the patient for the treatment of their cancer. The antibody
may be in the form
of a CAR, a BiTE, an ADC, a bispecific antibody, with or without an FC region
or a portion
of an Fc region, a bi-scFv, a di-scFv, a tandem di-seFv, a diabody, triabody,
tribody,
tetrabody and other antibody-like molecules that are multi-valent and multi-
specific.
[00700] We previously reported that it is the MUC1 transmembrane cleavage
product,
called MUC1* (mukl star), that mediates tumor growth and not full-length MUC1
(Mahanta
et al 2008). MUC1* is a growth factor receptor that is activated by ligand
induced
dimerization of its short extra cellular domain (Fig. IA). Dimerization of the
MUC1* extra
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cellular domain activates the MAP kinase signaling cascade and stimulates
growth and
survival of cancer cells (Fessler et al 2009). Bivalent antibodies that
dimerize the MUC1*
extra cellular domain stimulate cancer cell growth while the monovalent Fab of
the same
antibody, which cannot dimerize, inhibits cancer cell growth. We demonstrated
this in vitro
(Fig. 1B) and in vivo (Fig. 7A-7B).
[00701] We then identified the natural ligands that dimerize and activate
MUC1* growth
factor receptor function. Dimers of NME1 bind to and dimerize the MUC1* extra
cellular
domain and stimulate growth (Fig. 1C and Smagghe et al 2013). NME1 can turn
its growth
factor properties off. NME1 is secreted by MUC1* positive cells. Dimeric NME1
binds to
MUC1* to stimulate growth. However, as the cell population grows, more and
more NME1
is secreted from the cells. At high concentrations, the NME1 dimers
multimerize and form
hexamers, which do not bind to MUC1*, but likely bind to some unknown
receptor, as the
addition of NME1 hexamers turns off growth. NME1 is an adult form. The
embryonic form
is NME7AB (Carter et al 2016). Each NME7AB monomer has two binding sites for
MUC1* so
as a monomer it dimerizes MUC1* (Fig. 1D), stimulates growth and cannot turn
itself off. In
the developing embryo, BRD4 turns off NME7 and its co-factor JMJD6 turns on
the self-
regulating form, NME1. However, in cancers, NME7, which should be silenced in
adult life,
is aberrantly expressed again, where is renders the MUC1* growth factor
receptor
constitutively active.
[00702] In vitro, NME1 (SEQ Ill NO:4) and NME7AB (SEQ ID NO:827) bind to the
PSMGFR portion of the MUC1* extra cellular domain. Both growth factors can
bind to the
PSMGFR peptide (SEQ ID NO:2) even if the 10 N-terminal amino acids are
deleted, referred
to herein as N-10 (SEQ ID NO:3). However, neither NME1 nor NME7AB can bind to
the
PSMGFR peptide if the 10 membrane proximal amino acids are deleted (Fig. 2A-
2D),
referred to herein as C-10 (SEQ ID NO:825). In summary, the epitope to which
NME1 and
NME7AB bind includes all or part of the 10 membrane proximal amino acids:
PFPFSAQSGA
(SEQ ID NO:1743). We tested various antibodies that were generated in animals
by
immunizing with the PSMGFR peptide for their ability to recognize cancer cells
but not
healthy cells. Among the most cancer selective were the MNC2 and MNE6
monoclonal anti-
MUC1* antibodies. Two other monoclonal antibodies that were generated from
immunizing
animals with the PSMGFR peptide are MNC3 and MNC8. Although MNC2, MNE6, MNC3
and MNC8 all bind to the PSMGFR peptide, like NME1 and NME7AB, MNC2 and MNE6
bind strongly to the N-10 peptide but not to the C-10 peptide (Fig. 2B-2C). In
fact, MNC2
and MNE6 competitively inhibit the binding of NME1 and NME7AB to PSMGFR (Fig.
3A-
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3C). Conversely, MNC3 and MNC8 bind to the C-10 peptide, bind less well to the
N-10
peptide and do not compete with NME1 nor NME7AB for binding to MUC1* peptides,
including PSMGFR (Fig. 2E-2F). MNC3 and MNC8 are far less cancer specific than
MNC2
and MNE6. MNC3 and MNC8 recognize stem and progenitor cells, such as
hematopoietic
stem cells, whereas MNC2 and MNE6 do not (Fig. 39-41). Because hematopoietic
stem cells
are the progenitor cells for the blood cells, it would be problematic to have
a cancer
therapeutic that would also target such an important normal cell type.
[00703] Because MUC1* is generated by enzymatic cleavage of MUC1, we
researched
which cleavage enzymes cleave MUC1 to a MUC1* and whether or not we could
identify
antibodies that would recognize a MUC1* generated by a first cleavage enzyme
but not
MUC1* generated by a second cleavage enzyme. We found that MNC2 and MNE6
recognized a MUC1* generated by cleavage of MUC1 by MMP9 but not by cleavage
by
other enzymes such as MMP2 (Fig. 37 and Fig. 75). We note that MMP9 is
overexpressed in
cancers and is a predictor of poor prognosis (vant Veer et al 2002; Dufour et
al 2011) and has
been implicated in metastasis (Owyong et al, 2019), whereas MMP2 is expressed
in bone
marrow. One antibody binding to a MUC1* generated by cleavage by a first
enzyme but not
by cleavage by a second enzyme implies that the antibody recognizes a
conformational
epitope rather than a linear epitope.
[00704] We reasoned that the most cancer specific antibodies would be those
antibodies
that are characterized by some combination of most or all of the following:
[00705] (i) Antibody binds to PSMGFR peptide;
[00706] Antibody does not bind to full-length MUC1;
[00707] (ii) Antibody binds to N-10;
[00708] (iii) Antibody does not bind to C-10;
[00709] (iv) Antibody competitively inhibits binding of NME1 or NME7AB to
MUC1*
extra cellular domain or a PSMGFR peptide;
[00710] (v) Antibody recognizes a MUC1* generated by cleavage by MMP9;
[00711] (vi) Antibody recognizes a conformational epitope not a linear
epitope.
[00712] MNC2 and MNE6 are cancer specific.
[00713] Our experiments show that both MNC2 and MNE6: a) Bind to tumor cells;
b)
monovalent forms block tumor growth in vitro and in vivo; c) have minimal to
no binding of
normal tissue while having robust binding to a wide panel of tumor tissues; d)
when
incorporated into CAR T cells, MNC2 and MNE6 directed CAR T cells do not
recognize
full-length MUC1 and do not kill cells that only express full-length MUC1; e)
MNC2 and
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MNE6 directed CAR T cells cluster then kill tumor cells expressing MUC1*; and
f) MNC2
and MNE6 recognize a MUC1 cleavage product when it is cleaved by MMP9.
[00714] MNC2 directed CAR T cells do not recognize normal, healthy cells that
are
MUC1* positive. A panel of normal cell lines, as well as primary cells, were
co-cultured with
huMNC2-CAR44 T cells. The normal cell populations were analyzed to determine
whether
or not the MNC2 directed CAR T cells killed them. The CAR T cells were
analyzed to see if
co-culture with the MUC1 positive normal cells activated the killing function
of the CAR T
cells, as measured by secretion of IL-2 or interferon gamma. As Figures 50-52
show, the
MNC2 directed CAR T cells did not kill the normal cells, nor was there
cytokine secretion,
indicative of T cell activation. In addition, over 2,000 human tissue
specimens were analyzed.
The results showed that neither MNC2 nor MNE6 showed any significant binding
to normal
tissues but showed robust staining of a wide panel of cancerous tissues. For
example, MNC2
stained 93% breast cancer specimens, 83% ovarian, 78% pancreatic and 71% lung
cancer
specimens. In addition, patient-matched primary tumors (Fig. 54) and
subsequent metastases
showed that the amount of MNC2-reactive MUC1* increased with tumor progression
and
metastases. In summary, MNC2 is a highly cancer specific antibody.
[00715] Characterization of MNC2 and MNE6
[00716] Our gold standard, cancer-specific antibodies MNC2 and MNE6: 1) bind
to N-10
peptide but not to the C-10 peptide; 2) compete with NME7An and dimeric NME1
for the
same binding site near the C-terminus of the PSMC1FR peptide, which is the
membrane
proximal portion of MUC1* on cells; 3) do not work in a Western blot assay
indicating that
they recognize a conformational rather than linear epitope; 4) recognize a MUC
I* generated
when MUC1 is cleaved by MMP9; 5) do not bind to full-length MUC1 but only to
the
cleaved form, MUC1*, in model cell lines as well as cancer cell lines; 6) show
little to no
binding to normal tissues but robustly stain a wide variety of tumor tissues;
and 7) share some
consensus sequences in their Complementarity Determining Regions, CDRs.
[00717] In an effort to identify other antibodies that are highly cancer-
specific, like MNC2
and MNE6, we subjected new antibodies to a set of seven (7) characterization
experiments:
1) epitope binding assays; 2) functional assays such as the ability to
displace activating
growth factor NME7An or dimeric NME1 from binding to MUCI* peptides PSMGBR or
N-
10; 3) Western blots to determine whether or not the antibodies recognized a
linear epitope
versus a conformational epitope, in which case the antibodies would not work
in a Western;
4) binding assays to see if the antibodies recognized a cleaved MUC1 that was
dependent on
cleavage by MMP9; 5) FACS analysis to measure the ability of the antibodies to
recognize
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MUC 1* positive cells but not full-length MUCl; and FACS analysis to measure
the ability of
the antibodies to recognize MUC1/MUC1* positive cancer cells; 6)
immunohistochemistry,
1HC, assays of normal tissues versus cancerous tissues to determine true
cancer specificity;
and 7) aligning antibody sequences to determine if subsets of antibodies
shared consensus
sequences that could predict their cancer specificity or lack thereof.
[00718] Monoclonal antibodies were produced by immunizing animals with
peptides
derived from a MUC1 that is devoid of tandem repeats. These antibodies
included PSMGFR
and peptides that were extended at the N-terminus of PSMGFR. Immunizing
peptides were:
[00719] PSMGFR (SEQ ID NO:2))
[00720] N+9/C-9 (9 amino acids added onto the N-terminus and 9 amino acids
deleted
from the C-terminus) (SEQ ID NO:824)
[00721] N+20/C-27 (20 amino acids added onto the N-terminus and 27 amino acids
deleted from the C-terminus) (SEQ ID NO:823)
[00722] These monoclonal antibodies were then tested to determine which
satisfied the
seven (7) characterization criteria cited above, which we reasoned would
identify the most
cancer specific antibodies.
[00723] Epitope binding assays
[00724] ELISA assays were performed to determine if, in addition to
recognizing their
immunizing peptide, they recognized PSMGFR, N-10 or C-10. In addition, they
were tested
for their ability to bind to N+20/C-27, N+9/C-9. We first did the ELISA assay
on our set of
reference antibodies, MNC2, MNE6, which we know are cancer-specific plus MNC3,
which
we know recognizes stem cells and progenitor cells (Fig. 63A-63B). None of the
reference
antibodies bound to the N+20/C-27 peptide. MNC2 and MNE6 cannot bind to PSMGFR
peptides with 27, 10 or 9 C-terminal deletions, however, MNC3 binds to C-10
and to N+9/C-
9 peptides.
[00725] This same ELISA assay was performed on the antibodies of the invention
(Fig.
64-66 and Fig. 201). The binding patterns of the antibodies that were
generated by
immunizing with the PSMGFR peptide are shown in Fig. 64A-64B. Note that only
20A10
exactly matches the binding profile of MNC2 and MNE6. 25E6, 28F9 and 18G12 are
all able
to bind to the N-10 peptide. 18B4 is the only antibody raised against the
PSMGFR peptide
that requires the 10 most N-terminal amino acids of the peptide. The color of
the bars for
each antibody in the ELISA graph are color coded to match the deductive
cognate sequence,
or a portion thereof, of that antibody. In addition, another set of antibodies
was assayed by
ELISA (Fig. 201). Of this set, B12, B2, B7, B9, 8C7F3, and H11 bound to the
PSMGFR
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peptide, bound to the N-10 peptide, but not to the C-10 peptide (Fig. 201).
The binding
patterns of the antibodies that were generated by immunizing with the N+20/C-
27 peptide are
shown in Fig. 65A-65B. Although these antibodies were raised against the
N+20/C-27
peptide, all but one, 45C11, still bind to the PSMGFR peptide, albeit at the N-
terminal
portion of PSMGFR. The binding of 45C11 is weak but deductive reasoning shows
that all or
some of the cognate epitope must lie within SNIKERPGSVV (SEQ ID NO:1744).
[00726] Of the antibodies generated by immunizing with the N+9/C-9 peptide,
8A9 and
17H6 do not bind to the PSMGFR peptide, so must bind to the 9 additional N-
terminal amino
acids. Antibodies 3C5 and 39H5 appear to bind to the 10 most N-terminal amino
acids of the
PSMGFR peptide.
[00727] In order to further refine the epitopes to which each antibody binds,
a series of
smaller peptides derived from the PSMGFR sequence were synthesized: N-30 (SEQ
ID
NO:7). N-26 (SEQ ID NO:6), N-19 (SEQ ID NO:4), N-10/C-5 (SEQ ID NO:8), N-19/C-
5
(SEQ ID NO:9). Each of the antibodies was tested in an ELISA assay for their
ability to bind
to this refined set of peptides, plus PSMGFR. N-10 and C-10 peptides (Fig. 67-
69).
[00728] In Fig. 67A-67D, antibodies generated by immunization with the PSMGFR
peptide were assayed. As can be seen in the figure, amino acids ASRYNLT (SEQ
ID
NO:1745), which are essentially in the middle of the PSMGFR peptide, are
important or
essential for the binding of 28F9, 18G12, 25E6, and MNC3 antibodies. Amino
acids
CiTIN VHDVET (SEQ Ill NO:1746), which comprise the most N-terminal part of the
PSMGFR peptide are important or essential for the binding of the 18B4
antibody. Amino
acids FPFS (SEQ ID NO:1747) are important or essential for the binding of
20A10, MNC2
and MNE6. We note that these three antibodies recognize a conformational
epitope, not a
linear epitope. Because the proline in the FPFS sequence significantly alters
the conformation
of nearby portions of the PSMGFR peptide, it is also possible that the
antibodies do not bind
directly to these four amino acids, but that the absence of the proline alters
the fold of the
remaining peptide such that the conformation to which20A10, MNC2 and MNE6
bind, is no
longer present.
[00729] In Fig. 68A-68D, antibodies generated by immunization with the N+20/C-
27
peptide were assayed. As can be seen in the figure, amino acids GTINVHDVET,
which
comprise the most N-terminal part of the PSMGFR peptide are important or
essential for the
binding of the 29H1, 32C1, and 31A1 antibodies. Amino acids
SNIKFRPGSVVVQLTLAFRE (SEQ ID NO: 748), which is 20 additional amino acids N-
terminal to the PSMGFR peptide and outside of the PSMGFR peptide, are
important or
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essential for the binding of antibody 45C11. However, referring back to Fig.
65, antibody
45C11 was not able to bind to the N+9/C-9 peptide, therefore we conclude that
amino acids
within the SNIKFRPGSV V sequence are essential for the binding of 45C11. Amino
acids
QFNQYKTEA (SEQ ID NO:1749), which are still within the sequence of PSMGFR, are
important or essential for the binding of antibody 1E4.
[00730] In Fig. 69A-69D, antibodies generated by immunization with the N+9/C-9
peptide
were assayed. As can be seen in the figure, amino acids GTINVHDVET, which
comprise the
most N-terminal part of the PSMGFR peptide are important or essential for the
binding of the
39H5 and 3C5 antibodies. As can be seen in the figure, amino acids VQLTLAFRE
(SEQ ID
NO:1750), which is 9 additional amino acids N-terminal to the PSMGFR peptide
and outside
of the PSMGFR peptide, are important or essential for the binding of
antibodies 17H6 and
8A9. Because the 17H6 and 8A9 antibodies do not bind to any of the smaller
peptides shown
in this figure, refer to Figure 66A-66C, which shows that these two antibodies
only bind to
the peptide that has 9 additional amino acids N-terminal to the PSMGFR
peptide.
[00731] Table 2 below lists antibodies of the invention and their cognate
epitopes.
Table 2
Immunizing Peptide Antibody Name Cognate
Sequence
PSMGFR MNC2 FPFS or PFPFSAQSGA
MNE6 FPFS or PFPFSAQSGA
20A10 FPFS or PFPFSAQSGA
3C2B 1 FPFS or PFPFSAQSGA
5C6F3 SVSDV
MNC3 ASRYNLT
25E6 ASRYNLT
28F9 ASRYNLT
18G12 ASRYNLT
18B4 GTINVHD VET
N+20/C-27
45C11 SNIKFRPGSVV
29H1 GTINVHDVET
32C1 GTINVHD VET
31A1 GTINVHD VET
1E4 QFNQYKTEA
N+9/C-9
17H6 VQLTLAFRE
8A9 VQLTLAFRE
39H5 GTINVHD VET
3C5 GTINVHD VET
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[00732] Ability to displace NME7AB binding to the MUC1* extra cellular domain
peptide PSMGFR
[00733] We previously reported that dimeric NME1 dimerizes MUC1* extra
cellular
domain and stimulates growth. Monomeric NME7AB has two binding sites for MUC1*
so
that as a monomer it dimerizes MUC1* and mediates cancer cell growth. We
showed that
NME1 and NME7AB can bind to the MUC1* extra cellular domain. In vitro, NME1
and
NME7AB bind to the PSMGFR peptide even if the 10 N-terminal amino acids are
deleted,
referred to herein as N-10 (SEQ ID NO:3). However, neither NME1 nor NME7AB can
bind to
the PSMGFR peptide if the 10 membrane proximal amino acids are deleted,
referred to
herein as C-10 (SEQ ID NO:825). In summary, the epitope to which NME1 and
NME7AB
bind includes all or part of the 10 membrane proximal amino acids: PFPFSAQSGA
(SEQ ID
NO:1743). We tested various antibodies that were generated in animals by
immunizing with
the PSMGFR peptide for their ability to recognize cancer cells but not healthy
cells. Among
the most cancer selective were the MNC2 and MNE6 monoclonal anti-MUC1*
antibodies.
Two other monoclonal antibodies that were generated from immunizing animals
with the
PSMGFR peptide are MNC3 and MNC8. Although MNC2, MNE6, MNC3 and MNC8 all
bind to the PSMGFR peptide, like NME1 and NME7AB, MNC2 and MNE6 bind strongly
to
the N-10 peptide but not to the C-10 peptide. In fact, MNC2 and MNE6
competitively inhibit
the binding of NME1 and NME7AB to PSMGFR. Conversely, MNC3 and MNC8 are able
to
bind to the C-10 peptide, bind less well to the N-10 peptide and do not
compete with NME1
nor NME7AB for binding to MUC1* peptides, including PSMGFR (Fig. 70). MNC3 and
MNC8 are less cancer specific than MNC2 and MNE6. MNC3 and MNC8 recognize stem
and progenitor cells, such as hematopoietic stem cells, whereas MNC2 and MNE6
do not.
Because hematopoietic stem cells are the progenitor cells for the blood cells,
it would be
problematic to have a cancer therapeutic that would also target such an
important normal cell
type.
[00734] In this experiment, antibodies of the invention were tested for their
ability to
displace NME7AB from binding to the PSMGFR peptide. In this experiment, a
multi-well
plate was coated with the PSMGFR peptide. Recombinant NME7AB was allowed to
bind to
the surface-immobilized PSMGFR peptide. Wash steps followed. Various
antibodies were
added, followed by wash steps. The amount of NME7AB that remained attached to
the
PSMGFR coated plate, after antibody competition, was measured by detecting a
tag on the
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NME7AB. As a control, anti-NME7AB antibodies were also tested for their
ability to displace
NME7AB from the PSMGFR. Figure 70 shows a graph of an ELISA displacement
assay. The
bar graph is color coded to indicate the cognate epitope to which each
antibody binds. As can
be seen in the figure, the antibodies that bind to the more C-terminal
portions of PSMGFR
are the most potent at disrupting the binding of onco-embryonic growth factor
NME7AB to
the MUC1* extra cellular domain or the PSMGFR peptide. The rank order of
potency for
disrupting binding of NME7AB to PSMGFR according to their cognate epitope is
as follows:
FPFS> ASRYNLT> QFNQYKTEA>GTINVHDVET. Antibodies that bind to epitopes
outside of the PSMGFR peptide, such as 45C11, 8A9 and 17H6 did not compete
with
NME7AB for binding.
[00735] Western blot assay to determine linear versus conformational cognate
epitope
[00736] Antibodies were tested to determine whether they recognize a linear or
a
conformational epitope. Only antibodies that recognize a linear epitope work
in Western blots
when using denaturing gels. For comparison, known antibodies were tested for
their ability to
bind to HCT-116, a MUC1 negative cancer cell line, HCT-MUC1-18, which is a
cleavage
resistant clone of HCTs transfected with full-length MUC1, and HCTs
transfected with
MUC1*, wherein the extra cellular domain comprises only the PSMGFR sequence.
The
antibodies tested for comparison are MNC2 and MNE6, which were known to only
recognize
a conformational epitope, SDIX which is a polyclonal antibody raised against
PSMGFR and
VU4H5, which is a commercially available monoclonal antibody that recognizes
the tandem
repeats of full-length MUC1 (Fig. 71A-71D). As can be seen, neither MNC2 nor
MNE6
recognize a MUC1 or MUC1* specific linear epitope. Fig. 71A and 71E show that
antibody
20A10 also does not recognize a MUC1 or MUC1* specific linear epitope. The
SDIX
polyclonal antibody recognizes HCT-MUC1* but not full-length MUC1 and VU4H5
only
recognizes full-length MUC1. These same antibodies were also tested for their
ability to work
in Western blots of two breast cancer cell lines 1500, aka Zr-75-1, and T47D
cells and show
the same binding pattern (Fig. 71E-71H).
[00737] Antibodies that were raised against the PSMGFR peptide were tested the
same
way in Western blots (Fig. 72A-72P). As can be seen, antibodies 25E6 and 18B4
recognize
linear epitopes but 20A10 (Fig. 72A; 721), 3C2B1 (Fig. 72F; 72N), 5C6F3 (Fig.
72G; 720),
18G12 and 28F9 do not, indicating that they bind to a conformational epitope.
Antibodies
that were raised against the N+20/C-27 peptide were tested the same way in
Western blots
(Fig. 73A-73J). As can be seen, antibodies 31A1 and 32C1 recognize linear
epitopes.
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Antibodies 1E4 and 45C11 may recognize a conformational epitope. Antibodies
that were
raised against the N+9/C-9 peptide were tested the same way in Western blots
(Fig. 74A-
74H). As can be seen, none of these antibodies recognize linear MUC1 or MUC1*
specific
epitopes. These antibodies may recognize a conformational epitope. However, an
alternative
interpretation is that the lack of binding in a Western blot means that they
do not specifically
recognize MUC1 or a MUC1 cleavage product or that the concentration used in
this assay
was insufficient.
[00738] Recognition of a MUC1 cleavage product after cleavage by MMP9
[00739] We previously demonstrated that MNC2 recognizes a MUC1* that is
generated
when full-length MUC1 is cleaved by matrix metalloprotease 9, MMP9 (Fig. 37).
MMP9 is
expressed by tumor tissues and is a predictor of poor prognosis for breast
cancers (vant Veer
et al 2002; Dufour et al 2011). MMP9 has also been implicated in metastasis
(Owyong et al
2019). Recall also that MNC2 competitively inhibits the binding of onco-
embryonic growth
factor NME7An to the MUC1* extra cellular domain (Fig. 3). Therefore, it
follows that onco-
embryonic growth factor, which activates growth and survival functions of
MUC1*, also
recognizes a MUC1* generated by cleavage by MMP9. It then follows that the
most cancer
specific antibodies are those that recognize a conformational epitope formed
when MUC1 is
cleaved to MUC1* by MMP9.
[00740] Antibodies generated by immunization with PSMGFR, N+20/C-27, or N+9/C-
9
were tested for their ability to recognize M1JC1 after it is cleaved by MMP9.
To do this, we
transfected HCT-116, a MUC1 negative colon cancer cell line, with full-length
MUC1 and
isolated a single cell clone that is cleavage resistant; this cleavage
resistant cell line is called
HCT-MUC1-18. To HCT-MUC1-18 cells was added either a catalytically active MMP9
or
MMP2. The enzymes, added over a range of concentrations, were incubated with
the cells for
24 hours. The resultant cells were then incubated with the various antibodies
and analyzed by
FACS to determine which bound to a MUC1 cleavage product produced by cleavage
by
MMP9 (Fig. 75A-75N). Note that the first bar of each graph shows that none of
the
antibodies binds to full-length MUC1 in the absence of cleavage. Each bar
graph is labeled
with both the name of the antibody used in that assay and its cognate epitope.
The order of
the graphs from right to left corresponds to the distance from the cell
surface of the
antibody's cognate epitope. The antibodies that bind to the more C-terminal
epitopes within
PSMGFR peptide, such as 20A10 (Fig. 75E), 3C2B1 (Fig. 750) and 5C6F3 (Fig.
75P),
showed a concentration dependent increase in the binding to a MUC1 cleavage
product after
cleavage by MMP9 but not MMP2. Antibody 45C11, which binds to the SNIKFRPGSVV
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epitope, which is outside of the PSMGFR portion of MUC1, does not recognize a
MUC1
cleavage product after cleavage by MMP9 or MMP2 (Fig. 75K). Similarly,
antibodies 8A9
and 17H6 bind to the VQLTLAFRE epitope, which is also outside of the PSMGFR
sequence,
and they do not bind to a MUC1 cleaved by MMP9 or MMP2. This result is
consistent with
the idea that MMP9 cleaves MUC1 such that the extra cellular domain of the
remaining
transmembrane cleavage product comprises essentially the amino acids of the
PSMGFR
peptide. For the greatest degree of cancer specificity, the antibody should
recognize a
conformational epitope of a MUC1 cleavage product created when MUC1 is cleaved
by
MMP9. Of the antibodies shown in Fig. 75A-75N, only 20A10 recognizes the MUC1
cleavage product produced by cleavage by MMP9 and also does not work in a
Western blot,
indicating it recognizes a conformational epitope, as do MNC2 and MNE6.
Cleavage and
release of the massive tandem repeat domain of MUC1 unmasks the ectopic
binding site on
MUC1*; linear epitopes will be unmasked in addition to conformational
epitopes.
[00741] FACS analysis of binding to a panel of cancer cell lines
[00742] Fluorescence Activated Cell Sorting, FACS, was performed on reference
antibodies as well as new antibodies of the invention. FACS analyses of
reference antibodies
MNC2, "C2", and VU4H5 binding to either the MUC1-negative cell line HCT-116,
HCTs
transfected with MUC1*, "HCT-MUC1*", a cleavage resistant single cell clone of
HCTs
transfected with MUC1 full-length, "HCT-MUC1-18", and MNC2 binding to breast
cancer
cells line 14713 or breast cancer cell line 1500 also known as ZR-75-1, was
performed (Fig.
76A-76J). This analysis shows that MNC2 binds to an ectopic binding site on
the extra
cellular domain of MUC1*, which is only available after cleavage and release
of the bulk of
the extra cellular domain comprising the tandem repeat domain. VU4H5 binds to
hundreds of
repeating epitopes in the tandem repeat domain of full-length MUC1 and does
not bind to
MUC1*. Although we know that cancer cell lines express both full-length MUC1
and
MUC1*, antibodies against full-length MUC1 have, as yet, been shown to have no
therapeutic value. Stimuvax, ImMucin, IMGN242, SAR566658, PankoMab and AS1402
were all antibodies that bound to full-length MUC1 and all failed to show
efficacy in clinical
trials. MUC1*, and not full-length MUC1, is a potent growth factor receptor
that mediates the
growth of cancer cells (Mahanta et al 2008) and their resistance to
chemotherapy agents
(Fessler et al 2009). These studies showed that full-length MUC1 had no tumor
promoting
activity. Further, IHC studies show that as tumor stage increases, the amount
of MUC1*
increases as the amount of full-length MUC1 decreases (Fig. 54). In fact,
studies with tissue
micro arrays of breast cancers show that nearly 30% of breast cancer specimens
had no
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detectable full-length MUCI, compared to only 5% that were negative for MUC1*
(Fig. 10-
11). A point to consider for therapeutics that target full-length MUC1 is that
if cells
expressing full-length MUC1 are eliminated, that would simply enrich the tumor
population
for the more virulent MUCI* growth factor receptor expressing cells, which
would make the
cancers worse.
[00743] Reference antibody MNC2, "C2", was analyzed by FACS for its ability to
bind to
a panel of cancer cell lines that are all MUC1* positive, with the exception
of MDA-MB-231,
which expresses MUC1 and MUC1* at a level that is so low that it is often used
as a negative
control (Fig. 77A-77N). The panel of cancer cells that was probed with MNC2
included
T47D and 1500 breast cancer cells, NCI-H292 and NCI-H1975 lung cancer cells,
SKOV-3
ovarian cancer cells, HPAF-II and Capan-1 pancreatic cancer cells, DU145
prostate cancer
cells, and MDA-MB-231, breast cancer cells, which are nearly MUC1 and MUCI*
negative.
MNC2 robustly recognized a wide range of cancer cell lines. We note that
although MNC2
recognized HPAF-II pancreatic cells, it did not recognize another pancreatic
cell line, Capan-
1, as well. Similarly, MNC2 did not recognize prostate cancer cell line DU145
very well. In
IHC tissue studies, we found that MNC2 recognized about 57% of prostate cancer
tissues and
78% of pancreatic tissues, albeit with significant tumor heterogeneity.
[00744] Figure 78A-78C shows a color coded schematic of the PSMGFR sequence
that
has been extended or deleted at both the N- and C-termini. Antibodies of the
invention were
tested against this subset of peptides to further refine the epitopes to which
each antibody
binds or the critical amino acids within the epitope to which each antibody
binds. Fig. 78A is
an aligned schematic of the various subsets of peptides. Fig. 78B lists the
antibodies that bind
to each of the color coded sequences. Fig. 78C lists the cancer cell lines
that each antibody
recognizes.
[00745] Figures 80-87 show graphs of FACS analyses wherein antibodies of the
invention
are compared for their ability to specifically recognize different types of
cancer cells. Percent
cells recognized as well as the Mean Fluorescence Intensity, MFI, was
measured.
Considering only these FACS experiments, they show that only antibodies that
recognize the
PSMGFR peptide are able to recognize cancer cell lines. Antibodies that bind
to epitopes
outside of the PSMGFR sequence do not specifically recognize these cancer cell
lines.
[00746] IHC tissue studies of normal versus cancerous tissues to determine
true
cancer specificity
[00747] Immunohistochemistry, IHC, tissue studies of tissue micro arrays,
"TMAs", are a
more stringent test of the cancer specificity of antibodies than FACS analysis
of a single
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cancer cell line. Cancer cell lines are a single cell from a single patient
that have been
expanded in a lab for decades. Cell lines are limited in that they are not
representative of a
cross section of the human population. Further, after culturing the cell line
in vitro for
decades it may no longer look like the original cell. Also, there are no real
normal cell lines
for comparison, as they have to be made immortal. Tissue studies are more
informative
because each tissue micro array comprises tissues from multiple donors and the
cells are in
their natural environment, without years of culturing under non-physiologic
conditions.
Additionally, tissues provide information regarding tumor heterogeneity as
well as
information regarding normal patterns of expression. Each antibody of the
invention was
used to probe a normal tissue micro array, FDA Normal Array MN01021. In
addition the
antibody was also used to probe a panel of cancerous tissue arrays. In some
cases, antibodies
that showed strong staining of normal tissues, especially of critical organs
such as heart or
lung, were tested on a limited number of cancerous tissue arrays, since their
cross reactivity
to normal tissues eliminated them from consideration as anti-cancer
therapeutics.
[00748] Figures 113-200 show photographs of the IHC staining of normal TMAs
versus
cancerous TMAs for each antibody of the invention.
[00749] Figure 113-120 show photographs of tissues studies probed with
antibody 20A10.
Recall that 20A10 binds to the PSMGFR peptide, binds to the N-10 peptide, but
does not
bind to the C-10 peptide. Refined epitope mapping shows that like MNC2 and
MNE6, the
binding of 20A10 depends on amino acids PPP'S being present in the PSMGFR
peptide.
20A10 binds to the most membrane proximal part of the MUC1* extra cellular
domain. An
overview of FDA Normal Tissue Array MN01021 is shown in Figure 113. Figure
114A-
114X show that there is little to no cross reactivity of 20A10 for normal
tissues. We note that
MNC2, MNE6 and 20A10 all react with the MUC1* that is expressed on the luminal
edge of
the terminal breast ducts, luminal edge of the fallopian tubes, luminal edge
of about 10% of
the distal collecting ducts of normal kidney, and luminal edge of ureter.
Because the staining
is strictly limited to the luminal edge of a subset of ducts and glands, these
antibodies are
considered to be safe as therapeutics as the inside of ducts and glands are
protected from
large entities carried by blood, such as antibodies or CAR T cells.
Importantly, MNC2,
MNE6 and 20A10 show no staining of critical organs, such as heart, lung and
brain. In stark
contrast, 20A10, like MNC2 and MNE6, robustly binds to cancerous tissues.
20A10 stains
nearly all specimens of the BR1141 breast cancer array (Fig. 115-116). In
addition to robust
staining of the breast cancer tissue, the staining is membrane staining,
indicating that 20A10
recognizes an extra cellular portion of MUC1*, which is critical for an
effective antibody-
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based anti-cancer therapeutic. 20A10 also showed robust and membranous
staining of
pancreatic cancer tissues (Fig. 117-118) and esophageal cancer tissues (Fig.
119-120). In
summary, 20A10 shows great cancer specificity and as an anti-cancer
therapeutic offers a
large therapeutic window because of the vast difference between staining of
normal tissues
and cancerous tissues, in terms of the location and intensity of staining.
[00750] Anti-MUC1* antibody 3C2B1 is an antibody that like MNC2, MNE6 and
20A10,
binds to N-10 but not to C-10. More refined epitope mapping shows that like
these three
other highly cancer-specific antibodies, 3C2B1 requires the FPFS sequence for
binding to a
MUC1* extra cellular domain peptide. Figure 121 shows the photograph of the
FDA normal
array MN01021_ Figure 122A-122X shows photographs of specific tissues from FDA
normal tissue array MN01021 stained with the anti-PSMGFR antibody 3C2B1 at
20ug/mL.
As can be seen, there is no binding of 3C2B1 to any critical normal organs.
Figure 123
shows photograph of pancreatic cancer tissue array PA1003 stained with the
anti-PSMGFR
antibody 3C2B1 at 1-20ug/mL. Figure 124 shows photographs of specific tissues
from
pancreatic cancer tissue array PA1003 stained with the anti-PSMGFR antibody
3C2B1 at
20ug/mL. Figure 125 shows photograph of breast cancer tissue array BR1141
stained with
the anti-PSMGFR antibody 3C2B1 at 20ug/mL. Figure 126A-126F shows magnified
photographs of specific tissues from breast cancer tissue array BR1141 stained
with the anti-
PSMGFR antibody 3C2B1 at 20ug/mL. As can be seen in the figure, 3C2B1 robustly
stains
breast cancer tissues.
[00751] Anti-MUC1* antibody 5C6F3 binds to the N-10 peptide, does bind to the
C-10
peptide, although binding is reduced somewhat. Its cognate epitope comprises
all or some of
the sequence SVSDV (SEQ ID NO:1751). Figure 127 shows photograph of FDA normal
tissue array MN01021 stained with the anti-PSMGFR antibody 5C6F3 at lug/mL.
Figure
128 shows photographs of specific tissues from FDA normal tissue array MN01021
stained
with the anti-PSMGFR antibody 5C6F3 at lug/mL. Figure 129 shows photograph of
pancreatic cancer tissue array PA1003 stained with the anti-PSMGFR antibody
5C6F3 at 1-
20ug/mL. Figure 130 shows photographs of specific tissues from pancreatic
cancer tissue
array PA1003 stained with the anti-PSMGFR antibody 5C6F3 at lug/mL. Figure 131
shows
photograph of breast cancer tissue array BR1141 stained with the anti-PSMGFR
antibody
5C6F3 at lug/mL. Figure 132 shows photographs of specific tissues from breast
cancer
tissue array BR1141 stained with the anti-PSMGFR antibody 5C6F3 at lug/mL. As
can be
seen in the figure 5C6F3 is a high affinity antibody that has great cancer-
specificity and with
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the exception of adrenal, which may be an artefact of that tissue, did not
show binding to
normal tissues.
[00752] In contrast to 20A10, which binds to the most membrane proximal part
of the
MUC1* extra cellular domain, 18B4 binds within the GTINVHDVET sequence, which
is the
most distal part of the PSMGFR sequence. Unlike antibodies MNC2, MNE6 or
20A10, 18B4
cannot bind to the N-10 peptide but does bind to the C-10 peptide. Figure 133-
134 show the
binding of antibody 18B4 to normal tissues. In contrast to 20A10, antibody
18B4 shows
strong binding to a wide range of normal tissues (Fig. 134), including lung
(Fig. 134K).
Figure 135-138 show 18B4 staining of breast cancer tissues and esophageal
cancer tissues.
Because of the strong binding of 18B4 to normal tissues, there is less cancer
specificity to
this antibody.
[00753] Figure 139-144 show the binding of PSMGFR antibody 18G12 to normal
tissues,
breast cancer tissues and esophageal cancer tissues. 18G12 is able to bind to
the N-10
peptide, but is also able to bind to the C-10 peptide. 18G12 binds to the
ASRYNLT epitope
within the PSMGFR peptide. Antibody 18G12 binds to the luminal edge of many of
the
collecting ducts of normal kidney (Fig. 140D), binds to normal heart muscle
(Fig. 1401) as
well as to normal skeletal muscle (Fig. 140X). However, there is a clear
cancer specificity in
that 18G12 binds much more strongly to cancerous tissues than to the few
normal tissues. In
addition, 18G12 stains the entire cancerous tissues rather than just a luminal
edge here or
there. Figure 141-146 show 18612 staining of breast cancer tissues, pancreatic
cancer tissues
and esophageal cancerous tissues. The contrast between the staining of the
normal tissues and
the cancer tissues clearly demonstrates cancer specificity.
[00754] Figure 147-148 show the binding of PSMGFR antibody 25E6 to normal
tissues.
25E6 is able to bind to the N-10 peptide, but is also able to bind to the C-10
peptide. 25E6
binds to the ASRYNLT epitope within the PSMGFR peptide. Like MNC2, MNE6 and
20A10, antibody 25E6 binds to the luminal edge of terminal breast ducts,
luminal edge of
fallopian tubes, to the luminal edge of a subset of the distal collecting
ducts of normal kidney
and to the luminal edge of ureter. Unlike MNC2, MNE6 and 20A10, 25E6 binds,
albeit very
weakly, to normal heart muscle (Fig. 1481) as well as to normal skeletal
muscle (Fig. 148X).
However, there is a clear cancer specificity in that 25E6 binds much more
strongly to
cancerous tissues than to the few normal tissues. In addition, 25E6 stains the
entire cancerous
tissues rather than just a luminal edge here or there. Figure 149-152 show
25E6 staining of
breast cancer tissues and pancreatic cancerous tissues. The contrast between
the staining of
the normal tissues and the cancer tissues clearly demonstrates cancer
specificity.
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[00755] Figure 153-156 show the binding of PSMGFR antibody 28F9 to normal
tissues
and breast cancer tissues. 28F9 is able to bind to the N-10 peptide, but is
also able to bind to
the C-10 peptide. 28F9 binds to the ASRYNLT epitope within the PSMGFR peptide.
Like
MNC2, MNE6 and 20A10, antibody 25E6 binds to the luminal edge of terminal
breast ducts,
luminal edge of fallopian tubes, to the luminal edge of a subset of the distal
collecting ducts
of normal kidney and to the luminal edge of ureter. Figure 155-156 show 28F9
staining of
breast cancer tissues..
[00756] Figure 157-158 show the binding of the N+20/C-27 antibody 1E4 to
normal
tissues. 1E4 is able to bind to the N-10 peptide but also is able to bind to
the C-10 peptide.
1E4 binds to the QFNQYKTEA sequence which is within the PSMGFR sequence.
Examination of the entire normal tissue micro array (Fig. 157A) shows that
antibody 1E4
binds to many normal tissues, including brain, cerebellum, all 3 liver
specimens, pancreas,
parathyroid, spinal cord and skeletal muscle. Magnified images show that 1E4
stains heart
(Fig. 1581) as well. 1E4 staining of a breast cancer array (Fig. 159-160)
shows that there is
some cancer specificity.
[00757] Figure 161-162 show the binding of the N+20/C-27 antibody 29H1 to
normal
tissues. 29H1 binds within the GTINVHDVET sequence, which is the most distal
part of the
PSMGFR sequence. Unlike antibodies MNC2, MNE6 or 20A10, 29H1 cannot bind to
the N-
peptide but does bind to the C-10 peptide. Examination of the entire normal
tissue micro
array (Fig. 157A) shows that even at concentration as low as 0.5 ug/mL,
antibody 29H1
strongly stains a wide range of normal tissues, including brain, heart, liver
and lung. 29H1
staining of a breast cancer array (Fig. 163-164) and staining of a pancreatic
cancer tissue
array (Fig. 165-166) shows that there is no cancer specificity.
[00758] Antibody 31A1 is similar to 29H1 in that they are both N+20/C-27
antibodies that
bind within the GTINVHDVET (SEQ ID NO:1746) sequence, which is the most distal
part
of the PSMGFR sequence. Unlike antibodies MNC2, MNE6 or 20A10, neither 31A1
nor
29H1 can bind to the N-10 peptide but do bind to the C-10 peptide. Examination
of the entire
normal tissue micro array and the magnified images (Fig. 167-168) shows that
even at
concentration as low as 0.5 ug/mL, antibody 31A1 strongly stains a wide range
of normal
tissues, including brain, heart, lung, spleen, bone marrow, and skeletal
muscle. 31A1 was
used to stain a breast cancer array, (Fig. 169-170). 31A1 was used over a
range of
concentrations to stain a pancreatic cancer tissue array (Fig. 171-172). These
figure shows
that 3 1A1 has insufficient cancer specificity.
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[00759] Antibody 32C1 is similar to 29H1 and 31A1 in that they are all N+20/C-
27
antibodies that bind within the GTINVHDVET sequence, which is the most distal
part of the
PSMGFR sequence. Unlike antibodies MNC2, MNE6 or 20A10, none of 32C1, 31A1 or
29H1 can bind to the N-10 peptide but all do bind to the C-10 peptide.
Examination of the
entire normal tissue micro array and the magnified images (Fig. 173-174) shows
that even at
concentration as low as 0.25 ug/mL, antibody 32C1 strongly stains a wide range
of normal
tissues, including brain, heart, lung, liver, spleen and bone marrow. 32C1 was
also used to
probe a breast cancer array (Fig. 175-176). 32C1 was used over a range of
concentrations to
stain an esophageal cancer tissue array (Fig. 177-178). Taken together, these
figures show
that 32C1 has insufficient cancer specificity.
[00760] Antibody 45C11 is an N+20/C-27 antibody that binds to epitope
SNIKFRPGSVV
(SEQ ID NO: 744) that is 20 amino acids outside of the PSMGFR sequence at the
N-
terminal end. 45C11 does not bind to the N-10 peptide. Normal tissue array FDA
MN01021
was stained with 45C11 at 12.5ug/mL (Fig. 179-180). As can be seen in the
figures, 45C11
shows strong binding to many normal tissues, including brain, heart, lung,
liver, spleen,
skeletal muscle and bone marrow. 45C11 was used over a range of concentrations
to stain a
breast cancer tissue array (Fig. 181-182). 45C11 was also used to stain a
pancreatic cancer
tissue array (Fig. 183-184). Taken together, these figures show that 45C11 has
no cancer
specificity.
[00761] Antibody 3C5 is an N+9/C-9 antibody that binds to epitope GT1N VHD
VET. Like
the other antibodies that bind to this epitope such as 32C1, 29H1 and 31A1,
they bind to the
most distal, that is to say the most N-terminal, part of the PSMGFR sequence.
Unlike
antibodies MNC2, MNE6 or 20A10, none of 3C5, 32C1, 31A1 or 29H1 can bind to
the N-10
peptide but all do bind to the C-10 peptide. Examination of the entire normal
tissue micro
array, where 3C5 was used at lOug/mL, and the magnified images (Fig. 185-186)
shows that
antibody 3C5 strongly stains some normal tissues, including brain, heart,
adrenal gland and
bone marrow. 3C5 was also used to probe a pancreatic cancer array at lOug/mL,
(Fig. 187-
188). Taken together, these figures show that 3C5 has no cancer specificity.
[00762] Antibody 8A9 is an N+9/C-9 antibody that binds to epitope VQLTLAFRE
which
is outside of the PSMGFR sequence. Antibody 8A9 cannot bind to the N-10
peptide. Normal
tissue array FDA MN01021 was stained with 8A9 (Fig. 189-190). As can be seen
in the
figures, like antibody 45C11, which also binds an epitope that is N-terminal
beyond the
PSMGFR sequence, antibody 8A9 shows strong binding to many normal tissues,
including
adrenal, brain, heart, lung, liver, spleen, skeletal muscle and bone marrow. A
pancreatic
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cancer array stained with antibody 8A9 showed weak binding to a subset of
pancreatic cancer
tissues (Fig. 191-192). Taken together, these figures show that 8A9 has no
cancer specificity.
[00763] Antibody 17H6 is an N+9/C-9 antibody that binds to epitope VQLTLAFRE,
which is outside of the PSMGFR sequence. 17H6 was used to stain normal tissue
array
MN01021. Examination of the entire normal tissue micro array and the magnified
images
(Fig. 193-194) shows that antibody 17H6 stains some normal tissues, including
brain, heart,
adrenal gland, bone marrow and skeletal muscle. 17H6 was used to probe a
pancreatic cancer
array and showed weak binding to most pancreatic cancer tissues (Fig. 195-
196). However,
the binding of 17H6 to several normal tissues of critical organs shows that
17H6 has little
cancer specificity.
[00764] Antibody 39H5 is an N+9/C-9 antibody that binds weakly to the intact
PSMGFR
peptide but not significantly to any of the subset peptides. 39H5 may bind to
the
GTINVHDVET, which is the most distal part of the PSMGFR sequence. Examination
of the
entire normal tissue micro array and the magnified images (Fig. 197-198) shows
that
antibody 39H5 stains some normal tissues, including brain, heart, liver and
bone marrow.
39H5 was used to probe a pancreatic cancer array, (Fig. 199-200). Although
39H5 stained a
good percentage of the pancreatic cancer specimens, considering the normal
tissues that
39H5 stained, 39H5 has little cancer specificity.
[00765] Summary of FACS analysis
[00766] Determining the cancer specificity of antibodies using cell lines is
difficult, as
these cells were obtained from a single patient's tumor decades ago, and then
propagated in
culture for decades. Even if the patient's tumor was at one point
heterogeneous, the decades
of in vitro culture have essentially made the cell line a single cell clone.
Antibodies of the
invention were assayed by FACS to determine if they bound to MUC1 or MUC1*
positive
cancer cells but not MUC1 negative cells. The results of these experiments are
shown in
Figures 76-87. What is very clear is that antibodies that bind to epitopes of
the MUC1
sequence that are outside of and N-terminal to PSMGFR sequence show no cancer
specificity. Referring now to the readings of Mean Fluorescence Intensity
(MFI) it appears
that antibodies with cognate epitopes at the very N-terminus of the PSMGFR
sequence, such
as those that bind to an epitope within GTINVHDVET, show far less cancer
specificity than
the antibodies that recognize more C-terminal epitopes. For example, antibody
MNC2 that
will not bind to the C-10 peptide binds strongly to nearly every MUC1*
positive cell line
(Fig. 76-77). However, closer examination reveals that MNC2 binds lung cancer
line NCI-
H1975 much more strongly than NCI-H292. Similarly, MNC2 binds pancreatic cell
line
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HPAF-II much better than Capan-1 or prostate cancer line DU145. PCR
measurements show
that the expression levels of cleavage enzymes varies greatly across a panel
of cancer cell
lines (Fig. 43 and Fig. 44 ). The fold of the MUC1* extra cellular domain can
vary greatly
depending on which cleavage enzyme clips it, which likely accounts for
differences between
cancer cell lines that a single antibody recognizes. This variation in
antibody recognition of
various cell lines, even within a cancer sub-type is apparent in the figures.
[00767] Summary of IHC data
[00768] IHC analysis of real tissues, including both normal and cancerous
tissues, is more
informative than the study of cultured cell lines, as is necessary in FACS
analysis. Each
antibody was first tested over a range of concentrations to determine optimal
concentration.
Antibody concentration was increased until the stroma also picked up stain,
which indicates
non-specific background binding. The optimal concentration for that particular
antibody was
then deemed to be just below the concentration at which the antibody stained
the stroma.
[00769] An overview of the IHC tissue studies is shown in Figure 88-112. Here,
we
focused on the binding of antibodies to critical organ tissues, since binding
to certain normal
tissues would likely eliminate therapeutic use of that antibody. In these
figures, the antibodies
were grouped according to their cognate epitope. What is evident from the
tissue studies is
that the further the epitope is from the cell membrane, the more it binds to
normal MUC1 on
normal tissues. For example, binding to normal heart tissue by representative
antibodies that
recognize a specific epitope are shown in Figure 88A-88L. As the figure
illustrates,
antibodies that bind to epitopes that are N-terminal to the PSMGFR peptide
such as epitope
within SNIKFRPGSVV or VQLTLAFRE show such strong binding to normal heart that
they could not be used in therapeutics. In addition, antibodies that bind to
the more N-
terminal portion of PSMGFR, such as 29H1, also show binding to normal heart.
The
antibodies with the least binding to normal tissues and the strongest binding
to cancerous
tissues bind to epitopes within the FPFS or PFPFSAQSGA. Some antibodies that
bind to
epitopes within the ASRYNLT portion may also be suitable as therapeutics.
These antibodies
and others that recognize the same epitopes are desirable as anti-cancer
therapeutics because
they have a large therapeutic window, meaning that because of the low binding
to normal
tissues, and low side effects, patients can be dosed with antibody levels high
enough to
effectively kill the tumor cells. More detailed photographs of antibodies of
the invention
binding, or not binding, to other critical tissues are also shown. Figures 89-
94 show
magnified photographs of each antibody binding to normal heart tissue, where
the antibodies
have been categorized according to which epitope they bind. Figures 95-100
show magnified
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photographs of each antibody binding to normal liver tissue, where the
antibodies have been
categorized according to which epitope they bind. Figures 101-106 show
magnified
photographs of each antibody binding to normal lung tissue, where the
antibodies have been
categorized according to which epitope they bind. Figures 107-112 show
magnified
photographs of each antibody binding to normal bone marrow, where the
antibodies have
been categorized according to which epitope they bind.
[00770] The results of the 1HC studies (Fig. 88-Fig. 200) are summarized in
Table 3.
Table 3: Summary of Antibody Cross-Reactivity to Normal Tissues
Antibody BONE
SKELETAL
Name HEART BRAIN LUNG LIVER SPLEEN MARROW KIDNEY MUSCLE
ADRENAL
PSMG FR
MNC2
MNE6
20A10
25E6 ¨+ ¨+
18B4 +-F-'-
18G12 ++ +++ +++
28F9 ¨+
3C2B1
5C6F3 ¨+ ++
++++
N+20/C-
27
1E4 ++ ++
31A1 ++ +++ ++ ++++ ++++
32C1 +-F+ +++ +++ ++ +++ ++++
++
29H1 + ++++ ++++ ++++ ++
++++
45C 11 +++++ ++++ ++ +++++ ++ +++ + õ +++++
"''mm""mmmmmmmMAIMMonnmmmumommmpammmpemnvnffffffnffHEaNiAgEnagEME
N+9/r---
8A9 +++++ +++++ +++ +++++ ++++ ++++ ++++
+++
17H6 +++ ++++
++
3C5 +++ ++++ ++++
+++
39H5 ++++ +++++ + +++ +++ ++++
++++
[00771] As can be clearly seen in the table, the further away from the cell
membrane that
the antibody binds, the more non-specific binding there is. Although these
antibodies were
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generated by immunizing with the PSMGFR peptide, N+20/C-27 peptide or the
C+9/C-9
peptide, some of the antibodies generated by immunizing with an extended
peptide still bind
within the PSMGFR sequence, see Figures 63-69 for the details of epitope
binding for each
antibody. Some binding to normal tissues can be tolerated if the antibody is
incorporated into
an appropriate therapeutic format. For example, cellular therapies, such as
CAR T, are carried
by the blood and meet with physiological barriers including lamina propia and
blood-brain
barrier that limits the cell's access to luminal edge of ducts and glands.
Other antibodies that
bind much more strongly to cancerous tissues but do show some binding to
normal tissues
could also be safe and useful therapeutics if administered locally or if
cancer-specificity is
enhanced by incorporating into a bispecific antibody. However, widespread
antibody binding
to many normal organs or to essential organs for which there is no physical
barrier could be
lethal to the patient.
[00772] The most cancer-specific antibodies with little to no binding to
normal tissues are
MNC2, MNE6, 20A10, 3C2B1 and 25E6. An ideal antibody therapeutic is one that
stains no
normal tissues but robustly stains cancer cells. Unfortunately, cancer
antigens are also
expressed on normal tissues, so zero staining of normal tissue is not
possible. The aim is to
identify an antibody that binds much more strongly to tumor tissue than normal
tissue and
that either binds to non-critical normal tissues or binds to them in a way
that would not be
physiologically possible in an intact organ. For example, CAR T cells are
carried by the
blood and the lamina propia is a barrier to their getting to the luminal edge
of a duct or gland.
Similarly, the blood brain barrier prevents the passage of large molecules
like antibodies from
the blood into the brain. The usefulness of an antibody as a therapeutic also
depends on the
format of the therapeutic. As mentioned, cell based therapies have natural
barriers that
prevent the CAR T cells from getting to some normal tissues. Antibody Drug
Conjugate
(ADC) based therapies sometimes depend on a local, cancer-specific molecule to
activate the
toxin attached to the antibody, minimizing the importance of whether or not a
naked antibody
binds to some normal tissue. In another example, antibodies and antibody-based
therapeutics
can be administered locally, including intraperitoneally, to maximize the
effect on tumor cells
while minimizing their effect on normal tissues. In yet another example, an
antibody that is
not purely cancer-specific can be made more cancer-specific if it is
incorporated into a
bispecific antibody where a first side of the molecule binds to a first cancer
antigen and the
second side of the molecule binds to a second antigen that may be a tissue
specific antigen,
another cancer specific antigen or even an antigen on a cell such as a T cell,
which are called
BiTES, bispecific T cell engagers. In yet another example, the less cancer-
specific antibody
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can be incorporated into a cell-based therapy where its expression is induced
only after the
cell recognizes a tumor. In one aspect, a CAR T cell can express a first CAR
that recognizes a
first antigen which recognition induces expression of a second antibody, or
CAR
incorporating the second antibody. In one aspect the cell expresses a CAR
directed by an
antibody fragment that is cancer-specific and a second antibody or CAR
expressing the
second antibody is induced to be expressed in an NFAT inducible system. In one
aspect the
nucleic acids encoding the second antibody or second CAR are down stream of
NFAT
response elements. The NFAT inducible gene may be inserted into a Foxp3
enhancer or
promoter.
[00773] Figure 202 shows photographs of pancreatic cancer tissues, each from a
different
patient. As can be seen, the staining pattern of 1E4 is very different from
that of 18B4 and the
polyclonal antibody SDIX. 18B4 and SDIX antibodies were generated by
immunizing
animals with the same peptide (PSMGFR), while the 1E4 antibody was generated
from
immunization with a different peptide (N+20). Figures 203-207 show magnified
images of
selected tissues from this array to highlight the differences between these
antibodies. Figure
208 compares the staining of polyclonal antibody SDIX to monoclonal antibody
20A10,
which were both generated from immunization with the PSMGFR peptide. Also
shown is the
difference in staining pattern for antibody 29H1 which was generated by
immunization with
an N+20 peptide. Although the antibody staining is lighter, antibody 29H1
recognizes more
pancreatic cancer tissue specimens than the SDIX polyclonal or 20A10. Figure
209 shows
that esophageal cancers are better recognized by antibodies that bind to a
MUC1* peptide
with an extended N-terminus, such as antibody 29H1 and antibody 31A1.
Similarly, Figure
210 shows that prostate cancers are better recognized by antibodies that bind
to a MUC1*
peptide with an extended N-terminus, such as antibody 29H1.
[00774] Below Table 4 shows a summary of the test criteria to determine the
cancer-
specificity of the various monoclonal antibodies.
Table 4: Cancer-Specificity Test Criteria
1 2 3 4 5
6 7
mAb Name Binds
Binds Does Displaces Does not Recognizes Cancer Cancer
PSMGFR N-10 not NME7AB recognize MUC1
selective selective
bind from linear after by by IHC
C-10 MUC1* epitope cleavage FACS
by MMP9
LUSDVSNSDVP ..;AQSGA
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MNC2 El El El El El El
El Et Et Et
MNE6 0 0 0 0 0 0 0
000
20A10 0 0 0 0 0 0 0
000
3C2B1 0 0 0 0 171 0 0
000
SV SD V
5C6F3 0 0 ¨23 0 0 0 0
00
ASRVNLT
25E6 0 0 Li 0 0 0 00
MNC3 0 --Al z E 0 21
ND 0
18G12 0 0 I 0 0 21 -
4 0
28F9 0 0 El 0 E --
V 0
QIINQYKTE.A.
1E4 0 0 Z El .R' 0 -
4 0
ii:I=11'1A1:11a ',-'.1:1I'
18B4 0 g Z --V 0 0
0
29H1 0 g 21 --\/
31A1 E1 .g E El N 0 E
Z
32C1 RI g :..1 --V N 0
..,,.3
39H5 0 g 0 ---\/ N23 E
23
3C5 El g El
8A9 g 0 N213
213 0
17H6 E g 0 E 21 E
23
SNIKFRPGSVV
45C11 g 0 i'.1 0 23
Z fg
[00775] To summarize, we found that antibodies that bound to sequences that
are N-
terminal to the PSMGFR sequence had no cancer-specificity. Further, the closer
to the cell
membrane that the antibody binds, the more cancer-specific is the antibody.
More
importantly, test criteria 1-4 or even 1-5 provide a set of rapid, multiplexed
and inexpensive
tests that can be performed on hundreds or thousands of impure hybridoma clone
supernatants to identify antibodies that are highly selective for cancer-
specific forms of
MUC1*.
[00776] Satisfies test criteria
[00777] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of a MUC1* positive cancer based on satisfying four (4) of the seven
(7) criteria set
out in Table 4. In a more preferred embodiment an antibody is chosen for the
treatment,
prevention or diagnosis of cancer based on satisfying five (5) of the seven
(7) criteria set out
in Table 4. In a yet more preferred embodiment an antibody is chosen for the
treatment,
prevention or diagnosis of cancer based on satisfying six (6) of the seven (7)
criteria set out in
Table 4. In a more preferred embodiment an antibody is chosen for the
treatment, prevention
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or diagnosis of cancer based on satisfying all seven (7) of the criteria set
out in Table 4. An
antibody selected for suitability as a treatment for MUC1* positive cancers by
virtue of
satisfying four or more of the criteria set out in Table 4 can be incorporated
in part or in
whole into several therapeutic formats. In one aspect of the invention, the
antibody or
antibody fragment is incorporated into a CAR that is then expressed in an
immune cell, which
may be a T cell, then administered to a patient who has been diagnosed with or
is at risk of
developing a MUC1* positive cancer. In another aspect of the invention, a
fragment of the
antibody which may be an scFv, is incorporated into a BiTE, then administered
to a patient
who has been diagnosed with or is at risk of developing a MUC1* positive
cancer. In yet
another aspect of the invention, a fragment of the antibody which may be an
scFv, is
incorporated into a bi-specific antibody, then administered to a patient who
has been
diagnosed with or is at risk of developing a MUC1* positive cancer. In yet
another aspect of
the invention, the antibody or antibody fragment, is conjugated to a toxin or
an ADC,
antibody drug conjugate, then administered to a patient who has been diagnosed
with or is at
risk of developing a MUC1* positive cancer.
[00778] Bind to N-10
[00779] We have demonstrated that a MUC1 transmembrane protein, devoid of
tandem
repeats and having an extra cellular domain of 45 amino acids of PSMGFR
sequence, is
sufficient to function as a growth factor receptor and confers oncogenic
characteristics to the
cell (Mahanta et al 2008). Antibodies that bind to the PSMGFR peptide or
portion of a
transmembrane MUC1 cleavage product can be cancer specific but may also bind
to stem or
progenitor cells. Antibodies that bind to the N-10 peptide are more cancer-
specific. In a
preferred embodiment an antibody is chosen for the treatment, prevention or
diagnosis of
cancer based on the ability of the antibody to bind to the N-10 peptide.
[00780] Do not bind to C-10
[00781] We have demonstrated that the MUC1 extra cellular domain contains an
ectopic
binding site that is only exposed if the tandem repeat domain is missing,
which can occur as a
consequence of alternative splice variant or cleavage and release of the extra
cellular domain.
Cancer-specific antibodies MNC2 and MNE6 will not bind to full-length MUC1,
but do bind
to the remaining portion when MUC1 is cleaved and the tandem repeat domain is
shed.
MNC2 and MNE6 will bind to a MUC1*-like protein if it is devoid of tandem
repeats, for
example if a MUC1 negative cell is transfected or transduced with an
engineered MUC1 that
is devoid of tandem repeats, especially if extra cellular domain comprises the
PSMGFR.
Thus, the ectopic site to which MNC2 and MNE6 bind is unmasked when tandem
repeat
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domain is missing or removed. Both MNC2 and MNE6 require the 10 membrane
proximal
amino acids of a MUC1* extra cellular domain for binding; they do not bind to
the C-10
peptide. That means that the ectopic binding site for MNC2 and MNE6 is within
or contains
all or part of the 10 C-terminal amino acids of the PSMGFR: PFPFSAQSGA. In a
preferred
embodiment an antibody is chosen for the treatment, prevention or diagnosis of
cancer based
on the inability of the antibody to bind to the C-10 peptide. In a preferred
embodiment an
antibody is chosen for the treatment, prevention or diagnosis of cancer based
on the ability of
the antibody to bind to the N-10 peptide and the inability of the antibody to
bind to the C-10
peptide.
[00782] Compete with NME7AB or NME7-X1 for binding to MUC1* positive cell,
PSMGFR peptide or N-10 peptide
We have demonstrated that cancer-specific antibodies MNC2 and MNE6 bind to an
ectopic
epitope that comprises all or part of the 10 C-terminal amino acids of the
PSMGFR peptide:
PFPFSAQSGA. We have shown that growth factors, dimeric NME1 and NME7AB, also
bind
to an ectopic epitope that comprises all or part of the 10 C-terminal amino
acids of the
PSMGFR peptide. MNC2 and MNE6 compete with dimeric NME1 or NME7AB for binding
to the PSMGER peptide and the N-10 peptide. In a preferred embodiment an
antibody is
chosen for the treatment, prevention or diagnosis of cancer based on the
ability of the
antibody to disrupt the binding of NME1, NME7AB, or NME7-X1 to the PSMGFR
peptide,
the N-10 peptide, or to the surface of a M1JC1* positive cancer cell.
[00783] Recognize a conformational epitope rather than a linear epitope
[00784] Antibodies that are cancer-specific will be chosen based on their
ability to bind to
a MUC1 that is devoid of tandem repeats and for their inability to bind to
full-length MUC1.
Most often, MUC1* is generated when MUC1 is cleaved by a cleavage enzyme and
the
tandem repeat domain is released from the cell surface. Cleavage and release
of the tandem
repeat domain may also unmask portions of MUC1*-like cleavage products that
exist on
normal tissues. However, antibodies that recognize a conformation, rather than
a linear
epitope, are more selective. Antibodies that recognize a conformational
epitope rather than a
linear epitope can be identified by a variety of means. In particular,
antibodies that recognize
a conformational epitope will not work in a denaturing Western blot assay. In
a preferred
embodiment an antibody is chosen for the treatment, prevention or diagnosis of
cancer based
on the ability of the antibody to recognize a conformational epitope.
[00785] Recognize a MUC1* generated by cleavage by MMP9 or other tumor-
associated cleavage enzyme
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[00786] The fold, or conformation, of the MUC1* truncated extra cellular
domain differs
depending on which enzyme cleaves MUC1. Cleaved MUC1* or MUC1 *like cleavage
products can function as growth factor receptors on normal healthy tissues.
More than one
cleavage enzyme is able to cleave MUC1 to a MUC1*-like form. Cleavage by first
enzyme
may produce a conformation or a fold that is not the same as that produced by
cleavage by a
second enzyme. Support for this can be found in this application and is
illustrated in Figures
39-41. These figures show that although a polyclonal antibody that binds to
PSMGFR
recognizes a cleaved MUC1 on hematopoietic stem cells, some monoclonal
antibodies that
bind to the PSMGFR peptide can bind to this MUC1*-like form on hematopoietic
stem cells
while others cannot. For example, MNC3 readily recognizes this cleaved form of
MUC1 on
hematopoietic stem cells, but MNC2 and MNE6 do not. We know that MNC2 and MNE6
recognize a MUC1* that is produced by cleavage by MMP9 but not when it is
cleaved by
MMP2. MNC2 and MNE6 are cancer-specific while MNC3 is not, as it recognizes
stem and
progenitor cells. We also know that MMP9 is overexpressed in cancers. Bone
marrow, where
hematopoietic stem cells are made expresses nearly 2,500-times more MMP2 than
MMP9
(Fig. 65). MMP14 is another enzyme that cleaves MUC1 to a MUC1* growth factor
receptor
form (Fig. 38). In one aspect of the invention, an antibody is chosen for the
treatment,
prevention or diagnosis of cancer based on the ability of the antibody to
recognize a MUC1
cleavage product generated when MUC1 is cleaved by MMP14. In a preferred
embodiment
an antibody is chosen for the treatment, prevention or diagnosis of cancer
based on the ability
of the antibody to recognize a MUC1 cleavage product generated when MUC1 is
cleaved by
MMP9. In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on the ability of the antibody to recognize a MUC1
cleavage
product generated when MUC1 is cleaved by MMP9 and also recognizes a
conformational
epitope.
[00787] Binds to cancer cells more than normal cells
[00788] A traditional approach to identifying antibodies that are cancer-
specific involves
testing a panel of antibodies against a panel of different cancer cell lines
and determining, by
FACS, IF, immunoprecipitation or other method, if the antibody binds to cancer
cells.
Although this approach is traditional, it is sequential and time-consuming,
and thus limits the
analysis of large numbers of monoclonal antibody clones, which is required to
find an ideal
antibody suitable for cancer therapeutic or diagnostic. In addition, there are
no real normal
cell lines and the selection of normal primary cells is limited. The selection
criteria presented
above provide a rapid, multiplexed method for identifying monoclonal antibody
clones that
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are specific for MUC1* positive cancers. For many of the selection criteria,
hybridoma
supernatants can be used. This provides a huge advantage over state of the art
methods for
identifying antibodies that are specific for MUC1* positive cancers. The
ability to select
antibodies from assay performed using the impure hybridoma supernatants means
that much
of the selection can be done on hundreds or thousands of clones rapidly and at
very little cost.
Methods such as FACS analysis ans IHC tissue studies require the use of
purified antibodies
which limits the number of clones that can be tested to tens, not even
hundreds.
[00789] However, selecting an antibody based on its ability to bind to cancer
cells, or a
cancer cell type or to a cell engineered to express a certain antigen is
important for antibody
selection. In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on the ability of the antibody to bind to MUC1*
positive cancer
cells.
[00790] Binds to tumor tissue more than normal tissue
[00791] Immunohistochemistry, IHC, tissue studies of cancerous versus normal
tissues is a
more stringent test of the cancer specificity of antibodies than FACS
analysis. Cancer cell
lines are a single cell from a single patient that have been expanded in a lab
for decades and
are not representative of a cross section of the human population. Further,
analysis of cell
lines is blind to the heterogeneity of actual tumors. Tissue studies require
purified antibody,
are very expensive, time-consuming and require a skilled pathologist to
analyze each stained
tissue specimen. However, antibody staining of tissues from normal tissues
versus cancerous
tissues can reveal which antibodies cannot be used as therapeutics or
diagnostics because of
their cross-reactivity with normal tissues. Our systematic studies of numerous
antibodies with
thousands of human normal tissues or cancerous tissues, across several cancer
sub-types
showed that antibodies that bind to N-10, not C-10, disrupt the binding of
NME1 or
NME7An, or NME7-X1 to the PSMGFR peptide, the N-10 peptide, or to the surface
of a
MUC1* positive cancer cell, recognize a conformational epitope, and recognize
a
conformational epitope created by cleavage by MMP9 are the most cancer-
specific.
[00792] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on the ability of the antibody to bind to MUC1*
positive tumor
tissue at least 2-times more than it binds to normal tissues. In a preferred
embodiment an
antibody is chosen for the treatment, prevention or diagnosis of cancer based
on the ability of
the antibody to bind to MUC1* positive tumor tissue at least 5-times more than
it binds to
normal tissues. In a preferred embodiment an antibody is chosen for the
treatment, prevention
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or diagnosis of cancer based on the ability of the antibody to bind to MUC1*
positive tumor
tissue at least 10-times more than it binds to normal tissues.
[00793] Antibodies that bind to refined epitopes
[00794] In a preferred embodiment, an antibody, or fragments thereof, that
binds to a
peptide comprising the sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA are
incorporated into anti-cancer therapeutics or diagnostics for the diagnosis,
treatment or
prevention of a MUC1* positive cancer.
[00795] In a more preferred embodiment, an antibody, or fragments thereof,
that binds to a
peptide comprising the sequence ASRYNLTISDVSVSDVPFPFSAQSGA are incorporated
into anti-cancer therapeutics or diagnostics for the diagnosis, treatment or
prevention of a
MUC1* positive cancer.
[00796] In a yet more preferred embodiment, an antibody, or fragments thereof,
that binds
to a peptide comprising the sequence SDVSVSDVPFPFSAQSGA are incorporated into
anti-
cancer therapeutics or diagnostics for the diagnosis, treatment or prevention
of a MUC1*
positive cancer.
[00797] In a still more preferred embodiment, an antibody, or fragments
thereof, that binds
to a peptide comprising the sequence SVSDV are incorporated into anti-cancer
therapeutics
or diagnostics for the diagnosis, treatment or prevention of a MUC1* positive
cancer.
[00798] In a yet still more preferred embodiment, an antibody, or fragments
thereof, that
binds to a peptide comprising some or all of the sequence PI-PFSAQSGA are
incorporated
into anti-cancer therapeutics or diagnostics for the diagnosis, treatment or
prevention of a
MUC1* positive cancer. As an anti-cancer treatment, the selected antibodies or
fragments
thereof are incorporated into a CAR, BiTE, ADC or bi-specific and then
administered to a
patient diagnosed with or at risk of developing a MUC1* positive cancer.
[00799] Consensus Sequences
[00800] Antibodies of the invention were categorized according to cognate
epitope.
Sequences of their respective heavy chain CDRs are shown in Table 5. Sequences
of their
respective light chain CDRs are shown in Table 6. Consensus sequences for
CDR1, CDR2
and CDR3 for each epitope-specific set of antibodies were computer generated.
Figure 215
and Figure 216 show how the CDR consensus sequences change as the position of
the
antibodies' cognate epitope moves from the membrane-proximal portion of PSMGFR
toward
the more distal portions.
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[00801] As can be seen in Table 5 and Table 6, the sequences for CDR1 and CDR2
for
antibodies that bind to epitopes within the 10 membrane-proximal (C-terminal)
portion of
PSMGFR peptide closely adhere to the consensus sequence.
[00802] Table 5: HEAVY CHAIN CDRs
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA
Epitope Name CDR1 CDR2 CDR3
FPFS MNC2 FTFSGYAMS TI
SSGGTYIYYPDSVKG -LGGDNYYEYFDV- -
FP F S MNE6 FTFSRYGMS TI
SGGGTYIYYPDSVKG DNYGRNYDYGMDY- -
S 20A1 FTFSTYAMS -
SIGRAGSTYYSDSVKG -- -GP IYNDYDEFAY
0
FPFS 3C23 ITFSTYTMS TT
STGGEKTYYSDSVKG -GTTAMYYYANDY-
1
Consensus 11;:74NY A'
Sequence I I IVI=;,1:9P.05.1 E
= -5
SVSDV 5C 6E' FTFSTYAMS AI
SNGGGYTYYPDSLKG RYYDHYFDY
3
ASRYNLT 25E6 FTFSSYGMS TI
SNGGRHTFYPDSVKG QTGTEGWFAY
ASRYNLT MNC3 YRFTDYAMN VT
STFSGNTNFNOKFKG SDYYGPYFDY
ASRYNLT 18G1 YTFTGYFLY GINPDNGGIDFNEKERN --
LIGNY
2
ASRYNLT 28F9 YTFTGYFLY GI HP
SNGDTDFNEKFKN --LI GVY
Consensus F 2:
I
'Pek
Sequence ;;r7- - ,t;1µ = =
-
QFNQYKTEA 1E4 YAESTYWMN QIYPGDSDTNYNGKFKG GNHASMDY
GTINVHDVET 18B4 FTFNDAWMD EIRSTANIHTTYYAESVQ LLYGFAY
GTINVHDVET 29H1 FTF SDAWMD E I RSKATNHATYYAE SVK LLYGFAY
GTINVHDVET 31A1 YTFTSYWMH AYIDY--
YINP STGYTEYNQKFKD
GTINVHDVET 32C1 FTFSNYWMN EIRLKSNNYAIHYAESVK VPGLDAY
GTINVHDVET 39E5 YTFTNYGMN --GI HGYVDY--
WINTYTGEP TYVGDFKG
GTINVHDVET 3C5 YTFTNYGMN -GGLDGYYGY-
WINTYTGHP TYADDFKG
Consensus
El I 4x. A-0 Fki" A
7 110,.:LiiLit)'01
Sequence '
Table 6: LIGHT CHAIN CDRs
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA
Epitope Name CDR1 CDR2
CDR3
FPFS MNC2 PASKS --VS TSGYSYMH
LASNLES QHSPELPFT
FPFS MNE 6 SATS SVSY TH STSNLAS
QQRSSSPFT
FPFS 20A10 KSSOSVLYSSNOKNYLA WAS
TRES -HOYLSSLT
FPFS 3C:2B1 RASKS - LASNLES OHSPELP LT
I ST SDYNYI
Consensus A
11 Sequence õ-
SVSDV 5C6F3 RSSQTIVHSNGNTYLE KVSNRF S FQD SHVP
LT
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ASRYNLT 25E6 KS SQS LLD SDGKTYLN LVSKLDS
WQGTHFPQT
ASRYNLT MNC3 RS SQT IVHSNGNTYLE KVSNRFS FQGSHVPF T
ASRYNLT 18G12 KS SQS LLHSDGKTYL I LVSKLDS
CQGTHFPWT
ASRYNLT 28F9 HS SQS LLITSDGKTYL I LVSKLDS
CQGTHFDWT
Consensus AU:1:1 ,e1
Sequence v
, .
QFNQYKTEA 1E4 RS SQS LVHSNGNTYLH KVSNRFS SQKTHVPWT
GTINVHDVET 18B4 RTSQSLVHSNGNTYLH KVSSRFS SQNTHVPYT
GT INVHDVET 29H1 RSGOSLVHSNGHTYLH KVSNRFS SOTTHVPWT
GT INVHDVET 31A1 RS SQS IVHSNGNTYLE KVSNRFS
FQVSHFPir7T
GT INVHDVET 32C1 RS SQS LVHSNGNTYLH KVSNRFS SQ I
THVPYT
GT INVHDVET 39H5 RS SQS IVHRNGNTYL- KVSNRFS FQGSHLPWT
GT INVHDVET 3C5 KS SQS LLHSKGKTYLN LVSKLES
LQTTHFPir7T
Consensus
.
Sequence
[00803] Whereas Heavy Chain CDR1 for MNC2 is FTFSGYAMS, with the amino acids
numbered from left to right 1 through 9, the consensus of other antibodies
that bind to that
portion of PSMGFR is: F or I at position 1, T at position 2, F at position 3,
S at position 4, T,
G, or R at position 5, Y at position 6, A, G or T at position 7, M at position
8 and S at
position 9.
[00804] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a heavy chain CDR1 that is at least 90%
identical to a
CDR1 comprising the following amino acids at the specified positions: F or I
at position 1, T
at position 2, F at position 3, S at position 4, T, G, or R at position 5, Y
at position 6, A, G or
T at position 7, M at position 8 and S at position 9.
[00805] Whereas Heavy Chain CDR2 for MNC2 is TISSGGTYIYYPDSVKG, with the
amino acids numbered from left to right 1 through 17, the consensus of other
antibodies that
bind to that portion of PSMGFR is: T at position 1, I or S at position 2, I or
S at position 3, G
or R at position 5, G or A at position 6, T or I at position 9, Y at position
10, Y at position 11,
P or S at position12 and DSVKG for positions 13-17.
[00806] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a heavy chain CDR2 that is at least 90%
identical to a
CDR2 comprising the following amino acids at the specified positions: T at
position 1, I or S
at position 2, I or S at position 3, G or R at position 5, G or A at position
6, T or I at position
9, Y at position 10, Y at position 11, P or S at position12 and DSVKG for
positions 13-17.
[00807] Whereas Heavy Chain CDR3 for MNC2 is -LGGDNYYEYFDV--, with the
amino acids numbered from left to right 1 through 15, the consensus of other
antibodies that
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bind to that portion of PSMGFR is: G, L, or N at position 2, G or T at
position 4, Y at
position 7, D or E at position 12, A at position 14, and Y at position 15.
[00808] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a heavy chain CDR3 that is at least 90%
identical to a
CDR3 comprising the following amino acids at the specified positions: G, L, or
N at position
2, G or T at position 4, Y at position 7, D or E at position 12, A at position
14, and Y at
position 15.
[00809] Whereas Light Chain CDR1 for MNC2 is RASKS--VSTSGYSYMH, with the
amino acids numbered from left to right 1 through 17, the consensus of other
antibodies that
bind to that portion of PSMGFR is: K or R at position 1, A or S at position 2,
S at position 3,
K or Q at position 4, S at position 5, V at position 6, L at position 7. T or
S at position 10, Y
at position 15, and I, L or M at position 16.
[00810] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a light chain CDR1 that is at least 90%
identical to a
CDR1 comprising the following amino acids at the specified positions: K or R
at position 1,
A or S at position 2, S at position 3, K or Q at position 4, S at position 5,
L or V at position 6,
L at position 7, T or S at position 10, Y at position 15, and I, L or M at
position 16.
[00811] Whereas Light Chain CDR2 for MNC2 is LASNLES, with the amino acids
numbered from left to right 1 through 7, the consensus of other antibodies
that bind to that
portion of PSMCiPR is: L or W, or S at position 1, A or '1 at position 2, S at
position 3, N or
T at position 4, L or R at position 5, E or A at position 6, and S at position
7.
[00812] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a light chain CDR2 that is at least 90%
identical to a
CDR2 comprising the following amino acids at the specified positions: L or W,
or S at
position 1, A or T at position 2, S at position 3, N or T at position 4, L or
R at position 5, E or
A at position 6, and S at position 7.
[00813] Whereas Light Chain CDR3 for MNC2 is QHSRELPFT, with the amino acids
numbered from left to right 1 through 9, the consensus of other antibodies
that bind to that
portion of PSMGFR is: Q at position 1, H or Q at position 2, S, Q or R at
position 3, R, S or
Y at position 4, E, L, or S at position 5, L or S at position 6, P or S at
position 7, F or L at
position 8 and T at position 9.
[00814] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a light chain CDR3 that is at least 90%
identical to a
CDR3 comprising the following amino acids at the specified positions: Q at
position 1, H or
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Q at position 2, S, Q or R at position 3, R, S or Y at position 4, E, L, or S
at position 5, L or S
at position 6, P or S at position 7, F or L at position 8 and T at position 9.
[00815] Another set of antibodies was generated and resultant clones were
tested for their
ability to bind to PSMGFR, N-10 and C-10 peptides. Antibody clones that bound
to
PSMGFR and N-10 peptides, but not to the C-10 peptide were selected. These
antibodies
were sequenced. Table 7 shows the sequences of the heavy chain CDRs for cancer-
specific
antibodies MNC2, MNE6, 20A10, 3C2B1, plus new antibodies B2, B7, 8C7F3, H11
and B9.
Table 8 shows the sequences of the light chain CDRs for cancer-specific
antibodies MNC2,
MNE6, 20A10, 3C2B1, plus new antibodies B2, B7, 8C7F3, H11 and B9. Consensus
sequences for the heavy and light chain CRDs were generated and are shown in
Table 7 and
Table 8. Although antibodies 5C6F3 and 25E6 showed great cancer specificity in
IHC tissue
studies and they both bound to the PSMGFR and N-10 peptides, but not to the C-
10 peptide,
epitope mapping showed that they bound to epitopes that were a bit N-terminal
to the
epitopes to which MNC2, MNE6, 20A10 and 3C2B1 bound. For this reason,
consensus
sequences were generated for MNC2, MNE6, 20A10, 3C2B1 and the new antibodies
plus
consensus sequences were generated for all the antibodies that bound to N-10
but not to C-10.
[00816] As can be seen in Table 7 and Table 8, the sequences for CDR1, CDR2
and CDR3
for antibodies that require for binding the 10 membrane-proximal (C-terminal)
amino acids of
PSMGFR peptide closely adhere to a common consensus sequence.
[00817] Table 7: HEAVY CHAIN CDRs for antibodies that sharc broader epitope in
that they cannot bind to the C-10 peptide
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GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA
Epitope Name CDR1 CDR2 CDR3
FPFS MNC2 FTFSGYAMS TISSGGTYIYYPDS -
LGGDNYYEY7DV--
VKG
FPFS MNE6 FTFSRYGMS TISGGGTYIYYPDS
DNYGRNYDYGMDY--
VKG
FPFS 20A10 FTFSTYAMS ---GPIYNDYDEFAY
SIGRAGSTYYSDSV
KG
FPFS 3C2B1 ITFSIYTMS TISTGGDKTYYSDS -
GTTAMYYYAMDY--
VKG
PFPFSAQS D2 FAFSTFAMS AISNGGGYTYYPDT RYYDLYFDL--
GA LKG
PFPFSAQS B7 FTFSRYGMS TISSGGTYIYYPDS DNYGSSYDYAMDY--
GA VKG
PFPFSAQS 8C7F3 FTFSIYAMS AISNGGGYTYYPDS RYYDHYFDY--
GA LKG
PFPFSAQS 1111 7A7STFAMS AISNGGGYTYYPDT RYYDLYFDL--
GA LKG
PFPFSAQS B9 FTFSRYGMS TISSGGTYIYYPDS DNYGSSYDYAMDY--
GA VKG
Consensu
AIVI Al
Sequence
- all of
antibodi
es above
SVSDV 5C6F3 FTFSIYAMS ATSNGGGYTYYPDS RYYDHYFDY
LKG
ASRYNLT 25E6 FTFSSYGMS TISNGGRHTFYPDS QTGTEGWFAY
VKG
Consensu I
Sequence
- all
ant ibodi
es
Table 8: LIGHT CHAIN CDRs for antibodies that share broader epitope in that
they
cannot bind to the C-10 peptide
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA
Epitope Name CDR1 CDR2 CDR3
FPFS MNC2 RASKS-- LASNLES QHSRELPFT
VSTSGYSYMH
FPFS MNE6 SISNLAS QQRSSSPFT
SATSSVSY:H
FPFS 20A10 KSSQSVLYSSNQKNYL WASTRES -HQYLSSLE
A
FPFS 3C2B2 RASKS--- LASNLES QHSRELPL:
ISTSDYNY:H
PFPFSAQSGA B2 RSSQNIV- KVSNRFS FQDSHVPLE
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HSNGNTYLE
PFPFSAQSGA 37 RSSQTIV- KVSNRFS FQDSHVPLE
HSNGNTYLE
PFPFSAQSGA 8C7F3 LASTLDS OONNEDPPE
RASE SVATYGNNFMQ
PFPFSAQSGA H11 RSSQNIV- KVSNRFS FQDSHVFLE
HSNGNTYLE
PFPFSAQSGA B9 TTSNLAS QQRSSYPF-
SASSSVSYMH
_
Consensus = , ,.õ:i;;:! :
j)k*Ho ,T
Sequence -
k
all of
antibodies
above
SVSDV 5C6F3 RSSQTIVHSNGNTYLE
KVSNRFS FQDSHVPLE
ASRYNLT 25E6 KSSOSLLDSDGKTYLN
LVSKLDS WOGTHFP0F.
Consensus
V
Sequence -
all
antibodies
[00818] Whereas Heavy Chain CDR1 for MNC2 is FTFSGYAMS, with the amino acids
numbered from left to right 1 through 9, the consensus sequence of MNC2, MNE6,
20A10,
3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is: F or I at position 1, T
or A at
position 2, F at position 3, S at position 4, T, G, or R at position 5, Y or F
at position 6, A, G
or T at position 7, M at position 8 and S at position 9. The underlined amino
acids at
positions 2 and 6 are the only additional variants to the consensus sequence
generated for
cancer-specific antibodies MNC2, MNE6, 20A10, 3C2B1 alone.
[00819] As can be seen in Table 7, the inclusion of antibodies 5C6F3 and 25E6
into the
generation of consensus sequence did not change in any way the consensus
sequence for
heavy chain CDR1 that describes a cancer-specific anti-MUC1* antibody.
[00820] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a heavy chain CDR1 that is at least 90%
identical to a
CDR1 comprising the following amino acids at the specified positions: F or I
at position 1, T
or A at position 2, F at position 3, S at position 4, T, G, or R at position
5, Y or F at position
6, A, G or T at position 7, M at position 8 and S at position 9.
[00821] Whereas Heavy Chain CDR2 for MNC2 is TISSGGTYIYYPDSVKG, with the
amino acids numbered from left to right 1 through 17, the consensus sequence
of MNC2,
MNE6, 20A10, 3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is:
[00822] T or A at position 1, I or S at position 2, I or S at position 3, N,
S, T or G at
position 4, G or R at position 5, G or A at position 6, G, T, or D at position
7, Y, K or S at
position 8, T or I at position 9, Y at position 10, Y at position 11, P or S
at position12 and D
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at position 13, S or T at position 14, V or L at position 15 and KG for
positions 16-17. The
underlined amino acids indicate how this more inclusive consensus sequence
differs from the
consensus sequence generated for MNC2, MNE6, 20A10 and 3C2B1 alone. Of the 17
amino
acids in heavy chain CDR2, the consensus sequence for all nine antibodies
differs from the
consensus sequence for the original cancer-specific four by only 4 amino
acids. Note that 2 of
the 4 variants are homologous changes, T for S and L for V, which generally do
not
significantly impact the structure or specificity of a protein.
[00823] As can be seen in Table 7, the inclusion of antibodies 5C6F3 and 25E6
into the
generation of consensus sequence for heavy chain CDR2 only changed the
consensus
sequence by the addition of two other possible amino acids: a possible H at
position 8, and a
possible F at position 10, for a heavy chain CDR2 that describes a cancer-
specific anti-
MUC1* antibody. We note that the change of Y to F at position 10 is a
homologous change,
which generally does not significantly impact the structure or specificity of
a protein.
[00824] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a heavy chain CDR2 that is at least 90%
identical to a
CDR2 comprising the following amino acids at the specified positions: T or A
at position 1, I
or S at position 2, I or S at position 3, N, S, T or G at position 4, G or R
at position 5, G or A
at position 6, G, T, or D at position 7, Y, K, H or S at position 8, T or I at
position 9, Y or F at
position 10, Y at position 11, P or S at position12 and D at position 13, S or
T at position 14,
V or L at position 15 and KG for positions 16-17.
[00825] Whereas Heavy Chain CDR3 for MNC2 is LGGDNYYEYFDV, with the amino
acids numbered from left to right 2 through 13, the consensus sequence of
MNC2, MNE6,
20A10, 3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is:
[00826] G, L, or N at position 2, G, T, or Y at position 3, G or T at position
4, A, D, P, R,
or S at position 5, Y, M, I or S at position 6, Y at position 7, D, Y, or N at
position 8, E, D,
Y, L or H at position 9, Y, A, or G at position 10, M, D or F at position 11,
D or E at position
12, V. F, Y or L at position 13, and AY at position 14-15. The underlined
amino acids
indicate how this more inclusive consensus sequence differs from the consensus
sequence
generated for MNC2, MNE6, 20A10 and 3C2B1 alone. Of the 15 amino acids in
heavy chain
CDR3, the consensus sequence for all nine antibodies differs from the
consensus sequence
for the original cancer-specific four by 7 amino acids, with 3 of the 7
substitutions at position
6. For this reason, we conclude that the amino acid at position 6 can be
varied without
altering the specificity of the antibody.
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[00827] Analysis of the consensus sequence generated with the inclusion of
antibodies
5C6F3 and 25E6 highlighted which amino acids were conserved among all eleven
antibodies.
For this reason, our preferred consensus sequence for heavy chain CDR3 defines
amino acids
at positions 2, 3, 4, 7, 10, 11, 12, 14 and 15, where for 11 antibodies, there
were 3 or less
variants at these positions.
[00828] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a heavy chain CDR3 that is at least 90%
identical to a
CDR3 comprising the following amino acids at the specified positions: G, L, or
N at position
2, G, T, or Y at position 3, G or T at position 4, Y at position 7, Y, A, or G
at position 10,
M, D or F at position 11, D or E at position 12 and AY at position 14-15.
[00829] Whereas Light Chain CDR1 for MNC2 is RASKS--VSTSGYSYMH, with the
amino acids numbered from left to right 1 through 17, the consensus sequence
of MNC2,
MNE6, 20A10, 3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is:
[00830] K or R at position 1, A or S at position 2, S or R at position 3, K, Q
or A at
position 4. S, N or T at position 5, V. I, E, or K at position 6, L, V or S at
position 7, S. Y, I
or V at position 8, A, S. or H at position 9, T or S at position 10, N, S. or
Y at position 11, G,
S, D, or Q at position 12, V, Y, K or N at position 13, N, S, or T at position
14, Y or F at
position 15, and I, L or M at position 16, and H, A, E or Q at position 17.
The underlined
amino acids indicate how this more inclusive consensus sequence differs from
the consensus
sequence generated for MNC2, MNE6, 20A10 and 3C2B1 alone. Of the 17 amino
acids in
light chain CDR1, the consensus sequence for all nine antibodies differs from
the consensus
sequence for the original cancer-specific four by 13 amino acids. 4 of the 13
are homologous
substitutions, which in general do not significantly alter the structure or
specificity of the
protein. Of the remaining 9 substitutions, 1 is at position 4, 1 is at
position 5, 3 are at position
6, 1 is at position 7, 1 is at position 11, and 2 are at is at position 17.
The inclusion of the 5
new antibodies did not alter the amino acids, excluding homologous
substitutions, at
positions 1, 2, 3, 8, 9, 10, 12, 13, 14, 15 or 16. For this reason, we
conclude that the
conserved consensus sequence for light chain CDR1 that defines a MUC1* cancer-
specific
antibody comprises the amino acids given above for positions 1, 2, 3, 8, 10,
12, 13, 14, 15
and 16.
[00831] Analysis of the consensus sequence generated with all the antibodies,
including
5C6F3 and 25E6 further altered the consensus sequence for light chain CDR1
with amino
acid substitutions as follows: L at position 6; D at position 9; D at position
11 and N at
position 17. We note that none of these substitutions were at positions that
were invariant for
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the original four cancer-specific antibodies plus the five new antibodies.
Thus, we conclude
that a conserved consensus sequence for light chain CDR1 that defines at least
90% identity
of a cancer-specific antibody comprises amino acids defined above at positions
1, 2, 3, 8, 10,
12, 13, 14, 15 and 16.
[00832] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a light chain CDR1 that is at least 90%
identical to a
CDR1 comprising K or R at position 1, A or S at position 2, S or R at position
3, S, Y, I or V
at position 8, T or S at position 10, G, S, D, or Q at position 12, V, Y, K or
N at position 13,
N, S, or T at position 14, Y or F at position 15, and I, L or M at position
16.
[00833] Whereas Light Chain CDR2 for MNC2 is LASNLES, with the amino acids
numbered from left to right 1 through 7, the consensus sequence of MNC2, MNE6,
20A10,
3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is: L, W, S, T or K at
position 1, A,
T or V at position 2, S at position 3, N or T at position 4, L or R at
position 5, E, A, F or D at
position 6, and S at position 7. The underlined amino acids indicate how this
more inclusive
consensus sequence differs from the consensus sequence generated for MNC2,
MNE6,
20A10 and 3C2B1 alone.
[00834] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a light chain CDR2 that is at least 90%
identical to a
CDR2 comprising the following amino acids at the specified positions: L, W. S,
T or K at
position 1, A, '1 or V at position 2, S at position 3, IN or '1' at position
4, L or R at position 5,
E, A, F or D at position 6, and S at position 7. Of the 7 positions, the
inclusion of the five
new antibodies introduced 5 substitutions of which only 2 were not homologous
substitutions.
[00835] Analysis of the consensus sequence generated with all the antibodies,
including
5C6F3 and 25E6 further altered the consensus sequence for light chain CDR2
with amino
acid substitutions as follows: K at position 4, which is a substitution that
is homologous to N.
[00836] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a light chain CDR2 that is at least 90%
identical to a
CDR2 comprising: A, T or V at position 2, S at position 3, N, T, or K at
position 4, L or R at
position 5, E, A, F or D at position 6, and S at position 7.
[00837] Whereas Light Chain CDR3 for MNC2 is QHSRELPFT, with the amino acids
numbered from left to right 1 through 9, t the consensus sequence of MNC2,
MNE6, 20A10,
3C2B1 and new antibodies B2, B7, 8C7F3, H11 and B9 is: Q or F at position 1, H
or Q at
position 2, S, Q, R, D or N at position 3, R, S, Y or N at position 4, E, L, S
or H at position 5,
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L, S, V, D or Y at position 6, P or S at position 7, F, L or P at position 8
and T at position 9.
The underlined amino acids indicate how this more inclusive consensus sequence
differs
from the consensus sequence generated for MNC2, MNE6, 20A10 and 3C2B1 alone.
[00838] Analysis of the consensus sequence generated with all the antibodies,
including
5C6F3 and 25E6 further altered the consensus sequence for light chain CDR2
with amino
acid substitutions as follows: W at position 1; G at position 3; T at position
4; F at position 5;
Q at position 8.
[00839] In a preferred embodiment an antibody is chosen for the treatment,
prevention or
diagnosis of cancer based on having a light chain CDR3 that is at least 90%
identical to a
CDR2 comprising: Q, F or W at position 1, H or Q at position 2, R, S. T, Y or
N at position
4, E, L, S or H at position 5, L, S. V, D or Y at position 6, P or S at
position 7, and T at
position 9.
[00840] Other general strategy for using antibodies, antibody fragments and
CARs
that target the extracellular domain of MUC1*
[00841] In another aspect, the invention is directed to a composition that
includes at least
two different plasmids transfected into the same immune cell, wherein the
first encodes a
CAR comprising an antibody fragment, scFv, or peptide that binds to a tumor
antigen and the
other encodes a gene that is not a CAR, wherein the gene that is not a CAR is
expressed from
an inducible promoter that is activated by elements of an activated immune
cell. In one
aspect, the immune cell is a '1 cell or an INK cell. In one aspect the immune
cell is derived
from a stem cell that has been directed to differentiate to that immune cell
type in vitro. In
another aspect, a CAR containing sequences of the antibody are expressed in a
stem cell,
which then may be differentiated into an immune cell. In one aspect the CAR
comprises an
antibody fragment, scFy or peptide that binds to the extra cellular domain of
MUC1*. In one
aspect the CAR comprises an scFy derived from MNC2, MNE6, 20A10, 3C2B1, 5C6F3,
25E6, 18G12, 28F9, 1E4, B12, B2, B7, B9, 8C7F3, or H11. In one aspect the non-
CAR
species is a cleavage enzyme. In one aspect the cleavage enzyme is MMP2, MMP3,
MMP9,
MMP13, MMP14, MMP16, ADAM10, ADAM17, ADAM28 or catalytically active
fragments thereof. In another aspect the non-CAR species is a cytokine. In one
aspect, the
Cytokine is IL-7. In one aspect the cytokine is IL-15. In one aspect the
cytokine is IL-12. In
one aspect the cytokine is IL-18. The sequence of an activated IL-18 is given
(SEQ ID
NOS:1637-1638). Two examples of NFAT-inducible IL-18 embedded in the Foxp3
enhancer
region are given (SEQ ID NOS:16391640). Two examples of NFAT-inducible IL-18
embedded in the IL-2 enhancer region are given (SEQ ID NOS:1641-1642). In one
case,
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there are three (3) NFAT response elements and in the other acse there are six
(6) NFAT
response elements. The number of NFAT response elements can be varied in order
to get the
desired amount of 1L-18 expressed upon CAR T cell recognition of the target.
Examples of
antibodies of the invention incorporated into CARS with inducible IL-18 are
shown as:
murine or human MNC2 in a CAR with a 4-1BB or CD28 co-stimulatory domain plus
inducible IL-18 (SEQ ID NOS:1643-1646), or also with a 1XX mutated CD3-zeta
(SEQ ID
NOS:1647-1650); murine or human MNE6 in a CAR with a 4-1BB or CD28 co-
stimulatory
domain plus inducible IL-18 (SEQ ID NOS:1651-1654), or also with a 1XX mutated
CD3-
zeta (SEQ ID NOS:1655-1658); murine or human 20A10 in a CAR with a 4-1BB or
CD28
co-stimulatory domain plus inducible IL-18 (SEQ ID NOS:1659-1662), or also
with a 1XX
mutated CD3-zeta (SEQ ID NOS:1663-1666); murine or human 25E6 in a CAR with a
4-
1BB or CD28 co-stimulatory domain plus inducible IL-18 (SEQ ID NOS:16671670),
or also
with a 1XX mutated CD3-zeta (SEQ ID NOS:1671-1674). In another aspect the
cytokine is
IL-7 and IL-15. In one case expression of the non-CAR species is induced by
elements of an
activated immune cell. In one aspect the element of an activated immune cell
is an NFAT. In
one aspect the NFAT is NFATcl, NFATc3 or NFATc2. Cytokines IL-7, IL-15, IL-12
and IL-
18 are known to promote T cell persistence. In one aspect of the invention an
immune cell
described above is administered to a patient for the treatment or prevention
of cancer. In one
aspect of the invention, the cancer is a MUC1 positive cancer or a MUC1*
positive cancer.
[00842] In addition to making CAR '1 cells that also induce expression of a
cleavage
enzyme, we made CAR T cells that also induce local and transient expression of
IL-18. Many
of the T cell based inducible systems reported insert the gene to be inducibly
expressed into
an IL-2 promoter or enhancer. We compared inducible expression off an IL-2
promoter/enhancer to inducible expression off of a portion of the Foxp3
enhancer. In this
particular example, human T cells were transduced with both huMNC2-CAR44 and
an
NFAT inducible IL-18, wherein the 11-18 gene was either inserted into an IL-2
promoter or
the Foxp3 enhancer region. It is known in the field that a major problem with
CAR Ts with
inducible second factors is that the second factor is leaky, meaning that
significant expression
of the second factor occurs without activation of the CAR T cell. The other
problem with
existing inducible systems is the length of time that goes by between when the
CAR T cell is
activated and the second factor is induced is typically very long so that the
cell secreting the
second factor may be far away from the tumor by the time the second factor is
expressed.
[00843] Figure 211A- 211C show graphs of an ELISA experiment measuring the
amount
of IL-18 secreted into the condition media of huMNC2-CAR44 T cells, which also
bear an
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NFAT inducible IL-18, co-cultured with MUCI* positive cancer cells. As a
method of
inducing varying levels of IL-18 expression, we co-cultured the CAR T cells
with cancer
cells doped with increasing amounts of cells that were engineered to express
even more
MUCI*. In these figures we show T47D cancer cells that are either wild-type,
or doped with
5%, 10% or 30% of the T47D cells expressing more MUCI*. Fig. 211A shows the
graph of
IL-18 secreted into the supernatant of T47D breast cancer cells co-cultured
with untransduced
human T cells. Fig. 211B shows the graph of 1L-18 secreted into the
supernatant of T47D
breast cancer cells co-cultured with huMNC2-CAR44 T cells that also bore an
NFAT
inducible IL-18 gene inserted into a portion of the Foxp3 enhancer. Fig. 211C
shows the
graph of IL-18 secreted into the supernatant of T47D breast cancer cells co-
cultured with
huMNC2-CAR44 T cells that also bore an NFAT inducible IL-18 gene inserted into
a portion
of the IL-2 enhancer. As can be seen in the figure, the Foxp3 system induces
rapid and robust
expression of IL-18, which is significantly faster and higher than that of the
same construct in
an IL-2 promoter. In this example, the IL-18 gene is inserted downstream of
six (6) NFAT
response elements, however one can attenuate the amount of the second factor
by using a
lesser number of response elements or enhance the amount by increasing the
number of
NFAT response elements.
[00844] It has been reported that IL-18 increases persistence of CAR T cells
in vivo.
However, we observed an unexpected result. In a dose-dependent manner,
secretion of IL-18
increased the killing of low antigen density cells by the CAR '1 cells. We
differentially
labeled the T47D-wt cells (red: mCherry) and those T47Ds that were transduced
to express
more MUCI* (green: GFP). Figure 212A- 212X shows photographs of T47D breast
cancer
cells (red) doped with varying percentages of T47D cells engineered to express
more MUCI*
(green). The target cancer cells have been co-cultured with huMNC2-CAR44 T
cells with
NFAT inducible IL-18 wherein the IL-18 gene has been inserted into either the
Foxp3
enhancer/promoter or the IL-2 enhancer/promoter. Fig. 212A-212C, 212I-212K,
and 212Q-
212S show the cancer cells co-cultured with untransduced T cells. Fig. 212D-
212F, 212L-
212N, and 212T-212V show the cancer cells co-cultured with hiMNC2-CAR44 T
cells with
the NFAT inducible IL-18 gene inserted into the Foxp3 enhancer/promoter. Fig.
212G-212H,
2120-212P, and 212W-212X show the cancer cells co-cultured with hiMNC2-CAR44 T
cells
with the NFAT inducible IL-18 gene inserted into the IL-2 enhancer/promoter.
As can be
seen in the figure, the low antigen density T47D-wt type cells (red) are being
killed when
doped with higher percentages of cells that express more MUC1* and thus
secrete more IL-
18. The experiment shows that this is not just a bystander effect, because the
cells expressing
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IL-18 off of the IL-2 promoter, which expresses much lower levels of IL-18, do
not kill the
low antigen density cells even when they are doped with 30% cells expressing
more MUC1*.
[00845] We then showed that the CAR T mediated killing is specific for the CAR
T
specific antigen. We performed a similar experiment, wherein control,
MUC1/MUC1*
negative cells were doped with 5%, 10% or 30% of the T47D cells expressing
more MUC1*,
and co-cultured with MUC1* specific CAR T cells. Figure 213A- 213B shows
graphs of
EL1SA experiments in which levels of 1L-18 secreted into the conditioned media
are
measured for huMNC1-CAR44 T cells with NFAT inducible IL-18 gene, inserted
into the
Foxp3 enhancer or promoter, co-cultured with either MUC1* positive cancer
cells or MUC1
negative non-cancerous cells. Fig. 213A shows IL-18 secretion from huMNC2-
CAR44 T
cells with NFAT inducible IL-18 in co-culture with T47D breast cancer cells
where the
population has been doped with 5%, 10% or 30% T47D cells that had been
transfected with
even more MUC1*. Fig. 213B shows IL-18 secretion from huMNC2-CAR44 T cells
with
NFAT inducible IL-18 in co-culture with non-cancerous, MUC1 negative HEK293
cells
where the cell population has been doped with 5%, 10% or 30% T47D cells that
had been
transfected with more MUC1*. As can be seen in the figure, the amount of EL-18
secreted
into the media can be attributed to the MUC1* positive cells that the
population was doped
with. Time course fluorescent photographs of the experiment show that even
when doped
with significant percentages of high antigen density MUC1* positive cells, the
MUC1
negative cells are not killed by the M1JC1* targeting CAR '1 cells. Figure
214A-214X shows
photographs of T47D breast cancer cells (red) or non-cancerous HEK293 cells
(also red),
where both cell types have been doped with varying percentages of T47D cells
engineered to
express more MUC1* (green). These target cancer cells have been co-cultured
with
huMNC2-CAR44 T cells with NFAT inducible 1L-18 wherein the 1L-18 gene has been
inserted into the Foxp3 enhancer/promoter. Fig. 214A-214F shows either T47D
cells or
HEK293 cells that have not been doped with T47D cells engineered to express
high MUC1*
density. Fig. 214G-214L shows either T47D cells or HEK293 cells that have been
doped with
5% T47D cells engineered to express high MUC1* density. Fig. 214M-214R shows
either
T47D cells or HEK293 cells that have been doped with 10% T47D cells engineered
to
express high MUC1* density. Fig. 214S-214X shows either T47D cells or HEK293
cells that
have been doped with 30% T47D cells engineered to express high MUC1* density.
Fig.
214A-B, G-H. M-N, and S-T show T47D breast cancer cells. Fig. 214C-F, I-L, O-
R, and U-X
show HEK293 cells. As can be seen in the figures, the induced secretion of IL-
18 resulted in
low MUC1* density T47D cells being killed but did not induce non-specific
killing of the
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MUC1* negative HEK293 cells. Taken together these results show that the Foxp3
system is a
superior system for the inducible expression of a second factor and especially
useful in CAR
T systems. Further we have demonstrated the unexpected result that 1L-18
increases the
killing of low antigen density cells without the unwanted effect of killing
nearby
MUCl/MUC 1* negative cells.
[00846] In another aspect, the invention is directed to a composition that
includes at least
two different plasmids transfected into the same immune cell, wherein the
first encodes a
CAR comprising an antibody fragment, scFv or peptide that binds to the extra
cellular
domain of an antigen on the surface of a B cell and the other encodes a gene
that is not a
CAR, wherein the gene that is not a CAR is expressed from an inducible
promoter that is
activated by elements of an activated immune cell. In one aspect, the immune
cell is a T cell
or an NK cell. In one aspect the immune cell is derived from a stem cell that
has been
directed to differentiate to that immune cell type in vitro. In another
aspect, a CAR containing
sequences of the antibody are expressed in a stem cell, which then may be
differentiated into
an immune cell. In one aspect the CAR comprises an antibody fragment, scFv or
peptide that
binds to CD19. In another aspect the antibody fragment, scFv or peptide binds
to a surface
antigen of a B cell or a B cell precursor, or binds to CD19, CD20, CD22, BCMA,
CD30,
CD138, CD123, CD33 or LeY antigen. In one aspect the non-CAR species is a
cleavage
enzyme. In another aspect the non-CAR species is a cytokine. In one aspect,
the Cytokine is
1L-7. In one aspect the cytokine is IL-15. In another aspect the cytokine is
1L-7 and 1L-15. In
one case expression of the non-CAR species is induced by elements of an
activated immune
cell. In one aspect the element of an activated immune cell is an NFAT. In one
aspect the
NFAT is NFATc 1, NFATc3 or NFATc2. that is not a CAR, wherein the gene that is
not a
CAR is expressed from an inducible promoter wherein expression is induced by
elements of
an activated immune cell. In one aspect the immune cell transfected or
transduced with the
composition is administered to a patient for the treatment or prevention of
cancer. In one case
the cancer is a leukemia, lymphoma or blood cancer.
[00847] It is not intended for the invention to be limited by a specific
method or
technology for inserting the gene or plasmid comprising a sequence encoding a
CAR or
activated T cell inducible protein or peptide there encoded. For example, the
gene encoding
the CARs and activated T cell induced genes described herein can be virally
transduced into
an immune cell using viruses, which may or may not result in the CAR gene
being integrated
into the genome of the recipient cell. Virus delivery systems and viral
vectors include but are
not limited to retroviruses, including gamma-retroviruses, lentivirus,
adenoviruses, adeno-
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associated viruses, baculoviruses, poxvirus, herpes simplex viruses, oncolytic
viruses, HF10,
T-Vec and the like. In addition to viral transduction, CARs and activated T
cell induced
genes decribed herein can be directly spliced into the genome of the recipient
cell using
methods such as CRISPR technology, CRISPR-Cas9 and -CPF1, TALEN, Sleeping
Beauty
transposon system, and SB 100X.
[00848] Bulky cell surface proteins such as MUCl-FL can also cause a steric
hindrance
problem for BiTEs. A BiTE is a two-headed bispecific antibody wherein one head
binds to a
T cell and the other head binds to a tumor-associated antigen. In this way,
the BiTE links
together the T cell and the tumor cells. The antibody that binds to the T cell
should be an
antibody that activates the T cell, such as an antibody against CD3 or CD28_
To solve the
steric hindrance problem, the linker between the T cell specific antibody and
the tumor
specific antibody is lengthened.
[00849] In another aspect of the invention, an anti-MUC1* single chain
molecule is fused
to a cleavage enzyme or a catalytically active fragment of a cleavage enzyme.
In one aspect
of the invention, the cleavage enzyme is MMP9 (SEQ ID NO:643). In another
aspect of the
invention, the enzyme is a catalytically active fragment of MMP9 (SEQ ID
NO:645). In some
cases, the antibody fragment of the CAR is chosen for its ability to recognize
MUC1* when
cleaved by that specific cleavage enzyme. In one embodiment, the cleavage
enzyme is
MMP9, MMP3, MMP14, MMP2, ADAM17, ADAM TS16, and/or ADAM28. In one
embodiment, the antibody or antibody fragment binds to a peptide having the
sequence of
(PSMGFR)
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQS GA,
PSMGFR N-10, QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA, or PSMGFR N+20
SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFP
FSAQSGA. In another embodiment, cleavage enzymes MMP9 and MMP3 are transduced
into a T cell that is also transduced with a CAR with an antibody fragment
that is a fragment
of MNC2.
[00850] In many cases it is desirable to have the cleavage enzyme expressed
only after an
immune cell recognizes the tumor-associated target on a solid tumor. In this
way, the
cleavage enzyme will not freely move throughout the body, cleaving MUC1, MUC16
or
other proteins, wherein their cleavage could actually promote cancer. However,
there are
cancers that are physically accessible to direct application of chemotherapy
agents, CAR T
cells and other anti-cancer agents. For example, types of brain cancers,
prostate cancer and
ovarian cancers have all shown the benefit of direct application of anti-
cancer agents into the
local vicinity of the cancer. CAR T cells have been injected directly into the
brain and/or
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cerebral spinal fluid of glioblastoma patients. Radiation has been directed to
the prostate area
for the treatment of prostate cancers, including those that have metastasized.
Hot chemo
therapy agents have been directly injected into the intraperitoneal cavity for
the treatment of
ovarian cancers. In these and other cases, where the cancers that are
physically accessible to
direct application of chemotherapy agents, a cleavage enzyme is administered
in the presence
or absence of another anti-cancer agent, which could be a CAR T cell, an
immune cell
engineered to recognize a tumor-associated antigen, a BiTE, an ADC, a
biological or a
standard chemotherapy agent. Although ovarian cancer can metastasize to
anywhere in the
body, it usually stays in the abdomen as it spreads to adjacent organs, such
as the intestines,
liver and stomach. This makes ovarian cancer an ideal test case for improving
the effect of
anti-cancer agents by administering a cleavage enzyme in combination with
other anti-cancer
agents, including a platinum-based drug such as carboplatin (Paraplatin) or
cisplatin, and/or a
taxane such as paclitaxel (Taxol) or docetaxel (Taxotere). Alkeran
(Melphalan), Avastin
(Bevacizumab), Carboplatin, Clafen (Cyclophosphamide), and Cytoxan have all
been
approved for the treatment of ovarian cancer. Other treatments that are being
tested for the
treatment of ovarian cancers include agents that target MUC1, MUC16 and as
described
herein, MUC1*.
[00851] Other cleavage enzymes can be used in addition to or in place of MMP9.
MMP14
for example, has been shown to efficiently cleave MUCI to MUCI* (Fig. 38). In
one aspect
of the invention, MMP14 is expressed in an immune cell that is also engineered
to express a
CAR. In one case the CAR is an anti-MUCI* CAR. For example, it can be an MNC2-
CAR44 transduced T cell. In another aspect of the invention, the MMP14 is
directly
administered to the patient either in the location of the tumor or by i.v.
[00852] In yet another aspect of the invention, the cancer is an ovarian
cancer and either
MMP9 or MMP14 is directly injected into the abdominal area along with an anti-
cancer
agent, which can be a chemotherapy agent, a biological, an anti-MUCI* CAR T or
an anti-
MUC16 CAR T.
[00853] In addition to local administration of the cleavage enzyme, + iv
administration
alone or secreted from an immune cell, which may be a CAR T cell, which
further may be
expressed off of an inducible promoter is contemplated.
[00854] Methods used in carrying out experimentation in relation to the
present
invention
[00855] 1. Lentivirus production and viral transduction of immune cells
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[00856] HEK293 or HEK293T cells (ATCC) were used to produce lentivirus. The
day
prior transfection plates (6well plate) were coated with poly-D-lysine and
cells seeded so that
cell density reaches 90-95% at the time of transfection and cultures in a 5%
CO2 atmosphere.
The next day cells were transfected with Lipofectamine 3000 (life
technologies) and Opti-
MEM I Reduced Serum Medium according to the manufacturer instructions (0.75ug
of
lentiviral expression vector and 2.25ug of pPACKH1 packaging mix was used).
After 6h
incubation, the media was changed and media containing lentivirus was
harvested after 24
and 48 hours. Lentivirus was concentrated with Lenti-X concentrator (Clontech)
and titer was
calculated using the Lenti-X p@4 Rapid Titer Kit (Clontech). Lentivirus was
store at -80C in
single-use aliquots.
[00857] Transduction of immune cells with constructs including CARs
[00858] Human T cells, if frozen, were thawed and pre-warmed in 100-200 units
IL-2 and
TexMACS medium, 20 ml, and pelleted by centrifugation. Cells were resuspended
in 10 ml
of medium and cultured at 37 C, 5% CO2 at 1x106 cells/ml in complete medium
with anti-
CD3/anti-CD28 beads (TransAct kit).
[00859] After 4 days in culture, cells were counted and 450 ul of cell
suspension was
placed in single well of a 24-well plate at a density of approximately 1x106
cells/ml. Cells
were allowed to settle. 150 ul was carefully removed from the top of each
well. To each well
was added an appropriate dilution of lentiviral vector, diluted in plain
TexMACS medium,
along with protamine sulfate to a final concentration of 10 ug/ml, in a 150 ul
volume, for a
final total volume of 450 ul per well and incubated for 24 hrs. Transduced
cells were
removed, pelleted by centrifugation, and resuspended in fresh medium,
adjusting cell density,
not to exceed 1.0 x 106 cells/ml. Transduced T cells can be expanded and
frozen or used
directly. Typically transduced T cells are used or frozen between Day 7 and
Day 20 post
activation with IL-2 and TransAct media.
[00860] 2. Comparing anti-MUC1* CAR T cell activity in the presence or absence
of
exogenous cleavage enzymes
[00861] Human T cells (ALLCELLS) were transduced with huMNC2-CAR44 or
huMNC2-CAR50. CAR44 is huMNC2-scFv-CD8-CD8 (transmembrane-41BB-3z). CARS 0
is the same as CAR44 except that CAR50 has a murine MNC2-scFv and a CD4
transmembrane domain. The CAR T cells were incubated for 18 hours with target
and non-
target cells that have been dyed red using CMTMR. When T cells recognize a
target cell,
they cluster the target cells and begin to kill them. As can be seen in
Figures 45-47 the CAR
T cells effectively cluster and kill the target MUC1* positive cancer cells.
Figure 45 shows
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huMNC2-CAR44 or huMNC2-CAR50 T cells being co-cultured with HCT-116 cells
transduced to express MUC1*, "HCT-MUC1*" or with HCT-116 cells transduced with
a
full-length MUC1, "HCT-MUC1-41TR". Recall that MNC2 recognizes an ectopic
epitope
that is only revealed after cleavage and release of the MUC1 tandem repeat
domain. Neither
huMNC2-CAR44 nor huMNC2-CAR50 T cells recognize the cells expressing full-
length
MUC1 (Fig. 45F-45H). However, when MMP9 plus activator APMA is added, the CAR
T
cells recognize the cells, cluster and kill them (Fig. 45J-45L). The addition
of cleavage
enzyme ADAM-17 did not affect the recognition of either CAR T cell for full-
length MUC1
(Fig. 45N-45P). The reason could be that ADAM-17 doesn't cleave MUC1 or the
cleavage
product is not recognized by MNC2. A similar experiment was performed (Fig.
46) that
showed that MMP2 was only weakly effective at either cleavage MUC1 or that the
MMP2
cleavage product was only weakly recognized by MNC2. Figure 47 shows the
contrast
between huMNC2-CAR44 recognition of HCT-MUC1* cells, T47D-wt breast cancer
cells,
and T47D cells with added MMP9 which presumably cleaves the full-length MUC1
to an
MNC2 recognizable MUC1*.
[00862] 3. Confocal imaging of CAR T cells giving the "kiss of death" to MUC1*
positive cancer cells.
[00863] Confocal images of Human T cells that were transduced with huMNC2-
CAR44,
co-cultured for 24 hours with MUC1* positive DU145 prostate cancer cells
showed the CAR
'1' cells inserting Granzyme B into the target cancer cells. Figure 55 shows
fluorescent images
of the huMNC2-CAR44 T cells secreting Granzyme B when co-cultured with the
prostate
cancer cells, FACS analysis showing increased expression of Granzyme B by the
CAR T
cells and an xCELLigence experiment showing that the target prostate cancer
cells were in
fact killed.
[00864] 5. Analysis of CAR T cell induced killing of MUC1* positive cancer
cells by
FACS analysis
[00865] We have demonstrated the killing effect of huMNC2-CAR44 T cells on
T47D
MUC1* positive breast cancer cells, wherein the breast cancer cells have been
transfected
with increasing amounts of additional MUC1*. The killing effect of the huMNC2-
CAR44 T
cells increases as the amount of target MUC1* expressed on the cells
increases.
[00866] IFN-y secretion in media was measured using a human IFN-y ELISA kit
(Biolegend). Plates were coated with an anti- IFN-y antibody (capture
antibody, 1X in
coating buffer). After overnight incubation at 4 C, the plate was washed 4
times with PBS-T
and blocking solution was added to block remaining binding site on the well.
After lh at RT
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(shaking at 500rpm) the plate was washed 4 times with PBS-T and conditioned
media (CM)
and IFN-y standard, was added. After 2h at RT with shaking, the plate was
washed 4 times
with PBS-T and detection antibody (Ix), was added. After lh at RT with
shaking, the plate
was washed 4 times with PBS-T and Avidin-HRP (1x) was added. After 30min at RT
with
shaking, the plate was washed 5 times with PBS-T (soak lmin each wash) and TMB
substrate
solution was added. The reaction was stopped after 20min by adding the stop
solution and
absorbance was read at 450nm (minus absorbance at 570nm) within 15 min of
stopping.
[00867] 6. Analysis of CAR T cell induced killing of MUC1* positive cancer
cells by
xCELLigence
[00868] In addition to FACS analysis, many researchers now use an xCELLigence
instrument to measure CAR T killing of cancer cells. The xCELLigence
instrument uses
electrode arrays upon which cancer cells are plated. The adherent cancer cells
insulate the
electrode and so cause an increase in impedance as they grow. Conversely, T
cells are not
adherent and remain in suspension so do not contribute to insulation of the
electrode which
would increase impedance. However, if the T cells or CAR T cells kill the
cancer cells on the
electrode plate, the cancer cells ball up and float off as they die, which
causes the impedance
to decrease. The xCELLigence instrument measures impedance as a function of
time, which
is correlated to cancer cell killing. In addition, the electrode plates also
have a viewing
window. When CAR T cells effectively kill the adsorbed target cancer cells,
there is a
decrease in impedance but also one can see that there are no cancer cells left
on the plate
surface.
[00869] In most of the XCELLigence experiments, 5,000 cancer cells were plated
per well
of a 96-well electrode array plate. Cells were allowed to adhere and grow for
24 hours. CAR
T cells were then added at an Effector to Target ratio (E:T) of 0.5:1, 1:1,
2:1, 5:1, 10:1 and
sometimes 20:1. The E:T ratio assumes 100% transduction of the CAR into the T
cells, when
the actual transduction efficiency is 40%.
[00870] The xCELLigence instrument records impedance as a function of time and
experiments can go on for up to 7 days.
[00871] Fig. 48, Fig. 49, Fig. 55H, Fig. 56H, Figs. 57A-57C, all show results
of CAR T
and cancer cell experiments performed on an xCELLigence instrument.
[00872] 7. Anti-MUC1* CAR T cell therapy in mice bearing human tumors
[00873] Female NOD/SCID/GAMMA (NSG) mice between 8-12 weeks of age were
implanted with 500,000 human cancer cells, wherein the cancer cells had
previously been
stably transfected with Luciferase. Mice bearing Luciferase positive cells can
be injected with
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the enzyme's substrate Luciferin just prior to imaging, which makes the cancer
cells
fluoresce. The cancer cells are imaged in live mice within 10-15 minutes after
injection with
Luciferin on an IVIS instrument. The readout is flux or photons per second.
Tumors were
allowed to engraft until tumors were clearly visible by IVIS.
[00874] Figures 58A-58F show fluorescent photographs of mice taken on an IVIS
instrument. 10 minutes prior to IVIS photographs, mice were injected
intraperitoneally (IP)
with Luciferin, which fluoresces after cleavage by Luciferase, thus making
tumor cells
fluoresce. NSG (NOD/SCID/GAMMA) immune compromised mice that on Day 0 were
subcutaneously implanted on the flank with 500,000 human MUC1* positive cancer
cells that
had been stably transfected with Luciferase. Tumors were allowed to engraft.
On Day 7 after
IVIS measurement, animals were tail vein injected with either PBS, 10 million
untransduced
human T cells or 8.5M huMNC2-scFv-CAR44 T cells. As can be seen in the figure,
control
mice had to be sacrificed on Day 20 due to excess tumor burden (Fig. 58A-58B).
huMNC2-
CAR44 T cell treated mice were tumor free after a single CAR T cell injection
until Day 100
when they were sacrificed (Fig. 58C). Figure 58E shows Kaplan-Meier survival
curves that
demonstrate the efficacy of T cell therapy guided by anti-MUC1* antibody.
Figure 58F
shows a table summarizing the characteristics of the human T cells that were
collected from
the test mice upon sacrifice. The starting Car T cell population was 50% CD4
positive helper
T cells and 50% CD8 positive killer T cells. As can be seen in the table, the
percent of CD8
positive cells has increased in the CAR 'I treated group, indicating in vivo
expansion of that
group of cells, which is an indicator of efficacy. We also note that in the
treated group, the
CAR T cells express higher levels of PD1 which is a marker of T cell
exhaustion.
[00875] In another animal experiment, NSG mice were sub-cutaneously implanted
into the
flank with 500,000 tumor cells then injected on Day 7 and again on Day 14 with
either saline
solution, PBS, or 10M huMNC2-CAR44 T cells (Fig. 59A-59C). In this experiment
the
amount of MUC1* expressed on the tumor cells was varied. In one case, the
tumor cells that
were implanted were T47D-wildtype (Fig. 59B). In another case, the T47D cells
were doped
with 95% T47D cells that had been transfected to express even more MUC1* (Fig.
59C). As
can be seen, the tumors comprised of cells expressing more MUC1* were
eliminated more
quickly and did not recur. In a similar experiment, the tumor cells were doped
with a
relatively small amount of cells that expressed more MUC1*. Figure 60A-60C
shows NSG
mice implanted with T47D-wt breast cancer cells that have been doped with 30%
of T47D
cells transfected to express more MUC1*. As can be seen, even a small
percentage of cells
expressing high levels of MUC1* is sufficient to trigger CAR T cell mediated
killing of the
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entire tumor. Naturally occurring tumors are heterogeneous and are comprised
of both high
and low antigen expressing cells. This experiment indicates that huMNC2-CAR44
T cells
would be effective in eradicating naturally occurring tumors.
[00876] Figures 61A-61J show fluorescent photographs of mice taken on an IVIS
instrument. NSG (NOD/SCID/GAMMA) immune compromised mice that on Day 0 were
subcutaneously injected into the flank with 500K human BT-20 cells which are a
MUC1*
positive triple negative breast cancer cell line. The cancer cells had been
stably transfected
with Luciferase. Tumors were allowed to engraft. On Day 6 after IVIS
measurement, animals
were given a one-time injection of 10 million of either human T cells
transduced with
huMNC2-scFv-CAR44 or untransduced T cells. 5 million T cells were injected
intra-tumor
and 5 million were injected into the tail vein. 10 minutes prior to IVIS
photographs, mice
were IP injected with Luciferin. In one case the huMNC2-CAR44 T cells were
first incubated
with beads to which was attached the PSMGFR peptide to pre-stimulate the T
cells and in the
figure is marked Protocol 1. In Protocol 2, the huMNC2-CAR44 T cells were pre-
stimulated
with live tumor cells, which likely injected more tumor cells into the
animals' circulation.
[00877] Figures 62A-62M show fluorescent photographs of mice taken on an IVIS
instrument. NSG (NOD/SCID/GAMMA) immune compromised mice that on Day 0 were
injected into the intraperitoneal cavity (IP) with 500K human SKOV-3 cells
which are a
MUC1* positive ovarian cancer cell line. The cancer cells had been stably
transfectekl with
Luciferase. Tumors were allowed to engraft. On Day 3 after IVIS measurement,
animals were
IP injected with 10M either human T cells transduced with huMNC2-CAR44 T
cells,
untransduced T cells or PBS. Animals were IVIS imaged again on Day 7. 10
minutes prior to
IVIS photographs, mice were IP injected with Luciferin. As can be seen in the
figure the anti-
MUC1* CAR T cells effectively reduced ovarian tumor volume by Day 15.
[00878] 9. NFAT-induced IL-18 sequences and cloning
[00879] Cloning of IL18 in pGL4-14 3xNFAT:
[00880] An activated IL18 (SEQ ID NO:1644) was synthesized with the CD8 leader
sequence. The pGL4-14 3xIL2 NFAT and pGL4-14 3xFoxP3 NFAT were digested with
XhoI and HindIII restriction enzymes (New England Biolabs). The purified
plasmids and the
synthesized IL18 sequences were assembled using the Gibson assembly cloning
kit (New
England Biolab). The resulting constructs (pGL4-14 3xIL2NFAT-IL18 and pGL4-14
3xFoxP3NFAT-IL18) contains 3 repeats of NFAT response element (IL2 or FoxP3)
followed
by a minimum promoter (mCMV: SEQ ID NO:1634) and IL18 (SEQ ID NOS:1752-1753)
with CD8 leader sequence.
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[00881] Cloning of MNC2 CAR with IL18 in pCDNA vector:
[00882] MNC2 CAR sequence was amplified from previously made vector by
polymerase
chain reaction (PCR) using the following
primers: 5' -
agggagacccaagctggctagttaagatggatggccttaccagtgaccgccttgc-3' (SEQ ID NO:1754)
and 5'-
taggccagagaaatgttctggcattatcagcgagggggcagggcctgc-3' (SEQ ID NO:1755).
[00883] IL18 sequence including NFAT response element was amplify from pGL4-14
3xNFAT-IL18 by polymerase chain reaction (PCR) using the following primers: 5'-
tgccagaacatttctctgg-3 ' (SEQ ID NO:1756) and
5' -
acagtcgaggctgatcagegggataaacttatcagtectcgttctgcacgg-3' (SEQ ID NO: 1757). The
purified
PCR fragments and digested pCDNA 3.1 V5 (ThennoFisher scientific) were
assembled using
the Gibson assembly cloning kit (New England Biolab) to create the construct
pCDNA
MNC2CAR-3xIL2NFAT-IL18 and pCDNA MNC2CAR-3xFoxP3NFAT-IL18.
[00884] Cloning of MNC2 CAR-NFAT-IL18 in lentivector:
[00885] MNC2 CAR-NFAT-IL18 sequence was amplified from pCDNA MNC2CAR-
3xIL2NFAT-IL18 and pCDNA MNC2CAR-3xFoxP3NFAT-IL18.by polymerase chain
reaction (PCR) using the following primers:
5'-
atgcaggccctgcccectcgctgataagtttaaactgccagaacatttctctggcctaac-3' (SEQ ID
NO:1758) and 5'-
accggagcgatcgcagatccttcgcggccgcttatcagtcctcgttctgcacggtgaac-3' (SEQ ID
NO:1759). The
purified PCR fragments and digested pCDH Dual Hygro (System Biosciences, CA)
were
assembled using the Gibson assembly cloning kit (New England Biolab) to create
the
construct pCDH MNC2CAR-3xIL2NFAT-IL18 and pCDH MNC2CAR-3xFoxP3NFAT-
IL18.
[00886] Creation of lentivector with MSCV promoter
[00887] MSCV promoter sequence was amplified from pCDH-MSCV-MCS-EFla-GFP
(System Biosciences).by polymerase chain reaction (PCR) using the following
primers: 5'-
attgcactagttgaaagaccccacctgtagg-3' (SED ID NO:1760) and 5'-
aatgctctagaatacgggtatccagg-
3' (SEQ ID NO:1761). After digestion with SpeI and XbaI restriction enzymes
(New England
Biolabs), the purified fragment was cloned into pCDH CMV MCS (System
Bioscience)
digested with the same restriction enzymes to create the construct pCDH MSCV
MCS.
[00888] Cloning of MNC2 CAR-NFAT-IL18 in pCDH MSCV MCS:
[00889] MNC2 CAR-IL2NFAT-IL18 sequence was amplified from pCDNA MNC2CAR-
3xIL2NFAT-IL18 by polymerase chain reaction (PCR) using the following primers:
5'
atagcgaattcgtaccgagggccaccatgg-3' (SEQ ID NO: 762) and
5' -
taggcctcccaccgtacacgcctaggtaccacgccttctgtatg-3' (SEQ ID NO:1763) MNC2 CAR-
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IL2NFAT-IL18 sequence was amplified from pCDNA MNC2CAR-3xFoxP3NFAT-IL18 by
polymerase chain reaction (PCR) using the following primers: 5'
atagcgaattcgtaccgagggccaccatgg -3' (SEQ ID NO:1762) and
5' -
taggcctcccaccgtacacgcctaggtacctctgcagtaaatgg-3' (SEQ ID NO:1764). After
digestion with
EcoRI and KpnI restriction enzymes (New England Biolabs), the purified
fragment was
cloned into pCDH MSCV MCS digested with the same restriction enzymes to create
the
construct pCDH MSCV MNC2CAR-3x1L2NFAT-IL18 and pCDH MSCV MNC2CAR-
3 xFoxP3NFAT-IL18.
[00890] Cloning of 6xNFAT response elements:
[00891] 6xNFAT (IL2 and FoxP3) response element were synthesized followed by
different minimal promoter: mCMV (SEQ ID NO:1634), mlL2P (SEQ ID NO:1635) and
miniP (SEQ ID NO:1636). A total of six 6 sequences were synthesized: SEQ ID
NOS: 1768-
1779.
[00892] 6xNFAT sequences were amplified by polymerase chain reaction (PCR)
using the
following primers: 5' -
tgccagaacatttctctgg-3' (SEQ ID NO:1756) and 5'-
taaggccatggtggctagc-3' (SEQ ID NO:1765). The purified PCR fragments and
digested (KpnI
and XhoI) pCDNA MNC2CAR 3XNFAT IL18 were assembled using the Gibson assembly
cloning kit (New England Biolab) to create constructs with 6x NFAT response
elements in
place of the 3x NFAT response elements.
[00893] 6xNFAT sequences were amplified, from the pCDNA vector created above,
by
polymerase chain reaction (PCR) using
the following primers: 5' -
aataagtttaaactgccagaacatttctctgg-3' (SEQ ID NO:1766) and
5'-
atatagcggccgcttatcagtcctcgttctgcacgg-3' (SEQ ID NO:1767). After digestion with
PmeI and
Notl restriction enzymes (New England Biolabs), the purified fragments were
cloned into
pCDH MSCV MNC2CAR digested with the same restriction enzymes to create the
construct
pCDH MSCV MNC2CAR-6xIL2NFAT-IL18 and pCDH MSCV MNC2CAR-
6xFoxP3NFAT-IL18. For each construct 3 minimal promoter were tested.
[00894] Sequence Listing Free Text
[00895] As regards the use of nucleotide symbols other than a, g, c, t,
they follow the convention set forth in W=P0 Standard ST.25, Appendix 2,
Table l, wherein k represents t or g; n represen-2s a, c, t or g; m
represents a or c; r represents a or g; s represents c or g; w represents a
oL L arid y represenLs c or L.
MUC1 Receptor
(Mac:in 1 pLecursor, Genbank Accession numbeL: P15941)
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MTPOTQSPFELLLLLTVLTVVTGSGHASSTPOGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQG
QDVTLAPATEPASGSAATWGQDVTSVPVTKPALGSTTPPAHDVTSAPDNKPAPGSTAPPAHGVTSAPDiRPAPGS
TAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGV7SAPDTR
PAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTS
APDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPA
HGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGS
TAPPAHGVTSAPDTaPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTKPAPGSTAPPAHGViSAPDTR
PAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVISAPDTRPAPGSTAPPAHGVTS
APDTRPAPOSTAPPAHGVTSAPDTRPAPOSTAPPAHOVTSAPDTRPAPOSTAPPAHGVTSAPDTRPAPOSTAPPA
HGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGS
TAPPAHGVTSAPDTKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGV7SAPDTR
PAPGSTAPPAHGVTSAPDTKPAPGSTAPPAHGVTSAPDTKPAPGSTAPPAHGVISAPDTRPAPGSTAPPAHGVTS
APDTRPAPGSTAPPAHGVTSAPDTRFAFGSTAPPAHGVTSAPDNRFALGSTAPPVHNVTSASGSASGSASTLVHN
GTSARATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFELSFHIS
NLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNUK
TEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVOQCRRKNYGQLDIFPAR
DTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL (SEQ ID NO:1)
PSMGFR
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 2)
[00896] N-10 peptide
[00897] QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:3)
[00898] N-19
[00899] ASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:4)
[00900] N-23
[00901] NLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:5)
[00902] N-26
[00903] ISDVSVSDVPFPFSAQSGA (SEC ID NO:6)
[00904] N-30
[00905] SVSDV2IPPIPSAQSGA (SEQ ID NO:7)
[00906] N-10/C-5
[00907] QFNQYKTEAASRYNL7ISDVSVSDVPFPFS (SEQ ID NO:8)
[00908] N-19/C-5
[00909] ASRYNLTISDVSVSDVPFPFS (SEQ ID NO:9)
[00910] N-36
[00911] FPFSAQSGA (SEQ :D NO:10)
Mouse E6 Heavy chain variable region sequence:
(DNA)
gaggtgaaggtggtggagtctgggggagacttagtgaagoctggagggtccctgaaactctoctgtgtagtctct
ggaILLcdcLLLcalgLagalLalLggcalLgLcLAggLLcgccagalcLccalggcaldgalggcAgalgLgggLcgc
alatcc
attagtggtggcggtacttacatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgcc
aagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtatcactgtacaagggataac
tacggtaggaactacgactacggtatggactactggggtcaaggaacctcagtcaccgtctcctca (SEQ ID
NO:12)
(amino acids)
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EVKVVESGGDLVKPGGSLKLSCVVSGFIFSRYGMSWVRQITGKRLEWVATISGGGTYIYYPDSVKGRFTISRDNA
KNTLYLQMSSLKSEDTAMYHCTRDNYGRNYDYGMDYWGQGTSVTVSS (SEQ ID NO: 13)
Mouse E6 heavy chain variable framework region 1 (FWR1) sequence:
(DNA)
gaggtgaaggtggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgtagtctct
(SEQ ID NO:14)
(amino acids)
EVKVVESGGDLVKPGGSLKLSCVVSGETES (SEQ ID NO:15)
Mouse E6 heavy chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
ggattcactttcagtagatatggcatgtct (SEQ ID NO:16)
(amino acids)
RYGMS (SEQ ID NO:17)
Mouse E6 heavy chain variable framework region 2 (FWR2) sequence:
(DNA)
tgggttcgccagactccaggcaagaggctggagtgggtcgca (SEQ ID NO: 18)
(amino acids)
WVRQIPGKRLEWVA (SEQ ID NO:19)
Mouse E6 heavy chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
accattagtggtggcggtacttacatctactatocagacagtgtgaagggg (SEQ ID NO:20)
(amino acids)
TISGGGTYIYYPDSVKG(SE0 ID NO:21)
Mouse E6 heavy chain variable framework region 3 (FWR3) acid sequence:
(DNA)
cgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacaca
gccatgtatcactgtacaagg (SEQ ID NO:22)
(amino acids)
RETISRDNAKNTLYLQMSSLKSEDTAMYHCIR (SEQ ID NO:23)
Mouse E6 heavy chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
gaLaacLacggLaggaacLacgacLacggLaLggacLac (SEQ :D NC:24)
(amino acids)
DNYGRNYDYGMDY (SEQ ID NO:25)
Humanized E6 heavy chain variable region sequence from IGHV3-21*03:
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaacc
attagtggcggaggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgcc
aagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataac
tatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagc (SEQ ID
NO:38)
(amino acids)
198
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
EVQLVESGGGLVKPGGSLRLSCAASGFIFSRYGMSWVRQAPGKRLEWVSTISGGGIYIYYPDSVKGRFTISRDNA
KNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVIVSS (SEQ ID NO: 39)
Humanized E6 heavy chain variable framework region 1 (FWR1) acid sequence:
(DNA)
gaggtgcagctggtggagtotgggggaggcctggtcaagcctggggggtccctgagactctoctgtgcagcctct
ggattcaccttcagt (SEQ ID NO:40)
(amino acids)
EVOLVESGGGLVKPGGSLRLSCAASGETES (SEQ ID NO:41)
Humanized E6 heavy chain variable complementarity determining regions 1
(CDR1) sequence:
(DNA)
aggtatggcatgagc (SEQ ID NO:42)
(amino acids)
RYGMS (SEQ ID NO:43)
Humanized E6 heavy chain variable framework region 2 (FWR2) acid sequence:
(DNA)
tgggtccgccaggctccagggaagaggctggagtgggtctca (SEQ ID NO :44)
(amino acids)
WVRQAPGKRLEWVS (SEQ ID NO:45)
Humanized E6 heavy chain variable complementarity determining regions 2
(CDR2) sequence:
(DNA)
accattaqtqqcqqaqqcacctacatatactacccaqactcaqtqaaqqqc (SEQ ID NO:46)
(amino acids)
TISGGGTYIYYPDSVKG (SEQ ID NO:47)
Humanized E6 heavy chain variable framework region 3 (FWR3) acid sequence:
(DNA)
cgattcaccatctccagagacaacgccaagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacg
gctgtgtattactgtaccaga (SEQ ID NO:48)
(amino acids)
RETISRDNAKNTLYLQMNSLRAE=AVYYCIR (SEQ ID NO:49)
Humanized E6 heavy chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
gataactatggccgcaactatgattatggcatggattat (SEQ ID NO:50)
(amino acids)
DNYGRNYDYGMDY (SEQ ID NO:51)
Humanized E6 IgG2 heavy chain synthesized by Genescript:
(DNA)
gaattctaagcttgggccaccatggaactggggctccgctgggttttccttgttgctattttagaaggtgtccag
tgtgaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcc
tctggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctca
accattagtggcggaggcacctacatatactacccagactcagtgaagggccgatcaccatctccagagacaac
gccaagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctg:-.gtattactgtaccagagat
aactatqqccgcaactatgattatqqcatqqattattqqqqccaqqqcaccctqqtqaccqtqaqcaqcqcctcc
accaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgc
ctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacacc
LLoccagcLgLccLacagLccLcaggacLcLacLccoLcagcagcgLggLgaccg_gccoLccagcaacLLoggc
acccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgt
199
CA 03187555 2023- 1- 27
WC)2022/027039
PCT/US2021/071017
tqtqtcgagtqcccaccgtqcccagcaccacctqtqqcaggaccgtcagtcttcc7_cttccccccaaaacccaag
gacacccUcaUgaLcUcceggaccccUgaggl-cacgUgcgUggUggLggacglgagccacgaagaccccgaggl_c
cagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagc
accittccgtqtqqtcagcgtcctcaccgttgtqcaccaqqactqgctgaaccmcaaggagtacaagtqcaaggtc
tccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtg
tacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctac
cccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctg
gactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttc
tcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatag
taagtttaaactctaga (SEQ ID NO:52)
(amino acids)
EF*AWATMELGLRWVFLVAILEGVQCEVOLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVS
TISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSAS
TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFG
TQTYTCNVDIMPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKw*V*TLX (SEQ ID NO:53)
Human IgG2 heavy chain constant region sequence:
(DNA)
gcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctg
ggctqcctqqtcaaggactacttccccgaaccqqtgaccmtgtcgtggaactcacmcgctctgaccagcggcgtg
cacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggr.gaccgtgccctccagcaac
ttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaagg7.ggacaagacagttgagcgc
aaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcag7_cttcctcttccccccaaaa
cccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagacccc
gaggtccagttcaactqqtacqtqqaccmcgtggaggtqcataatqccaagacaaagccacgcmaggagcagttc
aacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgc
aaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaacca
caggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagccgacctgcctggtcaaaggc
ttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctccc
atqctqqactccgaccmctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcacycaggcmaac
gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt
aaatag (SEQ ID NO:54)
(amino acids)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL7SGVHTFPAVLQ3SGLYSLSSVVTVPSSN
FGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:55)
Humanized E6 IgG1 heavy chain sequence:
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaacc
attagtggcggaggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgcc
aagaacccactgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtcccagagataac
tatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcgctagcacc
aagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctg
gtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttc
ccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgacagtgccctccagcagcttgggcacc
cagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgt
gacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttcccccca
aaacccaaqqacaccctcatqatctcccqqacccctqacmtcacatqcqtqqtqqr.qqaccrtgagccaccraagac
cctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag
tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggc7_gaatggcaaggagtacaag
UgcaaggLcUccaacaaagcccUcccagccoccaUcgagaaaaccaLcUccaaagccaaagggcagccccgagaa
ccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaa
200
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
ggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcct
cccgLgcLggacLccgacggcLccl.L.cl.LccLcLacagcaagcLcaccgLggacaagagcaggLggcagcagggg
aacgtcttctcatgctccgtqatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccg
qqtaaatqataa (SEQ ID NO:56)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGETFSRYGMSWVRQAPGKRLEWVSTISGGGIYIYYPDSVKGRFIISRDNA
KNPLYLQMNSLRAEDTAVYYCPRDNYGRNYDYGMDYWGQGTLVIATSSASTKGPSVFPLAPSSKSTSGGEAALGCL
VKDYFPEPVIVSWNSGALTSGVHTFPAVLQSSGLYSLSSVITEVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTFITCPPCPAPELLC4C4PSVFLEPPKPKDILMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE0
YNSTYRVVSVLTVL:IQDWLNGHEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLEPSREEMTKNQVSLICLVK
GFYPSDIAVEWESNGQPENNYKTIPPVLDSOGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK** (SEQ ID NO:57)
Human IgG1 heavy chain constant region sequence:
(DNA)
gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctg
ggcLgccLggLcaaggacLaci,LccccgaaccggLgacggLgLegLggaacLcaggcgcccLgaccagcggcgLg
cacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtgg:.gacagtgccctccagcagc
ttqqqcacccaqacctacatctqcaacqtqaatcacaagcccaqcaacaccaaqq7,gqacaagaaaqttgagccc
aaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctc
ttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagc
cacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgg
qaqqaqcaqtacaacagcacqtaccqtqtqqtcaqcqtcctcaccqtcctqcaccaqqactqqctqaatqqcaaq
gagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccactccaaagccaaagggcag
ccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgc
ctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaag
accacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtgg
cagcaqqqqaacqtcttctcatqctccqtqatqcatqaqqctctqcacaaccactacacqcagaagagcctctcc
ctgtctccgggtaaatgataa (SEQ ID NO:58)
(amino acids)
ASTKGPSVIPPLAPSSKSTSGGTAALGCLVKDYIPPEPVIVSWNSGALTSGVIITIPPAVLOSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKESNTKVDKKVEPKSCDKTHICEPCPAPELLGGPSVFLEPPKEKDILMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSIYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNOVSLICLVKGEYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFELYSKL7VDKSRW
QQGNVESCSVMHEALHNNYTQKSLSLSPGK** (SEQ ID NO:59)
Mouse E6 Light Chain variable region sequence:
(DNA)
caaattg-ttctcacccagtotccagcaatcatgtctgcatctccaggggaggagg::caccctaacctgcagtgcc
acctcaagtgtaagttacatacactggttccagcagaggccaggcacttctcccaaactctggatttatagcaca
tccaacctggcttctggagtccctgttcgcttcagtggcagtggatatgqgacctcttactctctcacaatcagc
cgaatggaggctgaagatgctgccacttattactgccagcaaaggagtagttocccattcacgttcggctcgggg
acaaagttggaaataaaa (SEQ =D NO:65)
(amino acids)
QIVLIQSPAIMSAS?GEEVTLICSATSSVSYIHWFQQRPGTSPKLWEYSTSNLASGVPVRFSGSGYGTSYSLTIS
RMEAEDAATYYCQQRSSSPFTEGSGTKLEIK (SEQ ID NO:66)
Mouse E6 light chain variable framework region 1 (FWR1) sequence:
(DNA)
caaattgttctcacccagtctccagcaatcatqtctgcatctccaggggaggagg::caccctaacctgc (SEQ
ID NO:67)
(amino acids)
QIVLIQSPAIMSASIGEEVTLIC (SEQ ID NO:68)
Mouse E6 light chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
201
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
AGTGCCACCTCAAGTGTAAGTTACATACAC (SEQ ID NO:69)
(amino acids)
SATSSVSYIH (SEQ ID NO:70)
Mouse E6 light chain variable framework region 2 (FWR2) sequence:
(DNA)
tggttccagcagaggccaggcacttctcccaaactctggatttat (SEQ ID NO: 71)
(amino acids)
TAIFOORPGTSPKLWIY (SEQ ID NO:72)
Mouse E6 light chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
agcacatccaacctggottot (SEQ ID NO:73)
(amino acids)
STSNLAS (SEQ ID NO:74)
Mouse E6 light chain variable framework region 3 (FWR3) sequence:
(DNA)
ggagtccctgttcgcttcagtggcagtggatatgggacctcttactctctcacaaTacagccgaatggaggctgaa
gatgctgccacttattactgc (SEQ ID NO:75)
(amino acids)
GVPVRFSGSGYGTSYSLTISRMEAEDAATYYC (SEQ ID NO:76)
Mouse E6 light chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
cagcaaaggagtagttccccattcacg (SEQ ID NO:77)
(amino acids)
QQRSSSPFT (SEQ ID NO:78)
Humanized E6 light chain variable region sequence from IGKV3-11*02:
(DNA)
gaaattgtgttgacacagtotccagccaccctgtctttgtctccaggggaaagagccaccctcacctgcagcgcc
accagcagtgttagctacatccactggtaccaacagaggcctggccagagccccaggctcctcatctatagcacc
tccaacctggccagcggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagc
agcctagagcctgaagattttgcagtttattactgtcagcagcgtagcagctcccctttcacctttggcagcggc
accaaagtggaaattaaa (SEQ 7D NO:93)
(amino acids)
EIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGOSPRLLPYSTSNLASGIPARFSGSGSGSDYTLTIS
SLEPEDFAVYYCQQRSSSPFTFGSGTKVEIK (SEQ ID NO:94)
Humanized E6 light chain variable framework region 1 (FWR1) acid sequence:
(DNA)
gaaattgtgttgacacagtctccagccaccctgtotttgtctccaggggaaagagccaccctcacctgc (SEQ
ID NO:95)
(amino acids)
EIVLTQSPATLSLSRGERATLTC (SEQ ID NO:96)
Humanized E6 light chain variable complementarity determining regions 1
(CDR1) sequence:
(DNA)
agcgccaccagcagtgttagctacatccac (SEQ ID NO:97)
(amino acids)
SATSSVSYIH (SEC ID NO:98)
202
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Humanized E6 heavy light variable framework region 2 (FWR2) acid sequence:
(DNA)
tqqtaccaacagaggcctqqccagagccccaqqctcctcatctat (SEQ IC NO: 99)
(amino acids)
WYQQRPGQSPRLLIY (SEQ ID No:100)
Humanized E6 light chain variable complementarity determining regions 2
(CDR2) sequence:
(DNA)
agcacctccaacctggccagc (SEQ ID NO:101)
(amino acids)
STSNLAS (SEQ ID NO:102)
Humanized E6 light chain variable framework region 3 (FWR3) acid sequence:
(DNA)
ggcatcccagccaqqttcagtggcaqtqqqtctgggagcgactacactctcacca7.cagcagcctagagcctgaa
qattttqcaqtttattactqt (SEQ ID NO:103)
(amino acids)
GIPARFSGSGSGSDYILTISSLEPEDFAVYYC (SEQ ID NO:104)
Humanized E6 light chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
cagcagcgtagcagctcccctttcacc (SEC) ID NO:105)
(amino acids)
OORSSSFET (SEQ ID NO:106)
Humanized E6 Kappa light chain synthesized by Genescript:
(DNA)
gaattctaagottqggccaccatqqaagdcccagcgdagcttctottcctcctgdzactctggctcccagatacc
actggagaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctcacctgc
agcgccaccagcagtgttagctacatccactggtaccaacagaggcctggccagagccccaggctcctcatctat
agcacctccaacctggccagcggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcacc
atcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcagctcccctttcacctttggc
aqcqgdaccaaagtggaaattaaaaqqacqqtqgctgcaccatctqtcttcatctcccgccatctgatgagdag
LLgaaaLcLqqaacLqccLcLgLLqLqLqccLqcLqaaLaacl.LcLaLcccagagaqqccaaagLacaqLqqaaq
gtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagc
ctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcag
ggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagtaagtttaaactctaga (SEC ID
NO: 10/)
(amino acids)
EF*AWATMEAPAQLLFLLLLWLPDITGEIVLIQSPATLSLSPGERAILICSATSSVSYIHWYQQRPGQSPRLLIY
STSNLASGIPARFSGSGSGSDYILTISSLEFEDFAVYYCQQRSSSPFTFGSGTKVEIKRIVAAPSVFIFFFSDEO
LKSGTASVVOLLN=PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSILTLSKADYEKHKVYACEVTHQ
GLSSEWIKSFNRGEC**V*TLX (SEQ ID NO:108)
Human Kappa light chain constant region sequence:
(DNA)
aqqacggtggctgcaccatctqtcttcatcttcccgccatctqatgagcagttgaaatctggaactgcctctqtt
gtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggt
aactcccaqqaqaqtqtcacagaqcaqqacaqcaaqqacagcacctacaqcctcaqcaqcaccctqacqctqaqc
aaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcc7.gagctcgcccgtcacaaag
agcttcaacaggggagagtgttag (SEC ID NO:109)
(amino acids)
203
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS
KADYEKHKVYACEVTHQGLSSPVTKSENRGEC (SEQ ID NO:110)
Humanized E6 lambda light chain sequence:
(DNA)
gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctcacctgcagcgcc
accagcagtgttagctacatccactggtaccaacagaggcctggccagagccccaggctcctcatctatagcacc
tccaacctggccagcggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagc
agcctagagcctgaagattttgcagtttattactgtcagcagcgtagcagctoccctttcacctttggcagcggc
accaaagtggaaattaaaggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagctt
caagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccg:gacagtggcctggaaggca
gatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagc
agctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagc
accgtggagaagacagtggcccctacagaatgttcatagtaa (SEQ ID NO:111)
(amino acids)
EIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLL=YSTSNLASGIPARFSGSGSGSDYTLTIS
SLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKGQPKAAPSVTLFPFSSEELQANKATLVOLISDFYPGAVTVAWKA
DSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS** (SEQ ID
NO :112)
Humanized lambda light chain constant region sequence:
(DNA)
ggtcagcccaaggctgccccctcggtcactctgttoccgccctcctctgaggagcttcaagccaacaaggccaca
ctggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaag
gcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacg
cctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtg
gcccctacagaatgttcatagtaa (SEQ ID NO:113)
(amino acids)
GOPKAAPSVTLEPPSSEELOANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKOSNNKYAASSYLSLT
PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS** (SEQ ID NO:114)
Mouse C2 heavy chain variable region sequence:
(DNA)
gaggtccagctggaggagtcagggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctct
ggattcactttcagtggctatgccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaacc
attagtagtggtggtacttatatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgcc
aagaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtattactgtgcaagacttggg
qqqqaLaaLLacLacciaaLacLLcgaALcLqqqgclgcaqgqaccacqqLcaccq_cLccLccqccaaaacqaca
cccccatctgtctat (SEQ ID NO:118)
(amino acids)
EVQLEESGGGLVKPGGSLKLSCAASGFIFSGYAMSWVRQIPEKRLEWVATISSGGTYIYYPDSVKGRF=SRDNA
KNTLYLQMSSLRSEDTAMYYCARLGGDNYYEYEDVWGAGTTVTVSSAKTTPPSVY (SEQ ID NO:119)
Mouse C2 heavy chain variable framework region 1 (FWR1) sequence:
(DNA)
gaggtccagctggaggagtcagggggaggcttagtgaagcctggagggtccctgaaactctoctgtgcagcctct
ggattcactttcagt (SEQ ID NO:120)
(amino acids)
EVOLEESGGGLVKFGGSLKLSCAASGFTFS (SEQ ID NO: 121)
Mouse C2 heavy chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
ggctatgccatgtct (SEQ ID NO:122)
(amino acids)
GYAMS (SEQ ID NO:123)
204
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Mouse C2 heavy chain variable framework region 2 (FWR2) sequence:
(DNA)
tgggttcgccagactccggagaagaggctggagtqggtcgca (SEQ ID NO: 124)
(amino acids)
WVRQTPEKRLEWVA (SEQ ID NO:125)
Mouse C2 heavy chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
accattagtagtggtggtacttatatctactatccagacagtgtgaagggg (SEQ ID NO:126)
(amino acids)
TISSGGTYIYYPDSVKG (SEQ ID NO:127)
Mouse C2 heavy chain variable framework region 3 (FWR3) sequence:
(DNA)
cgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacg
qccatqtattactqtqcaaqa (SEQ ID NO:128)
(amino acids)
RFTISRDNAKNTLYLQMSSLRSEDDAMYYCAR (SEQ ID NO:129)
Mouse C2 heavy chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
cttgggggggataattactacgaatacttcgatgtc (SEQ ID NO:130)
(amino acids)
LGGDNYYEYFDV (SEQ ID NO::31)
humanized derived from IGHV3-21*04:
Humanized C2 heavy chain variable region sequence:
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaacc
attagtagtggcggaacctacatatactaccccgactcagtgaagggccgattcaccatctccagagacaacgcc
aagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttggg
ggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccqz:ctcctcc (SEQ ID
NO:144)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFIFSGYAMSWVRQAPGKGLEWVSTISSGGIYIYYPDSVKGRFTISRDNA
KNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYEDVWGKGITVIVSS (SEC ID NO: 145)
Humanized C2 heavy chain variable framework region 1 (FWR1) sequence:
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagt (SEQ ID NO:146)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGETFS (SEQ ID NO: 147)
Humanized C2 heavy chain variable complementarity determining regions 1
(CDR1) sequence:
(DNA)
ggctatqccatqaqc (SEQ ID NO:148)
(amino acids)
GYAMS (SEQ ID NO:149)
205
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Humanized C2 heavy chain variable framework region 2 (FWR2) sequence:
(DNA)
tgggtccgccaggctccagggaaggggctggagtgggtctcaa (SEQ ID NO:150)
(amino acids)
WVRQAPGKGLEWVS (SEQ ID NO:151)
Humanized C2 heavy chain variable complementarity determining regions 2
(CDR2) sequence:
(DNA)
accattagtagtggcggaacctacatatactaccccgactcagtgaagggc (SEQ ID NO:152)
(amino acids)
TISSGGTYIYYPDSVKG (SEQ ID NO:153)
Humanized C2 heavy chain variable framework region 3 (FWR3) sequence:
(DNA)
cgaLLcaccaLcLccagagacaacgccaagaacLcacLgLaLcLgcaaaLgaacagccLgagagccgaggacacg
gccgtgtattactgtgcgaga (SEQ ID NO:154)
(amino acids)
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:155)
Humanized C2 heavy chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
cttgggggggataattactacgaatacttcgatgtc (SEQ ID NO:156)
(amino acids)
LGGDNYYEYFDV (SEQ ID NO:L57)
Humanized C2 IgG1 heavy chain sequence
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctectgtgcagcctct
ggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaacc
attagtagtggcggaacctacatatactaccccgactcagtgaagggccgattcaccatctccagagacaacgcc
aagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttggg
ggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccg7Ictcctccgctagcaccaag
ggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtc
aaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccg
gcLqLccLacagLccLcaggacLcLacLcccLcagcaqcgLqqLqacaql.gcccLccaqcaqcLLqqqcacccaq
acctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagftgagcccaaatottgtgac
aaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcag7..cttcctcttccccccaaaa
cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccct
gaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac
aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc
aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacca
caggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagccgacctgcctggtcaaaggc
ttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctccc
gtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac
gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctocctgtotccgggt
aaatgataa (SEQ ID NO:157)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFIFSGYAMSWVRQAPGRGLEWVSTISSGGIYIYYPDSVKGRF7ISRDNA
KNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTIVIVSSASTKGPSVFPLAFSSKSTSGGTAALGCLV
KDYFPEPVIVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIVPSSSLGTQTYIONVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPFYKENWYVDGVEVHNAKTKPREFOY
NSTYRVVSVLTVLHQDWLNGKEYKOKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQRSLSLSPG
K** (SEC) ID NO:1O8)
206
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Humanized C2 IgG2 heavy chain sequence
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactrtcctgtgcagcctct
ggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaacc
attagtagtggcggaacctacatatactaccccgactcagtgaagggccgattcaccatctccagagacaacgcc
aagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttggg
ggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccg7;ctcctccgcctccaccaag
ggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtc
aaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttccca
gctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccag
acctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagT.tgagcgcaaatgttgtgtc
gagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacacc
ctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttc
aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttc
cgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaac
aaaggccLcccagcccccaLcgagaaaaccaLcLccaaaaccaaagggcagccccgagaaccacaggLgLacacc
ctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctgg7.caaaggcttctaccccagc
gacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactcc
gacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgc
tccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatagtaa
(SEQ ID NO:163)
(amino acids)
EVOLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNA
KNSLYLQMNSERAEDTAVYYCAREGGDNYYEYFDVWGKG1TVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLV
KDYFPEPVIVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVIVPSSNFGTOTYTCNVDHKPSNTKVDKTVERKCCV
ECPPCPAPPVAGPSVFLFPFKPKDILMISRTPEVICVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF
RVVSVLIVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTEPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGOPENNYKTIPPMEDSDGSFFLYSKETVDKSRWOOGNVFSCSVMHEALHNHYTOKSESESPGK**
(SEQ ID NO:164)
Mouse C2 light chain variable region sequence:
(DNA)
gacattgtgatcacacagtctacagcttccttaggtgtatctctggggcagagggccaccatctcatgcagggcc
agcaaaagtgtcagtacatctggctatagttatatgcactggtaccaacagagaccaggacagccacccaaactc
ctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttc
accctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgttc
acqLLcqqacmqqqaccaagcLqqacjaLaaaacqqqcLqaAcLqcaccaacLq_aLcc (SEQ ID NO:168)
(amino acids)
DIVITQSTASEGVSLGQRATISCRASKSVSTSGYSYMHWYQQRPGQFFKLLIYLASNLESGVFARFSGSGSGTDF
TENIHPVEEEDAATYYCQHSRELPFTEGGGTKLEIKRADAAPTVS (SEQ ID NO:169)
Mouse C2 light chain variable framework region 1 (FWR1) sequence:
(DNA)
gacattgtgatcacacagtctacagcttccttaggtgtatctctggggcagagggccaccatctcatgc (SEQ
ID NO:170)
(amino acids)
DIVITQSTASLGVSLGQRATISC (SEQ ID NO:171)
Mouse C2 light chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
aqggccagcaaaaqtgtcagtacatctggctataqttatatgcac (SEQ ID NO:172)
(amino acids)
RASKSVSTSGYSYMH (SEQ ID NO:173)
207
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Mouse C2 light chain variable framework region 2 (FWR2) sequence:
(DNA)
tggtaccaacagagaccaggacagccacccaaactcctcatctat (SEQ ID NO:174)
(amino acids)
WYQQRPGQPPKLLIY (SEQ ID NO:175)
Mouse C2 light chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
cttgcatccaacctagaatc (SEQ ID NO:176)
(amino acids)
LASNLES (SEQ ID NO:177)
Mouse C2 light chain variable framework region 3 (FWR3) sequence:
(DNA)
LggggLeccLgccaggLLcagLggcagLgggLcLgggacagacLLcaccci_cadcaLccaLccLgLggaggagga
ggatgctgcaacctattactgt (SEQ ID NO:178)
(amino acids)
GVPARFSGSGSGTDFILNINPVEEEDAATYYC (SEQ ID NO:179)
Mouse C2 light chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
cagcacagtagggagcttccgttcacg (SEQ ID NO:180)
(amino acids)
QHSRELPFT (SEQ ID NO:181)
Humanized derived from IGKV7-3*01
Humanized C2 light chain variable region sequence:
(DNA)
gacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgcagagcc
agtaagagtgtcagtaccagcggatactcctacatgcactggtatcagcagaaaccaggacaacctcctaaactc
ctgatttacctggcatccaatctggagagcggggtcccagccaggttcagcggcagtgggtctgggaccgatttc
accctcacaattaatcctgtggaagctaatgatactgcaaatt;Ittactgtcagcacagtagggagctgcctttc
acattcggcggagggaccaaggtggagatcaaacgaact (SEQ ID NO: 194)
(amino acids)
DIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGIDF
ILTINPVEANDTANYYCQHSRELPFTFGGGIKVEIKRT (SEQ ID NO:195)
Humanized C2 light chain variable framework region 1 (FWR1) acid sequence;
(DNA)
gacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgc (SEQ
ID NO:196)
(amino acids)
DIVLTQSPASLAVSPGQRATITC (SEQ ID NO:197)
Humanized C2 light chain variable complementarity determining regions 1
(CDR1) sequence:
(DNA)
agagccagtaagagtgtcagtaccagcggatactcctacatgcac (SEQ ID NO :198)
(amino acids)
RASKSVSTSGYSYMH (SEQ ID NO:199)
Humanized C2 heavy light variable framework region 2 (FWR2) acid sequence:
(DNA)
tggtatcagcagaaaccaggacaacctcctaaactcctgatttac (SEC ID No:200)
208
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
(amino acids)
WYQQKPGQPPKLLIY (SEQ ID NO:201)
Humanized C2 light chain variable complementarity determining regions 2
(CDR2) sequence:
(DNA)
ctggcatccaatctggagagc (SEQ ID NO:202)
(amino acids)
LASNLES (SEQ ID NO:203)
Humanized C2 light chain variable framework region 3 (FWR3) acid sequence:
(DNA)
ggggtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaaT_taatcctgtggaagctaat
gatactgcaaattattactgt (SEQ ID NO:204)
(amino acids)
GVPARFSGSGSGTD7TLTINPVEANDTANYYC (SEQ ID NO:205)
Humanized C2 light chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
cagcacagtagggagctgcctttcaca (SEQ ID NO:206)
(amino acids)
QNSRELPFT (SEQ ID NO:207)
Humanized C2 light chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
ctgcagagtaagaattttcctcccaca (SEQ ID NO:208)
(amino acids)
LQSKNEPPT (SEQ ID NO:209)
Murine Ig kappa chain leader sequence
(DNA)
atggagacagacacactcctgctatgggtactgctgctctgggttccaggttocactggtgac (SEQ ID
NO:222)
(amino acids)
METDTLLLWVLLLWVPGSTGD (SEQ ID NO:223)
Interleukin-2 (IL-2) leader sequence
(DNA)
atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagt (SEQ ID NO:224)
(amino acids)
MYRMQLLSCIALSLALVTNS (SEQ ID NO:225)
CD33 leader sequence
(DNA)
atgcctcttctgottctgcttcctctgctttgggctggagctcttgct (SEQ ID NO:226)
(amino acids)
MPLLLLLPLLWA@ALA (SEQ ID NO:227)
IGHV3-21*03 leader sequence
(DNA)
209
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
atqqaactqqqqctccgctqqqttttccttgttqctattttagaaggtqtccagtqt (SEQ ID NO :228)
(amino acids)
MELGLRWVFLVAILEGVQC (SEQ ID NO:229)
IGHV3-11*02 leader sequence
(DNA)
atggaagccccagcgcagettctettectcctgctactctggctcccagataccactgga (SEQ ID NO:230)
(amino acids)
MEAPAQLLFLLLLWLPDTTG (SEQ ID NO:231)
Humanized E6 single chain GS3
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtetcaacc
attagtggcggaggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgcc
aagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataac
LaUggccgcaacLaLgal_LaUggcaUggaLUaLLggggccagggcacccUgglgaccgUgagcagcggcggUggc
ggatccggcggtggcggatccggcggtggcggatccgaaattgtgttgacacagtctccagccaccctgtctttg
tctccaggcmaaagagccaccctcacctqcagcgccaccagcacitgttagctaca7.ccactgqtaccaacagagg
cctggccagagccccaggctcctcatctatagcacctccaacctggccagcggca7.cccagccaggttcagtggc
agtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagatttgcagtttattactgtcag
cagcgtagcagctcccctttcacctttggcagcggcaccaaagtggaaattaaa (SEQ ID NO: 232)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRF7ISRDNA
KNTLYLOMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTOSPATLSL
SPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQ
QRSSSPFTFGSGTKVEIK (SEQ 7D NO:233)
Humanized E6 single Chain IgGlnoC
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaacc
attagtcmcggaggcacctacatatactacccagactcagtgaacmccgattcaccatctccagagacaacgcc
aagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataac
tatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcgataaaacc
catactaaaccgccaaaaccggcgccggaactgctgggtggtcctggtaccggtgaaattgtgttgacacagtct
ccagccaccctgtctttgtctccaggggaaagagccaccctcacctgcagcgccaccagcagtgttagctacatc
cactggtaccaacagaggcctggccagagccccaggctcctcatctatagcacctccaacctggccagcggcatc
ccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagatttt
gcagULUaLLacUgUcagcagcqUagcagcLccccULUcaccULUggcagccylcaccaaagUggaaaLLaaa
(SEQ ID NO:234)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFIFSRYGMSWVRQAPGKRLEWVSTISGGGIYIYYPDSVKGRF7ISRDNA
KNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSDKTHTKPPKPAPELLGGPGTGEIVLIQS
PAILSLSPGERATLICSATSSVSY=HWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDF
AVYYCQQRSSSPFTFGSGTKVEIK (SEQ ID NO:235)
Humanized E6 single chain X4 (linker is /gG1 and IgG2 modified hinge
region)
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaacc
attagtggcggaggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgcc
aagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataac
tatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcgataaaacc
catactaaaccgccaaaacccmcgccqqaactqctcmcitqqtcctqqtaccqqtactqqtcptccciactattaaa
ectecgaaacctecgaaacctgetccgaacctgetgggtggtecggaaattgtgt7.gacacagtetecagecacc
ctgtctttgtctccaggggaaagagccaccctcacctgcagcgccaccagcagtg7_tagctacatccactggtac
caacagaggcclggccagagccccaggcUccUcaLcUaLagcaccUccaacclggccageggcaUcccagccagg
ttcagtggcagtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttat
210
CA 03187555 2023- 1- 27
VVCO 2022/027039
PCT/US2021/071017
tactqtcagcagcgtagcagctcccctttcacctttcmcacicqqcaccaaagtqqaaattaaa (SEQ ID
NO :236)
(amino acids)
EVOLVESGGGLVKPGGSLRLSCAASGFIFSRYGMSWVRQAPGKRLEWVSTISGGGIYIYYPDSVKGRFTISRDNA
KNTLYLQMNSLRAEDTAVYYCIRDNYGRNYDYGMDYTAIGQGTLVIVSSDKTHTKPPKPAPELLGGPGIG7GGPTIK
PPKPPKPAPNLLGGPEIVLIQSPACLSLSPGERATLICSATSSVSY=HWYQQRPGQSPRLLIYSTSNLASGIPAR
FSGSGSGSDYILTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIK (SEQ ID NO: 237)
Humanized C2 single chain GS3
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaacc
attagtagtggcggaacctacatatactaccccgactcagtgaagggccgattcaccatctccagagacaacgcc
aagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttggg
ggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccg7.ctcctccggcggtggcgga
tccggcggtggcggatccggcggtggcggatccgacattgtgctgacccagtctccagcctccttggccgtgtct
ccaggacagagggccaccaUcaccLgcagagccagUaagagUgUcagUaccagcggaLacUccUacaUgcacUgg
tatcagcagaaaccaggacaacctcctaaactcctgatttacctggcatccaatcr.ggagagcggggtcccagcc
aggttcagccmcagtqcmtctqqqaccgatttcaccctcacaattaatcctqtqqaagctaatgatactqcaaat
tattactgtcagcacagtagggagctgcctttcacattcggcggagggaccaagg7.ggagatcaaacgaact
(SEQ ID NO:238)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFIFSGYAMSWVRQAPGKGLEWVSTISSGGIYIYYPDSVKGRFCISRDNA
KNSLYLOMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVLTOSPASLAVS
PGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFILTINPVEANDTAN
YYCQHSRELPFTFGGGTKVEIKRT (SEQ ID NO:239)
Humanized C2 single Chain IgGlnoC
(DNA)
Gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaacc
attagtagtcmcqqaacctacatatactaccccgactcagtgaacmccgattcaccatctccagagacaacgcc
aagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttggg
ggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccgrxtcctccgataaaacccat
actaaaccgccaaaaccggcgccggaactgctgggtggtcctggtaccggtgaca7tgtgctgacccagtctcca
gcctccttggccgtgtctccaggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagcgga
tactcctacatgcactggtatcagcagaaaccaggacaacctcctaaactcctgattacctggcatccaatctg
gagagcggggtcccagccaggttcagcggcagtgggtctgggaccgatttcacccr.cacaattaatcctgtggaa
gcLaaUgaLaclqcaaaLUaLLacLqUcagcacaqUagggagcLqccULUcacal¨cggcqqacmaccaaggl_g
gagatcaaacgaact (SEQ ID NO:240)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFIFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFCISRDNA
KNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGITVIVSSDKIHTKPPKPAPELLGGPGIGDIVLTUP
ASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVE
ANDTANYYCQHSRELPFTFGGGTKVEIKRT (SEQ ID NO:241)
Humanized C2 single chain X4 (linker is IgG1 and IgG2 modified hinge region)
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaacc
attagtagtggcggaacctacatatactaccccgactcagtgaagggccgattcaccatctccagagacaacgcc
aagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttggg
ggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccg7.ctcctccgataaaacccat
actaaaccgccaaaacccmcgccqqaactqctqcmtqcitcctqqtaccqqtactqqtqqtccgactattaaacct
cegaaacctecgaaacctgetecgaacctgctgggtggtceggacattgtgetgacceagtctecagectecttg
gccgtgtctccaggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctac
aUgcacUggUalcagcagaaaccaggacaaccUccUaaacUccUgaLULacclggcaUccaaLcUggagagcggg
gtcccagccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgat
211
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
actgcaaattattactqtcagcacagtagggacactgcctttcacattcqgcggagggaccaagcatggagatcaaa
cgaacl. (SEQ ID NO:242)
(amino acids)
EVOLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSC;GTYIYYPDSVKGRFTISRDNA
KNSLYLOMNSLRAEDTAVYYCARLGGDNYYEY7DVWGKGTTVTVSSDKINTKPPKPAPELLGGPGIGIGGPTIKP
PKPPKPAPNLEGGPDIVETOSPASLAVSPGQRATITORASKSVSTSGYSYMHWYQQKPGQPPKELIYLASNLESG
VPARFSGSGSGTEFTLTINPVEANDTANYYCQHSRELPFIEGGGTKVEIKRT (SEQ ID NO:243)
Humanized C3 single chain GS3
(DNA)
caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttct
ggttacacctttaccgactacgccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggagtg
atcagcaccttcagcggtaacacaaacttcaaccagaagttcaagggcagagtcaccatgaccacagacacatcc
acgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagaagcgac
tactacggcccatacttcgactactggggccagggcaccaccctgaccgtgtccagcggcggtggcggatccggc
ggtggcggatccggcggtggcggatccgatattgtgatgacccagactccactctctctgtccgtcacccctgga
cagccggccLccaLcLccLgcaggLcLagLcagaccaLLgLccaLagLaaLggaaacaccLaLLLggagLggLac
ctgcagaagccaggccagtctccacagctcctgatctataaggtttccaaccggttctctggagtgccagatagg
ttcaqtqqcaqcqqqtcaqqqacagatttcacactqaaaatcagccqqqtqqaqqctqaqqatqttqqqqtttat
tactgottccaaggtagccacgtgcctttcaccttcggcggagggaccaaggtggagatcaaacgaact (SEQ
ID NO:244)
(amino acids)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGVISTFSGNINFNQKFKGRV2ATTDTS
TSTAYMELRSLRSDDTAVYYCARSDYYGPYFDYWGOGTTLTVSSGGGGSGGGGSGGGGSDIVNTOTPLSESVTPG
OPASISCRSSOTIV4ISNGNTYLEWYLOKPGQSPOLLIYKVSNRFSGVPDRFSGSGSGTDFILKISRVEAEDVGVY
YCFQGSHVPFTFGGGTKVEIKRT (SEC ID NO:245)
Humanized C3 single chain X4 (linker is IgG1 and IgG2 modified hinge region)
(DNA)
caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtotcctgcaaggcttct
qqttacacctttaccqactacqccatqaactqqqtqcqacaqqcccctqqacaaqqqcttqaqtqqatqqqaqtq
atcagcaccttcageggtaacacaaacttcaaccagaagttcaagggcagagtcaccatgaccacagacacatcc
acgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagaagcgac
tactacggcccatacttcgactactggggccagggcaccaccctgaccgtgtccagcgataaaacccatactaaa
ccgccaaaaccggcgccggaactgctgggtggtoctggtaccggtactggtggtccgactattaaacctccgaaa
cctccgaaacctgctccgaacctgctgggtggtccggatattgtgatgacccagactccactctctctgtccgtc
acccctggacagccggcctccatctcctgcaggtctagtcagaccattgtccatagtaatggaaacacctatttg
gagLqqLaccAcacjaalgccaqqccagLcLccacacicLcoLgaLcLaLaaggLLLccaaccqqLLcLcLqqaoLy
ccagataggttcagtggcagegggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatqtt
ggggtttattactgcttccaaggtagccacgtgcctttcaccttcggcggagggaccaaggtggagatcaaacga
act (SEQ ID NO:248)
(amino acids)
OVOLVOSGAEVKKEGASVKVSCKASCYTFITYAMNWVROARGQGLEWMGVISTFSGNTNFNQKFKORVTATTDTS
ISTAYMELRSLREDDTAVYYCARSDYYGPYFDYWGQGTTLIVSSDKMTKPPKPAPELLGGPGIGIGGPTIKPPK
FFKFAFNLLGGFDIVMTOTFLSLSVTFGQPASISCRSSOTIVHSNGNTYLEWYLOKPGOSFOLLIYKVSNRFSGV
PDRFSGSGSGTDFILKISRVEAEDVGVYYCFQGSHVPFTFGGGTKVEIKRT (SEQ ID NO:249)
Humanized C8 single chain GS3 (linker is [Gly4Seri]3)
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtetcaacc
attagtagtggcggaacctacatatactaccctgactcagtgaagggccgattcaccatctccagagacaacgcc
aagaactcactqtatctqcaaatqaacagcctqaqaqccgaqqacacqqccqtqtattactqtqcgagactqqqc
ggogataactattatgaatattggggcaaagggaccacggtcaccgtctoctccggcggtggcggatcoggcggt
ggcggatccggcggtggcggatccgacatcgtgatgacccagtctccagactocctggctgtgtctctgggcgag
agggccaccaLcaacLgcagggccagcaagagLgLLagcaccagcggcLacagcLacaLgcacLggLaccagcag
aaaccaggacagcctcctaagctgctcatttacctgotgtctaacctggaatccggggtccctgaccgattcagt
212
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US202 V071017
qqcaqcqqqtctqqqacagatttcactctcaccatcaqcaqcctqcaqqctqaagatqtqqcaqtttattactqt
caacacal_LcgggaacUgaccaggagUgaaLUcggcggagggaccaaggUggaga_caaacgaaci_ (SEQ ID
NO :250)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRF7ISRDNA
KNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYWGKGTTVIVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGE
RATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLVSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC
QHIRELTRSEFGGGTKVEIKRT (SEQ ID NO:251)
pSECTag2 E6 acFV¨FC
(DNA)
atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacgcggcccagccg
gccgaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccc7gagactctcctgtgcagcc
tctggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctca
accattagtggcggaggcacctacatatactacccagactcagtgaagggccgatcaccatctccagagacaac
gccaagaacacccUgUaLcUgcaaaUgaacagccUgagagccgaggacacggcl_gd_cicUgUaccagagaL
aactatggccgcaactatgattatggcatggattattggggccagggcaccctggr.gaccqgagcagcggcggt
qqcqqatccqqcqqtqqcqqatccqqcqqtqqcqqatccgaaattqtqttqacacaqtctccaqccaccctqtct
ttgtctccaggggaaagagccaccctcacctgcagcgccaccagcagtgttagctacatccactggtaccaacag
aggcctggccagagccccaggctcctcatctatagcacctccaacctggccagcggcatcccagccaggttcagt
ggcagtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttattactgt
cagcaqcqtagcaqctcccctttcacctttqqcaqcqqcaccaaaqtqqaaattaaagagcccaaatcttqtqac
aaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagrxttcctcttccccccaaaa
cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccct
gaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac
aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc
aaqqtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaaqqqcaqccccgagaacca
caggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagccr.gacctgcctggtcaaaggc
ttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctccc
gtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac
gtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt
aaatqataa (SEQ ID NO:256)
(amino acids)
METDTLLLWVLLLWVPGSTGDAAQPAEVOLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVS
TISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGG
GGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFS
GSGSGSDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK** (SEQ ID NO:257)
Human IgG1 Fc sequence:
(DNA)
gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaaccctggggggaccgtcagtc
ttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggr.cacatgcgtggtggtggac
gtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaag
ccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtccr.gcaccaggactggctgaat
ggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaa
gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctg
acctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaac
tacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc
aqqtqqcaqcaqqqqaacqtcttctcatqctccqtqatqcatqaqqctctqcacaaccactacacqcaqaaqaqc
ctctecctgtetccgggtaaatgataa (SEQ ID NO:272)
(amino acids)
213
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQINSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM7KNQVSL
TCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGK** (SEQ ID NO:273)
Human IgG1 CH2-CH3 domain sequence:
(DNA)
ccgtgcccagca
gdactectggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatg
atctcccggacccc:_gaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactgg
tacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtg
gtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagcc
ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccc
ccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatc
gccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggc
tccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgetccgtg
atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgataa (SEQ ID
NO :2/4)
(amino acids)
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQINSTYRV
VSVLTVLHQDWLNGXEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGUENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK**
(SEQ ID NO:275)
Human IgG1 CH3 domain sequence:
(DNA)
gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctg
acctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaac
tacaaqaccacqcctcccqtqctqqactccqacqqctccttcttcctctacaqcaaqctcaccqtqqacaaqaqc
aggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagc
ctctccctgtctccgggtaaatgataa (5E0 ID NO:276)
(amino acids)
GQPREPQVITLPPSREEMTKNQVSLTCLVKGFIPSDIAVEWESNGUENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK** (SEQ ID NO; 277)
CAR-T E6 CD8/CD8/CD28/CD3z sequence:
N-CD81s-huMNE6scFv-CD8ecd fragment- CD8 transmembrane- CD28- CD3zeta-C
(DNA)
al_qqcccUqcccqUqaccqcULUqcl_qcUcccccUqqcqcl_qcl_qcLqcacqccqccaqqccaqaqql_ccaqcl
_q
gttgagagtggcggtgggctggttaagcctggcggctccctgcggctgagctgcgccgcgagtggatttactttc
agccgatatgggatgagttgggtgcggcaagctcccgggaagaggctggaatggg7ctcaacaatctccgggggg
ggcacttacatctattaccccgactcagtcaaggggagatttaccatttcacgagacaacgctaagaataccctg
tatttgcagatgaattctctgagagcagaggacacagctgtttactattgtacccgcgacaactatggcaggaac
tacgactacggtatggactattggggacaagggacattggttacagtgagcagtggcggcgggggcagcggagga
ggaggcagcggtggggggggcagcgagatagtgctcacgcagtcacccgcgactcr.cagtctctcacctggggaa
cgagctaccctgacgtgctctgctacctcctcagtgtcatatattcactggtatcagcaacggcccgggcagtcc
cctagattgctcatttatagtacctctaatctggcctcaggtatccctgcacgatttctggatctggttcaggt
tctgattacaccctcactatctctagcctggagcctgaagactttgccgtttattactgccagcagaggtctagc
tccccattcacctttgggagtgggaccaaggttgaaattaaaacgacaaccccggcccccagaccaccaacgcca
gcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcac
acaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctc
tccctggtgattaccctgtactgcagaagcaagcggtctcggctcctgcattctgattacatgaacatgacccca
agaagaccaggccccaccaggaaacattaccagccctacgctccgccacgcgactr.cgctgcctaccggtcccgc
gttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaac7.gtacaacgagctgaatctc
qqtaqacqqqaaqaqtacqacqtqttqqacaaacqqaqaqqccqcqacccaqaaar.qqqcqqcaaqcctcqcaqq
aaaaacceccaggagggactgtacaatgagttgcagaaagataagatggcagaagettatagcgagatcggaatg
aagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtat
gacgcccUccalaUgcaggcacLLccaccacggLgaLaa
(SEQ ID NO:291)
214
CA 03187555 2023- 1- 27
VVCO 2022/027039
PCT/US2021/071017
(amino acids)
MALPVTALLLPLALLLHAARPEWLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGG
GTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGG
GGSGGGGSEIVLTOSPATLSLSPGERATLTCSATSSVSYTHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSG
SDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH
TRGLDFACDIYIWAPLAGTOGVLLLSLVITLYORSKRSRLLBSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR
VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM
KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR** (SEQ ID NO: 298)
CAR-T E6 CD8/CD8/4-1BB/CD3z sequence:
N-CD81s-huMNE6scFv-CD8ecd fragment- CD8 transmembrane- 4-1BB- CD3zeta-C
(DNA)
atggccctgcccgtgaccgctttgctgctccccctggcgctgctgctgcacgccgccaggccagaggtccagctg
gttgagagtggcggtgggctggttaagcctggcggctccctgcggctgagctgcgccgcgagtggatttactttc
agccgatatgggatgagttgggtgcggcaagctcccgggaagaggctggaatgggctcaacaatctccgggggg
ggcacULacaLcUaLLaccccgacLcagUcaaggggagaULLaccaLLUcacgagacaacgcLaagaaLacccUg
tatttgcagatgaattctctgagagcagaggacacagctgtttactattgtacccgcgacaactatggcaggaac
tacgactacqqtatqqactattgqqqacaagqqacattggttacagtqaqcagtqqcqqcqgqqqcagcqqaqqa
ggaggcagcggtggggggggcagcgagatagtgctcacgcagtcacccgcgactc7.cagtctctcacctggggaa
cgagctaccctgacgtgctctgctacctcctcagtgtcatatattcactggtatcagcaacggcccgggcagtcc
cctagattgctcatttatagtacctctaatctggcctcaggtatccctgcacgat7.ttctggatctggttcaggt
tctgattacaccctcactatctctagcctggagcctgaagactttgccgtttattactgccagcagaggtctagc
tccccattcacctttgggagtgggaccaaggttgaaattaaaacgacaaccccggcccccagaccaccaacgcca
gcccccaccatcgccagccaacccctgtctctgagaccagaagcctgtaggcctgccgccggtggagctgtgcac
acaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcacatgtggagtgctcctcctc
tccctggtgattaccctgtactgcaaaaggggccgcaaaaaactcctttacattt7.taagcagccttttatgagg
ccagtacagacgactcaagaggaagacqggtqctcatqccgctttcctgaqqaqqaqqaaggagggtqcgaactq
cgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaat
ctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgc
aggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcgga
atgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacg
tatgacgccctccatatqcaggcacttccaccacqqtgataa
(SEQ ID NO:300)
(amino acids)
MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGG
GTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGG
GGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYWRPWSPRLLIYSTSNLASGIPARFSGSGSG
SDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH
TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG
MKGERRRGKGHDGLYQGLSTATKUTYDALHMQALPPR**
(SEQ ID NO:301)
CAR-T E6 CD8/CD8/CD28/4-1BB/CD3z sequence:
N-CD81s-huMNE6scFv-CD8ecd fragment- CD8 transmembrane- CD28- 4-1BB-
CD3zeta-C
(DNA)
atggccctgcccgtgaccgctttgctgctccccctggcgctgctgctgcacgccgccaggccagaggtccagctg
gttgagagtggcggtgggctggttaagcctggcggctccctgcggctgagctgcgccgcgagtggatttactttc
agccgatatgggatgagttgggtgcggcaagctcccgggaagaggctggaatgggctcaacaatctccgggggg
ggcacttacatctattaccccgactcagtcaaggggagatttaccatttcacgagacaacgctaagaataccctg
tatttgcagatgaattctctgagagcagaggacacagctgtttactattgtacccgcgacaactatggcaggaac
tacgactacqqtatqqactattgqqqacaagggacattggttacagtgagcagtggcggcqgqggcagcggagga
ggaggcagcggtggggggggcagcgagatagtgctcacgcagtcacccgcgactc7.cagtctctcacctggggaa
cgagctaccctgacgtgctctgctacctcctcagtgtcatatattcactggtatcagcaacggcccgggcagtcc
ccUagal_l_gcl_caLLUaLagUaccLcUaaUcUggccUcaggLaUccoUgcacgal_l_l_cUggaUcUggLUcagg
L
tctgattacaccctcactatctctagcctggagcctgaagactttgccgtttattactgccagcagaggtctagc
215
CA 03187555 2023- 1- 27
LZ -Z0Z GSSL9t0 VO
9TZ
dMOONScialIOUUM(MACASHUUSWISNAZONOOOMAVdVaVSUSZNAU7S3OOSSESdZE3S3OCISSOMMOAdU
HadON3=7,77MMUSUMSUAVVICIUddVAdOAHMUMdOdUUdINNNAGSWIZUSUMSUOATLIA7S777A00MOV7
dYMIAIODVE(T10)3MHAVDOVVdHOYEdErIS'IdOSVIM(DidIddHdVaMM.L.DIMIZAMMOSSEMEdrIEHSH
ODAA
NVIONVaAdNIMTlaaMOSOSSSaUVdAOSTINSWIAIVINddOOdMOCIAMHWASAOSMSASMSVUOMIMVUOOd
SATISVdSOIZAIOS9999S9D59S9959SSAIAII9M9MACHX2AANCIDSZUVOAAAILIOTaTISNNOZA7SNM
liNOUSIZAUOMASOciXAIX/OOSSIMSAMarlOMOdInUAMSGWADSZJAOSII,VOS7U7SOOdMA7000SaArnA
a
(yrrpu, OUTW12)
(90E:ON (II Os) PP1P616.60P00P00110e05bP061P1P0010
Doboabqeqboequbeeeepeepbppeopqbwobbbeoqe1Twobboeboepebbeeebbbeboebebbeeeb
bbb5evbqeebbolebebobeqeqlobeebeobbqebeeqebeevbvobqqbebqeeoeqbqoebbbebbe0000
ow21212125512obogoobuyobbob55.4121212512000s5oboo551251255ovvvol255.11.5.1.5oub
ovq51251212555o
e5eqbbololeeblo5eboee3elbweeooee5e3obbbeobevoelloblooboboeboobe3lvb000loll
bvt.11bobobloevboblbbbebbvvffmbbebbebloolqwbooblvowbqbbbovbevbbvbevolovbot.
beopqbepobbebqeqqqqopbeobeeqqqqqepeqqqopqpeepepeaboobbbbeeeepoqbbopewobwb
oqloeboboeooboowboeq000beooeqqeoeeebbeooe0000bbeooebeebee0000ebqeoeebqeoeq
qvbqoggyobqopqobbogoqbbobvvobvv5vobwygbqoopwqqp5.1.55.4000gogoogoogobgbp55.1.5.
4
eoeobbeobowb0000555444eoewqe4e545400boq44e554oebbebeeoeoeo6464o5v55455oo5
oobqoobbeqbqoobeebeooebebqoqoqbq0000eeoobeooboqeooe00000beooboevooeooebe000
33bb3333ve325o2332P526221122Pbolbbet,23235bbbbobbolqboelll0000lo226563331123
35532352
bbbwoqqqqpbobobqopbbobbooqbybbqqovvvoqqpboloqyqq.1.26.4.1.6.4p22poppopowvoybbyp
o
eevbeoeeoqegbtgoeofyLez)egobeoegobbobeooeobebqbobeeeeoowobbbogbqeoeqgeeoegob
oboveoobb0004o45454o564000wobe00004eeoeoeow54544e4e545ee55e56555555eo4555
obbebbqbbe3loeblbbPbbe553b223111b2ovblb33el3ebbb222bbbbblblbovbqqw21626321
3el3evoeb3553553-4335323515qqeq3eqeq5q3bq3e3ebevbe3555eqq3qbe3ee54eve33q33eq
3-4333-4322b22vob.42232b3b3l3qqq2532qqq2533552vvvqb332b33qvqq2q2-4232-4-4323bb
ebbeo4bwo424w43-45-Lbbb4bebo432bbweebbbboDwobbwowbww-45554004b4eoobo2-44552o4
qqqweo.11.45523woboobob.11.523.4o2523.43o52255.465o3o5225.455.43255555o55.45e5e
beq5
33535333235335333533332533533552
(Ima)
3-21azEco
-Elzap 9c0 -4uswfmag posEM-Aa:DvZONWILI-TEICO-N
:4301.10nbou zcao/5st-t/scao/8ao/8ao co X-UVO
(17oc:oN ai Os) .uaa
7V514H711OAMOXIVMS700A70OHOXOUUUa0X140IaSAVaVIAIXOX07aNAZOndNXUUEXDONadOUOUUXO7
AOAaaUUS'INgaNArIONOSONAlid5iMiSUSaMAUrlaOSSaaaadaUDSOSOaaOLLOAdUNadOMaIMMMUSU
NSUIllniaCIUddliAdOXHNII1dOdUUdINNNAOSH77USUNSUOMIA7S777A00.1.01i7liMIXIOOliaTI
OUM
HAVDOVValOVadU7S7dOSVIMdVdMddUdVdMMINIaAMMOSOLLMSSSU000AXAVEOadarISSIM7MAOS
OSOSOSIUlidIOSIEINSMSAIrrnlaSOOdU0OXMHIXSASSIMSOMTIVIMOdS7S7VidSOIZAIZSOODOSOO
00SOODOSSAMATIOOOMAUKOAOANUOANOILLOAAAILLOaVIMSNNOZATINXIiNOUSLIALIOXASOdAAIAMO
DOSIMSAM3ZUMOdVOUAMSKOAUS011010SVVOS7U7SOOdNAZOODS3A70A3dUVV117771i7d777VMAdril
ai
(sID10e ouTwe)
(E0E:ON ai ozs)
2225553233233
gwyobbeobgegyoog000boebgegboegebeeveoeeobeovooqbwobbbeogeggwobboeboeoebb
eeeffi5eb3ebebteee56556ee6qeebb3qebe53beqe4q3beebe3bbqebe8qebeeebe3bqqbebqee
oelbloebbbebbv00000eeeeebbeoboloobegobbobbbleevbe000eboboobbebebboeeeoebbll
bqbo2.632-452bevbffioPfmqbboqoqP2bqobeboP232-1.blovvo32262336652362232-4336-
433636
32533.623.485333.43.4.45eelq53535q3ee535.455526bee5525525525.433qq.435335.42oqo
b.4555
32522.65e5ee3g3e53e5e3eqf233.6525.42.11.4433523522.44.11.4232.4.433g3eeeeee3b33
55552
eyvoollbbooelloobllobo-
L11:)yboboyooboolloboell000beoovqqyoyeebbyooe0000bbyooybeyby
e0000vbleoeebqeoelqeblolleobwolobbololbbobeeobeebeobloelbl000eqleblbbl000l
oloolooloblbeff)151Povobbvoboqob0000bbblqqe32131P126161336311125bloeffm62232
opo616136e661E6Do600610366e161336peEpoop6e6131316l0000ppoo6e036olpoop000006
voobovvoovooyby00000bb0000vvoybovvyyggyvvbqqbbyvooybbbgbybbbqgwoyoggy0000g
LIOLLORZOZSallad
6COLZO/ZZOZ OM
WO 2022/027039
PCT/US2021/071017
RRKNPQEGLYNELQKDKMAEAYSEECMKGERRRGKGHDGLYQGLSTATKDTYDALRMQALPPR** (SEQ ID
NO:307)
Humanized E6 scFV sequence in CAR:
(DNA)
gaggtccagctggttgagagtggcggtgggctggttaagcctggcggctccctgcggctgagctgcgccgcgagt
ggatttactttcagccgatatgggatgagttgggtgcggcaagctcccgggaagaggctggaatgggtctcaaca
atctccggggggggcacttacatctattaccccgactcagtcaaggggagatttaccatttcacgagacaacgct
aagaataccctgtatttgcagatgaattctctgagagcagaggacacagctgtttactattgtacccgcgacaac
tatggcaggaactacgactacggtatggactattggggacaagggacattggttacagtgagcagtggcggcggg
ggcagcggaggaggaggcagcggtggggggggcagcgagatagtgctcacgcagtcacccgcgactctcagtctc
tcacctggggaacgagctaccctgacgtgctctgctacctcctcagtgtcatatattcactggtatcagcaacgg
cccgggcagtcccctagattgctcatttatagtacctctaatctggcctcaggtat.ccctgcacgattttctgga
tctggttcaggttctgattacaccctcactatctctagcctggagcctgaagactt.tgccgtttattactgccag
cagaggtctagctccccattcacctttgggagtgggaccaaggttgaaattaaa (SEQ ID NO:34-_)
(amino acids)
EVOLVESGGGLVKPGGSLRLSCAASGFIFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFDISRDNA
KNTLYLQMNSLRAEDTAVYYCIRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLIQSPAILSL
SPGERATLICSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYILTISSLEPEDFAVYYCQ
QRSSSPFTFGSGTKVEIK (SEC 7D NO:342)
CD8 leader sequence:
(DNA)
atggccctgcccgtgaccgctttgctgctccccctggcgctgctgctgcacgccgccaggcca (SEQ ID
NO:343)
(amino acids)
MALPVTALLLPLALLLHAARP (SEQ ID NO:344)
CD8 hinge domain sequence:
(DNA)
acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaa
gcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgat (SEQ ID NO :345)
(amino acids)
ITTPAPAPPIPAPTIASQPLSLRPEACRPAAGGAVNTRGLDFACD (SEQ ID NO:346)
CD4 hinge domain sequence:
(DNA)
Lcqqqacaqql.ccLqcLqgaaLccaacaLcaaggLLcLqcccacaLqqLccaccccqql.gcagcca (SEQ ID
NO:347)
(amino acids)
SGQVLLESNIKVLPTWSTPVQP (SEQ ID NO:348)
CD28 hinge domain sequence:
(DNA)
aaacacctttgtccaagtcccctatttcccggaccttctaagccc (SEQ ID NO:349)
(amino acids)
KHLCPSPLFPGPSKP (SEQ ID NO:350)
CD3 zeta transmembrane domain sequence:
(DNA)
ctctgctacctgctggatggaatcctcttcatctatggtutcattctcactgoctogttcctg (SEQ ID
NO:361)
(amino acids)
LCYLLDGILFIYGVILTALFL (SEQ ID No:362)
217
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
CD8 transmembrane domain sequence:
(DNA)
atctacatttgggccccgctcgcaggcacatgtggagtgctcctcctctccctgg7.gattaccctgtactgc
(SEQ ID NO:363)
(amino acids)
IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:364)
CD4 transmembrane domain sequence:
(DNA)
atggccctgattgtgctggggggcgtcgccggcctoctgcttttcattgggctaggcatcttcttc (SEQ ID
NO 365)
(amino acids)
MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO:366)
CD28 transmembrane domain sequence:
(DNA)
ttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttc
tqcmtg (SEQ ID NO:367)
(amino acids)
FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:368)
4-1BB transmembrane domain sequence:
(DNA)
atcatctccttctttottgcgctgacgtcgactgcgttgetcttcctgctgttctcctcacgctccgtttctct
gttgtt (SEQ ID NO:369)
(amino acids)
IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO:370)
0X40 transmembrane domain sequence:
(DNA)
gttgccgccatcctgggcctgggcctggtgctggggctgctgggccccctggcca7.cctgctggccctgtacctg
ctc (SEQ ID NO:371)
(amino acids)
VAAILGLGLVLGLLGFLAILLALYLL (SEQ ID NO: 372)
CD3 zeta domain sequence:
(DNA)
cgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactgtacaacgagctgaat
ctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgc
aggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagatcgga
atgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacg
tatgacgccctccatatgcaggcacttccaccacgg (SEQ ID NO:373)
(amino acids)
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG
MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPFR (SEQ ID NO:374)
CD3 zeta domain variant sequence:
(DNA)
agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaat
ctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgaga
aggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattggg
atgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacc
tacgacgcccttcacatgcaggccctgccccctcgc (SEQ ID NO:375)
(amino acids)
218
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKaRGRDPEMGGKPRIIKNPQEGLYNELQKDKMAEAYSEIG
MKGERRRGKGEDGLYQGLSTATKDEYDALEMQALPPR (SEQ ID NO:376)
CD28 domain sequence:
(DNA)
agaagcaagcggtctoggctoctgcattctgattacatgaacatgaccccaagaagaccaggccccaccaggaaa
cattaccagccctacgctccgccacgcgacttcgctgcctaccggtcc (SEQ ID NO:377)
(amino acids)
RSKRSRLLHSDYMNMTPRREGETRKHYQPYAPPRDFAAYRS (SEQ ID NO:378)
4-1BB domain sequence:
(DNA)
aaaaggggccgcaaaaaactoctttacatttttaagcagocttttatgaggccagacagacgactcaagaggaa
gacgggtgctcatgccgctttcctgaggaggaggaaggagggtgcgaactg (SEQ ID NO: 379)
(amino acids)
KRGRKKLLYIFKQFFMREWQTTQEEDGCSCRFFEEEEGGCEL (SEQ ID NO:380)
HumanizeE6 scFV (VH-VL) sequence:
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtaggtatggcatgagctgggtocgccaggctccagggaagaggctggagtgggtetcaacc
attagtggcggaggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgcc
aagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataac
tatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcggeggtggc
ggatccggcggtggcggatccggcggtggcggatccgaaattgtgttgacacagtctccagccaccctgtctttg
tctocaggggaaagagccaccctcacctgcagcgccaccagcagtgttagctacatccactqgtaccaacagagg
cctggccagagccccaggctcctcatctatagcacctccaacctggccagcggcacccagccaggttcagtggc
agtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcag
cagcgtagcagctcccctttcacctttggcagcggcaccaaagtggaaattaaa (SEQ ID NO:39:)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGETESRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFEISRDNA
KNTLYLOMNSLRAEDTAVYYCIRDNYGRNYDYGMDYWGOGILVTVSSGGGGSGGGGSGGGGSEIVLTOSPATLSL
SPGERATLICSATSSVSYINWYQQAPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYILTISSLEPEDFAVYYCQ
QRSSSPFTFCrSGTKVEIK (SEC) ID NC:392)
HumanizeE6 scFV (VL-VH) sequence:
(DNA)
gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctcacctgcagcgcc
accagcagtgttagctacatccactggtaccaacagaggcctggccagagccccaggctcctcatctatagcacc
tccaacctggccagcggcatcccagccaggttcagtggcagtgggtctgggagcgactacactctcaccatcagc
agcctagagcctgaagattttgcagtttattactgtcagcagcgtagcagctoccctttcacctttggcagcggc
accaaagtggaaattaaaggcggtggcggatccggcggtggcggatccggcggtggcggatccgaggtgcagctg
gtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttc
agtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaaccattagtggcgga
ggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgccaagaacaccctg
tatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataactatggccgcaac
tatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagc (SEQ ID NO:393)
(amino acids)
EIVLTOSPATLSLSPGERATLICSATSSVSYIHWYOORPGQSPRLL=YSTSNLASGIPARFSGSGSGSDYTLTIS
SLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFIT
SRYGMSWVRQAPGKRLEWVSTISGGGIYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCIRDNYGRN
YDYGMDYWGQGTLVIVSS(SEQ ID NO:394)
HumanizeC2 scFV (VH-VL) sequence:
(DNA)
219
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
claggtgcagctggtggagtotgggggaggcctqgtcaagoctgggggqtccctgagactctoctqtgcagcctct
ggaLLcaccL.LcagLygcLaLgccaLgagcLgggLccgccaggcLccagggaaggggcLggagLgggLoLcaacc
attagtagtggcggaacctacatatactaccocgactcagtgaagggccgattcaccatctccagagacaacgcc
aagaactcactqtatctqcaaatgaacagcctgagagccgaggacacqgccgtqtattactqtqcgagacttgqg
ggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccg7Ictcctccggcggtggcgga
tccggcggtggcggatccggcggtggcggatccgacattgtgctgacccagtctccagcctccttggccgtgtct
ccaggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacatgcactgg
tatcagcagaaaccaggacaacctcctaaactcctgatttacctggcatccaatc:-.ggagagcggggtcccagcc
aggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaat
tattactgtcagcacagtagggagctgcctttcacattcggcggagggaccaagg=ggagatcaaacgaact
(SEQ ID NO:395)
(amino acids)
EVOLVESGGGLVKPGGSLRLSCAASGETFSGYAMSWVROAPGKGLEWVSTISSGGTYIYYPDSVKGRETISRDNA
KNSLYLOMNSLRAEDTAVYYCARLGGDNYYEYEDVWGKGTIVIVSSGGGGSGGGGSGGGGSDIVLTOSPASLAVS
PGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKELIYLASNLESGVPARFSGSGSGTDETLTINPVEANDTAN
YYCQIISRELDFTEGGGTKVEIKRT (SEQ ID NO:396)
HumanizeE6 scFV (VL¨VH) sequence:
(DNA)
gacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagggccaccatcacctgcagagcc
agtaagagtgtcagtaccagcggatactcctacatgcactggtatcagcagaaaccaggacaacctcctaaactc
ctgatttacctggcatccaatctggagagcggggtcccagccaggttcagcggcagtgggtctgggaccgatttc
accctcacaattaatcctgtggaagctaatgatactgcaaattattactgtcagcacagtagggagctgcctttc
acattcggcggagggaccaaggtggagatcaaacgaactggcggtggcggatccggcggtggcggatccggcggt
ggcggatccgaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgt
gcagcctctggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgg
gtctcaaccattagtagtggcggaacctacatatactaccccgactcagtgaagggccgattcaccatctccaga
gacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcg
agacttgggggggataattactacgaatacttcgatgtctggggcaaagggaccacggtcaccgtctcctcc
(SEC) ID NO:397)
(amino acids)
DIVETOSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPFKLLIYLASNLESGVPARFSGSGSGTDF
TLTINPVEANDTANYYCQHSRELPFTEGGGTKVEIKRIGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSC
AASGETFSGYAMSWVROAPGKGLEWVSTISSGGTYIYYPDSVKGRETISRDNAKNSLYLOMNSLRAEDTAVYYCA
RLGGDNYYEYEDVWGKGTIVIVSS (SEQ ID NO:398)
G4S1 linker sequence:
(DNA)
qqcqqLqqcqqaLcc (SEQ ID NO:399)
(amino acids)
GGGGS (SEQ ID NO:400)
[G4S1]x3 linker sequence:
(DNA)
ggcggtggcggatccggcggtggcggatccggcggtggcggatcc (SEQ ID NO:401)
(amino acids)
GGGGSGGGGSGGGGS (SEQ ID NO:402)
8 aa GS linker sequence:
(DNA)
ggcggttccggcggtggatccgga (SEQ ID NO:403)
(amino acids)
GGSGGGSG (SEQ ID NO:404)
12 aa GS linker sequence:
(DNA)
220
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
gqcqqttccggcggtggatccqqcqgtggcqqatccqqa (SEQ ID NO:405)
(amino acids)
GGSGGGSGGGSG (SEQ ID NO:406)
13 aa GS linker sequence:
(DNA)
ggcggtggatccggcggtggcggatccggcggtggatcc (SEC) ID NO :407)
(amino acids)
GGGSGGGGSGGGS (SEQ ID NO:408)
22 aa GS linker sequence:
(DNA)
ggcggtggaagcggcggtggcggatccggcagcggcggaagcggcggtggcggatccggcggtgga (SEQ ID
NO:409)
(amino acids)
GGGSGGGGSGSGGSGGGGSGGG (SEQ ID NO:4110)
24 aa GS linker sequence:
(DNA)
ggcggttccggcggtggatcoggcggtggcggatcoggaggcggttccggcggtggatccggcggtggcggatcc
qqa (SEQ ID NO:411)
(amino acids)
GGSGGGSGGGSGGGSGGGSGGGSG (SEQ ID NO:412)
Mouse C3 Heavy chain variable region sequence:
(DNA)
caggtccagctgcagcagtotgggcctgagctggtgaggcctggggtctcagtgaagatttcctgcaagggttcc
ggctacagattcactgattatgctatgaactgggtgaagcagagtcatgcaaagagtctagagtggattggagtt
attagtactttctctggtaatacaaacttcaaccagaagtttaagggcaaggccacaatgactgtagacaaatcc
tccagcacagcctatatqqaacttqccagattqacatctqaqqattctqccatqtattactqtqcaagatcqqat
tactacggcccatactttgactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO:413)
(amino acids)
QVQLQ0SGPELVRPGVSVKISCKGSGYRFTDYAMNWVKOSHAKSLEWIGVISTFSGNTN7NOKFKGKATMTVDKS
SSTAYMELARLTSEDSAMYYCARSDYYGFY7DYWGQGTTLIVSS (SEQ ID NO: 414)
Mouse C3 heavy chain variable framework region 1 (FWR1) sequence:
(DNA)
caggtccagctgcagcagtctgggcctgagctggtgaggcctggggtotcagtgaagatttcctgcaagggttcc
ggctacagattcact (SEQ ID NO:415)
(amino acids)
QVQLQQSGPELVRPGVSVKISCKGSGYRFT (SEQ ID NO:416)
Mouse C3 heavy chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
gattatgctatgaac (SEQ ID NO:417)
(amino acids)
DYAMN (SEQ ID NO:418)
Mouse C3 heavy chain variable framework region 2 (FWR2) sequence:
(DNA)
tgggtgaagcagagtcatgcaaagagtctagagtggattgga (SEC) ID NO: '119)
(amino acids)
221
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WVKQSHAKSLEWIG (SEQ ID NO:420)
Mouse C3 heavy chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
gttattagtactttctctggtaatacaaacttcaaccagaagtttaagggc (SEQ ID NO:421)
(amino acids)
VISTFSGNINFNQKFKG (SEQ ID NO:422)
Mouse C3 heavy chain variable framework region 3 (FWR3) acid sequence:
(DNA)
aaggccacaatgactgtagacaaatectccagcacagcctatatggaacttgccagattgacatctgaggattct
gccatgtattactgtgcaaga (SEQ ID NO:423)
(amino acids)
KATMTVDKSSSTAYMELARLTSEDSAMYYCAR (SEQ ID NO:424)
Mouse C3 heavy chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
tcggattactacggcccatactttgactac (SEQ ID NO:42b)
(amino acids)
SDYYGPYFDY (SEQ ID NO:426)
Humanized C3 heavy chain variable region sequence from IGHV1-18*04:
(DNA)
caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttot
ggttacacctttaccgactacgccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggagtg
atcagcaccttcagcggtaacacaaacttcaaccagaagttcaagggcagagtcaccatgaccacagacacatcc
acgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagaagcgac
tactacqqcccatacttcqactactqqqqccaqqqcaccaccctqaccqtqtccaqc (SEQ ID NO:439)
(amino acids)
QVQLVQSGAEVKKPGASVKVSCKASGYTFIDYAMNWVRQAPGQGLEWMGVISTFSGNINFNQKEKGRV7MTIDTS
TSTAYMELRSLRSDDTAVYYCARSDYYGPYFDYWOOGITLIVSS (SEC ID NO: 44G)
Humanized C3 heavy chain variable framework region 1 (FWR1) acid sequence:
(DNA)
caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttct
ggttacacctttacc (SEQ ID NO:441)
(amino acids)
QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO:442)
Humanized C3 heavy chain variable complementarity determining regions 1
(CDR1) sequence:
(DNA)
gactacgccatgaac (SEQ ID NO:443)
(amino acids)
DYAMN (SEQ ID NO:444)
Humanized C3 heavy chain variable framework region 2 (FWR2) acid sequence:
(DNA)
tgggtqcqacaqqcccctqqacaaqqqcttqaqtqgatqqqa (SEQ ID NO; 445)
(amino acids)
WVRQAPGQGLEWMG (SEQ ID NO:446)
222
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Humanized C3 heavy chain variable complementarity determining regions 2
(CDR2) sequence:
(DNA)
gtgatcagcaccttcagcggtaacacaaacttcaaccagaagttcaagggc (SEQ ID NO:447)
(amino acids)
VISTFSGNINFNQKFKG (SEQ ID NO:448)
Humanized C3 heavy chain variable framework region 3 (FWR3) acid sequence:
(DNA)
agagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacg
gccgtgtattactgtgcgaga (SEQ ID NO:449)
(amino acids)
RVIMITDISTSTAYMELRSLRSDDTAVYYCAR (SEQ ID NO:450)
Humanized C3 heavy chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
agogactactacggcccatacttcgactac (SEQ ID NO:451)
(amino acids)
SDYYGFYFDY (SEQ ID NO:452)
Humanized C3 IgG1 heavy chain sequence
(DNA)
caggttcagctggtgcagtotggagctgaggtgaagaagoctggggcctcagtgaaggtctoctgcaaggcttct
ggttacacctttaccgactacgccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggagtg
atcagcaccttcagcggtaacacaaacttcaaccagaagttcaagggcagagtcaccatgaccacagacacatcc
acgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagaagcgac
tactacggcccatacttcgactactggggccagggcaccaccctgaccgtgtccagcgctagcaccaagggccca
tcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccc7.gggctgcctggtcaaggac
tacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcggcacaccttcccggctgtc
ctacagtcctcaggactctactccctcagcagcgtggtgacagtgccctccagcagcttgggcacccagacctac
atctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaact
cacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcc7;cttccccccaaaacccaag
gacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtc
aagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagc
acgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtc
tccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtg
LacacccLqcccccaLcccqqqaqqacjaLqaccaagaaccaqqLcaqccLgaccLqccLggLcaaaqqcLLcLaL
cccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctg
gactocgacggctccttcttoctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttc
tcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtotccgggtaaatga
taa (SEQ ID NO: 453)
(amino acids)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAMNWVRQAPGQGLEWMGVISTYSGNINFNQKFKGRV?MTIDTS
TSTAYMELRSLRSDDTAVYYCARSDYYGFYFDYWGQGTTLIVSSASTKGPSVFFLAFSSKSTSGGTAALGCLVKD
YEPEPVIVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIVPSSSLG7QTYICNVNHKPSNTKVDKKVEPKSCDKT
HICFPCFAFELLGGPSVFLEFFKFKDTLMISRTPEVICVVVDVSHEDFEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPOVYTLPPSREEMTKNOVSLICLVKGFY
PSDIAVEWESNGQPENNYKTIPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
*(SEQ ID NO:454)
Humanized C3 IgG2 heavy chain sequence
(DNA)
caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttct
ggttacacctttaccgactacgccatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggagtg
aLcagcaccLLcagcggLaacacaaacLLcaaccagaagLLcaagggcagagLcaccaLgaccacagacacaLcc
acgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagaagcgac
223
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tactacggcccatacttcgactactggggccagggcaccaccctgaccgtgtccagcgcctocaccaagggccca
LoggLcl.LccccoLggcgoccLgcLccaggagcaccLccgagagcacagccgccc_gggoLgccLggLcaaggac
tacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtc
ctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctac
acctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgraaatgttgtgtcgagtgc
ccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatg
atctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactgg
tacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagtcaacagcacgttccgtgtg
gtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggc
ctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgccc
ccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatc
gccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggc
tccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg
atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatagtaa (SEQ ID
NO:455)
(amino acids)
QVQLVQSGAEVKKFGASVKVSCKASGYTFTDYAMNWVRQAFGQGLEWMGVISTFSGNTNFNQKFKGRV7MTTDTS
TSTAYMELRSLRSDDTAVYYCARSDYYGDYFDYNGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSOLYSLSSVVTVPSSNFG7QTYTCNVDHKPSNTKVDKTVERKCCVEC
PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVOFNWYVDGVEVHNAKTKPREEOFNSTFRV
VSVLTVVNQDWLNGKEYKOKVSNKOLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGOPENNYHTTFFMLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTOKSLSLSFGK**(SE
Q ID NO:456)
Mouse C3 Light Chain variable region sequence:
(DNA)
gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatct
agtcagaccattgtacatagtaatggaaacacctatttagaatggtacctgcagaaaccaggccagtotccaaag
ctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagat
ttcacactcaagatcaacagagtggaggctgaggatctgggagtttattactgctttcaaggttcacatgttcca
ttcacgttcggctcggggacaaagttggaaataaaa (SEQ ID NO:458)
(amino acids)
DVLMTQTPLSLPVSLODQASISCRSSQTIVHSNONTYLETNYLQKPOQSPKLLIYKVSNRFSGVPDRFSGSGSGTD
FTLKINRVEAEDLOVYYCFQGSHVPFTEGSGTKLEIK (SEQ ID NO:459)
Mouse C3 light chain variable framework region 1 (FWR1) sequence :
(DNA)
gatgttttgatgacccaaactccactctccctgcctgtcagtottggagatcaagcctccatctcttgc (SEQ
ID NO:460)
(amino acids)
DVLMTQTFLSLEWSLGDOASISC (SEQ ID NO:461)
Mouse C3 light chain variable complementarity determining regions 1 (CDR1)
sequence :
(DNA)
agatctagtcagaccattgtacatagtaatggaaacacctatttagaa (SEQ ID NO:462)
(amino acids)
RSSOTIVHSNONTYLE (SEQ ID NO:463)
Mouse C3 light chain variable framework region 2 (FWR2) sequence:
(DNA)
tggtacctgcagaaaccaggccagtctccaaagctcctgatctac (SEQ ID NO:464)
(amino acids)
WYLOKPOOSPKLLIY (SEQ ID NO:465)
Mouse C3 light chain variable complementarity determining regions 2 (CDR2)
sequence:
224
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(DNA)
aaagLLLccaaccgaLLLLcl. (SEQ ID NO:466)
(amino acids)
KVSNRFS (SEQ ID NO:467)
Mouse C3 light chain variable framework region 3 (FWR3) sequence:
(DNA)
ggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaaga7;caacagagtggaggctgag
gatctgggagtttattactgc (SEQ ID NO:468)
(amino acids)
GVFDRFSGSGSGTD7TLKINRVEAEDLGVYYC (SEQ ID NO:469)
Mouse C3 light chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
LLLcalaggi_LcalcaLgLLccaLLcacg (SEQ ID NO:470)
(amino acids)
FQGSHVPFT (SEC ID NO:471)
Humanized C3 light chain variable region sequence from IGKV2-29*03:
(DNA)
gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaggtct
agtcagaccattgtocatagtaatggaaacacctatttggagtggtacctgcagaagccaggccagtotccacag
ctcctgatctataaggtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagat
ttcacactgaaaatcagccggqtqqaggctgaggatqttgqggtttattactgctccaaggtagccacgtqcct
ttcaccttcggcggagggaccaaggtggagatcaaacgaact (SEQ ID NO: 486)
(amino acids)
DIVMTOTPLSLSVTPGQPASISCRSSOTIVIISNGNTYLEWYLOKPGOSPOLLIYKVSNRIPSGVPDRIPSGSGSGTD
FILKISRVEAEDVGVYYCFQGSHVPFTFGGGTKVEIKRT (SEQ ID NO:487)
Humanized C3 light chain variable framework region 1 (FWR1) acid sequence:
(DNA)
gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgc (SEQ
ID NO:488)
(amino acids)
DIVMTQTPLSLSVTPOQPASISC (SEQ ID NO:489)
Humanized C3 light chain variable complementarity determining regions 1
(CDR1) sequence:
(DNA)
gyLcLagLcagaccaLLgLccaLagLaaLggaaacaccLaLLAgag (SEQ ID NO:490)
(amino acids)
RSSOTIVHSNGNTYLE (SEQ ID NO:491)
Humanized C3 light chain variable framework region 2 (FWR2) acid sequence:
(DNA)
tggtacctgcagaagccaggccagtctccacagctcctgatctat (SEQ ID NO:492)
(amino acids)
WYLQKPGQSPQLLIY (SEQ ID NO:493)
Humanized C3 light chain variable complementarity determining regions 2
(CDR2) sequence:
(DNA)
aaggtttccaaccggttctct (SEC ID NO:494)
225
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(amino acids)
KVSNRFS (SEQ ID NO:495)
Humanized C3 light chain variable framework region 3 (FWR3) acid sequence:
(DNA)
ggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaa::cagccgggtggaggctgag
gatgttggggtttattactgc (SEQ ID NO:496)
(amino acids)
GVPDRFSGSGSGTD7ILKISRVEAEDVGVYYC (SEQ ID NO:497)
Humanized C3 light chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
ttccaaggtagccacgtgcctttcacc (SEQ ID NO:498)
(amino acids)
FQGSHVPFT (SEQ ID NO:499)
Humanized C3 lambda light chain sequence
(DNA)
gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaggtct
agtcagaccattgtccatagtaatggaaacacctatttggagtggtacctgcagaagccaggccagtotccacag
ctcctgatctataaggtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagat
ttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgct7:.ccaaggtagccacgtgcct
ttcaccttcggcggagggaccaaggtggagatcaaacgaactggtcagcccaaggctgcccoctcggtcactctg
ttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcaTaagtgacttctacccggga
gccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaa
agcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagc
tgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatagtaa (SEO ID
NO:500)
(amino acids)
DIVMTOTPLSLSVTPGQPASISCRSSOTIVHSNGNTYLETNYLOKPGQSPOLLIYKVSNRFSGVPDRFSGSGSGTD
FILKISRVEAEDVGVYYCFOGSHVPFTFGGGTKVEIKRTGOPKAAPSVELFPPSSEELQANKATLVCLPSDFYPG
AVIVAWKADSSPVKAGVETTIPSYQSNNKYAASSYLSLIPEQWKSHRSYSCQVINEGSTVEKTVAPTECS**
(SEQ ID NO:501)
Humanized C3 Kappa light chain
(DNA)
gatattgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgcaggtct
agtcagaccattgtccatagtaatggaaacacctatttggagtggtacctgcagaagccaggccagtctccacag
ctcctgatctataaggtttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagat
ttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgctccaaggtagccacgtgcct
ttcaccttcggcggagggaccaaggtggagatcaaacgaactacggtggctgcaccatctgtcttcatottcccg
ccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcc
aaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtg::cacagagcaggacagcaag
gacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgc
gaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagtaa (SEQ ID
NO:502)
(amino acids)
DIVMTOTPLSLSVTPGQPASISCRSSOTIVHSNGNTYLEWYLOKPGOSPOLLIYKVSNRFSGVPDRFSGSGSGTD
FILKISRVEAEDVGVYYCFQGSHVPFTFGGGIKVEIKRITVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALOSGNSQESVIEODSKDSTYSLSSILTLSKADYEKHKVYACEVIHOGLSSPVIKSFNRGEC**(SE
Q ID NO: 503)
Mouse C8 heavy chain variable region sequence
(DNA)
226
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qaagtqatqqtcgtqqaaagcqqcqgtggtctqgtaaagccqqqgqqatcccttaaqctttottqcgccgcatcc
gggl.LcacgL.LcLcoggcLaLgccaLgLccLgggLccgacagacLoccgaaaagcgcLLggaaLgggLggccacL
atctcctccggggggacgtacatctactaccccgacagtgtgaaaggaagatttacaatatctcgcgacaacgca
aaaaataccttqtatcttcaaatqaqctccctqcqqtcagaqqacactqccatqtactattqcqcccqcctqqqc
ggcgacaattactatgagtat (SE() ID NO:505)
(amino acids)
EVMVVESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQ2PEKRLEWVATISSGGTYIYYPDSVKGaFTISRDNA
KNTLYLQMSSLRSEDTAMYYCARLGGDNYYEY (SEQ ID NO:506)
Mouse C8 heavy chain variable complementarity determining region 1 (CDR1)
sequence:
(DNA)
ggctatgccatgtcc (SEQ ID NO:507)
(amino acids)
GYAMS (SEQ 2D NO:508)
Mouse C8 heavy chain variable complementarity determining region 2 (CDR2)
sequence:
(DNA)
actatctcctccggggggacgtacatctactaccccgacagtgtgaaagga (SEC ID NO:509)
(amino acids)
TISSGGTYIYYPDSVKG (SEQ ID NO:510)
Mouse C8 heavy chain variable complementarity determining region 3 (CDR3)
sequence:
(DNA)
ctqqqcqqcqacaattactatqaqtat (SEQ ID NO:511)
(amino acids)
LGGDNYYEY (SEQ ID NO:512)
Humanized C8 heavy chain variable region sequence from IGHV3-21*04:
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaacc
attagtagtggcggaacctacatatactaccctgactcagtgaagggccgattcaccatctccagagacaacgcc
aagaactcactqtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagactgggc
ggcgataactattatgaatattggggcaaagggaccacggtcaccgtctcctcc (SEQ ID NO: 525)
(amino acids)
EVOLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRF7ISRDNA
KNSLYLOMNSLRAEDTAVYYCARLGGDNYYEYWGKGTIVTVSS (SEQ ID NO: 526)
Humanized C8 heavy chain variable framework region 1 (FWR1) sequence:
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctct
ggattcaccttcagt (SEQ ID NO:527)
(amino acids)
EVOLVESGGGLVKPGGSLRLSCAASGFITS (SEQ ID NO:528)
Humanized C8 heavy chain variable complementarity determining region 1
(CDR1) sequence:
(DNA)
ggctatqccatqaqc (SEQ ID NO:529)
(amino acids)
GYAMS (SEQ ID NO:530)
227
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PCT/US2021/071017
Humanized C8 heavy chain variable framework region 2 (FWR2) sequence:
(DNA)
tgggtocgccaggctccagggaaggggctggagtgggtotca (SEC) ID NO: 531)
(amino acids)
WVRQAPGKGLEWVS (SEQ ID NO:532)
Humanized C8 heavy chain variable complementarity determining region 2
(CDR2) sequence:
(DNA)
accattagtagtggcggaacctacatatactaccctgactcagtgaagggc (SEQ ID NO:533)
(amino acids)
TISSGGTYIYYPDSVKG (SEQ ID NO:534)
Humanized C8 heavy chain variable framework region 3 (FWR3) sequence:
(DNA)
cgaLLcaccaLcLccagagacadcgccalagaacLcaoLgLaLcLgcalaaLgadcagccLgagagccgaggacdcg
gccgtgtattactgtgcgaga (SEQ ID NO:535)
(amino acids)
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:536)
Humanized C8 heavy chain variable complementarity determining region 3
(CDR3) sequence:
(DNA)
ctgggcggcgataactattatgaatat (SEQ ID NO:537)
(amino acids)
LGGDNYYEY (SEQ ID NO:538)
Humanized C8 IgG1 heavy chain sequence
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctectgtgcagcctct
ggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaacc
attagtagtggcggaacctacatatactaccctgactcagtgaagggccgattcaccatctccagagacaacgcc
aagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagactgggc
ggcgataactattatgaatattggggcaaagggaccacggtcaccgtctcctccgctagcaccaagggcccatcg
gtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactac
ttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtccta
cagLccLcaqqacLcLacLcccLcagcaqcqLqqLqacaqLqcccLccaycaqcL_qqqcacccaqaccLacaLc
tgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcac
acatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctct7_ccccccaaaacccaaggac
accctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaag
ttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacg
taccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctcc
aacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtac
accctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgccggtcaaaggcttctatccc
agcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggac
tccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctca
tgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctcc=gtctcogggtaaatgataa
(SEQ ID NO:539)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRF7ISRDNA
KNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH
TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD(WEVHNAKTKPREEOYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK**
(SEC) ID NO:540)
228
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
Humanized C8 IgG2 heavy chain sequence
(DNA)
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctoctgtgcagcctct
ggattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaacc
attagtagtggcggaacctacatatactaccctgactcagtgaagggccgattcaccatctccagagacaacgcc
aagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagactgggc
ggcgataactattatgaatattggggcaaagggaccacggtcaccgtctcctccgcctccaccaagggcccatcg
gtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactac
ttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtccta
cagtoctcaggactctactocctcagcagcgtggtgaccgtgccctccagcaact7;cggcacccagacctacacc
tgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgccca
ccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatc
tcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtac
gtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtc
agcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctc
ccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgccccca
LccegggaggagaLgaccaagaaccaggLcagccLgaccLgccLggLcaaaggcL5cLaccccagcgacaLcgcc
gtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctcc
ttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacg-tcttctcatgctccgtqatg
catgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatagtaa (SEQ ID
NO:541)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFIFSGYAMSWVRQAPGKGLEWVSTISSGGIYIYYPDSVKGRFEISRDNA
KNSLYL0MNSLRAEDTAVYYCARLGGDNYYEYWGKGTIVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVIVSWNSGALTSGVHTFPAVLQSSOLYSLSSVVIVPSSNFOTQTYTCNVDHKPSNIKVDKIVERKCCVECP
PCPAPPVAGPSVFL7PPKPKDILMTSRTPEVTCVVVDVSHEDPEVOFNWYVDGVEVHNAKTKPREEOFNSTFRVV
SVLIVVHQDWLNGKEYKOKVSNKGLPAPIEKTISKTKGQPREPQVY7LPFSREEMTKNQVSLICLVKGFYPSDIA
VEWESNGQPENNYKTIPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK"
(SEC) ID N0:542)
Mouse C8 light chain variable region sequence
(DNA)
gacatcgtcattacgcagacccctgccagtottgccgtttctctgggccagagggccactatcagttacagggcg
agtaagtctgtgagtaccagcggctatagttacatgcattggaaccagcagaaaccgggacagccaccacgcctg
cttatttatctggtgtctaatcttgagtccggggtgcccgccaggttcagcggcagcggctctgggaccgacttc
acactcaacattcatccagtggaagaagaggacgctgctacatactactgtcaacacattcgggaactgaccagg
agtgaa (SEQ ID NO:543)
(amino acids)
DIVITQTPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDF
TLNIHPVEEEDAATYYCQHIRELTRSE (SEQ ID NO:544)
Mouse C8 light chain variable complementarity determining region 1 (CDR1)
sequence:
(DNA)
agggcgagtaagtctgtgagtaccagcggctatagttacatgcat (SEQ ID NO :545)
(amino acids)
RASKSVSTSGYSYMH (SEQ ID NO: 546)
Mouse C8 light chain variable complementarity determining region 2 (CDR2)
sequence:
(DNA)
ctggtgtctaatcttgagtcc (SEQ ID NO:547)
(amino acids)
LVSNLES (SEQ ID NO:548)
Mouse C8 light chain variable complementarity determining region 3 (CDR3)
sequence:
229
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
(DNA)
caacacaLLcgggaacLgaccaggagLgaa (SEQ ID NO:549)
(amino acids)
QHIRELTRSE (SEQ ID NO:550)
Humanized C8 light chain variable region sequence from NCBI germline
z00023:
(DNA)
gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcagggcc
agcaagagtgttagcaccagcggctacagctacatgcactggtaccagcagaaaccaggacagcctcctaagctg
ctcatttacctggtgtctaacctggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttc
actctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacacattcgggaactgaccagg
agtgaattcggcggagggaccaaggtggagatcaaacgaact (SEQ ID NO: 565)
(amino acids)
DIVMTOSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLVSNLESGVPDRFSGSGSGTDF
TLTISSLOAEDVAVYYCQHIRELTRSEFOGGTKVEIKRT (SEQ ID NO:566)
Humanized C8 light chain variable framework region 1 (FWR1) sequence:
(DNA)
gacatcgtgatgacccagtotccagactcoctggctqtgtototgggcgagagggccaccatcaactqc (SEQ
ID NO:567)
(amino acids)
DIVMTOSPDSLAVSLGERATINC (SEQ ID NO:568)
Humanized C8 light chain variable complementarity determining region 1
(CDR1) sequence:
(DNA)
agggccagoaagagtgttagcaccagoggctacagctacatg (SEQ ID NO: 569)
(amino acids)
RASKSVSTSGYSYM (SEQ ID NO:570)
Humanized C8 light chain variable framework region 2 (FWR2) sequence:
(DNA)
cactggtaccagcagaaaccaggacagcctcctaagctgctcatttac (SEC) ID NO:571)
(amino acids)
HWYQQKPGQPPKLLIY (SEQ ID NO:572)
Humanized C8 light chain variable complementarity determining region 2
(CDR2) sequence:
(DNA)
ctggtgtctaacctggaatcc (SEQ ID NO:573)
(amino acids)
LVSNLES (SEQ ID NO:574)
Humanized C8 light chain variable framework region 3 (FWR3) sequence:
(DNA)
ggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaa
gatgtggcagtttattactgt (SEQ ID NO:575)
(amino acids)
GVPDRFSGSGSGTD7TLTISSLQAEDVAVYYC (SEQ ID NO:576)
Humanized C8 light chain variable complementarity determining region 3
(CDR3) sequence:
(DNA)
230
CA 03187555 2023- 1- 27
LZ -Z0Z GSSL9t0 VO
ICZ
(PT OP OUTWP)
(G8S:ON GI OSS) eele540540e454000elle545540004040040040545
ebb-4511eoyobbyobollobooz,o565111111yowItoqylly5115-
400bollItgebblloubbybeeoyouo511511obubb
qbbooboobloobbvlbloobevbvoovb2blowlbl0000vvoobvoobolvoov00000bvoobovvoovoo
ebpooppabboopoeepeboeepeqleeebllbbeuppebbblbebbblqlopeolleoppolobelolbbebeo
beoobqoeqqeqq1b33bq-Lqoebvebwobebbw35eqowlv3eowooeoeqqe5qoqqbbvoqqbbwq
1255-4o4-4-44sbosobwool2-45512oqoobbqoquviqoqool24512-412-4-4-412o4o5-4-41252-
40000qfmobbb000
55oveo5eolel5Eloeoqeqelvolbq5eoloolooeloblow515oe5l000elo5e5oee55551ooeol
ololfmolowebob000eolbeoboeowbqbelvbebobeobbbbbabblbbobeobbebbebbebbobeobb
bbbobbobblbepeeblbepeqqbbllepebbbeeoebbbbllelopbblelbboelpeboelpeebbeobblel
oevoebob000eqEqqeweqqqblobeoeoebbe5eobebeblowqqeebqebepErmeqbwooeqeebee
qobouvoubsbovoqqquoovqqq12512.6555svoqEvoqovb0000vq-412-
4oquovqqopo55555555ooqoqu
eoevowq.655-4ee55-4o5be5eebbbo3owbeeobbobqb56-4-45e5qe555-4eqeboobe3qqweqqqe55
qbveobooboblobeblobbobq000lobbobbloobeelq55135551bbobblbebe5115blobeoolbbeb
(VNG)
:epuenbas ago 9g y-)11r0
(ZSS:ON ai Os)
440SOUNZSMIAdSSZOOHIA2010,AMMISACIVMSTITISSZSAISCINSCIOSIASSOSNOSOZWICIAMMOAM
IfEEdLECTVIDAASILIOSW103aSddZIZASdkrVASZEMISAMInnOZESE.YIEUIHODAXAYAUENEMSSIZTI
ICIOSOSOSIUCMAOSM'INSNIXITIMddOOdMOOAMUNXSAOSMSASMSVUONIMVUMVISAWISCMSOMNADI
(14,Toe oupme)
(T8C:ON
ai 02s) eeq5eq-45.45e5eb5b5eoeeoqw5ebeeeoeolb000bowbe5.433555eoqe000eoqbee5
obloobowwqbvv2323922b2bovqovb2obvvvobvbwbovbwoovobvobvowob232.43323b2o2b
bewobeL)ebbeL)bwbellbebebbeww11555LY4ww5L)welle551155webbIlbeL)ellbewe
oobbe5e5e000qeqoqweeqeebqobwo5-45-45-4-45-4owob3ee5bqoqeeebqqbeobebqe5qoqeoo
b33ollolv3lloqbl3lPo3eobl3bbqbb3vlovvb32ve3lebvbblbbve3oveibbebbobbollP2b1b2
b5m3ovbweeb5Eoqqeoeoeeolbweqqe.4.4.4Eeobbqbqebev5.4355e35.4335eobeoqeooeowwe
3.4132bvovbbbqoqbbbobvobblbvoqq2boovbwooqbbbboo.422bb.43322.43.4bqbb.4332.4.4.4
23.43
511:)5welle.wLmTpbeLn)ewebeL).beLn.m,115511Lm,L)511eLm,11L)5eLm,11L)5eQfpw:x.m,
L)5w114646e5ww:Am,
oobbbeobweeoqeooeoo555ebebo555-4ow-45-46-4056woowebeoo-40-46e000eb-T-
b15oqeoef)
(vNG)
eDuenbas uTeqo 414bTT eddex 93 pazTuewmH
(08C:ON GI 02S)
44SOSITiAMMSAISOSHMAOOSASUHSXMOS&IZSZASSYVAXNNSOXS&IMMSADVXAdSSGIPAMVAMAV
OdAdaS=r1DNIDDINVOrlaaSSddaTLASdliliNdOaLUNIaAMISSOdaSULrlaUIHODAAANiAaaNfOrISS
ILTI,
aCLIOSOSOSZUCHAOSSZNSAZAITINddOOdMOOAMHWASAOSMSASMSlaIONISMUSOZSAWISadSOMNADI
(spToe ouTwe)
(6tc:ON
ai oas) et.452qe3q45qeebe3e43333bb4Ee3ebeebebb4b33e3bebbbeeb4e3b3e34ffm33b4
obeoe43beebe3e333q5ee5bqUeobeb433b3e54335e543qewbeobeoobboboeqbeeoeeoeeobe
eeoeevool000eoeooeopebebblbebbbobbeeolboopobeobelebeobbeebbloobblbeoeblboob
ebbboo32.43.4.43vbqb2P-
L23.4.315.16q55.432ovoobbveovepob223.4.43525b25.43qopqopoboopq.4
54343234.6534333335335522333.6234664322632223-4262664662e3326662663663-
44226462
5523325-43e25563-4-42323e23.45-43eqqe4 35535-
425ev5-4355235-433523523-423323-43-432
3qqqebe3855533q555358355.4De33qe1333et33331313513333eeb13333eeq3313qb13333e333e
333
6135ev.433133583855833et.8583.6833e15513835qe3el35e3e.4355358338358.4.151585883
58
3355523543883338335658585355546636633136
385646348325
(VNG)
aouanbes uTstio qqb-FT spqmva 80 pazTuywnH
(8LS:ON GI CGS) asuziauniO
(8p-ç32 ou-pup)
(LLS:ON ai OSS) 825358558338533885553338383883
LIOLLORZOZSahlad
6COLZO/ZZOZ OM
VVCO 2022/027039
PCT/US2021/071017
EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRFTISRDNA
KNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSL
SPGERATLTCSATSSVSYIHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCQ
QRSSSPFTFGSGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG
VLLLSLVITLYC** (SEQ ID NO:585)
CAR-T C2 CD8 CD8 sequence:
N-CD81s-huMNC2scFv-CD8ecd fragment- CD8 transmembrane-C
(DNA)
gaagtgcagctcgtagagagtggcgggggactggtgaagcccggtggaagcctcagactcagttgcgccgcctca
ggtttcactttttcaggttacgccatgtcctgggtaagacaggcaccggggaaaggactcgagtgggtgtctact
atcagctcaggaggcacttatatatattatcctgactctgtaaaaggccgatttacgatttctcgcgacaatgca
aagaactccctctacctccaaatgaacagtcttagggcagaagacactgctgtatactattgtgcacgcctcggc
ggcgacaactactacgagtactttgacgtgtgggggaaagggactaccgtgacagr.ttcaagcggaggaggtggc
tcaggtggaggcgggtcaggggggggaggaagtgatattgtgctcacacaatccccagcctccctggctgtgtct
cccggccaacgcgctacaattacatgtcgggcctccaaaagcgtgagcaccagcggctacagctacatgcactgg
tatcaacagaaaccaggacaaccccccaaactgttgatttatctcgcttcaaactggagtccggcgtgcctgcg
cgcLULUcagggagUgggagcggcacagaLLULacgcl_gacUaLcaaccccglagaagcaaacgaLacagcgaaL
tattattgtcaacattcccgggaactcccctttacgttcggcgggggcacaaaggr.cgaaattaagagaaccacg
acaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaaccccgtctctgagaccagaagcc
tgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccg
ctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgctgataa (SEQ ID NO:586)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSGGTYIYYPDSVKGRFTISRDNA
KNSLYLOMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVS
PGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFILTINPVEANDTAN
YYCQHSRELPFTFGGGTKVEIKRTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP
LAGTCGVLLLSLVITLYC** (SEQ ID NO: 587)
CD8/4-1BB sequence
N- CD8 transmembrane- 4-L33-C
(DNA)
acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaa
gcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggcc
ccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcaaaaggggccgcaaaaaa
ctcctttacatttttaagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgc
tttcctgaggaggaggaaggagggtgcgaactgtgataa (SEQ ID NO: 588)
(amino acids)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK
LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL** (SEQ ID NO: 589)
CD8/CD28 sequence
N- CD8 transmembrane- CD28-C
(DNA)
acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaa
gcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggcc
ccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcagaagcaagcggtctcgg
ctcctgcattctgattacatgaacatgaccccaagaagaccaggccccaccaggaaacattaccagccctacgct
ccgccacgcgacttcgctgcctaccggtcctgataa (SEQ ID NO: 590)
(amino acids)
TTTPAPRPPTPAPTIASQPLSIMPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR
LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS** (SEQ ID NO: 591)
CD8/CD3z sequence:
N- CDR transmembrane- CD3zeta-C
(DNA)
acgacaaccceggcceccagaccaccaacgccagcceccaccaUcgccagccaaccccUgUcUcUgagaccagaa
gcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggcc
232
CA 03187555 2023- 1- 27
VVCO 2022/027039
PCT/US2021/071017
ccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgccgcgttaagttctcccga
UcagccgacgcgccUgcULacaagcagggccagaaccaacUgUacaacgagclgaaLcUcggLagacgggaagag
tacgacgtgttggacaaacggagaggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggag
ggactgtacaatgagttgcagaaagataagatggcagaagcttatagcgagateggaatgaagggggaaaggaga
cgagggaaaggacacgacggcctttatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatg
caggcacttccaccacggtgataa (SEQ ID NO:592)
(amino acids)
TTTPAPRPPTPAPTIASULSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLACTCGVLLLSLVITLYCRVKFSR
SADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPIDEGLYNELQKDKMAEAYSEIGMKGERR
RGKGHDGLYQGLSTATKDTYDALHMQALPPR** (SEQ ID NO:593)
CD8/CD28/CD3z sequence:
N- CD8 transmembrane- CD28- CD3zeta-C
(DNA)
acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaa
gcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggcc
ccgcl_cgcaggcacaUgUggagUgcUccUccUcLcccUggl_gaLLacccUgUacUgcagaagcaagcggUcLcgg
ctcctgcattctgattacatgaacatgaccccaagaagaccaggccccaccaggaaacattaccagccctacgct
ccgccacgcgacttcgctgcctaccggtcccgcgttaagttctcccgatcagccgacgcgcctgcttacaagcag
ggccagaaccaactgtacaacgagctgaatctcggtagacgggaagagtacgacg7.gttggacaaacggagaggc
cgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagat
aagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttat
cagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacr.tccaccacggtgataa
(SEQ ID NO:594)
(amino acids)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR
LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG
RDPEMGGKPRRKNPUGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMALPPR**
(5E0 ID NO:595)
CD8/4-113B/CD3z sequence:
N- CD8 transmembrane- 4-L33- CD3zeta-C
(DNA)
acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaa
gcctgtaggcctgccgccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggcc
ccgctcgcaggcacatgtggagtgctcctcctctccctggtgattaccctgtactgcaaaaggggccgcaaaaaa
ctcctttacatttttaagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgc
tttcctgaggaggaggaaggagggtgcgaactgcgcgttaagttctcccgatcagccgacgcgcctgcttacaag
cagggccagaaccaacUqUacaacgagcl_gaaLcUcqgUagacqggaagagUacgacqUqUUggacaaacggaga
ggccgcgacccagaaatgggcggcaagcctcgcaggaaaaacccccaggagggac7.gtacaatgagttgcagaaa
gataagatggcagaagcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctt
tatcagggcctgtccacagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataa
(SEQ ID NO:596)
(amino acids)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK
LLYIFKOPFMRPVOTTUEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKOGONOLYNELNLGRREEYDVLDKRR
GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR**
(SEQ ID NO:597)
CD8/CD28/4-1BB/CD3z sequence:
N- CD8 transmembrane- CD28- 4-133- CD3zeta-C
(DNA)
acgacaaccccggcccccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaa
cfcctqtacmcctgccgccgcrtqqacfctqtqcacacaaciaggactqqatttcqcctqtqatatctacatttcmgcc
ccgctcgcaggcacatgtggagtgctectectctecctggtgattaccctgtactgcagaagcaageggtctegg
ctcctgcattctgattacatgaacatgaccccaagaagaccaggccccaccaggaaacattaccagccctacgct
ccgccacgcgacLUcgcl_gccUaccggUccaaaaggggccgcaaaaaacUcclULacaULLULaagcagccULUL
atgaggccagtacagacgactcaagaggaagacgggtgctcatgccgctttcctgaggaggaggaaggagggtgc
233
CA 03187555 2023- 1- 27
VVCO 2022/027039
PCT/US2021/071017
gaactqcgcgttaagttctcccgatcagccgacqcgcctqcttacaagcacmccagaaccaactqtacaacciag
cUgaaLcUcggLagacgggaagagLacgacgUgULggacaaacggagaggccgcgacccagaaaUgggeggcaag
cctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagar.ggcagaagcttatagcgag
atcqqaatgaaggcmaaaggagacgagggaaaggacacqacqgcctttatcacmcctqtccacagcaacaaaa
gatacgtatgacgccctccatatgcaggcacttccaccacggtgataa (SEQ ID NO: 598)
(amino acids)
TTTPAPRPPTPAPTIASULSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSR
LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC
ELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE
IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR** (SEQ ID NO: 599)
CAR-T C2 CD8/CD8/CD28/CD3z sequence:
N-CD81s-huMNC2scFv-CD8ecd fragment- CD8 transmembrane- CD28- CD3zeta-C
(DNA)
aUggccUl_gccagUgacggcccUgcl_gcl_gccaLUggcLcLUcUgULgcacgcl_gccaggccUgaagl_gcagcL
c
gtagagagtggcgggggactggtgaagcccggtggaagcctcagactcagttgcgccgcctcaggtttcactttt
tcaggttacgccatqtcctqqqtaagacaggcaccqqqqaaaggactcgagtqqq7.qtctactatcagctcagga
ggcacttatatatattatcctgactctgtaaaaggccgatttacgatttctcgcgacaatgcaaagaactccctc
tacctccaaatgaacagtcttagggcagaagacactgctgtatactattgtgcacgcctcggcggcgacaactac
tacgagtactttgacgtgtgggggaaagggactaccgtgacagtttcaagcggaggaggtggctcaggtggaggc
cmtcaggcmcmgaggaagtqatattgtqctcacacaatccccagcctccctqqctqtqtctcccggccaacqc
gctacaattacatgtcgggcctccaaaagcgtgagcaccagcggctacagctacar.gcactggtatcaacagaaa
ccaggacaaccccccaaactgttgatttatctcgcttcaaacttggagtccggcg7.gcctgcgcgcttttcaggg
agtgggagcggcacagattttacgctgactatcaaccccgtagaagcaaacgatacagcgaattattattgtcaa
cattcccgggaactcccctttacgttcggcgggggcacaaaggtcgaaattaagagaaccacgacaaccccggcc
cccagaccaccaacgccagcccccaccatcgccagccaacccctqtctctgagaccagaagcctqtaggcctqcc
gccggtggagctgtgcacacaagaggactggatttcgcctgtgatatctacatttgggccccgctcgcaggcaca
tgtggagtgctcctcctctccctggtgattaccctgtactgcagaagcaagcggtctcggctcctgcattctgat
tacatgaacatgaccccaagaagaccaggccccaccaggaaacattaccagccctacgctccgccacgcgacttc
gctgcctaccggtcccgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaactg
tacaacgagctgaatctcqqtagacgcmaagagtacgacgtqttggacaaacqqagaggccgcgacccagaaatg
ggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaagct
tatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggccr.ttatcagggcctgtccaca
gcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataa (SEQ ID NO:608)
(amino acids)
MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSG
GTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGSGGG
GSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSG
SGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRTT7TPAPRPPTPAPTIASQPLSLRPEACRPA
AGGAVHTRGLDFACDIYIWAPLAG7CGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF
AAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA
YSEIGMKGERRRGKGHDGLYQGLS7ATKDTYDALHMQALPPR** (SEQ ID NO:609)
CAR-T C2 CD8/CD8/4-18B/CD3z sequence #13:
N-CD81s-huMNC2scFv-CD8ecd fragment- CD8 transmembrane- 4-1BB- CD3zeta-C
(DNA)
atggccttgccagtgacggccctgctgctgccattggctcttctgttgcacgctgccaggcctgaagtgcagctc
gtagagagtggcgggggactggtgaagcccggtggaagcctcagactcagttgcgccgcctcaggtttcactttt
tcaggttacgccatgtcctgggtaagacaggcaccggggaaaggactcgagtggg7.gtctactatcagctcagga
ggcacttatatatattatcctgactctgtaaaaggccgatttacgatttctcgcgacaatgcaaagaactccctc
tacctccaaatgaacagtcttagggcagaagacactgctgtatactattgtgcacgcctcggcggcgacaactac
tacgagtactttgacgtgtgggggaaagggactaccgtgacagtttcaagcggaggaggtggctcaggtggaggc
cmtcacmcmcmclaqqaaqtqatattqtqctcacacaatccccagcctccctqqctqtgtctccccmccaacqc
gctacaattacatgtcgggcctccaaaagcgtgagcaccageggctacagctaca7.gcactggtatcaacagaaa
ccaggacaaccccccaaactgttgatttatctcgcttcaaacttggagtccggcg7_gcctgcgcgcttttcaggg
agUgggageggcacagaLLULacgcl_gacUaLcaaccccgUagaageaaacgaLacagcgaaLUaLUaLUgUcaa
cattcccgggaactcccctttacgttcggcgggggcacaaaggtcgaaattaagagaaccacgacaaccccggcc
234
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
cccagaccaccaacgccagcccccaccatcgccagccaacccctgtctctgagaccagaagcctqtaggcctgcc
gccggLggagcLgLgcacacaagaggacLggaLLLcgccLgLgaLaLcLacaLLLgggccccgcLcgcaggcaca
tgtggagtgctcctcctctccctggtgattaccctgtactgcaaaaggggccgcaaaaaactcctttacattttt
aagcagccttttatgaggccagtacagacgactcaagaggaagacgggtgctcatgccgctttcctgaggaggag
gaaggagggtgcgaactgcgcgttaagttctcccgatcagccgacgcgcctgcttacaagcagggccagaaccaa
ctgtacaacgagctgaatctcggtagacgggaagagtacgacgtgttggacaaacggagaggccgcgacccagaa
atgggcggcaagcctcgcaggaaaaacccccaggagggactgtacaatgagttgcagaaagataagatggcagaa
gcttatagcgagatcggaatgaagggggaaaggagacgagggaaaggacacgacggcctttatcagggcctgtcc
acagcaacaaaagatacgtatgacgccctccatatgcaggcacttccaccacggtgataa (SEQ ID
NO: 610)
(amino acids)
MALPV-TALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFIFSGYAMSWVRQAPGKGLEWVSTISSG
GIYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGITVIVSSGGGGSGGG
GSGGGGSDIVLIQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSG
SGSGTDFILTINPVEANDTANYYCQHSRELPFTFGGGIKVEIKRTITTPAPRPPIPAPTIASQPLSLRPEACRPA
AGGAVHIRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP7MRPVQTTQEEDGCSCRFPEEE
EGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNDQEGLYNELQKDKMAE
AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALFPR'* (SEQ ID NO; 611)
MUC1 truncated extra cellular domain sequence
(amino acids)
SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 520)
MUC1 truncated extra cellular domain sequence
(amino acids)
SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO:621)
MUC1 truncated extra cellular domain sequence
(amino acids)
VOLTLAFREGTINVHDVETOFNOY (SEC ID NO:622)
mUC1 truncated extra cellular domain sequence
(amino acids)
SNIKFRPGSVVVQLTLAFREGTIN (SEQ ID NO:623)
Primers
attctaagcttgggccaccatggaactg (SEQ ID NO:624)
tctagagtttaaacttactatttacccggagacagggagag (SEQ ID NO:625)
agtatggcccagccggccgaggtgcagctggtggagtctgg (SEQ ID NO:626)
tagaaggcacagtcgaggctgatcag (SEQ ID NO:627)
attctaagcttgggccaccatggaagc (SEQ ID NO:628)
tctagagtttaaacttactaacactctcccctgttgaagc (SEQ 7D NC: 629)
agtatggcccagccggccgaaattgtgttgacacagtctccag (SEQ ID NO; 630)
tagaaggcacagtcgaggctgatcag (SEQ ID NO:631)
acLgLcaLaLggaggLgcagcLggLggagLcLg (SEQ ID NO:632)
actgtctcgagtttaatttccactttggtgccgctgc (SEQ ID NO :633)
actgtcatatggaggtgcagctggtggagtctg (SEQ ID NC: 634)
actgtaccggttttaatttccactttggtgccgctgc (SEQ ID NO; 635)
cttcttcctcaggagcaagctcaccgtgg (SEQ ID NO:636)
gagccgLcggagLccagc (SEQ :D NC:637)
gcacctgaactcctgggg (SEQ :D NO:638)
tttaatttccactttggtgccg (SEQ ID NO:639)
cgcggctagcttaagcttggtaccgagggcca (SEQ ID NO; 640)
cgcggcggccgcctgatcagcgggtttaaacttatc (SEQ ID NO: 641)
MMP9
(DNA)
atgagcctctggcagcccctggtcctggtgctcctggtgctgggctgctgctttgctgcccccagacagcgccag
LccacccLLgLgcLcLLcccLggagaccLgagaaccaaLcLcaccgacaggcagc_ggcagaggaaLaccLgLac
cgctatggttacactcgggtggcagagatgcgtggagagtcgaaatctctggggcctgcgctgctgcttctccag
235
CA 03187555 2023- 1- 27
LZ -Z0Z GSSL9t0 VO
SICZ
daaSZINQUCIANVILVDMHAS3SU5aLIDYSASOSOZI3d300dXSCWNSCIOLArlURSdaiSZUCMICIANILLIS
D
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dAOCIOHSVA030IAICMIUSAAUMarldZAVSITIVaVUVaVaaIAVUdgaSSANOIMAMINHHWOMOS,InaN
(PT- v ouTum)
(17179:0N (II OSS) eeleblbeeleb
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412512-4-4o555-4obob
oe3o5bollbe5leo5o55o55q53qooq151115eoele5beeooe550005lollo5555gbee5eeoe5o5e
ot.6111ovvboloovoovqoboblbblowobobb5lvembboboobbfmbobeoovlblooebolovlbvffmv
lbberlopqqweoggpoppagogbobqbwbubbbbbobbolpepobbabbbgebqbboebogoebwbeboope
b000bqoqqobbolqowbeeoebbbooeboeweeoobooeoovoobobqbbwbooewbboeboowboqbb
oubbovoovobwobooqouqooqvvooffmsooqqoquoqquooqq-4512oobwool2122555-4.125-4o5-
41212o55
oebbe000eoelowebebebobe0000blollo551115600eboebooeoeboeweeoobboeooelbeobq
bbl000bllobbovboolobolbbovboovoovoblooblowvlooloboobffmbolloleoll0000llovo
obwabbabobbqubeoboeeebbqqqbboweepoqqbbgbogbobbbeepbbbqopoqbbqbqqbeboebgeb
oeboqqqe000boebebbbeoqqeobb0000bbwowoqqwobovoeobbwowbbboebbeebbboeboqq
000qvq55.6ovbvEbovobvbbobo-45-455-4-4-45vooqvoqboqwovbvobovbbboobvovqb-
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uTemop DT4ATIr4s0 6d1414
(E179:ON GI Os) ..xaaaaxAaaacipOrliaLLAA:DAMAONaasussAumAaua
00dAliMaUA0dAGEIG1dA0dINIUGAaSVSUdaANOVNACUUMMUDSMWXOUOSUZVOZAOVAQVOZO'Dlaq
UUdOZASVOIAAMA0U9SaaarIMMSZUSEJASTIMUdqVaMVIrladOOdUSOUOSSaUMANOMIZIAZONOIE
IfIlfaZINANOIMaAdSrldAIDLISdOlf.IddaldOYSddaidOlfIdU2SdHAZddald0AZEdIf.Ind.12.1
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Hd2d11,30AZHUIONACKIMHZdd02.1311ANdANZVEdASSIVIZOZVH932HVVArlarlSA00ad030MMMUSC
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sAs000diadacoaxoavNonmAguasdpaoduaamamvimsomdzoasuoammovsAsuoadiadaHolivoa
VN9aUJAAAAOMMSWIECITIIHVUO0I0dOddaYHVTIOaXpaadApapHSVA030IADIVaUSXAUIAIrldIA
IfSboVIILDDWalfaclIAIdUeICIESMOIMXZINHHHMMMIOE3I0ZUnqadAnnUEIEWIPATIMSGIEnZEd'I
S'ION
OTIVIVdOrISMSESUN2VAILIXOXIIIZA2ZWIOUCLIZNIAMOdZIATISOUOIldliV3009ZAVININMONYIS
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(z179:0N ai ins) ppqvfoqbwvqvbowboebqvbbppovqqvbovbbveqopper4bvpbqopqv
peblewoeb.453e.43555-Lbeeooebbqbbeooee5.4.45ebqfm55333qq5e5.45353b5wewqwbooe5
b2o3b13111213obv22b2beboovlbvoollolbovbovobovovbblqlooblbbbb0000llblvbboovb
bqbbebobeopboEebboopoqebbqbbqebeoboEbeebqboeboqqabebbwqopbobbobbbobeoqqbqo
bwbgebee5555Eeobbqbe5boog000bbbbooebqbbe00055-45oeboobebbbwobbbqobeeoebbqo
46:36bwb000bbec4obqb5D-Oparx,bbwowowqbqbbbqbqbbs,Dobobbbqoqoqq:3-4-4D-4-4-4-
4obwwbwwooq
owbbobebbebqlqoqbbowebbqobeeob0000bwbob000bbqbeeoebooboqeqqooqw000bbbeo
boo5boobvbbbbEvobbbvblowlqvbobbloelbvvbbblvbbvvolqb111vlblobvoovvbbblqvbvb
bo6o1voo6oP6311o1P3eP6163PP3613361PE3P661663316P61110061613P13P33663P131133
obbqobwe000000qbbeoe0000bbqobeog000000qbbeov0000bbqobeoe0000bobebeog0000eo
oqb4ov000000t=Ebooe0000bwqbboeb00000qobboe000fmobooeoeooeooeooeeoo400bboeoo
be6woeebwoof:owoq554eqoqooeobbooqeobbqee5.45oeboebbeeqeobqw000005555e5we
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b3bb3bb.163.4331.45.4.4q5232.4v5b2P33ebboo3b3q.1.36566.462262e32636Po25.4.4.4o2
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ooloelooleeooEbeeoo-Lloqeolleoolllbmobl000eeebbbleblobleeobboebbe000eoelow
ebv52.63623333Z-43.4.4356-
4.4.46b33Pb3eb3ov3P532.1.32e33bb3233e.46236.466q3335.4.4366326
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205oeve5.6-4-4-455oweeooqq55-450-45obbbevobbbwoo-455-45-4-4525o25-
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bobobvbblbloeobollopbob000blqlooboeboPfilqeblbbobbboboob11325225bolo21322223
oly6611eloopoleopeopoppoopo6616epolooe63666e6illoopepoolle6e0666133e6p000l6
bbbobqbbov0000vvbobvoobbvvbwbovoobobt-42bbqobvbqbboovbvb000bwooqbqovvobvt,
LIOLLORZOZSahlad 6COLZO/ZZOZ OM
LZ -Z0Z GSSL9t0 VO
LCZ
DoznYmb0000ptoqqbDDobbiplboDolbobbeoloebblbobooDeDebboolooDlobbboobbbeobeo
olill0000b000000b00000bb00000660000l0000eop6oloillo66600bleobeo-noeblepleop
qqqqebobqoobevbbbbbobeoeooqqqeeeooqqqqobobeebqbeoobqoobbbeqoqqqooqqqeebbbqe
bbbpbe1144vegbe-4454D-Leeoboeoobbovbbboopqboebpweegeeoebgeeeevqevbeoy-444e4ebe
obvloolbbylolboob2b9323obl0000bboobov0000qoobbb0000qbobobvbqbb2obbbvlobbobbb
powoopaboeepuleoppbobolbeabobolbeebeolbooppooloabbbbbboobblbebgblbeeebbebe
bevlboebebbbeoboebebeeb000bobqobebqobeob5.4.453boobqbbobqeeeebeeoeeoobeeooqeo
oo30000vvooqoovv-movovfmoovovovqoqqqbec42-4-4.4vb3qvvobqboqbqbbbvvqvovoqvvvq-
44
beqq1.65511looEeq115e5oeoebeo5eob0000555loolooe55a6go155555l000ge515eoeo5555
ov000vobbbbbbqbllobobbebbvaffmobbop000loobooeb000bobvoblolobbbobolloovobobo
o6610106660P6bov666o63e666oloo66Poo6P661Peboo6606666016P610616111P331166660
obvvevbbqbbobloobbeboobbvoowwqboeobbbooqeelob0000eooeopbbobbebobvbbeoboob
ouboqoo5.6-4121212E5w000l2512512-45.612ob00000555512-45oobobboqobbqoofopoo5-
4.5obo5555-4o4
obqbb000bobb000g0000lool0000bbob000loolbobbbwobbooe000b00000eb000000ebl000
polbvbbloboloobbobobbbbblbobbbbebbbbbvbbUmbbUmbbqllebvve6bobbobolovovbbbb
10366v3333661331PPPo3633331313PPP66oP6666666066660666P66PP6P6666166P6613316
Do3ob3ooboo31bebobboop3owooqqebb5owobbopoebeoob0000bobobbbqobbooebqboeo
ooqoqfmoqo55551250-45555-
4.obbobool255125oqobbb000qbboqbfmooboqoobvobbfmwoo5125512o
w3wbb0000bb0000b00000boobboobebeebbebbebbe555o5.655e555bee5bebbobyobbobbeb
bebbebbeebggegggeggeeeeeveciLbbbgeobqoqqqqpbebbbeeeebbepebeobebebqqqobboebbe
elvbabbbb3bbb33323qb3oqqvbbbo33ebvpv2Pfibbeb2o2vP1123653223bbo3pqbblb33233bb
o5353.65.433535133owee5633133533533555.43.436o53355.435e535553355e5e35355553355
boobbvbvpboobEqbbob000bbbbboobvb2obvbbbbowbobbbvpbobbbb222582522bbvbbvpbby
(yria)
6d1191¨dT0MYMI
(9179:ON aI Os) Umbeoloebe
b3bvbvo1b3b13o3bb3b3beb23bb2bb23bbvvvvvqqq3bv33qqqqb1bb2bbbbb3bb2bbbb3bb33b
65:Dobopbe555oZobbbbbeb5beobbpeo-454e5-45:)555bepbeobbb000bbbz)bpboopobbbb0000-
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3 3333133 33353 33313333333 35 3313333333535 333 33335355533
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Loo3333535.453333.433333o3535.4533o3.4333333535q63333.4333353
fp.453333.43333.4v3333353ew000b000qq53333qq2bb00000bb000bw00000bobv0000bvoobo
Do=4-4115o3oopZo4-45ppob5434Lopo45obbeo43eb54bpb000epeb5oo4op4ob55opbbbeo5eo
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(vNa)
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LIOLLORZOZSahlad 6COLZO/ZZOZ OM
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(vNa)
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6163333133331y3p33363p1333363331163o3311e66o333o663336133333o6o6p33336p3363
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(speroe ouTme)
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(vNa)
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136ploobbobobebbbl6pb6bbebbbbbebbbbubbbbebbqqlebeebbbobbobolpepebbbbloobbeo
(vNa)
6411,1KEdT*MILEN
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44UddrIVONIVIV0AI0MIVISZOOArlDaHOYOUIDIaDMNOIaSAV
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aS2d3U0S000SHOMMOAdUNIdONZIATIMMUOUNOMINISTIZADOZOIrldliMIAI001i30q0UMHAVOOli
vauovaduricrinsvizavamaduavamaimuNiaAmmossamaarlausuODAANviaNvEnaNimrimaamoses
OSZUVdAOSTINSVIAIIIMdd00,33100AAHNASA5SMSASMSVUOMIMVUOOdSAVISVSOMIAI0S0000S0
DODSOODOSSAIAZIONOMACHAaAAN000qUVOAXAVI0alaVISNNOZAZSNMVN0USII3HOMASCHAXIAID
OSSIISAMTIOMOdliOUAMSVIVAOSZIZOSVVOSWISOOdNAZOODSSAZOASdUliV=Prldr=IATIVAI
(spToe ouTwe)
(8TL:ON
ai Os) eeqe6qoboq000006q000t6eo6qeoeoqq0006oe6oeqooeoebbeeooeoo6eoe
.46volo.46.662ooellloo661e6oeo.6666e2o6566266006o6e6o.6622e612666112626162o2loo
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olleleqvlbloowevebevebeobabbovevobloellwooeolellbblovolblooloqloolbbbblbq
qoe66.600.66.4.4poo63656.4oqeoeqoqeoe6o6qoo6oqqqe66.4oe.66e6eooeoeo6.45oo6e6636
6.436
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obbobeollbbeoobe000-Lbbbbobebebblolevooleobblooelllebloolovvelooqooevoebbeoo
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bbebeoebbeooqoqbqboobbqqooqoobeooqoqbe000eblobqbqq2o2booqebbobbqbbobbooqebb
D66-46.6obboogeZ6D66-466o6boogoogogbooeo466oeope666eeeo66664:y464e5D4wegeeboe4
oeggeeqe.6666666-44oe6e6o6.46qoegge-46-
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46.43o4o4oe6e6woo.4656666.43o6evo.466.43366e6566633.46e66.46
bwbeobqbbebboobbepobooboepoqobwbqqopbbqoboobqopqobqqopboopbqbeopeqqopbbqe
(vNa)
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(sIDT0e ouTwe)
(099:0N (II 02s) 3boq3o3336qo33662obqeovoqq3o36326324
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(VN0)
uveutop imezem
(6(29:0Nai Os) risaaaadluDsDtmaaOLLOAdurtidONaixarixNut)ux
(spToe ouTwe)
(8g9:0N ai 028) 6lope6161e66e66eeEme6erEme6eoollie6=6106e161.1661e6
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(vNa)
4.06dPIK¨ZdipMYJN
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1.5355beeobbbwoo.455-L5-
41.5v53ebqe5oe5oqqqe333b3ebebbbe3qqe3bbopoob5.433.433.4.4.43
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LIOLLORZOZSahlad
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353-45.6oebboeooeobqoobooqoeqooqeeoo55e2ooqqoqwoqqeooqqqbeoobq000eee555-4e5qob
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(VNG)
6dWN-ZANOmMTIVAN
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(iNG)
aa4owoad TstuTuTiu AND
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(VNO)
sqeadaa quawaTe asuodsaa Iyam
(9LL:ON ai Os) 13505beebeoele01115.21leeeebbebee
(vNa)
quawaTa asuodsaa IyAN
(gLL:ON ai oas) eegebqbeegeboeboebq
ebbveoeqqe5.4poeebqopobo.433.165.4eqoqpoeobbooqe355.4eefr453e5pebbeeqe353.43333
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(Ima)
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35353
33511133bovbovb112b-Lbbobbboboobllovb2vbbowylovvvvoolvbb1121332312322323323
opobbqbepowovbobb8pbqqqoppepooqqwEqqqobw6qobbbqobqbbwowbqbbqopqbbqoppo
beobbqogoobebgeooeoobebegoqqqboggeoebogogobebogoobeoogebooebbbooeoybeebegeo
L'IlLn.lubliqqqfpliotL)eLnx4ewbebblibLY4wbeL)libLn)weblibeliliqbLY4L)bebeL)beel
lellelbbeb
bbqbboeqbqbobbeqqbe-Loeqqbobbeebeoeqeoqqqbqqqvevebbebeeqqbobbeebeoeqeoqqqbqq
qeveebbebeellEobbevbeoelvollqbqlleevebbebeellbobbeebeoelvc11151ilvevvbbebee
(IMO)
4e06M4101-ZANOwZaH/VAN
(6LL:ON GI OS) eeleblbeeleD.DeD.Deb4ebbee3e44e5
S 333 3 33 33 33 5 33 33 5 33315
bqb3b3bb.432.431.43b33ebb2335.43.4.4.4e33Z2P26262b33vq6233.4.43.4632b323b323266
.4.4.433
5-4555b3333-4-45125533e55-455eb3be335352553333-4255-455-425e3535be25-
463eb3qq55255-4
owobobbobbboEeo-4-45-5-4.o5-42.6e2555552o55-4525boowoobbbboo25-455200055-
45oeboob
U513 U5
31131131111obgebeeooloqobbobebbeb11131bbowebblobeeob0000blobob000bblbeeoeb
poboqvq.433.4w000bbbeoboobboobvbbbbbvobbb2b.4333.4.42.6366.432.4b2P665.4.265223
.4.46.4.4
-42.45-435233e25E5-4-426255353-423353e53-4-43-423225.453ye3b33b-4253255-46533-
4525-4-4-433
6-45-4o2weoobboeqoqq000bbqobwe000000qbbeoe0000ffy4obeow00000-455eoe0000ffy4o5
yovoo.00bobebeolloL)ooeLnx4bwe000000ebtooe0000bliollbboeb00000llobboe000beobooe
oe
oovoovooeeooloobboeoobeblooeebl000boloolbblelolooeobbooleobbleeblboeboebbee
.1.235113333335Z6526q323.4.13booPlblvl000Plbqeolobobbvbbooblbvolooqq23126Pllobb
61o6o6oPoo660116P6leo606606616olooll61116PoPiP66PPooP66000610110666616PPEPP
ovbobvovb-movvbowovoovwbobqbbqowobobbbqvbvbboboobbbvbobvoovqbwovbowvq.
LIOLLORZOZSahlad 6COLZO/ZZOZ OM
LZ -1 -Z0Z GSSL9t0 VO
gn
b0000lTholllb000bblIollbo:xy_lbobbeolloybbIlbob000eL)ebbooll000lobbeloobbbyobeo
oll-moo
336000000b00000bb00000bb0000l0000Poloboloillo6660061Po6Pollor.61PP1PoPilliP6o
5-43obvebbbbbo5eoeooqqeevooqqqqobobvebqbeoofy400bbbeqoqqqooqqqeebbbqebbbobeq
gllyellbellItbIlogyeoboyoobboybbb000gboyZolloyegyeoebIleyeyegyebyoy-
mellybeobebollb
bylolboobb2ovoobloopobboobov0000loobbb0000lbobobvbqbbvobbbylobbobbbool00000
boppeuleoppboeolbeobobolbeebeolbooppooloobbbbbboabblbublblbeeebbebebeelboeb
ebbbeoboebebeeb000bobqobv5wbeobb.4.45.45335.4bbobqeeeebeeoeeoobeeepoqv00000000
vvooqoovv-movove.voovovovqoqqqbbqvqqqvboqvvobqboqbqbbbvvqvovoqvvvqqqbvqqqbb
blqqoo5e1115eEoeoeSeoBeob0000555loolooe55o5qo1555.65l000le515eoeo5555oe000eo
bbbe6.6.461136356vbbebbbeobb00000looboovboopbofmoblowbbbobollooeoboboobblow
66631066010666oE010666oloo66100061066-
00PEop66o666601610610616111epoll6666006101010106
33 335 Q33QQ35 33 Q5Q3QQQ3QQQ 35 353353 QQ
65.412121255w000l252512q5bwob00000555512-45oobobbowbbwobovoofy4bobobbbbqow5-
45boo
ob355333lo333lool000055o5oo3gool5o555lo35b33v333533333e5333333e5loo333lbebb
wboloobbobobbbbblbobbfAmbabbbvbbbembbbbebblqqvfmvbbbobbobolovoebbbbloobbvo
(liNa)
(d) luelamig 1040mold ToLYJN
(z8L:oN al OZS) 55v5e040e5e
bobebyogbobqopobbobobefmobbebbeobbeueeeqqqpbepoqqqqbqbbebbbbbobbebbbbobboob
553ob3ob25bboZ356552666v3bbovolbleZ1bobbbfmobvpbbboopbbboboboopobbbboopolb
oq3v53553.4353E5535555e65553bo553533E-43553625355e3=335.43333535q43.4335333353
boblboopoqoppoppobobqboopowooppoopEobqboopolooppopobobbboopowooppobobbboo
oollool,0000boblib000LY_Lnxxx,obobIlb0000ll0000000bobltb0000ll000000boblIb0000l
l0000bo
5463=o43=34B3333353e43333bo33445==44e5bo33335bo33b4333333535e33335e3353
333ollqb3333pe3llb333bbl3lb333lb3bbv3l3vbblbob333232bb33l333l35bb33bbb23b23
3.413.4333353333335333336533333553333.43333e3253.43.4.4.43555335.4e3be3qq325.42
2.4232
.4.4132bobwobvvbbbbbobeovooqqq22v33.4.4.4.43bobv2bqbvoobqoobbb2-
43.4.4q33.43.42vbbb.42
55-4-4-4-452-4-45-45-43-45-42-452-4-4-42-425k
obeboqbbegogboobe5eoeoobw000bbooboe0000goobbb33ooqbobobebqbbe3bbbygob53555
33lo333353vveglP33353bolfm35ob3lbvetv3lboo3333l33bbbbbb3obblEmbqb1622Pbb262
bet,35325e555eoboebebeeboo3535.435e5w5e355.4.4535335.45535.4eeeebeeoeeoobeeooqe
o
33333032233.43ovvqqq3232bv332323E,43.4.4.4bb.42.4.41vb3.422obqboqbqbbb22.4.2ovo
qv22.4.4.4
bellq-45511-4woZell11115ewbeLpobber,55:AltLY4556611oLny_lebIlbeLmq.)5555
oe000eo5.65555-45-44o5o55ebbebbbeobb00000goobooeb000bobeobwqobbboboggooeobobo
366.43.4obbbovbtoetibbobovbbfoqopbbvpotvbeiqevboobbobbbboqbvbqobqbqqqvpoqqbbbbo
obvvet,55.455obloobbeboobbv33.43.43.453eobbbooqeelob0000eooeopbbobbebobv55e3533
5
ovbowobbqvveEbq0000vbebvq&Emob000pobbbbeqboobobbowbbwobovoobqbobobbbbwq
obqbboopbobboopqoppowoqoppobboboopqopqbobbbwobbooppoobooppowboopopowbqopo
ooqbe.65-
4o5ogoofthobo55555.45o.6555e55555e556bebbbbeafrrnebeebbbobboboweoe5555
11.LnAbebbqoLY_Iweeny_w_IL)wwwbboubbbbbbbL)bbbb:AbbebbewbebbbblibbebbIlLny_lb
33335333533 3 158535533313313331185553 13355333858335333353535551355338515383
pololbeolobbbEe5315555.4.355obooebbebolobbbooplbbolbbeooboloobeobbbeeoobebbeo
13313bb3333bb33336333336336bo36P6PPE6106610661066606666106666101066106606190660
66106
68558.65885-4-481-41eqqeeeee851.65551e3b13qqqq3bebbbeeeebbe3ebe3bebeb13bb3ebbe
eqe5b5bb53bbb333e34b33qqe5b5333ebe32eebbbebe3e2e1123663223663331.66463323366
35353.651335351333132255331336335335f:513135353355136253555335525235355553355
633558523533551553533366666036e6e06E666631060666e0606666eee66e6E.E.66e65e355e
(VNO)
(Zd) Wauthya3 me4owomd ToIYAN
(T8L:ON
ai OEs) bEobobbbeobeobe000lebepoboboobolbebbobbooboqoeeboolobbebeowebe
6352623.1636.4333553536262356266236622222.4q135233.4.4116.456265656356265663663
36
bbooboo52555oEo555652555yobboeo-45-4eEqbobbbbeobyobbb000bbbobob000055550000-45
owebobbowbo555o5555eb555obobboboo5qobbobebobbe00000fy40000bobqqowob0000bo
boblibl)000ll000000L)bobIlb0000ll00000000toblIbooLny_l0000000bobbbL)ooull000000
bobbboo
pol0000000bobqb0000-Loop000boblb000pl00000poboblb0000l000pooboblb0000l0000bo
blbopooloppolgooppoboeqoopob000llboopollybboopoobb000bloppopobobvpopobvpobo
3333111633333631163336613163331636610313P661636333P3P663313331366633666P36P3
oqqqw000b000000b00000bb00000bb0000w000vot,bow-mobbboobqvobvoqqovbqvvqvot,
LIOLLORZOZSahlad 6COLZO/ZZOZ OM
WC)2022/027039
PCTPUS2021A71017
tttoccccgccagccccagcgcccccctgcccggcccccggattccccgttoccgcccctacgcccccatcccct
ccccgLgcgccccLccccgLgcgccccccLccccgLgcgcccccccLccccgLgcgccccccLccccgLgcgccc
cccctccccgggcgcccccctccccgggcgccccccctccccgtgcgcccccccc7.ccccgtgcgccccccctcc
ccgtqcgcgccccgcctcttgcgcccctgcccccaggcgagcggctgccgcggcgcggggaggggcgggcgctcg
gcgactcgtccccggggccccgcgcgggcccgggcagraggggcgtgatgtcacggragggagggggcgcgggag
ccgccgggccggcggggaggcgggggaggtgttttccagctttaaaaaggcaggaggcagagcgcggccctgcgt
cagagcgagactcagaggctccgaactcgccggcggagtcgccgcgccagatcccagcagcagggcgcgg (SEQ
ID NO:783)
pNFAT¨MMP9cat-1 gBLOCK sequence
(DNA)
aagaggaaaatttgtttcatacagaaggcgttactagttaggcgtgtacggtgggaggcctatataagcagagct
cgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccga
tccagcctctcgacattcgtttctagagccaccatgagcctctggcagcccctgg:-.cctggtgctcctggtgctg
ggctgctgctttgctttccaaacctttgagggcgacctcaagtggcaccaccacaacatcacctattggatccaa
aactactcggaagacttgccgcgggcggtgattgacgacgcctttgcccgcgcct7..cgcactgtggagcgcggtg
acgccgctcaccttcactcgcgtgtacagccgggacgcagacatcgtcatccagtftggtgtcgcggagcacgga
gacgggLaLcceLLcgacgggaaggacgggcLccLggcacacgccLLLcci_ccLggccccggcaLLcagggagac
gcccatttcgacgatgacgagttgtggtccctgggcaagggcgtcgtggttccaactcggtttggaaacgcagat
ggcgcggcctgccacttccccttcatcttcgagggccgctcctactctgcctgcaccaccgacggtcgctccgac
ggcttgccctggtgcagtaccacggccaactacgacaccgacgaccggtttggct7Ictgccccagcgagagactc
tacacccaggacggcaatgctgatgggaaaccctgccagtttccattcatcttccaaggccaatcctactccgcc
tgcaccacggacggtcgctccgacggctaccgctggtgcgccaccaccgccaactacgaccgggacaagctcttc
ggcttctgcccgacccgagctgactcg (SEQ ID NO:784)
NFAT consensus sequence:
(A/T)GGAAA(A/N)(A/T/C)N (SEQ ID NO:80d)
Current NFAT RE (Form System Blosciences. The sequence is from the mouse
IL2 promoter
(DNA)
aagaggaaaatttgtttcatacagaaggcgtt (SEQ ID NO:805)
Mouse IL2 Promoter (highlighted in green the NFAT RE used, highlighted in
yellow is the start codon)
(DNA)
aactagagacatataaaataacaccaacatccttagatacaacccttcctgagaattattggacatcatactct
ttttaaaaagcataataaacatcaagacacttacacaaaatatgttaaattaaattaaaacaacaacgacaaaa
tagtacctcaagctcaacaagcattttaggtgtccttagcttactatttctctggctaactgtatgaagccatct
atcaccctgtgtgcaattagctcattgtgtagataagaaggtaaaaccatcttgaaacaggaaaccaatatcctt
cctgtctaatcaacaaatctaaaagatttattottttcatctatctcctcttgcgttgtccaccacaacaggct
gcttacaggttcaggatggttttgacaaagagaacattttcatgagttacttttggtctccaccccaaagagga
aaatttgtttcatacagaaggcgttcattgtatgaattaaaactgccacctaagtgtgggctaacccgaccaaga
gggatttcacctaaatccattcagtcagtgtatgggggtttaaagaaattccagagagtcatcagaagaggaaaa
acaaaaggtaatgctttctgccacacaggtagactctttgaaaatatgtgtaatagtaaaacatcgtgacaccc
ccaLaLLaLLLLLccagcaLLaacagLaLaaaLLgccLcccaLgcLgaagagcLgccLaLcacccLLgcLaaLca
ctcctcacagtgacctcaagtcctgcaggcatgtacagcatgcagctcgcatcctgtgtcac (SEQ 7D
NO:806)
NFAT RE (Form PRomega. The sequence is from the humane IL2 promoter
(DNA)
ggaggaaaaactgtttcatacagaaggcgt (SEQ ID NO:807)
Possible NFAT RE from ET-1 promoter
(DNA)
tccagggaaaatcggagtagaacaagagggatg (SEQ ID NO:808)
Possible NFAT RE from ET-1 promoter
(DNA)
acALLggaaaacgLaaacacgLLaLLaaacggL (SEQ ID NO:809)
246
CA 03187555 2023- 1- 27
LZ -Z0Z GSSL9t0 VO
LVZ
4bvq1blilloeellqqoowbeeipolbllo1111b11D1be4eeyevloleolbeoeeoevebeleooleeleeo
0000billo1616666elo-lobey166opeepillio6666ep0000pobbille666yebe66poolobo616
ev3oovoyebqeb5-455bboebbqqeqeeeebeqoqobbe000boovbqbeoeeobe0000beebbwooboebqe
Dvoyogobgbbwoobv4eDw4bobbgebbeoofyLlogvo5DoDbovbv.664obob000bb000bboogobvobb
bloollobt.bllbobvbblvbbbovbbbovb2vbool000v000vobblbb2obbbbobov0000bvvvbblvbby
bloppobabebopelblbobopeobbaboepoppbulbbepobqoppobobblobbloppoblopeobobbblbb
bevvbbebbbbbelbeoqq5bobqoobbwqeobbqbeebbeofmb000qqqqebqbeeqqeqboqbeoeoeebe
312126fmoboq-
41231212buopubfmobqouvvvobquvibpoobbbqbwboovoosbqbvvoqoqobuvuubbbqub
q0000y5eveolooyeeeo5eblqbebqolle55155555eebye55eogoo5155ee551gb51q5l000llo
oobbabblbolvovbbloqolbefmvololbblvemooloqqqv000blvvvlevvlobbl000vbeoololfmq
op1pPiliPP106111661ol1eP1Plo1lillo6P1PPPlemPePlipP1116PoloiPePPePPePPPlipP6
wvoeoqqeoqqbEefrabwbbqeooeeoowbeo.43.4.4.43.4.433.4veebbbeeeqoeeqe.45.4bbboeoeb
qob
-4551212.612o-45121212E5212512-4-4ovfm0000vol2125512o400-4555-112-45-
4121212000puubqoqouvi2512-412-455-1.
eobvoyelbeeleolglbgebebevell5515o5555eebbbeool000eeobell000beebgelooeeebbbe
ovbellqbvemvloblvoq-Lblbbblffmbleoolbvlbbqbbleovfml.4111fmblolebqbbllvoolvblo
e6116vPP66P16661;13163poPoP66100061PP1001P61133661116P36P613616=6166PolP166
bev5.4056.65.16oebbbbebbbooloeoobbwobwoqq5bebbbbqbbeqqq.4355.45ebobwvbqeebebq
555-1.5v55-4=-1.5wvgooqovbqvbp5v5555-4-1.5355-1.vvbqowbqqv.6555-4-4ovobvpobvoo-
45555oovb
556y5ebbebbbebeo6551=4e6beogeebeeeeeeebeobebeoebeeo5400beooweobwboo54eo4
qbqopobebbbeptgobbbbqqbeybbeopobbbqqpeogebbebgyobbebqobbeebeogoegobeopoqbeg
blooblb123261Zblblffmoobv112P2le225t,11222evoevoP23223131231=25223521P32236
ebloobeooebeelqqbeffm000fraqbebe333.45.4e3oee.4133.43.4eoeee5.45.4.43eqqbqobqeo
beoeq
D-4135.6b2pqqbbEbqqqbpvqboo-
42.652bwoqqbqbbbbqolbvvvqoqqopqqqopfrapqqopyobvpqbq
54oLleobge4qbelggooD-L-4-4ebbeebeeebefowbebb-4466-
4eoobeDeowbeefo4eeecifo4beo4bee-44
44.4444q44e5e5gobeoeee4bee444goe444e4o5eeeee6y4e44eebeooeeeo5e44eee44eebe44e
bybbloqb2b2=e1111P-Lbleo6Pb1622e2vevvEtivve22b2vPb222be22222P222222e22P22223
.1.3153owebe5pEebeoeeobbbloobe0000e35.4.4e3oeoeoqvbebbqbe5.45e35.4.45bebb.455e5
553
oovvb.4.4323.42vb2bb2obbebqobbvbbbqww4ob2333.42w4b.4332323bobbqbbqbobbboobvq.42
2
yeeoe4beeee4e434o45=Doeeeb4bb4eoeeopeb43obwooebeeo4obeebeD455ebewo4ebbobb
bobbeggobbebbEggweobeoogeeqbeooboeogobbqbbobobbbweoogeggoeobebgeyebeeogq5
ebbeolob2b111.4312b5e2323662.6.436622bv11132362=3122.46132blv3lobbqb2oblbbblov
b5vowoobbebeee3.45q-Lbebebeobeeobbeb5e5boqwobbbeweoeooeooeeqqq.43-43.43355ebe
oobbvbqqqbbqbb23.43922322v3.423obb2.43.4.4b.423obbbqvbb233.433.4232w4b22323bob2
23.43
44y34 422 43&444442444223 42462
oevoeebwobbeebeoeeobBeeoeegebeobbeooeeoeeoegebbgeegeeoggweoowegyboqwebb
bbqvv.opfmovvouqoqq5vo6vvfmaobbvbqvvbvovoqovvvvvobvvbqvqoqvvwepoqobvfmfmvqvv
3f23b.453eqe3eeeqe5-
Lee3ev3eqebeee5e5.45.43qeel3e35eeeeqe5qee.453.4q45.4vebbebeee
qqvq111bbvvvvv2111q-Lobbylvvvvovb211blvvloblobblb2bblob222bbvolwybob2322332
opowbowbewpwbEbqwboppoewebeqpopopovqobeepoqopqoqqqopqpowbebbebeqoqboobqurn.
(VNG)
abaTaT aagowoad ToyydN
(V[8:0N ciiOas) DIELV=TVV,100,1,1011V
qouoyyraott buTsvitioreouottyaog x4qmoxi5 uywnq woig INfabl
eTwFssod
(ETB:ON GI CGS) eLe4c,4044D4eeDDL6ee
(VNC)
90do tremnq W02; auJf TqTssod
(78:0M (II 030 54555222551200542
(vNa)
iaqoulosd Ern usnura( woig au IvaN arqTasod
(TT8:0N GI )SS) 0-4-46-42-42-4beeeeebbeee
(vNa)
AEaD usumm mo=g au mum etcassod
(0T8:0N
2222=.1PPPPP6602P11001
(vNa)
Acao uming 1)10=3
aTc[Tecod
LIOILOITZOZSIIMOd
6COLZO/ZZOZ OM
LZ -Z0Z GSSL9t0 VO
(618:ON cii OS) 4v00444444vo
(lam)
sosuasuoo quamaTa asuodsaa ToyvaN
(818:0N
ai Os) ploboloboogglwabepollopplebolowebweelbbeeooebblblogbbbbleplep
(VNO)
aouanbas 1a4owo1d zoviam
(L18:0N ai OaS) eeobeo
1061111066oloPoblooll-bool01011161066-
pEopeeloeloboloile66610666polow0666peopiolo
3.413.4s5bebbbolbqoqbaoobbsq000qqebbboobbs000lbqbeebbbsobbbbboboeqqqossosbb.4.4
qos5o651212555-4-
4o2o125551212555soobb00005ob000bo5555125.6ob000bbbbobsqbbsosobobboq
000bolob0000sE555loobbebbbobqbbolbbebeobb000qobeolglosobsobbb000lb000loblob
qoolll000bb000looloboloboollqloobsooll000qabololoabloselbbasoosbblblolbbbbq
Poleoboolo00olloo0610100666610106100116061066106106106100
(Ima)
T sousnbou soviomosd zoxym
(918:0N
ai Os) boobosoobbbl000sobsoblobsbbebbebloobooboobooboobqobooblloboob
ooblobqobebbsEqqbobbobeefrasobb000bbobob000bblqoqoosboboqbe55.45=5.43.45esebbo
op6opoobbbobolbobboobossbbossbboqqqobspobbobobobbowbwbqbbwoqqbbobwbbob
bbL)ffy4e.booyobte411obbebb-4bsoyboobeygobb-4-4eoyebbbbobbobbbbbbblIb-
4oybobbobblIbbo
bbobbob000sobobebebobobb6sobbobobobbboobbbbo55555egbobobo655456beobbsoobsob
(VNO)
aouanbas aa4omoad EoIyaN
(SIU:ON ai
oas) bqebow0005ososobobobosoob000055boobboboobbbewoqbbeoboboosoobebbow55
bsobbbobobbbboosobbbobobbbsobsobsopolsbsooboboobolbsbbobbooboloesboolobbsby
oq3absbobebsolbobwoobbobobebsobbebEsobbessesqqqobsooqqqqbqbbebb55bobbe5555
obboobbbooboobsbbbobobbbbbsbbbsobbosoqbqvbqbobbbbsobsobbb000bbbobob0000bbbb
L'000-4bo-4owbobZo4o5L)556o5655s555b0boZ5L)boob-4obbobsbobbsoopoob-40000bob-4-
4o40ob
0000bobobgb0000g0000000bobgb0000g00000000bobgb0000g0000000bobbb0000g000000b
obbb0000qopop000tiobaboopoqopoopobobqboopoqoopoopobobqboopoqopoopobobqboopoq
3333535 333313333 333353331 333
3335333333535p3333
bsoob00000qqqE00000boqqb000bbqoqb000qbobbsoloabbqbob000soabbooq000lobbboobb
33 13555335
segeoeqqqqaboEgoobeeb5b5bobeosooqqqessooqqqqobobeebqbeoobwobbbegoqqqooqqqa
ebbbliebbbobsqliqwwq5eqqblioqueobosoobtosbbbL)oolibosbollowslissoublisessequebs
owliq
qeqabsobeboqbbegoqboobbsosoobw000bboobosoopowobbb0000qbobobebqbbsobbbeqob
5355b33133333E3sses-Le3335353qbe35353lbsebsolb000000loobbbbbboo55lbeblblbses
661061061010160106126661006010610610106opo6o6lobe6lo610066116160061660611010101
06ep0elo006ee
sooqe00000000esooqooseqqqoeoebeoososoeqolq-465-4eqqqaboqesobqbo-45-
4555seqeosoq
ses4qqbeqqqbbEqqqoofm-11.152bosoefmobsob0000f)551oogooebbo6131656651000gebgbeo
23655.63233323ff)555615443535.6ebbe5552355333333335332533353523633435553533333
23535335 313 532 32555353 31335 335255 331
.1.665boof)22225Z-45535-
Looff'sboobbsoogogoqbosobbboo.4221obooposoosoobbo562636266
2353353e53133E5122255l333325252-455e05333335555sqboobobboqobb33532335153535
b55-4o4o5-45b000bobb000q0000qooq0000bEob000qoo-45355.6-
400bboos000b00000eb000000
ebw3333abs5513533335535355.66535355t5s655b5s5555e.6655855333abseb3b35535313e
325 33 23333 133 3313333313132 325 5 51f'f'PS
5woqb3333533o533q3-L5e53bb333q33q33oqq2bb531o3bb33325e33533335353bbbq355332
5-1.532333131be3135565253-4555.6-4355353325525313555333-
4553155233531335235552233
52552313313b50333bb3333533333533bb305252255255s5525553555525555225525535235
bobbebbebbebbyebqqw_qqyggyeyeeybqbbtbgeob4o4-41111obebbbeyeebbeoebyobebybqqqob
bosbbselebbbbbbobbb000solbooqqabbb000sbaossebbbebsossellsobboseobb000lbblbo
osoobbobobobbqopbob-L000lossbbooloobooboobbblowboboobblobsbobbboobbsbsobobb
66306660066P6Po60066166o60006666600EP6Po6P6666olo6o666Po6o6666PPP66P6PP66P6
k23552bba532133222322212135535215213333131532215153332222122221331223323123
LIOLLORZOZSahlad
6COLZO/ZZOZ OM
WO 2022/027039
PCT/US2021/071017
NFATcl response element consensus
(DNA)
tttttcca (SEQ ID NO:820)
NFAT response elements contained within the Forp3 enhancer region
(DNA)
acttgaaaatgagataaatgttcacctatgttggcttctagtctcttttatggctr.catt
ttttccatttactatagaggttaagagtgtgggtactggagccagactgtctgggacaa (SEQ ID NO: 821)
N+20
SNIKFRPGSVVVQLTLAFREGTINVHDVETUNQYKTEAASRYNLT:SDVSVSEVPFPFSAQSGA (SEQ ID
NO:822)
N+20/C-27
SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTE (SEQ ID NO:823)
N+9/C-9
VQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVP(SEQ ID NO:824)
C-10
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDV(SEQ ID NO: 825)
NME1-AB
(DNA)
atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaa7.tatcgaaattatcaacaaa
gcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccc7_ggattttcatgtcgaccac
cagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgatta7.cgcaatggaaattctgcgt
gatgacgctatctqcgaatqqaaacgcctqctcmcccqgcaaactcaggtqttqcgcgtaccgatqccagtgaa
tccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagct
cgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacg
tgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgaggcaatccgtgatgctggc
tttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagast7.ctacgaagtttacaaaggc
qtqqttaccgaatatcacgatatqqttacqqaaatqtactccqqtccgtqcgtcgcgatqqaaattcagcaaaac
aatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtacc
ctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatcr.gccggaagacggtctgctg
gaagttcaatactttttcaaaattctggataatctcgagcaccaccaccaccaccactga(SEQ ID NO:826)
NME7AB
(amino acids)
MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPI=AMEILR
DDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCT
CCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQN
NATKTFREFCGPADPEIARHLRPG7LRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDNLEHHHHHH(SEQ
ID NO:827)
Human NME7 xl
(DNA)
atgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctg
atccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaaga
ctgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagat
ggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaa7.ggagttgttttttccttca
agtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcatgttaaaccccatgctgtc
agtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaarxtcagctatgcagatgttc
aatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtg
acagaaatqtattctcmcccttqtqtaqcaatqqacrattcaacagaataatqctacaaagacatttccracraattt
tgtggacctgctgatcctgaaattgcccggcatttacgccctggaactetcagagcaatctttggtaaaactaag
atccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttg
gaLaaLcUcgagcaccaccaccaccaccacUga(SEQ ID NO:828)
249
CA 03187555 2023- 1- 27
WC) 2022/027039
PCT/US2021/071017
(amino acids)
MMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARIDASESIRALFGTD
GIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFE=SAMOMF
NMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGILRAIFGKIK
IONAVHCTDLPEDGLLEVOYFFKILDNLEHHHHHHA(SEO ID NO:829)
Mouse Antibody 17H6 Heavy chain: DNA sequence
Signal segucnce-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
AIGAAGTIGTGGCTGAACIGGATITICCTIGIAACACTITIAAAIGGIATCCAGIGIGAGGTGAAGOIGGTGGAG
TCTGGAGGAGGOTTGGTACAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAACTTCTGGGTTCACCTTCACTGAT
TACTACATGAGCTGGGTOOGOCAGCCTOCAAGAAAGGCACTTGAGTGGTTGGGITTTATTAGAAACAAAGCTAAT
GGTTAGACAGCAGAGTACAGTGCGTOTGTGAAGGGTCGGITCACCA7CTOCAGAGATGTTTCCCAAAACCTCOTC
TATCTTCAAATGAACATCCTGAGAGCTGAGGACAGTGCCACTTATTACTGTGCAAAAGATTACTACGGTAGTAAC
OOTGOCIGGITIGOITACIGGGGCCAAGGGACTCIGGTCACIGTCICIGCA (SEQ ID NO :830)
Mouse Antibody 17H6 Heavy chain: Amino acid sequence
Signal pepLide-FR1-CDR1-FR2-CDR2-FRS-CDR3-FR4
MKLWLNWIFLVILLNGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVROPPRKALEWLGFIRNKAN
GYTAEYSASVKGRFTISRDVSONLLYLOMNILRAEDSATYYCAKDYYGSNPAWFAYWGQGTLVIVSA (SEQ ID
NO:831)
Mouse 17H6 heavy chain variable framework 1 (FW1) sequence:
(DNA)
GAGGTGAAGCTOGIGGAGICTGGAGGAGGCTIGGTACAGCCTGGGGGITCTCTGAGACTCTOCTGTGCAACTICT
GGGITCACCTICACT (SEQ ID NO:832)
(amino acids)
EVKLVESGGGLVQPGGSLRLSCATSGFTFT(SEQ ID NO:833)
Mouse 17H6 heavy chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
GATTAGTAOATGAGC (SEQ ID NO:834)
(amino acids)
DYYMS(SEQ ID NO:835)
Mouse 17116 heavy chain variable framework 2 (FW2) sequence:
(DNA)
GAGGTGAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAACTTCT
GGGTTCACCTTCACT (SEQ ID NO:836)
(amino acids)
WVROPPRKALEWLG(SEQ ID NO:837)
Mouse 17H6 heavy chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
TTTATTAGAAACAAAGCTAATGGT7ACACAGOAGAGTACAGTGCGTOTGTGAAGGGT (SEQ ID NO :838)
(amino acids)
FIRNKANGYTAEYSASVKG (SEQ ID NO:839)
Mouse 17H6 heavy chain variable framework 3 (FW3) sequence:
(DNA)
CGGTTCACCATCTCCAGAGATGTT7CCCAAAACCTCCTCTATCTTCAAATGAACATCCTGAGAGCTGAGGACAGT
GCCACTTATTACTGTGCAAAA (SEQ ID NO:840)
(amino acids)
RFTISRDVSQNLLYLOMNILRAEDSATYYCAK (SEC ID NO:841)
250
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
Mouse 17H6 heavy chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
GATTACTACGGTAGTAACCCTGCCEGGITTGCTTAC (SEQ ID NO :842)
(amino acids)
DYYGSNPAWFAY (SEQ ID NO:)
Mouse 17H6 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGCCAAGGGACTOTGGTCACTGTOTCTGCA (SEQ ID NO:843)
(amino acids)
WGQGTLVTVSA (SEQ ID NO:1)
Mouse Antibody 17H6 Light chain: DNA sequence
Signal sequence-FRi-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAAGITGCCTGTGAGGCTGITGGIGCTGATGITCTGGATTCCIGCTICCAACAGTGATAITTTGATGACCCAG
ACTCCACTCTCCCTGCCIGICAGICTIGGAGATCAAGCCTCCATCTCTIGCAGATCTAGICAGAGCATEGTACAT
AGTAGTGGAAACACCITITTAGAAIGGTACCIGCAGAAACCTGGCCAGICTCCAAAGCTCCTGATCTACAAAGIT
TCCAACCGATITICTGGGGICCCAGACAGGITCAGIGGCAGTGGATCAGGGATAGATTICACACTCAAGATCAGC
AGAGTGGAGGCTGAGGATCTGGGAGITTATTACTGCTITCAAGGITCACATGITCCITICACGTICGGCTCGGGG
ACAAAGTIGGAAATAAAA (SEQ ID NO:844)
Mouse Antibody 17H6 Light chain: Amino acid sequence
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MKLPVRLLVLMFWIPASNSDILMTQTPLSLPVSLGDQASISCRSSQSIVHSSGNIFLEWYLQKPGQSPKLLIYKV
SNRFSGVPDRFSGSGSGIDFILKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEC ID NO:845)
Mouse 17H6 light chain variable framework 1 (FW1) sequence:
(DNA)
GATATITTGATGACCCAGACTCCACTCTCCCTGCCIGICAGICTIGGAGATCAAGCCTCCATCTCTIGC (SEQ
ID NO:846)
(amino acids)
DILMTOTPLSLEWSLGDQASISC (SEQ ID NO:847)
Mouse 17H6 light chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
AGATCTAGICAGAGCATIGTACATAGTAGIGGAAACACCETTITAGAA (SEQ ID NO :848)
(amino acids)
RSSQSIVHSSGNTFLE (SEQ ID NO:849)
Mouse 1/H6 lignt chain variable framework 2 (FW2) sequence:
(DNA)
TGGTACCTGCAGAAACCTGGCCAGECTCCAAAGCTCCTGATCTAC (SEQ ID NO:850)
(amino acids)
WYLQKPGQSPKLLIY (SEQ ID NO:851)
Mouse 17H6 light chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
AAAGTTTCCAACCGATTTTCT (SEQ ID NO:852)
(amino acids)
KVSNRFS (SEQ ID NO:853)
Mouse 17H6 lignL chain variable framework 3 (FW3) sequence:
(DNA)
251
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
GOGGTCCCAGACAGOTTCAGTGOCAGTOGATCAGGGATAGATTICACACTCAAGATCAGCAGAGTOGAGGCTGAG
GATCTGGGAGITTATTACTGC (SEQ ID NO:854)
(amino acids)
GVPDRFSGSGSGIDFTLKISRVEAEDLGVYYC (SEQ ID NO:855)
Mouse 17H6 light chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
TTICAAGGITCACATGITCCITTCACG (SEQ ID NO:856)
(amino acids)
FQGSHVPFT (SEQ ID NO:857)
Mouse 17H6 light chain variable framework 4 (FW4) sequence:
(DNA)
TTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO:858)
(amino acids)
FGSGTKLEIK (SEQ ID NO:859)
Mouse antibody 39115 Heavy chain: DNA sequence
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGCTIGGGIGIGGACCITGCTATTCCTGATGGCAGCTGCCCAAAGTGCCCAAGCACAGATCCAGTTGGIGCAG
ICIGGACCTGAGCTGAAGAAGCCIGGAGAGACAGICAAGATCTCCIGCAAGGCITCIGGGIATACCTICACAAAC
TATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGITTAAAGTGGATGGGCTGGATAAACACCIACACTGGA
GAGCCAACATATGTTGGTGACTTCAAGGGACGGTTTGCCITCTCTT7GGAGACCTCTGCCAGCACTGCCTATTTG
CAGATCAACAACCICAAAAATGAGGACACGGCTACATATTTTTGTG7TAGAGGIATCCACGGCTACGIGGACTAC
TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:860)
Mouse antibody 39H5 Heavy chain: Amino acid sequence
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MAWVWTLLIPLMAAAOSAOAQTQLVOSGPELKKPGETVKISCKASGYTIPTNYGMNWVKOAPGKGLKWMGWINTYTG
EPTYVGDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCVRGIHGYVDYWGQGTTLTVSS (SEQ ID
NO:861)
Mouse antibody 39115 heavy chain variable framework 1 (FW1) sequence:
(DNA)
CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCT
GGGIATACCTICACA (SEQ ID NO:862)
(amino acids)
QIQLVQSGPELKKPGETVKISCKASGYTFT (SEQ ID NO:863)
Mouse antibody 39H5 heavy chain variable compiementarity determining
regions 1 (CDR1) sequence:
(DNA)
AACTATGGAATGAAC (SEQ ID NO:864)
(amino acids)
NYGMN (SEQ ID NO:865)
Mouse antibody 39H5 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGC (SEQ ID NO:866)
(amino acids)
WVKQAPGKGLKWMG (SEQ ID NO:867)
Mouse antibody 39115 heavy chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
TGGATAAACACCTACACTGGAGAGCCAACATATGTTGGTGACTTCAAGGGA (SEC ID NO:868)
252
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
(amino acids)
WINTYTGEPTYVGDFKG (SEQ ID NO:869)
Mouse antibody 39H5 heavy chain variable framework 3 (FW3) sequence:
(DNA)
CGGTTTGCCTTCTCTTTGGAGACC7CTGCOAGOACTGCCIATTTGOAGATCAACAACCTOAAAAATGAGGACACG
GCTACATATTTTTGTGTTAGA (SEQ ID NO:870)
(amino acids)
RFAFSLETSASTAYLQINNLKNEDTATYFCVR (SEQ ID NO:871)
Mouse antibody 39H5 heavy chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
GGTATCCACGGCTACGTGGACTAC (SEQ ID NO:872)
(amino acids)
GINGYVDY (SEQ ID NO:873)
Mouse antibody 39H5 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEC ID NO:874)
(amino acids)
WGQGTTLTVSS (SEQ ID NO:875)
Mouse antibody 39H5 Light chain: DNA sequence
Signal sequence-FR1-CDR1-FR2-CDR2-FR2-CDR2-5R4
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAA
ACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATEGTACAT
AGAAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTT
TCCAACCGATTTTCTGGGGTCCCAGACAGGTICAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGC
AGAGTGGAGGCTGAGGATCTGGGAGITTATTACTGCTITCAAGGITCACATCTTCCGIGGACGTICGGTGGAGGC
ACCAAGCTGGAAATCAAA (SEQ ID NO:876)
Mouse antibody 39H5 Light chain: Amino acid sequence
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MKLEVRLLVLMFWIPASSSDVLMTOTPLSLEWSLGDQASISCRSSOSIVERNGNTYLEWYLQKPGQSPKLLIYKV
SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHLPWTEGGGTKLEIK (SEQ ID NO: 877)
Mouse antibody 39H5 light chain variable framework 1 (FW1) sequence:
(DNA)
GAIGTITTGATGACCCAAACTCCACTCTCCCTGCCIGICAGICTIGGAGAICAAGCCTCCATCTCTIGC (SEQ
ID NO:878)
(amino acids)
DVLMTQTELSLEWSLGDQASISC (SEQ ID NO:879)
Mouse antibody 39H5 _Light chain variable compiementarity determining
regions 1 (CDR1) sequence:
(DNA)
AGATCTAGTOAGAGCATTGTACATAGAAATGGAAACACCIATTTAGAA (SEQ ID NO :880)
(amino acids)
RSSQSIVHRNGNTYLE (SEQ ID NO:881)
Mouse antibody 39H5 light chain variable framework 2 (Fw2) sequence:
(DNA)
TGGTACCTGCAGAAACCAGGCCAGECTCCAAAGCTCCTGATCTAC (SEQ ID NO: B82)
(amino acids)
WYLOKPGQSPKLLIY (SEC ID NO:883)
Mouse antibody 39H5 lighL chain yailable complemenLariLy determining
regions 2 (CDR2) sequence:
253
CA 03187555 2023- 1- 27
WC) 202/1127039
PCT/US2021/071017
(DNA)
AAAGTTTCCAACCGATTTTCT (SEQ ID NO:884)
(amino acids)
KVSNRFS (SEQ ID NO:885)
Mouse antibody 39H5 light chain variable framework 3 (FW3) sequence:
(DNA)
GGGGTCCCAGACAGGETCAGEGGCAGEGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGEGGAGGCTGAG
GATCTGGGAGTTTATTACTCC (SEQ ID NO:886)
(amino acids)
GVEDRFSGSGSGTD7TLKISRVEAEDLGVYYG (SEQ ID NO:887)
Mouse antibody 39H5 light chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
TTTCAAGGTTCACATCTTCCGTGGACG (SEQ ID NO:888)
(amino acids)
FQGSHLFWT (SEQ ID NO:889)
Mouse antibody 39H5 light chain variable framework 4 (FW4) sequence:
(DNA)
TTCGOTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:890)
(amino acids)
FGGGTKLEIK (SEQ ID NO:89:)
Antibody 3C5 Heavy chain: DNA sequence
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGCTTGGGTGTGGACCITGCIGTICCTGATGGCAGCTGCCCAAAGTGCCCAAGCACAGATCCAGITGGTGCAG
TCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAAC
TATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTITAAAGEGGATGGGCTGGATAAACACCTACACTGGA
AAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTEGGAGACCTCTGCCAGCACTGCCTATTTG
CAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAGGGGGACTAGATGGTTACTACGGC
TACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO :892)
Antibody 3C5 Heavy chain: Amino acid sequence
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MAWVWTLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGY7FTNYGMKWVKQAPGKGLKWMGWINTYTG
KPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGGLDGYYGYWGQGTTLIVSS (SEQ ID
NO: 893)
Mouse antibody 3C5 heavy chain variable framework 1 (FW1) sequence:
(DNA)
CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTOCTGCAAGGCTTCT
GGGTATACCTTCACA (SEQ ID NO:894)
(amino acids)
QIQLVQSGPELKKPGETVKISCKASGYTFT (SEQ ID NO:895)
Mouse antibody 3C5 heavy chain variable complement arity determining regions
1 (CDR1) sequence;
(DNA)
AACTATGGAATGAAC (SEQ ID NO:896)
(amino acids)
NYGMN (SEQ ID NO:897)
Mouse antibody 3C5 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGC (SEQ ID NO:898)
(amino acids)
WVKQAPGKGLKWMG (SEQ ID NO:899)
254
CA 03187555 2023- 1- 27
WC) 202/1127039
PCT/US2021/071017
Mouse antibody 3C5 heavy chain variable complementarity determining regions
2 (CDR2) sequence:
(DNA)
TGGATAAACACCTACACTGGAAAGCCAACATATGCTGATGACTTCAAGGGA (SEQ ID NO:900)
(amino acids)
WINTYTGKPTYADD7KG (SEQ ID NO:901)
Mouse antibody 3C5 heavy chain variable framework 3 (FW3) sequence:
(DNA)
CGGTTTGCCTTCTCTTTGGAGACC7CTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACG
GCTACATATTICTGIGCAAGA (SEQ ID NO:902)
(amino acids)
RFAFSLETSASTAYLQINNLKNEDEATYFCAR (SEQ ID NO: 903)
Mouse antibody 3C5 heavy chain variable complement arity determining regions
3 (CDR3) sequence:
(DNA)
GGGGGACTAGATGGTTACTACGGCTAC (SEQ ID NO: 904)
(amino acids)
GGLDGYYGY (SEQ ID NO:905)
Mouse antibody 3C5 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:906)
(amino acids)
WGOGTTLTVSS (SEQ ID NO:907)
Antibody 3C5 Light chain: DNA sequence (393 bp)
Signal sequence-Fa1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAGTCCTGCCCAGTTCCTGTTTCTGCTAGTGCTCTCGATTCAGGAAACCAACGGTGATGITGTGATGGCTCAG
ACCCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAATCAAGTCAGAGCCTCTTACAT
AGTAAAGGAAAGACATATTTGAATEGGITATTACAGAGGCCAGGCCAGICTCCAAAGCTCCTAATCTATCTGGTG
TCTAAACTGGAATCTGGAGICCCTGACAGGITCAGTGGCAGTGGATCAGGGACAGATTICACACTGAAAATCAGC
AGAGTGGAGGCTGAAGATTTGGGAGTTTATTACTGCTTGCAAACTACACATTTTCCGTGGACGTTCGGTGGAGGC
ACCAAGCTGGAAATCAAA (SEQ ID NO:908)
Antibody 3C5 Light chain: Amino acid sequence (131 aa)
Signal pcptidc-FR1-CDR1-7R2-CDRE-FRS-CDR3-FR4
MSPAQ7L7LLVLSIQETNGDVVMAQTFLTLSVTIGWASISCKSSQSLLIISKGHTYLNWLLQRFGQSFKLLIYLV
SKLESGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCLQITHFPWTEGGGTKLEIK (SEQ ID NO: 909)
Mouse antibody 3C5 light chain variable framework 1 (FW1) sequence:
(DNA)
GAIGTIGTGAIGGCTCAGACCCCACTCACTITGICGGITACCATTGGACAACCAGCCTCCATCTCTIGC (SEQ
ID NO:910)
(amino acids)
DVVMAQTPLILSVTIGQPASISC (SEQ ID N :911)
Mouse antibody 39H5 light chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
AAATCAAGTCAGAGCCTCTTACATAGTAAAGGAAAGACATATTTGAAT (SEQ ID NO :912)
(amino acids)
KSSQSLEHSKGKTYLN (SEQ ID 110:913)
Mouse antibody 3C5 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGTTATTACAGAGGCCAGGCCAGECTCCAAAGCTCCTAATCTAT (SEQ ID NO:914)
(amino acids)
WLLORPGQSPKLLIY (SEQ ID NO:915)
255
CA 03187555 2023- 1- 27
WC) 202/1127039
PCT/US2021/071017
Mouse antibody 3C5 light chain variablc complementarity determining regions
2 (CDR2) sequence:
(DNA)
CTGGTGTCTAAACTGGAATCT (SEQ ID NO:916)
(amino acids)
LVSKLES (SEQ ID NO:917)
Mouse antibody 3C5 light chain variable framework 3 (FW3) sequence:
(DNA)
GGAGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAA
GATTTGGGAGTTTATTACTGC (SEQ ID NO:918)
(amino acids)
GVPDRFSGSGSGTDD.TLKISRVEAEDLGVYYC (SEQ ID NO:919)
Mouse antibody 3C5 light chain variable complement arity determining regions
3 (CDR3) sequence:
(DNA)
TTGCAAACTACACATTTTCCGTGGACG (SEQ ID NO:920)
(amino acids)
LQTTHFPWT (SEQ ID NO:921)
Mouse antibody 3C5 light chain variable framework 4 (FW4) sequence:
(DNA)
TTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:922)
(amino acids)
FGGGTKLEIK (SEQ ID NO:923)
Mouse antibody 8A9 Heavy chain: DNA sequence (420 bp)
Signal sequence-Fa1-CDR1-Fa2-CDR2-Fa3-CDR3-Fa4
ATGAAGTTGTGGCTGAACTGGATTiTCCTTGIAACACTTITAAATGGTATCCAGTGTGAGGIGGAGCIGGTGGAG
TCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAACTTCTGGGTTCACCTTCACTGAT
CACTACATGAGCTGGGTCCGCCAGCCTCCAGGAAAGGCACTTGAGTGGTTGGGATTTATTAGAAACAAAGCTAAT
GGTTACACAACAGAGTACAGTGCATCTGTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCCAAAGCATCCTC
TATCTTCAAATGAAAACCCTGAGAACTGAGGACAGTGCCACTTATTACTGTGCAAGACCTTCTGACTGGGACTCC
TGGITTGCTTACTGGGGCCAAGGGACTCTGGICACTGICICTGCA (SEQ ID NO: 924)
Mouse antibody 8A9 Heavy chain: Amino acid sequence (140 aa)
Signal peptide-FR1-CDR1-7R2-CDR2-FR3-CDR3-7R4
MKLWLNWIFLVTLLNCIOCEVELVESCGGLVOPCGSLRLSCATSCF7FTDAYMSWVROPPCKALEWLCFIRNKAN
GYTTEYSASVKGRFTISRDNSQSILYLQMKTLRTEDSATYYCARPSDWDSWFAYWGQGTLVTVSA (SEQ ID
NO: 925)
Mouse antibody 8A9 heavy chain variabic framework 1 (FW1) sequence:
(DNA)
GAGGTGGAGCTGGTGGAGTOTGGAGGAGGCTTGGTACAGCCTGGGGGTTOTOTGAGACTOTCOTGTGCAACTTOT
GGGTTCACCITCACT (SEQ ID NO:926)
(amino acids)
EVELVESGCGLVOPGGSLRLSCATSGFTFT (SEQ ID NO: 927)
Mouse antibody 8A9 heavy chain variable complement arity determining regions
1 (CDR1) sequence:
(DNA)
GATCACTACATGAGC (SEQ ID NO:928)
(amino acids)
DHYMS (SEQ ID NO:929)
Mouse antibody 8A9 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGTCCGCCAGCCTCCAGGAAAGGCACTTGAGTGGTTGGGA (SEC ID NO:930)
(amino acids)
WVRQPPGKALEWLG (SEQ ID NO:931)
256
CA 03187555 2023- 1- 27
WC) 2022/027039
PCT/US2021/071017
Mouse antibody 8A9 heavy chain variable complementarity determining regions
2 (CDR2) sequence:
(DNA)
TTTATTAGAAACAAAGCTAATGGTEACACAACAGAGTACAGTGCATCTGTGAAGGGT (SEQ ID NO:932)
(amino acids)
FIRNKANGYTTEYSASVKG (SEQ ID NO:933)
Mouse antibody 8A9 heavy chain variable framework 3 (FW3) sequence:
(DNA)
CGGTTCACCATCTCCAGAGATAATECCCAAAGCATCCTCTATCITCAAATGAAAACCCTGAGAACTGAGGACAGT
GCCACTTATTACTGTGCAAGA (SEQ ID NO:934)
(amino acids)
RFTISRDNSQSILYLOMKTLRTEDSATYYCAR (SEQ ID NO: 935)
Mouse antibody 8A9 heavy chain variable complement arity determining regions
3 (CDR3) sequence:
(DNA)
CCTICTGACTGGGACTCCIGGITTGCTTAC (SEQ ID NO:936)
(amino acids)
PSDWDSWFAY (SEQ ID NO:937)
Mouse antibody 8A9 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGCCAAGGGACTCTGGICACIGTCTCTGCA (SEQ ID NO:938)
(amino acids)
WGOGTLVTVSA (SEQ ID NO:939)
Mouse antibody 8A9 Light chain: DNA sequence (393 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAAGTTGCCIGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAA
ACTCCACTCTCCCTGCCIGICAGICTIGGIGATCAAGCCTCCATCTCTIGCAGATCTAGICAGAGCATEGTACAT
AGTAATGGCAACACCIATTTAGATTGGTACTTGCAGAAACCAGGCCAGICTCCAAAGCTCCTGATCTACAGAGIT
TCCAACCGAITTICTGGGGICQCAGACAGGITCAGIGGCAGIGGATCAGGGACAGATTICACACTCAAGATCAGC
AGAGTGGAGGCTGAGGATCTGGGACTITATTACTGITTICAAGGITCACATGTTCCGIGGGCGTICGGTGGAGGC
ACCAAGCTGGAAATCAAA (SEQ ID NO:940)
Mouse antibody 8A9 Light chain: Amino acid sequence (131 aa)
Signal peptide-FR1-CDR1-7R2-CDR2-FR3-CDR3-7R4
MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLDWYLQKPGQSPKLLIYRV
SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYYCFQGSHVPWAFGGGTKLEIK (SEQ ID NO: 941)
Mouse antibody 8A9 light chain variable framework 1 (FW1) sequence:
(DNA)
GAIGTITTGATGACCCAAACTCCACTCTCCCTGCCIGICAGICTIGGIGAICAAGCCTCCATCTCTIGC (SEQ
ID NO:942)
(amino acids)
DVLNITQTPLSLPVSLGDQASISC (SEQ ID NO:943)
Mouse antibody 8A9 light chain variable complement arity determining regions
1 (CDR1) sequence:
(DNA)
AGATCTAGTCAGAGCATTGTACATAGTAATGCCAACACCTATTTAGAT (SEQ ID NO:944)
(amino acids)
RSSOSIVHSNGNTYLD (SEQ ID NO:945)
Mouse antibody 8A9 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGTACTTGCAGAAACCAGGCCAGECTCCAAAGCTCCTGATCTAC (SEC ID NO: 946)
(amino acids)
WYLQKPGQSPKLLIY (SEQ ID No:947)
257
CA 03187555 2023- 1- 27
WC) 202/1127039
PCT/US2021/071017
Mouse antibody 8A9 light chain variable complcmcntarity dctcrmining rcgions
2 (CDR2) sequence:
(DNA)
AGAGTTTCCAACCGATTTTCT (SEQ ID NO:948)
(amino acids)
RVSNRFS (SEQ ID NO:949)
Mouse antibody 8A9 light chain variable framework 3 (FW3) sequence:
(DNA)
GGGGTCCCAGACAGGITCAGIGGCAGIGGATCAGGGACAGATTICACACTCAAGATCAGCAGAGIGGAGGCTGAG
GATCTGGGACTTTATTACTGT (SEQ ID NO:950)
(amino acids)
GVPDRFSGSGSGTD7TLKISRVEAEDLGLYYC (SEQ ID NO:951)
Mouse antibody 8A9 light chain variable complement arity determining regions
3 (CDR3) sequence:
(DNA)
TTTCAAGGTTCACATGTTCCGTGGGCG (SEQ ID NO:952)
(amino acids)
FQGSHVPWA (SEQ ID NO:953)
Mouse antibody 8A9 light chain variable framcwork 4 (FW4) sequence:
(DNA)
TTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:954)
(amino acids)
FGGGTKLEIK (SEQ ID NO:955)
Mouse antibody 18G12 Heavy chain: DNA sequence (399 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGGATGGAGCTATATCATCCICITTTTGGICGCAACAGCTACAGGTGTCCACTCCCAGGTCCAACTGCAGCAG
TCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCGGC
TACTTTTTGTACTGGGTGAAGCAGAGGCCTGGACAAGGCOTTGAGTGGATTGGGGGGATTAATCCTGACAATGGT
GGTATTGACTTCAATGAGAAGTTCAGGAACAAGGCCACACTGACTGTAGACAAATCCTOCAGCACAGCCTACATG
CAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTACATTACTAATAGGGAACTATTGGGGCCAA
GGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:956)
Mouse antibody 18G12 Heavy chain: Amino acid sequence (133 aa)
Signal bcptidc-FR1-CDR1-7R2-CDR2-FRS-CDR3-7R4
MGWSYIILFLVATATGVHSQVQLQQSGAELVKFGASVKLSCKASGY7FTGYFLYWVKQRFGQGLEWIGGINFDNG
GIDFNEKERNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTLLIGNYWGQGTTETVSS (SEQ ID NO: 957)
Mouse antibody 18G12 heavy chain variable framcwork 1 (FW1) sequence:
(DNA)
CAGGTCCAACTGCAGCAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTOCTGCAAGGCTTCT
GGCTACACCTTCACC (SEQ ID NO:958)
(amino acids)
QVQLQQSGAELVKPGASVKLSCKASGYTFT (SEQ ID NO:959)
Mouse antibody 18G12 heavy chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
GGCTACTTTTTGTAC (SEQ ID NO:960)
(amino acids)
GYFLY (SEQ ID NO:961)
Mouse antibody 18G12 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGIGAAGCAGAGGCCTGGACAAGGCCITGAGTGGATTGGG (SEQ ID NO:962)
(amino acids)
WVKQRPGQGLEWIG (SEQ ID NO:963)
258
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
Mouse antibody 18G12 hcavy chain variable complcmcntarity determining
L-egions 2 (CDR2) sequence:
(DNA)
GGGATTAATCCTGACAATGGTGGIATTGACTTCAATGAGAAGTTCAGGAAC (SEQ ID NO: 964)
(amino acids)
GINPDNGGIDENEK7RN (SEQ ID NO:965)
Mouse antibody 18G12 heavy chain variable framework 3 (FW3) sequence:
(DNA)
AAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCIACATGCAACTCAGCAGCCTGACATCTGAGGACTCT
GCGGTCTATTATTGTACATTA (SEQ ID NO:966)
(amino acids)
KATLTVDKSSSTAYMOLSSLTSEDSAVYYCTL (SEQ ID NO:967)
Mouse antibody 18G12 heavy chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
CTAATAGGGAACTAT (SEQ ID NO:968)
(amino acids)
LIGNY (SEQ ID NO:969)
Mouse antibody 18G12 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:970)
(amino acids)
WG0GTTLTVSS (SEQ ID NO:971)
Mouse antibody 18G12 Light chain: DNA sequence (393 bp)
Signal sequence-Fa1-CDa1-Fa2-CDR2-Fa3-CDR3-Fa4
ATGAGTCCTGCCCAGTTCCTGTTICTGTTAGTGCTCTGGATTCGGGAAACCAATGGTGATGITGTGATGACCCAG
ACTCCACTCACTTTGTCGGTAACCATTGGACAGCCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTACAT
AGTGATGGAAAGACATATTTGATTEGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTG
ICTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATITCACACTGAAAATCAGC
AGAGTGGAGGCTGAGGATTIGGGAGTITATTITTGCTGTCAAGGTACACATTITCCGTGGACGTTCGGEGGAGGC
ACCATGCTGGAAATCAAA (SEQ iD NC:972)
Mouse antibody 18G12 Light chain: Amino acid sequence (131 aa)
Signal peptide-FR1-CDR1-7R2-CDR2-FRS-CDR3-7R4
MSFAQ7L7LLVLWIRETNGDVVMTQTFLTLSVTIGQFASISCKSSQSLLIISDGKTYLIMLLQRFGQSFKRLIYLV
SKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYFCCQGTHFPWTEGGGTMLEI-,K (SEQ ID NO:973)
Mouse antibody 18G12 light chain variable framework 1 (FW1) sequence:
(DNA)
GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTAACCATTGGACAGCCAGCCTCCATCTCTTGC (SEQ
ID NO:9/4)
(amino acids)
DVVMTQTPLTLSVTIGQPASISC (SEQ ID NO:975)
Mouse antibody 18G12 light chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
AAGTCAAGTCAGAGCCTCTTACATAGTGATGGAAAGACATATTTGA7T (SEQ ID NO :976)
(amino acids)
KSSQSLLHSDGKTYLI(SEO ID NO:977)
Mouse antibody 18G12 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGTTGTTACAGAGGCCAGGCCAGECTCCAAAGCGCCTAATCTAT (SEQ ID NO: 978)
259
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
(amino acids)
WLLQRPGQSPKRLIY(SEQ ID NO:979)
Mouse antibody 18G12 light chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
CTGGTGTCTAAACTGGACTCT (SEQ ID NO:980)
(amino acids)
LVSKLDS(SEQ ID NO:981)
Mouse antibody 18G12 light chain variable framework 3 (FW3) sequence:
(DNA)
GGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAG
GATTTGGGAGTTTATTTTTGC (SEQ ID NO:982)
(amino acids)
CVPDRFTGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO:983)
Mouse antibody 18G12 light chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
TGTCAAGGTACACATTTTCCGTGGACG (SEQ ID NO:984)
(amino acids)
CQGTHFPWT (SEQ ID NO:985)
Mouse antibody 18G12 light chain variable framework 4 (FW4) sequence:
(DNA)
TTCGGTGGAGGCACCATGCTGGAAATCAAA (SEC ID NO:986)
(amino acids)
FGGGTMLEIK (SEQ ID NO:987)
Mouse antibody 20A10 Heavy chain: DNA sequence (417 bp)
Signal sequence-FR1-CDR1-7R2-CDR2-7R3-CDR3-7R4
ATGAACTTCGGGTTCAGCTTGATTETCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGATGCTGGTGGAG
TCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTCAGCCECTGGATTCACTETCAGTACCT
ATGCCATGTCTTGGATTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTGGICGTGCTGGTTCCA
CCTACTATTCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGTCCGGAACATGCTGTACCTGCAAA
TGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCTAGAGGCCCGATCTACAATGATTACGACGAGT
TTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:988)
Mouse antibody 20A10 Heavy chain: Amino acid sequence (139 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MNFGFSLIFLVLVLKGVOCEVMLVESGGGLVKPGGSLKLSCAASGF7FSTYAMSWIROTPEKRLEWVASIGRAGS
TYYSDSVKGRETISRDNVRNILYLQMSSLRSEDTAMYYCARGPIYNDYDEFAYWGQGTLVTVSA (SEQ ID
NO; 989)
Mouse antibody 20A10 heavy chain variable framework 1 (FW1) sequence:
(DNA)
GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCOCTGAAACTCTOCTGTGCAGCCTCT
GGATTCACTTTCAGT (SEQ ID NO:990)
(amino acids)
EVMLVESGGGLVKPGGSLKLSCAASGFTFS (SEQ ID NO:991)
Mouse antibody 20A10 heavy chain variable complementarity determining
r-egions 1 (CDR1) sequence:
(DNA)
260
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
ACCTATGCCATGTCT (SEQ ID NO:992)
(amino acids)
TYAMS (SEQ ID NO:993)
Mouse antibody 20A10 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGATTCGCCAGACTCCAGAGAAGAGGCTGGAGIGGGICGCA (SEQ ID NO: 994)
(amino acids)
WIRQTPEKRLEWVA (SEQ ID NO:995)
Mouse antibody 20A10 heavy chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
TCCATIGGICGTGCTGGTICCACCTACTATICAGACAGIGTGAAGGGC (SEQ ID NO:996)
(amino acids)
SIGRAGSTYYSDSVKG (SEQ ID NO:997)
Mouse antibody 20A10 heavy chain variable framework 3 (FW3) sequence:
(DNA)
CGATICACCATCTCCAGAGATAATGICCGGAACATCCIGTACCTGCAAATGAGCAGICTGAGGICTGAGGACACG
GCCATGTATTACTGTGCTAGA (SEQ ID NO:998)
(amino acids)
RFTISRDNVRNILYLQMSSLRSEDTAMYYCAR (SEQ ID NO:999)
Mouse antibody 20A10 heavy chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
GGCCCGATCTACAATGATTACGACGAGITTGCTTAC (SEQ ID NO:1000)
(amino acids)
GPIYNDYDEFAY (SEQ ID NO:l 01)
Mouse antibody 20A10 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGCCAAGGGACTOTGGICACTGICTCTGCA (SEQ ID NO:1002)
(amino acids)
WGQGTLVIVSA (SEQ ID N0:1003)
Mouse antibody 20A10 Light chain: DNA sequence (396 bp)
Signai sequence-FRi-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGAATCACAGACTCAGGICTICCICTCCCTGCTGCTCTGGGTATCTGGTACCIGIGGGAACATTATGATGACA
CAGICGCCATCATCTCTGGCTGIGTCTGCAGGAGAAAAGGTCACTATGAGCTOTAAGICCAGICAAAGTGITTTA
TACAGTTCAAATCAGAAGAACTAT=GGCCTGGTACCAGOAGAAACCAGGGCAGTOICCTAAACTGCTGATCTAC
TGGGCATCCACTAGGGAATCTGGIGICCCTGATCGCTICACAGGCAGTGGATCTGGGACAGATITTACTCTTACC
ATCAGCAGIGTACAAGCTGAAGACCTGGCAGITTATTACTGTCATCAATACCICTOCTCGCICACGTICGGIGCT
GGGACCAAGCTGGAGCTGAAA (SEQ ID NO:1004)
Mouse antibody 20A10 Light chain: Amino acid sequence (132 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-Fa3-CDR3-FR4
MESQTQVFLSLLLWVSGTCGNIMM7QSPSSLAVSAGEKVIMSCKSSQSVLYSSNQKNYLAWYQQKPGOSPKLLIY
WASTRESGVPDRFIGSGSGTDFTL7ISSVQAEDLAVYYCHQYLSSL=FGAGTKLELK (SEQ ID NO:1005)
Mouse antibody 20A10 light chain variable framework 1 (FW1) sequence:
(DNA)
AACATTATGATGACACAGTCGCCATCATCICIGGCTGIGTCTGCAGGAGAAAAGGICACTATGAGCTG7 (SEQ
ID NO:1006)
261
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
(amino acids)
NIMMTQSPSSLAVSAGEKVINISC (SEQ ID N0:1007)
Mouse antibody 20A10 light chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
AAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTATT7GGCC (SEQ ID NO :1008)
(amino acids)
KSSQSVLYSSNQKNYLA (SEQ ID NO:1009)
Mouse antibody 20A10 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTAC (SEQ ID NO:1010)
(amino acids)
WYQQKPGQSPKLLIY (SEQ ID NO:1011)
Mouse antibody 20A10 light chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
TGGGCATCCACTAGGGAATCT (SEQ ID NO:1012)
(amino acids)
WASTRES (SEC ID NO:1013)
Mouse antibody 20A10 light chain variable framework 3 (FW3) sequence:
(DNA)
GOTGTCCCIGATCGOTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTIACCATCAGCAGTOTACAAGCTGAA
GACCTGGCAGTTTATTACTGT (SEQ ID NO:1014)
(amino acids)
GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC (SEC) ID NO:1015)
Mouse antibody 20A10 light chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
CATCAATACCTCTCCTCGCTCACG (SEC ID NO:1016)
(amino acids)
HQYLSSLT (SEQ ID NO:1017)
Mouse antibody 20A10 light chain variable framework 4 (FW4) sequence:
(DNA)
TTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 1018)
(amino acids)
FGAGTKLELK (SEQ ID NO:10:9)
Mouse antibody 25E6 Heavy chain: DNA sequence (414 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAAOTTCGGGOTCAGCTTGAT=TOOTTGCCCTOATTITAAAAGGTGTCCAGTGIGAGGIGOAGOTGGTGGAG
TCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGTTTCACTTTCAGTAGT
TATGGAATGTOTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTASTAATGG7GGTAGA
CACACCTICIATCCAGACAGIGTGAAGGGGCGATICACCATCICCAGAGACAATGCCAAGAACACCCIGTAICIG
CAAATGAGCAGTCTGAAGTTGAGGACACAGCCATGTATTTATGTGTAAGACAGACTGGGACGGAGGGCTGGTTTG
CTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 1020)
Mouse antibody 25E6 Heavy chain: Amino acid sequence (138 aa)
Signal peptide-ER1-CDR1-ER2-CDR2-FR3-CDR3-ER4
MNEGLSLIFLALILKGVQCEVQLVESGGDLVKPGGSLKLSCAASGF7FSSYGMSWVRQTPDKRLEWVA7ISNGGR
HTFYPDSVKGRETISRDNAKNTLYLQMSSLKSEDTAMYLCVRQTGTEGWFAYWGQGTLVTVSA (SEQ ID
NO: 1021)
Mouse antibody 25E6 heavy chain variable framework 1 (FW1) sequence:
(DNA)
262
CA 03187555 2023- 1- 27
WC) 2022/027039
PCT/US2021/071017
GAGGTGCAGCTGGTGGAGTOTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGEGGAGCCTOT
GGTTTCACTTTCAGT (SEQ ID NO:1022)
(amino acids)
EVQLVESGGDLVKPGGSLKLSCAASGFITS (SEQ ID NO:1023)
Mouse antibody 25E6 heavy chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
AGTTATGGAATGTCT (SEQ ID NO:1024)
(amino acids)
SYGMS (SEQ ID NO:1025)
Mouse antibody 25E6 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGTTGGCCAGACTCCAGACAAGAGGCTGGAGTGGGICGCA (SEQ ID NO: 1026)
(amino acids)
WVRQTPDKRLEWVA (SEQ ID NO:1027)
Mouse antibody 25E6 heavy chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
ACCATTAGTAATGGTGGIAGACACACCTTCTATCCAGACAGTGTGAAGGGG (SEQ ID NO :1028)
(amino acids)
TISNGGRHTFYPDSVKG (SEQ ID NO:1029)
Mouse antibody 25E6 heavy chain variable framework 3 (FW3) sequence:
(DNA)
CGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTATCTGCAAATGAGGAGTCTGAAGTCTGAGGACACA
GCCATGTATTTATGTGTAAGA (SEQ ID N0:1030)
(amino acids)
RFTISRDNAKNTLYLOMSSLKSEDTAMYLCVR (SEC ID NO:1031)
Mouse antibody 25E6 heavy chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
CAGACTGGGACGGAGGGCTGGTTTGCTTAC (SEQ ID NO:1032)
(amino acids)
OTGTEGWFAY (SEQ ID NO:1033)
Mouse antibody 25E6 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:1034)
(amino acids)
WGQGTLVIVSA (SEQ ID NO::035)
Mouse antibody 25E6 Light chain: DNA sequence (393 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATGITOTGATGACCCAG
ACICCACTCACTITGTCGGTTACCATTGGAGAACCAGCCTCCATCTCTTGCAAGICAAGTCAGAGCCTCTTAGAT
AGTGAIGGAAAGAGATATTTGAATTGOTTOTTACAGAGGCCAGGCCAGTCICCAAAGGGCCIAATCTATGTGGIG
TCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGIGGATCAGGGACAGATTICACACTGAAAAICAGC
AGAGGGAGGCTGAGGATTTGGGAG7TTATTATTGCTGGCAAGGIACACATTTTCCTCAGACGTTCGGTGGAGGCA
CCAAGCTGGAAATCAAA (SEQ =D NC:1036)
Mouse antibody 25E6 Light chain: Amino acid sequence (131 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MSPAQFLELLVLWIRETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLV
SKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWOGTHEPOTEGGGTKLEIK (SEQ ID NO:1037)
Mouse antibody 25E6 1191-IL chain vailable framework 1 (FW1) sequence:
(DNA)
263
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
CATCTICTGATGACCCAGACTOCACTOACTITCTCCCITACCATTCGACAACCAGOCTOCATCTCTICC (SEQ
ID NO:1038)
(amino acids)
DVVMTQTPLTLSVTIGQPASISC (SEQ ID NO:1039)
Mouse antibody 25E6 light chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
AAGICAAGTCAGAGCCICITAGATAGTGATGGAAAGACATATTIGAAT (SEQ ID NO: 1040)
(amino acids)
KSSQSLLDSDGKTYLN (SEQ ID N0:1041)
Mouse antibody 25E6 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGITGITACAGAGGCCAGGCCAGECTCCAAAGCGCOTAATCTAT (SEQ ID NO: 10'J2)
(amino acids)
WLLQRPGQSPKRLIY (SEQ ID NO:1043)
Mouse antibody 25E6 lighL chain vailable complemenLariLy deLermining
regions 2 (CDR2) sequence:
(DNA)
CIGGIGICTAAACTGGACTCT (SEQ ID NO:1044)
(amino acids)
LVSKLDS (SEQ ID N0:1045)
Mouse antibody 25E6 light chain variable framework 3 (FW3) sequence:
(DNA)
GGAGICOCTGACAGGITCACTGGCAGIGGATCAGGGACAGATTICACACTGAAAATCAGCAGAGIGGAGGCTGAG
GATTIGGGAGITTATTATTGC (SEQ ID NO:1046)
(amino acids)
GVPDRFIGSGSGTDETLKISRVEAEDLGVYYC (SEQ ID NO:1047)
Mouse antibody 25E6 light chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
TGGCAAGGTACACATTITCCTCAGACG (SEQ ID NO:1048)
(amino acids)
WQGINFPQT (SEQ ID N0:1049)
Mouse antibody 25E6 light chain variable framework 4 (FW4) sequence:
(DNA)
TICGGIGGAGGCACCAAGCTGGAAATCAAA (SEQ ID N0:1050)
(amino acids)
FGGGTKLEIK (SEQ ID NO:1051)
Mouse antibody 28F9 Heavy chain: DNA sequence (399 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FRS-CDR3-FR4
ATGGGAIGGAGCTATATCATCCICTITTIGGTAGCAACAGCTACAGGIGTCCACTCCCAGGTCCAACTGCAGCAG
CCIGGGGCTGAACTGGIGCAGCCTGGGGCTICAGTGAAGITGICCTGCAAGGCTTCTGGCTACACCTICACCGGC
TACTITTIGTACTGGGIGAAGGAGAGGCCTGGACATGGCCITGAGTGGATIGGGGGAATICATCCTAGCAATGGT
GATACTGACTICAATGAGAAGTICAAGAACAAGGCCACACTGACTOTAGACATATCCTOCAGCACTGCCTACATG
CAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATIGTACATTACTAATAGGGGICTACTGGGGCCAA
GGCACCACTCTCACAGICTCCICA (SEQ ID N0:1052)
Mouse antibody 28F9 Heavy chain: Amino acid sequence (133 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MGWSYIILFLVATATGVHSQVQLQQPGAELVQPGASVKLSCKASGYEFTGYFLYWVKQRPGHGLEWIGGIHPSNG
DIDFNEKFKNKATLIVDISSSTAYMQLSSLISEDSAVYYCILLIGVYWGQGTTLIVSS (SEQ ID
NO: 1053)
Mouse antibody 28F9 heavy chain variable framework 1 (FWI) sequence:
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(DNA)
CAGGTOCAACTGOAGOAGOCIGGGGOTGAACIGGIGOAGCOTGGGGCTICAGTGAAGITGTOCTGOAAGGCTICT
GGCTACACCTTCACC (SEQ ID NO:1054)
(amino acids)
OVOLOOPGAELVOPGASVKLSCKASCrYTFT (SEC ID NO:1055)
Mouse antibody 28F9 heavy chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
GGCTACTITTIGTAC (SEQ ID NO:1056)
(amino acids)
GYFLY (SEQ ID NO:1057)
Mouse antibody 28F9 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGIGAAGCAGAGGCCIGGACATGGCCITGAGIGGATIGGG (SEQ ID NO:1058)
(amino acids)
WVKQRPGHGLEWIG (SEQ ID NO:1059)
Mouse antibody 28F9 heavy chain variable complementarity determining
regions 2 (0022) sequence:
(DNA)
GGAATICATCCIAGCAATGGIGATACTGACTICAATGAGAAGTICAAGAAC (SEQ ID NO :1060)
(amino acids)
GIHPSNGDIDENEKYKN (SEQ ID NO:1061)
Mouse antibody 28F9 heavy chain variable framework 3 (FW3) sequence:
(DNA)
AAGGCCACAOTGAOTGTAGAOATAIOOTCOAGCACTGCCIAOATGCAACTCAGCAGCCTGASATOIGAGGACIOT
GCGGTCTATTATIGIACATTA (SEQ ID NO:1062)
(amino acids)
KATLIVDISSSTAYMQLSSLISEDSAVYYCIL (SEQ ID NO:1063)
Mouse antibody 28F9 heavy chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
CTAATAGGGGTCTAC (SEQ ID NO:1064)
(amino acids)
LIGVY (SEQ ID NO:1065)
Mouse antibody 28F9 heavy chain variable framework 4 (FW4) sequence:
(DNA)
IGGGG00AAGGCACCACTOICACAGTOIC0T0A (SEQ ID NO:L066)
(amino acids)
WGQGTILTVSS (SEQ ID NO:1067)
Mouse antibody 28F9 Light chain: DNA sequence (393 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAGICOTGCCCAGTICCIGITTCTGITAGTGOTCTGGATTOGGGAAACCAACGGIGATGTIGTGATGACCCAG
ACTCCACTCACTITGICGGITACCATIGGAOAACCAGCCICCAICIOTTGCAAGICAAGICAGAGCCTOTTACAT
AGTGATGGAAAGACATATTTGATTIGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTAICTGGIG
TCTAAACTGGACTCTOGAGTCCOTGACAGGTICACCGGCAGTGCATCAGGGACAGATTTCACACTGAAAATCAGC
AGAGIGGAGG0TGAGGATTEGGGAGITIATITITGCTGTCAAGGTACACATITIOCGIGGACGITOGG7GGAGGC
ACCATGCTGGAAATCAAA (SEQ ID NO:1068)
Mouse antibody 28F9 Light chain: Amino acid sequence (131 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-F124
MSPAOFLFLLVLWIREINGDVVMTOTPLTLSVTIGQPASISCKSSOSLLHSDGKTYLIWLLORPGQSPKRLIYLV
SKLDSGVPDRFIGSGSGTDFILKISRVEAEDLGVYFCCOGTHFPWITGGGTMLEIK (SEQ ID NO:1069)
Mouse antibody 28F9 light chain variable framework 1 (FWI) sequence:
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(DNA)
GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTOTTGC (SEQ
ID NO:1070)
(amino acids)
DVVMTOTPLTLSVTIGGPASISC (SEQ ID NO:1071)
Mouse antibody 28F9 light chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
AAGTCAAGTCAGAGCCTCTTACATAGTGATGGAAAGACATATTTGATT (SEQ ID NO: 1072)
(amino acids)
KSSQSLLNSDGKTYLI (SEQ ID NO:1073)
Mouse antibody 28F9 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGTTGTTACAGAGGCCAGGCCAGECTCCAAAGCGCCTAATCTAT (SEQ ID NO:1074)
(amino acids)
WLLQRPGQSPKRLIY (SEQ ID NO:107b)
Mouse antibody 28F9 light chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
CTGGTGTCTAAACTGGACTCT (SEQ ID NO:1076)
(amino acids)
LVSKLDS (SEQ ID NO:1071)
Mouse antibody 28F9 light chain variable framework 3 (FW3) sequence:
(DNA)
GGAGTCCCTGACAGGTTCACCGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAG
GATTTGGGAGTTTATTTTTGC (SEQ ID NO:1078)
(amino acids)
GVPDRFTGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO:1079)
Mouse antibody 28F9 light chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
TGTCAAGGTACACATTTTCCGTGGACG (SEQ ID NO:1080)
(amino acids)
CQGTHFPWT (SEC ID NO:1081)
Mouse antibody 28F9 light chain variable framework 4 (FW4) sequence:
(DNA)
TTCGGTGGAGGCACCATGCTGGAAATCAAA (SEQ ID NO:1082)
(amino acids)
FGGGTMLEIK (SEQ ID NO:1083)
Mouse antibody 18B4 Heavy chain: DNA sequence (411 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGTACTIGGGACTGAACTATGTA=CATAGTTTTTOTCITAAATGGTGTOCAGAGTGAAGIGAAACT?GAGGAG
TOTGGAGGAGGOTTGGTGCAACCTGGGGGATCCATGAAACTOTOTTGTGOTGCCTOTGGATTCACITTTAATGAC
GOCTGGATGGACTGGGTOCGCCAGTOTCCAGAGAAGGGGOTTGAGTGGGTTGCTGAAATTAGAAGOACAGOTAAT
ATTCATACAACATACTATGCTGAG7CTGTCCAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTC
TACCTGCAAATGAACAGCTTGAGAGCTGAAGACACTGGCATTTATTATTGTACCCCATTACTCTACGGATTTGCT
TACTGGGGCCAAGGGACTCTGGICACTGTCTCTGCA (SEQ ID NO: 1084)
Mouse antibody 18B4 Heavy chain: Amino acid sequence (137 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MYLGLNYVFIVFLLNGVQSEVKLEESGGGLVQPGGSMKLSCAASGFTFNDAWMDWVRQSPEKGLEWVAEIRSTAN
IHTTYYAESVOGRETISRDDSKSSVYLOMNSLRAEDTGIYYCTPLLYGFAYWGOGTLVTVSA (SEQ ID
NO: 1085)
Mouse antibody 1884 heavy chain variable framework 1 (FWI) sequence:
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(DNA)
GAAGTGAAACTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGGGGATCCAIGAAACTCTOTTGTGCGCCTCT
GGATTCACTTTTAAT (SEQ ID NO:1086)
(amino acids)
EVKLEESGGGLVOPGGSMKLSCAASGETFN (SEQ ID NO:1087)
Mouse antibody 18B4 heavy chain variable complementarity determining
regions I (CDRI) sequence:
(DNA)
GACGCCTGGATGGAC (SEC) ID NO:1088)
(amino acids)
DAWMD (SEQ ID NO:1089)
Mouse antibody 1894 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCT (SEQ ID NO:1090)
(amino acids)
WVRQSPEKGLEWVA (SEQ ID NO:1091)
Mouse antibody 18B4 heavy chain variable complementarity determining
regions 2 (C222) sequence:
(DNA)
GAAATTAGAAGCACAGCTAATATTCATACAACATACTATGCTGAGTCTGTCCAAGGG (SEQ ID NO: 1092)
(amino acids)
EIRSTANIHTTYYAESVQG (SEQ ID NO:1093)
Mouse antibody 1894 heavy chain variable framework 3 (FW3) sequence:
(DNA)
AGGTTCACCATCTCAAGAGATGAT7CCAAAAGTAGTGTCTACCTGCAAATGAACAGCTTGAGAGCTGAAGACACT
GGCATTTATTATTGTACCCCA (SEQ ID NO:1094)
(amino acids)
RETISRDDSKSSVYLQMNSLRAEDTGIYYCTP (SEQ ID NO:1095)
Mouse antibody 18B4 heavy chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
TTACTCTACGGATTTGCTTAC (SEQ ID NO:1096)
(amino acids)
LLYGPAY (SEQ ID NO:1097)
Mouse antibody 18B4 heavy chain variable framework 4 (FW4) sequence:
(DNA)
IGGGGCCAAGGGACICIGGICACTGICICIGCA (SEQ ID NO:L098)
(amino acids)
WGQGTLVTVSA (SEQ ID NO:1099)
Mouse antibody 18B4 Light chain: DNA sequence (393 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTGTGATGACCCAA
AGTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGAACTAGTCAGAGCCT7GTACAC
AGTAATGGAAACACCTATTTACAT7GGCACCTGCAGAAGCCAGGCCAGTCTCCAAAGGTCCTGATCTACAAAGTT
TCCAGCCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCGGGGACAGATTTCACACTCAAGATCAGC
AGAGTGGAGGCTGAGGAICTGGGAGITIATITCIGCICT0AAAATACACATGITCCGIACACGTICGGAGGGGGG
ACCAAGCTGGAAATAAAA (5E0 ID NO:1100)
Mouse antibody 18B4 Light chain: Amino acid sequence (131 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MKLPVRLLVLMFWIPASSSDVVMTOSPLSLPVSLGDQASISCRTSOSLVHSNGNTYLHWHLOKPGQSPKVLIYKV
SSRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSONTHVPYTEGGGTKLEIK (SEQ ID NO:1101)
Mouse antibody 1884 light chain variable framework 1 (FWI) sequence:
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(DNA)
GATGTTGTGATGACCCAAAGTOCACTCTOCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGC (SEQ
ID NO:1102)
(amino acids)
DVVMTOSPLSLPVSLGDOASISC (SEQ ID NO:1103)
Mouse antibody 18B4 light chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
AGAACTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACAT (SEQ ID NO:1104)
(amino acids)
RTSQSLVHSNGNTYLH (SEQ ID N0:1105)
Mouse antibody 18B4 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGCACCTGCAGAAGCCAGGCCAGICTCCAAAGGTCCTGATCTAC (SEQ ID NO: 1106)
(amino acids)
WHLQKPGQSPKVLIY (SEQ ID NO:1107)
Mouse antibody 18B4 light chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
AAAGTTTCCAGCCGATTTTCT (SEQ ID NO:1108)
(amino acids)
KVSSRFS (SEQ ID NO:1109)
Mouse antibody 18B4 light chain variable framework 3 (FW3) sequence:
(DNA)
GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCGGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAG
GATCTGGGAGTTTATTTCTGC (SEQ ID NO:1110)
(amino acids)
GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO:1111)
Mouse antibody 18B4 light chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
TCTCAAAATACACATGTTCCGTACACG (SEC) ID NO:1112)
(amino acids)
SQNTHVPYT (SEC ID NO:1113)
Mouse antibody 1884 light chain variable framework 4 (FW4) sequence:
(DNA)
TTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID N0:1114)
(amino acids)
FGGGTKLEIK (SEQ ID NO:11:5)
Mouse Antibody 1E4 Heavy chain: DNA sequence (408 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGAATGGCCTTGTATCTTTCTOITCOTOCTGTCAGTAACTGAAGGTGTCCACTCCCAGGITCAGCTGCAGCAG
TCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATTTCCTGTAAGGCTTCTGGCTATGCATTCAGTACC
TACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTOTTGAGTGGATTGGACAGATTTATCCTGGAGATAGT
GATACTAACTACAATGGAAAGTTCAAGGGTAAAGCCACACTGACTGCAGACAAGTCCTCCAACACAGCCTACATG
CAGCTCAGCAGCCTAACATCTGAGGACTCTGCGGTCTTTTTCTGTGCAAGAGGTAACCACGCCTCTATGGACTAC
TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:1116)
Mouse Antibody 1E4 Heavy chain: Amino acid sequence (136 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MEWPCIFLELLSVTEGVHSQVQLQQSGAELVRPGSSVKISOKASGYAFSTYWMNWVKQRPGQGLEWIGQIYPGDS
DTNYNGKFKGKATLTADKSSNTAYMQLSSLTSEDSAVFECARGNHASMDYWGOGTSVTVSS (SEQ ID
NO:1117)
Mouse Antibody 1E4 heavy chain variable framework I (FWI) sequence:
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(DNA)
CAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGOGTCCTCAGTGAAGATTTOCTGIAAGGCTTCT
GGCTATGCATTCAGT (SEQ ID NO:1118)
(amino acids)
QVQLQQSGAELVRPGSSVKISCKASGYAFS (SEQ ID NO: 1119)
Mouse antibody 1E4 heavy chain variable complementarity determining regions
1 (CDR1) sequence:
(DNA)
ACCTACTGGATGAAC (SEQ ID NO:1200)
(amino acids)
TYWMN (SEQ ID NO:1201)
Mouse antibody 1E4 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGA (SEQ ID NO:1202)
(amino acids)
WVKQRPGQGLEWIG (SEQ ID NO:1203)
Mouse antibody 1E4 heavy chain variable complementarity determining regions
2 (C222) sequence:
(DNA)
CAGATTTATCCTGGAGATAGTGATAOTAAOTAOAATGGAAAGTTOAAGGGT (SEQ ID NO :1204)
(amino acids)
QTYPGDSDINYNGKYKG (SEQ ID NO:1205)
Mouse antibody 1E4 heavy chain variable framework 3 (FW3) sequence:
(DNA)
AAAGCCACACTGACTGCAGACAAGTCCTCCAACACAGCCTACATGCAGCTCAGCAGCCTAACATCTGAGGACTCT
GCGGTCTTTTTCTGTGCAAGA (SEQ ID NO:1206)
(amino acids)
KATLTADKSSNTAYMQLSSLTSEDSAVEECAR (SEQ ID NO:1207)
Mouse antibody 1E4 heavy chain variable complementarity determining regions
3 (CDR3) sequence:
(DNA)
GGTAACCACGCCTCTATGGACTAC (SEQ ID NO:1208)
(amino acids)
GNHASMDY (SEQ ID NO:1209)
Mouse antibody 1E4 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:1210)
(amino acids)
WGQGTSVTVSS (SEQ ID NO:1211)
Mouse Antibody 1E4 Light chain: DNA sequence (393 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTGTGATGACCCAA
ACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTEGTACAC
AGTAATGGAAACACCTATTTACATEGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTT
TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGC
AGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAAAACACATGTTCCGTGGACGTTCGGTGGAGGC
ACCAAGCTGGAAATCAAA (SEQ ID NO:1212)
Mouse Antibody 1E4 Light chain: Amino acid sequence (131 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FRS-CDR3-FR4
MKLPVRLLVLMFWIPASSSDVVMTOTPLSLPVSLGDIDASISCRSSOSLVHSNGNTYLHWYLOKPGQSPKLLIYKV
SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQKTHVPWTEGGGTKLEIK (SEQ ID NO:1213)
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Mouse antibody 1E4 light chain variable framcwork 1 (FW1) sequence:
(DNA)
GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGC (SEQ
ID NO:1214)
(amino acids)
DVVMTQTPLSLPVSLGDQASISC (SEQ ID NO:1215)
Mouse antibody 1E4 light chain variable complementarity determining regions
1 (CDR1) sequence:
(DNA)
AGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACAT (SEQ ID NO: 1216)
(amino acids)
RSSQSLVHSNGNIYLd (SEQ ID NO:1217)
Mouse antibody 1E4 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGTACCTGCAGAAGCCAGGCCAGECTCCAAAGCTCCTGATCTAC (SEQ ID NO :1218)
(amino acids)
WYLQKPGQSFKLLIY (SEQ ID NO:1219)
Mouse antibody 1E4 light chain variable complementarity determining regions
2 (C222) sequence:
(DNA)
AAAGTTTCCAACCGATTTTCT (SEQ ID NO:1220)
(amino acids)
KVSNRFS (SEQ ID NO:1221)
Mouse antibody 1E4 light chain variable framework 3 (FW3) sequence:
(DNA)
GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAG
GATCTGGGAGTTTATTTCTGC (SEQ ID NO:1222)
(amino acids)
GVPDRFSGSGSGTDRTLKISRVEAEDLGVYFC (SEQ ID NO:1223)
Mouse antibody 1E4 light chain variable complementarity determining regions
3 (CDR3) sequence:
(DNA)
TCTCAAAAAACACATGTTCCGTGGACG (SEQ ID NO:1224)
(amino acids)
SQKTHVPWT (SEQ ID NO:1225)
Mouse antibody 1E4 lighL chain variable framework 4 (FW4) sequence:
(DNA)
TICGGIGGAGGCACCAAGCTGGAAAICAAA (SEQ ID NO:1226)
(amino acids)
FGGGTKLEIK (SEQ ID NO:1227)
Mouse antibody 29H1 Heavy chain: DNA sequence (411 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-5R4
ATGTACTTGGGACTGAACTATGTAITCATAGTTTTTCTCTTAAATGGTGTCCAGAGTGAAGTGAAGCTIGAGGAG
TCTGGAGGAGGCTTGGTACAACCTGGAGGATCCATGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTEAGTGAC
GCCTGGATGGACTGGGTCCGCCAGECTCCAGAGAAGGGGCTTGAATGGGTTGCTGAAATTASAAGCAAAGCTACT
AATOAIGCAACATACTAIGCTGAG7CIGIGAAAGGGAGGTICACCATCICAAGAGAIGATIOCAAAAGTAGIGIC
TACCTGCAAATGAACAGCTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCCCCCTACTTTACGGGTTTGCT
TACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 1228)
Mouse antibody 29H1 Heavy chain: Amino acid sequence (137 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FRS-CDR3-FR4
MYLGLNYVFIVELLNGVOSEVKLEESGGGLVQPGGSMKLSCAASGFPFSDAWMDWVROSPEKGLEWVAEIRSKAT
NHATYYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCTPLLYGFAYWGQGTLVTVSA (SEQ ID
NO: 1229)
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Mouse antibody 29H1 heavy chain variable rramework 1 (FW1) sequence:
(DNA)
GAAGTGAAGOTTGAGGAGTOTGGAGGAGGCTTGGTACAACCTGGAGGATCCATGAAACTOTOTTGIGCTGCCICT
GGATICACTITTAGT (SEQ ID NO:1230)
(amino acids)
EVKLEESGGGLVQPGGSMKLSCAASGETES (SEQ ID NO:1231)
Mouse antibody 29111 heavy chain variabl3 complementarity determining
regions 1 (CDR1) sequence:
(DNA)
GACGCCTGGATGGAC (SEQ ID NO:1232)
(amino acids)
DAWMD (SEQ ID NO:1233)
Mouse antibody 29111 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGTCCGCCAGICTCCAGAGAAGGGGCTTGAATGGGITGCT (SEQ ID NO: 1234)
(amino acids)
WVRQSPEKGLEWVA (SEQ ID NO:1235)
Mouse antibody 29111 heavy chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
GAAATIAGAAGCAAAGCTACIAAICAIGCAACATACTAIGCTGAGICIGIGAAAGGG (SEQ ID NO: 1236)
(amino acids)
EIRSKATNNATYYAESVKG (SEQ ID NO:1237)
Mouse antibody 29H1 heavy chain variable framework 3 (FW3) sequence:
(DNA)
AGGITCACCATCTCAAGAGATGATECCAAAAGTAGTGICTACCTGCAAATGAACAGCTTAAGAGCTGAAGACACT
GGCATTTATTACTGTACCCCC (SEQ ID NO:1238)
(amino acids)
RFTISRDDSKSSVYL0MNSLRAEDEGIYYCIP (SEQ ID NO:1239)
Mouse antibody 29H1 heavy chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
CTACTITACGGGITTGCTTAC (SEQ ID NO:1240)
(amino acids)
LLYGFAY (SEQ ID NO:1241)
Mouse antibody 29H1 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGCCAAGGGACTCTGGICACTGICTOTGCA (SEQ ID NO:1242)
(amino acids)
WGOGILVIVSA (3E0 ID NO:1243)
Mouse antibody 29H1 Light chain: DNA sequence (393 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
AIGAAGITGCCTGITAGGCIGTIGGIGCTGATGIICTGGATICCIGCTICCAGCAGIGAIGTIGIGAIGACCCAA
ACTCCACTCTCCCTGCCIGICAGICTIGGAGATCAAGCCTCCATCTCTIGCAGATCTGGICAGAGCCTEGTACAC
AGTAATGGACACACCIATTTACATEGGTACCIGCAGAAGCCAGGCCAGICTCCAAGGCTCCTGATCTACAAAGTT
TCCAACCGATTTICTGGGGICCCAGACAGGITCAGIGGCAGTGGATCAAGGGCAGATTICACACTCAAGATCAGC
AGAGTGGAGGCTGAGGATCTGGGAGITTATTICTGCTOTCAAACTACACATGITCCGTGGACGTTOGGTGGAGGC
ACCAAGCTGGAAATCAAA (8E0 ID NO:1244)
Mouse antibody 29H1 Light chain: Amino acid sequence (131 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FRS-CDR3-FR4
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MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDOASISCRSGQSLVHSNGHTYLHWYLQKPCQSPRLLIYKV
SNRESGVPDRESGSGSRADEILKISRVEAEDLGVYFCSQ=VPWIEGGGIKEEIK (SEQ ID NO:1245)
Mouse antibody 29H1 light chain variable framework 1 (FW1) sequence:
(DNA)
GAIGTIGIGATGACCCAAACTCCACTCTCCCTGCCIGICAGICTIGGAGATCAAGCCTCCA1CICTIGC (SEQ
ID NO:1246)
(amino acids)
DVVMTQTPLSLPVSLGDQASISC (SEQ ID NO:1247)
Mouse antibody 29H1 light chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
AGATCTGGTCAGAGCCTTGTACACAGTAATGGACACACCTATTTACAT (SEQ ID NO: 1248)
(amino acids)
RSGQSLVHSNGHTYLH (SEQ ID NO:1249)
Mouse antibody 29111 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGTACCTGCAGAAGCCAGGCCAG7CTCCAAGGCTCCTGATCTAC (SEQ ID NO: 1250)
(amino acids)
WYLOKRGOSPRLLIY (SEQ ID NO:1251)
Mouse antibody 29H1 light chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
AAAGTTTCCAACCGATTTTCT (SEQ ID NO:1252)
(amino acids)
KVSNRFS (SEQ ID NO:1253)
Mouse antibody 29111 light chain variable framework 3 (FW3) sequence:
(DNA)
GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAAGGGCAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAG
GATCTGGGAGTTTATTTCTGC (SEQ ID NO:1254)
(amino acids)
GVPDRFSGSGSRADFTLKISRVEAEDLGVYFC (SEQ ID NO:1255)
Mouse antibody 29H1 light chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
TCTCAAACTACACATGTTCCGTGGACG (SEQ ID NO:1256)
(amino acids)
SOTTHVPWT (SEQ ID NO:1257)
Mouse antibody 29H1 light chain variable framework 4 (FW4) sequence:
(DNA)
TTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:1258)
(amino acids)
FGGGTKLEIK (SEQ ID NO:1259)
Mouse antibody 31A1 Heavy chain: DNA sequence (399 bp)
272
CA 03187555 2023- 1- 27
WC) 2022/027039
PCT/US2021/071017
Signal acquonco-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGAAAGGCACTGGATCTITCTCiTCCTGITTICAGTAACTGCAGGIGTCCACTCCCAGGTCCAGC=CAGCAG
TCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGC
TACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCACTGGT
TATACTGAGTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATG
CAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGCCTACATTGACTACTGGGGCCAA
GGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:1260)
Mouse antibody 31A1 Heavy chain: Amino acid sequence (133 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MERHWIFLFLFSVTAGVHSQVQLQQSGAELAKPGASVKMSCKASGYEFTSYWMAWVKQRPGQGLEWIGYINPSTG
YTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAYIDYWGQGTTLIVSS (SEQ IC NO: 26)
Mouse antibody 31A1 heavy chain variable framework 1 (FW1) sequence:
(DNA)
CAGGTCCAGCTTCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGAAGATGTOCTGCAAGGCTTCT
GGCTACACCTTTACT (SEQ ID NO:1262)
(amino acids)
QVQLQQSGAELAKPGASVKMSCKASGYTFT (SEQ ID NO:1263)
Mouse antibody 31A1 heavy chain variable complomontarity dctormining
regions 1 (CDR1) sequence:
(DNA)
AGCTACTGGATGCAC (SEQ ID NO:1264)
(amino acids)
SYWMH (SEC ID NO:1265)
Mouse antibody 31A1 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGA (SEQ ID NO:1266)
(amino acids)
WVKQRPGQGLEWIG (SEQ ID NO:1267)
Mouse antibody 31A1 heavy chain variable complcmcntarity dctormining
regions 2 (CDR2) sequence:
(DNA)
TACATTAATCCTAGCACTGGTTATACTGAGTACAATCAGAAGTTCAAGGAC (SEQ ID NO :1268)
(amino acids)
YINFSTGYTEYNQKFHD (SEQ ID NO:1269)
Mouse antibody 31A1 heavy chain variable framework 3 (FW3) sequence:
(DNA)
AAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCT
GCAGTCTATTACTGTGCAAGA (3E0 ID NO:1270)
(amino acids)
KATLTADKSSSTAYMQLSSLTSEDSAVYYCAR (SEQ ID N0:1271)
Mouse antibody 31A1 heavy chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
GCCTACATTGACTAC (SEQ ID NO:1272)
(amino acids)
AYIDY (SEQ ID NO:1273)
273
CA 03187555 2023- 1- 27
WC) 202/1127039
PCT/US2021/071017
Mouse antibody 31A1 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:1274)
(amino acids)
WGQGTTLTVSS (SEQ ID NO:l275)
Mouse antibody 31A1 Light chain: DNA sequence (393 bp)
Signal sequence-FR1-CDR1-7R2-CDR2-7R3-CDR3-7R4
ATGAAGITGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGITTTGATGACCCAA
ACTCCACTCTCCCTGCCTGICAGICTIGGAGATCAAGCCTCCTTCTCTIGCAGATCTAGICAGAGCAT7GTACAT
AGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGICTCCAAAGCTCCTGATCTACAAAGTT
TCCAACCGATTTICTGGGGICCCAGACAGGITCAGIGGCAGTGGATCAGGGACAGATTICACACTCAAGATCAAC
AGAGTGGAGGCTGAGGATCTGGGAGITTATTACTGCTITCAAGTTICACATTITCCGTGGACGTICGGTGGAGGC
ACCAAGOTGGAAATCAAA (SEQ ID NO:1276)
Mouse antibody 31A1 Light chain: Amino acid sequence (131 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MKLPVRLLVLMFWIRASSSDVLMTQTPLSLPVSLGDQASFSCRSSQSIVHSNGNTYLEWYLQKPCQSPICLLIYKV
SNRFSGVFDRFSGSGSGTDFILKINRVEAEDLGVYYCFQVSHFPWITGGGTKLEIK (SEQ ID NO: :277)
Mouse antibody 31A1 light chain variable framework 1 (FW1) sequence:
(DNA)
GAIGTITTGATGACCCAAACTCCACTCTCCCTGCCIGICAGICTIGGAGATCAAGCCTCCTICICTIGC (SEQ
ID NO:1278)
(amino acids)
DVLMTOTPLSLPVSLGDOASFSC (SEC) ID N0:1279)
Mouse antibody 31A1 light chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
AGATCTAGTCAGAGCATTGTACATAGAAATGGAAACACCTATTTAGAA (SEQ ID NO: 1280)
(amino acids)
RSSQSIVNRNGNTYLE (SEQ ID NO:1281)
Mouse antibody 31A1 lighL chain variable framework 2 (FW2) sequence:
(DNA)
TGGTACCTGCAGAAACCAGGCCAG7CTCCAAAGCTCCTGATCTAC (SEQ ID NO: 1282)
(amino acids)
WYLQKPGQSPKLLIY (SEQ ID NO:1283)
Mouse antibody 31A1 light chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
AAAGTTTCCAACCGATTTTCT (SEQ ID NO:1284)
(amino acids)
KVSNRFS (SEQ ID NO:1285)
Mouse antibody 31A1 light chain variable framework 3 (FW3) sequence:
(DNA)
GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAACAGAGTGGAGGCTGAG
GATCTGGGAGTTTATTACTGO (SEQ ID N0:1296)
(amino acids)
274
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
OVPDRFSGSGSOIDTTLKINRVEAEDLGVYYC (SEQ ID NO:1287)
Mouse antibody 31A1 light chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
TTICAAGITICACATTITCCGTGGACG (SEQ ID NO:1288)
(amino acids)
FQVSHFPWT (SEQ ID NO:1289)
Mouse antibody 31A1 light chain variable framework 4 (FW4) sequence:
(DNA)
TICGGIGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:1290)
(amino acids)
FGGGTKLEIK (SEQ ID NO:1291)
Mouse antibody 32C1 Heavy chain: DNA sequence (411 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGTACTIGGGACTGAACTGIGTATICATAGITTITCTCTTAAAAGGIGICCAGAGTGAAGTGAAGCTEGAGGAG
TCTOGAGGAGGCTIGGTGCAATCTOGAGGATCCATGAAACTCTCCIGTOTTOCCTCTOGATTCACTITCAGTAAT
TACTGGATGAACTGGGICCGCCAGECTCCAGAGAAGGGGOTTGAGTGGGITGCTGAAATTASATTGAAATCTAAT
AATTATGCAATACATTATGCGGAGECTGIGAAGGGGAGGTICACCATCTCAAGAGATGATTOCAAAAGTAGIGIC
TACCIGCAAAIGAACAACTIAAGAGUTGAAGACACTGGCATTIATTACIGIACCAGGGICCCGGGACIGGAIGCT
TACTGGGGCCAAGGGACICIGGICACTGICTCTGCA (SEQ ID NO: 1292)
Mouse antibody 32C1 Heavy chain: Amino acid sequence (137 aa)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MYLGLNCVFIVELLKGVQSEVKLEESGGGLVQSGGSMKLSCVASGF1FSNYWMNWVRQSPEKGLEWVAEIRLKSN
NYAIHYAESVKGRFTISRDDSKSSVYLOMNNLRAEDIGIYYCTRVPGLDAYWGOGILVIVSA (SE0 ID
NO: 1293)
Mouse antibody 32C1 heavy chain variable framework 1 (FW1) sequence:
(DNA)
GAAGTGAAGCTIGAGGAGICIGGAGGAGGCTIGGIGCAATCTGGAGGATCCATGAAACTCTCCIGTGTEGCCICT
GGATICACTITCAGT (SEQ ID NO:1294)
(amino acids)
EVKLEESGGGLVQSGGSMKLSCVASGFIFS (SEQ ID NO:1295)
Mouse antibody 32C1 heavy chain variablo complcmcntarity determining
regions 1 (CDR1) sequence:
(DNA)
AATIACTGGAIGAAC (SEQ ID NO:1296)
(amino acids)
NYWMN (SEQ ID NO:1297)
Mouse antibody 32C1 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGTOCGCCAGICTOCAGAGAAGGGGCTIGAGIGGGITGCT (SEC) ID NO: 1298)
(amino acids)
WVRQSPEKGLEWVA (SEQ ID NO:1299)
Mouse antibody 32C1 heavy chain variable complementarity determining
regions 2 (CDR2) sequence:
(DNA)
GAAATTAGATTGAAATCTAATAATTATGCAATACATTATGCGGAGICIGTGAAGGGG (SEC ID NO: 1300)
275
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
(amino acids)
EIRLKSNNYAIHYAESVKG (SEQ ID NO:1301)
Mouse antibody 32C1 heavy chain variable framework 3 (FW3) sequence:
(DNA)
AGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGIGTCIACCTGCAAATGAACAAOTTAAGAGCTGAAGACACT
GGCATTTATTACTGTACCAGG (SEQ ID N0:1302)
(amino acids)
RPTISRDDSKSSVYLQMNNLRAEDTGIYYCTR (SEQ ID NO:1303)
Mouse antibody 32C1 heavy chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
GTCCCGGGACTGGATGCTTAC (SEQ ID NO:1304)
(amino acids)
VPGLDAY (SEQ ID NO:1305)
Mouse antibody 32C1 heavy chain variablc framcwork 4 (FW4) sequence:
(DNA)
TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO::306)
(amino acids)
WGQGTLVTVSA (SEQ ID NO:L307)
Mouse antibody 32C1 Light chain: DNA sequence (393 bp)
Signal seguence-FR1-CDR1-FR2-CDR2-FR3-CDR3-ER4
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTGTGATGACCCAA
ACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCT7GTACAC
AGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTT
TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGC
AGTGTGGAGGCTGAGGATCTGGGAGTTTATTICTGCTCTCAAATTACACATGTTCCGTACACGTTCGGAGGGGGG
ACCAATCTGGAAATAAAA (SEQ ID NO:1308)
Mouse antibody 32C1 Light chain: Amino acid sequence (131 aa)
Signal peptide-FR1-CDR1-7R2-CDRE-FR3-CDR3-7R4
MKLPVRLLVLMFWI2ASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKV
SNRFSGVPDRFSGSGSGTDFTLKISSVEAEDLGVYFCSQITHVPYTEGGGTNLEIK (SEQ ID NO: :309)
Mouse antibody 32C1 light chain variable framework 1 (FW1) sequence:
(DNA)
GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGC (SEQ
ID NO:1310)
(amino acids)
DVVMTQTPLSLPVSLGDQASISC (SEQ ID NO:1311)
Mouse antibody 32C1 light chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
AGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACAT (SEQ ID NO: 1312)
(amino acids)
RSSOSLVHSNGNTYLH (SEQ ID NO:1313)
Mouse antibody 32C1 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGTAOCTGCAGAAGCCAGGCCAGICTCCAAAGCTCCTGATCTAC (SEC ID NO: 1314)
(amino acids)
WYLQKPGQSPKLLIY (SEQ ID NO:1315)
276
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Mouse antibody 32C1 light chain variable complcmcntarity dctcrmining
Legions 2 (CDR2) sequence:
(DNA)
AAAGTTTCCAACCGATTTTCT (SEQ ID NO:1316)
(amino acids)
KVSNRFS (SEQ ID NO:1317)
Mouse antibody 32C1 light chain variable framework 3 (FW3) sequence:
(DNA)
GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGTGTGGAGGCTGAG
GATCTGGGAGTTTATTTCTGC (SEQ ID NO:1319)
(amino acids)
GVFDRFSGSGSGTDFTLKISSVEAEDLGVYFC (SEQ ID NO:1319)
Mouse antibody 32C1 light chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
TCTCAAATTACACATGTTCCGTACACG (SEQ ID NO:1320)
(amino acids)
SQITHVPYT (SEQ ID NO:1321)
Mouse antibody 32C1 light chain variablc framcwork 4 (FW4) sequence:
(DNA)
TTCGGAGGGGGGACCAATCTGGAAATAAAA (SEQ ID NO:1322)
(amino acids)
FGGGTNLEIK (SEQ ID NO:1323)
Mouse antibody 45C11 Heavy chain: DNA sequence (423 bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAAATGCAGCTGGGITATCTIClICCTGATGGCAGTGGITACAGGGGTCAATICAGAGGITCAGCTGCAGCAG
TCTGGGGCAGACCTTGTGAAGCCAGGGGCCTCAGTCAAGITGTCCTGCACAGCTTCTGGCTICAACATTAAAGAC
ACCTTTATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGT
AATACIAAATATGACCCGAAATTCCAGGGCAAGGCCACIATAACAGCAGACACATCCICCAACACAGCCTACCIG
CAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAAACCGTATGGTAACTACGGCTATTAC
TATGCTITGGACTACTGGGGICAAGGAACCTCAGTCACCGICTCCTCA (SEQ ID NO: 1324)
Mouse antibody 45C11 Heavy chain: Amino acid sequence (141 aa)
Signal peptide-FR1-CDR1-7R2-CDR2-FRS-CDR3-7R4
MKCSWVIFFLMAVVTGVNSEVQLQQSGADLVKFGASVKLSCTASGFNIKDT7MIIWVKQRFEQGLEWIGRIDFANG
NTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCAKPYGNYGYYYALDYWGQGTSVTVSS (SEQ ID
NO: 1325)
Mouse antibody 45C11 heavy chain variable framework 1 (FW1) sequence:
(DNA)
GAGGITCAGCTGCAGCAGICIGGGGCAGACCTIGTGAAGCCAGGGGCCTCAGICAAGITGICCTGCACAGCTICT
GGCTTCAACATTAAA (SEQ ID NO:1326)
(amino acids)
EVQLQQSGADLVKPGASVKLSCTASGFNIK (SEQ ID NO:1327)
Mouse antibody 45C11 heavy chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
GACACCITTAIGCAC (SEQ ID NO:1328)
(amino acids)
DTEMH (SEQ ID NO:1329)
Mouse antibody 45C11 heavy chain variable framework 2 (FW2) sequence:
(DNA)
TGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGA (SEQ ID NO:1330)
(amino acids)
WVKORPEOGLEWIG (SEQ ID NO:1331)
277
CA 03187555 2023- 1- 27
VVC1 202/(027039
PCT/US2021/071017
Mouse antibody 45C11 heavy chain variable complemenLariLy determining
regions 2 (CDR2) sequence:
(DNA)
AGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAATTCCAGGGC (SEQ ID NC: 1332)
(amino acids)
RIDPANGNTKYDPKYQG (SEQ ID NO:1333)
Mouse antibody 45C11 heavy chain variable framework 3 (FW3) sequence:
(DNA)
AAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACT
GCCGTCTATTACTGTGCTAAA (SEQ ID NO:1334)
(amino acids)
KATITADTSSNTAYLQLSSLTSEDTAVYYCAK (SEQ ID NO:1335)
Mouse antibody 45C11 heavy chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
CCCTATCCTAACTACGGCTATTACTATGCTTTCCACTAC (SEQ ID NO: 1336)
(amino acids)
PYGNYGYYYALDY (SEQ ID NO:1337)
Mouse antibody 45C11 heavy chain variable framework 4 (FW4) sequence:
(DNA)
TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO::338)
(amino acids)
WGQGTSVTVSS (SEQ ID NO:L339)
Mouse antibody 45C11 Light chain: DNA sequence (381 bp)
Signal sequence-FRi-CDR1-FR2-CDR2-FR3-CDR3-ER4
ATGAGGTTCCAGGTTCAGGTTCTGGGGCTCCTTCTGCTCTGGATATCAGGTGCCCAGTGTGATGTCCAGATAACC
CAGTOTOCATCTTATCTTGOTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAGC
AAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGSATCCACTTTGCAA
TOTGGAATTOCATCAAGGITCAGTGGCAGTGGATCTGGTACAGATTTCACTOTCACCATCAGTAGCCTGGAGCCT
GAAGATITTGCAATGTAITACTGTCAACAGCATAATGAAITCCCGTGGACGTICGGTGGAGGCACCAAGCTGGAA
ATCAAA (SEQ ID NO:1340)
Mouse antibody 45C11 Light chain: Amino acid sequence (127 aa)
Signal peptide-FR1-CDR1-7R2-CDR2-FR3-CDR3-7R4
MREQVQVLGLLLLWISGAQCDVQ=QSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKINKLLIYSGSILQ
SGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEFPWTEGGG7KLEIK (SEQ ID N0:1341)
Mouse antibody 45C11 light chain variable framework 1 (FW1) sequence:
(DNA)
GATGICCAGATAACCCAGICTCCATCTTATCITGCTGCALCICCTGGAGAAACCATIACTAITAATTGC (SEQ
ID NO:1342)
(amino acids)
DVQITQSPSYLAAS?GETITINC (SEQ ID NO:1313)
Mouse antibody 45C11 light chain variable complementarity determining
regions 1 (CDR1) sequence:
(DNA)
ACCGCAACTAAGAGCATTACCAAATATTTAGCC (SEQ ID NO: 1344)
(amino acids)
RASKSISKYLA (SEQ ID NO:1345)
Mouse antibody 45C11 light chain variable framework 2 (FW2) sequence:
(DNA)
TGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTAC (SEC ID NC: 1346)
(amino acids)
WYQEKPGKTNKLLIY (SEQ ID No:1347)
278
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Mouse antibody 45C11 light chain variablc complementarity determining
negions 2 (CDR2) sequence:
(DNA)
TCTGGATCCACTTTGCAATCT (SEQ ID NO:1348)
(amino acids)
SGSTLQS (SEQ ID NO:1349)
Mouse antibody 45C11 light chain variable framework 3 (FW3) sequence:
(DNA)
GGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAA
GATTTTGCAATGTATTACTGT (SEQ ID NO:1350)
(amino acids)
GIFSRFSGSGSGTD7TLTISSLEFEDFAMYYC (SEQ ID NO:1351)
Mouse antibody 45C11 light chain variable complementarity determining
regions 3 (CDR3) sequence:
(DNA)
CAACAGCATAATGAATTCCCGTGGACG (SEQ ID NO :1352)
(amino acids)
QQHNEFPWT (SEQ ID NO:1353)
Mouse antibody 45C11 light chain variable framework 4 (FW4) sequence:
(DNA)
TTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID N0:1354)
(amino acids)
FGGGTKLEIK (SEQ ID N0:1355)
Mouse antibody 5C6F3 Heavy chain sequence
Signal peptide-Fa1-CDR1-Fa2-CDR2-FR3-CDR3-FR4
(DNA)
Gaagtgatgctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctct
ggattcactttcagtacctatgccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcagcc
attagtaatggtggtggttacacctactatccagacagtctgaaggggcgattcaccatctccagagacaatgcc
aagaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccacgtattactgtgcaagacgttac
tatgatcactactttgactactggggccaaggcaccgctctcacggtctcctca (SEQ ID N0:1816)
(amino acids)
EVMLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVROTPEKRLEWVAATSNGGGYTYYPDSLKGRFTISRDNA
KNTLYLQMSSLRSEDTATYYCARRYYDITY7DYWGQGTALTVSS (SEQ ID NO: 1817)
Mouse 5C6F3 heavy chain variable framework 1 (FW1) sequence:
(DNA)
Gaagtgatgctggtggagtotgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctct
ggattcactttcagt (SEQ ID N0:1356)
(amino acids)
EVMLVESGGGLVKPGGSLKLSCAASGFTFS (SEQ ID NO:1357)
Mouse 506F3 heavy chain variable compiementarity determining regions 1
(CDR1) sequence:
(DNA)
acctatgccatgtct (SEQ ID NO:1358)
(amino acids)
TYAMS (SEQ ID NO:1359)
Mouse 5C6F3 heavy chain variable framework 2 (FW2) sequence:
(DNA)
tgggttcgccagactccggagaagaggctggagtgggtcgca (SEQ ID NO: 1360)
(amino acids)
WVROTPEKRLEWVA (SEQ ID No:1361)
279
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Mouse 5C6F3 heavy chain variablc complementarity dctermining regions 2
(CDR2) sequence:
(DNA)
gccattaqtaatggtggtggttacacctactatccagacaqtctgaagggq (SEQ ID NO:1362)
(amino acids)
AISNGGGYTYYPDSLKG (SEQ ID NO:1363)
Mouse 5C6F3 heavy chain variable framework 3 (FW3) sequence:
(DNA)
cgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtotgaggtotgaggacacg
gccacgtattactgtgcaaga (SEQ ID NO:1364)
(amino acids)
RFTISRDNAKNTLYLQMS3LRSED7ATYYCAR (SEQ ID N0:1365)
Mouse 5C6F3 heavy chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
cyLLacLaLgaLcacLacLLLgacLac (SEQ ID NO:1366)
(amino acids)
RYYDHYFDY (SEQ ID NO:1367)
Mouse 5C6F3 heavy chain variablc framework 4 (FW4) sequence:
(DNA)
tggggccaaggcaccgctctcacqqtctcctca (SEQ ID NO:1368)
(amino acids)
WGOGTALTVSS (SEQ ID NO:1369)
Mouse antibody 5C6F3 Light chain sequence
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
(DNA)
Gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatct
agtcagaccattgtacatagtaatggaaacacctatttagaatggtacctgcagaaaccaggccagtctccaaag
ctcctgatctacaaagtttccaaccgattttctqqggtcccagacaggttcagtggcagtqqatcagggacagat
ttcacactcaagatcagcagggtggaggctgaggatctgggagtttattactgcttcaagattcacatgttcct
ctcacgttcggtgctgggaccaagctggagctgaaa (SEQ ID NO: 1818)
(amino acids)
DVLMTQTFLSLFVSLGDOASISCRSSQTIVIISNGNTYLEWYLQKFGQSFKLLIYKVSNRESGVFDRFSGSGSGTD
FTLKISRVEAEDLGVYYCFQDSHVPLTEGAGIKLELK (SEQ ID NO:1819)
Mouse 5C6F3 light chain variablc framework 1 (FR1) sequence:
(DNA)
gatgttttgatgacccaaactccactctccctgcctgtcagtottggagatcaagcctccatctcttgc (SEQ
ID NO:1310)
(amino acids)
DVLMTQTDLSLDVSLGDQASISC (SEQ ID NO:1371)
Mouse 5C6F3 light chain variable complementarity determining regions 1
(CDR1) sequence:
(DNA)
agatotagtcagaccattgtacatagtaatggaaacacctatttagaa (SEC) ID N0:1372)
(amino acids)
RSSQTIVHSNGNTYLE (SEQ ID NO:1373)
Mouse 5C6F3 light chain variable framework 2 (FR2) sequence:
(DNA)
tggtacctgcagaaaccaggccagtctccaaagctcctgatctac (SEC ID No:1374)
(amino acids)
WYLQKPGQSPKLLIY (SEQ ID NO:1375)
280
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Mousc 5C6F3 light chain variablc complcmcntarity dctcrmining rcgions 2
(CDR2) sequence:
(DNA)
aaaqtttccaaccqattttct (SEQ ID NO:1376)
(amino acids)
KVSNRFS (SEQ ID NO:1377)
Mouse 5C6F3 light chain variable framework 3 (FR3) sequence:
(DNA)
ggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaaga7cagcagggtggaggctgag
gatctgggagtttattactgc (SEQ ID NO:1378)
(amino acids)
GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC (SEQ ID NO: 1379)
Mouse 5C6F3 light chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
ULUcaagal_l_cacaUgUl_ccUcUcacg (SEQ ID NO:1380)
(amino acids)
FQDSHVPLT (SEQ ID NO:138:)
Mouse 5C6F3 light chain variablc framcwork 4 (FR4) sequence:
(DNA)
ttcqqtqctqqqaccaaqctqqaqctqaaa (SEQ ID NO:1382)
(amino acids)
FGAGTKLELK (SEQ ID NO:1383)
mu5C6F3 scFv sequence
(DNA)
gaagtgatgctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctct
ggattcactttcagtacctatgccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcagcc
attagtaatggtggtggttacacctactatccagacagtctgaaggggcgattcaccatctccagagacaatgcc
aagaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccacgtattactgtgcaagacgttac
tatqatcactactttqactactqqqqccaaggcaccgctctcacqqtctcctcaqqtqqcqqaqqatctqqcqqa
ggtggaagcggcggaggcggatccgatgttttgatgacccaaactccactctcccr.gcctgtcagtcttggagat
caagcctccatctcttgcagatctagtcagaccattgtacatagtaatggaaacacctatttagaatggtacctg
cagaaaccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttc
agtggcagtggatcagggacagatttcacactcaagatcagcagggtggaggctgaggatctgggagtttattac
tgctttcaagattcacatgttcctctcacgttcggtgctgggaccaagctggagczgaaa (SEQ ID
NO: 1384)
(amino acids)
EVMLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVAAISNGGGYTYYPDSLKGRFTISRDNA
KNTLYLQMSSLRSEDTATYYCARRYYDHYFDYWGQGTALTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSLGD
QASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYY
CFQDSHVPLTFGAGTKLELK (SEQ ID NO:1385)
3C2B1
/Abuse antibody 3C2B1 Heavy chain stagnance
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
(DNA)
Gaagtgatgctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctct
ggaatcactttcagtacctataccatgtcgtgggttcgccagactccggagaagaggctggagtgggtcgcaacc
attagtactggtggtgataaaacctactattcagacagtgtgaagggtcgattcaccatctccagagacaatgcc
aagaacaacctgtacctccaaatgagcagtctgaggtctgaggacacggccttgtattactgtgcaaggggaacc
acggctatgtattactatgctatggactactggggtcaaggaacctcagtcaccgrxtcctca (SEQ ID
NO:1820)
(amino acids)
EVMLVESGGGLVKPGGSLKLSCAASGITFSTYTMSWVROTPEKRLEWVATISTGGDKTYYSDSVKGRFTISRDNA
KNNLYLQMSSLRSEDTALYYCARGTTAMYYYAMDYWGQGTSVTVSS (SEQ ID NO: 1821)
281
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Mousc 3C2D1 heavy chain variable framework 1 (FW1) scqucncc:
(DNA)
gaagtgatgctggtggagtetgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctct
qqaatcactttcaqt (SEQ ID NO:1386)
(amino acids)
EVMLVESGGGLVKPGGSLKLSCAASGITFS (SEQ ID NO:1387)
Mouse 302B1 heavy chain variable complementarity determining regions 1
(CDR1) scqucncc:
(DNA)
acctataccatgtcg (SEQ ID NO:1388)
(amino acids)
TYTMS (SEQ ID NO:1389)
Mouse 3C2B1 heavy chain variable framework 2 (FW2) sequence:
(DNA)
tgggttcgccagactccggagaagaggctggagtgggtcgca (SEQ ID NO:1390)
(amino acids)
WVRQTDEKRLEWVA (SEQ ID NO:1391)
Mouse 3C2B1 heavy chain variable complementarity determining regions 2
(CDR2) sequence:
(DNA)
accattaqtactqqtqqtqataaaacctactattcaqacaqtqtqaaqqqt (SEQ ID NO:1392)
(amino acids)
TISTGGDKTYYSDSVKG (SEC) ID NO:1393)
Mouse 3C2B1 heavy chain variable framework 3 (FW3) sequence:
(DNA)
cgattcaccatctccagagacaatgccaagaacaacctgtacctccaaatgagcagtctgaggtctgaggacacg
gccttgtattactgtgcaagg (SEC ID NO:1394)
(amino acids)
RFTISRDNAKNNLYLQMSSLRSEDTALYYCAR (SEQ ID NO:1395)
Mouse 3C231 heavy chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
ggaaccacggctahgtattactatgctahggactac (SEC) ID NO: 1396)
(amino acids)
GTTAMYYYAMDY (SEQ ID NO:L397)
Mouse 3C201 heavy chain variable framework 4 (FW4) sequence:
(DNA)
tggggtcaaggaacctcagtcaccgtctcctca (SEQ ID NO:1398)
(amino acids)
WGQGTSVTVSS (SEQ ID NO:1399)
Mouse antibody 3C2B1 Light chain sequence
Signal peptide-FR1-CDR1-FR2-CDR2-FRS-CDR3-FR4
(DNA)
gacattgtgctgacacagtctcctqcttccttagctqtatctctggggcagagggccaccatctcatgcagggcc
agcaaaagtatcagtacatotgactataattatattcactggtaccaacagaaaccaggacagccacccaaactc
ctcatctatCTTGCATCCAACCTAGAATCTgggtccctgccaggttcagtggcag::gggtctgggacagacttca
ccctcaacatccatcctgtggaggaagaagatgctgcaacctattactgtcagcacagtagggagcttcctctca
cgttcggtgctgggaccaagctggagctgaaa (SEQ ID NO:1822)
(amino acids)
DIVLTOSPASLAVSLGQRATISCRASKSISTSDYNYIHWYQQKPGOPPKLLIYLASNLESGVPARFSGSGSGTDF
TLNIHPVEEEDAATYYCQHSRELPLIFGAGTKLELK (SEQ ID NO: 1823)
Mouse 3C2B1 light chain variable framework 1 (FW1) sequence:
282
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(DNA)
gacal_l_gl_gcl_gacacagUcUccUgcLUccLUagcUgUaLcUcUggggcagagggccaccaLcUcaLgc (SEQ
ID NO:1400)
(amino acids)
DIVLTQSPASLAVSLGQRATISC (SEQ ID NO:1401)
Mouse 3C231 light chain variable complementarity determining regions 1
(CDR1) sequence:
(DNA)
agggccagcaaaagtatcagtacatctgactataattatattcac (SEQ ID NO:1402)
(amino acids)
RASKSISTSDYNYIS (SEQ ID NO:1403)
Mouse 3C231 light chain variable framework 2 (FW2) sequence:
(DNA)
tggtaccaacagaaaccaggacagccacccaaactcctcatctat (SEQ ID NO:1404)
(amino acids)
WYQQKPGQPPKLLIY (SEQ ID NO:1405)
Mouse 3C231 light chain variable complementarity determining regions 2
(CDR2) sequence:
(DNA)
CTTGCATCCAACCTAGAATCT (SEQ ID NO:1406)
(amino acids)
LASNLES (SEQ ID NO:1407)
Mouse 3C231 light chain variable framework 3 (FW3) sequence:
(DNA)
gggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaagaag
atgctgcaacctattactgt (SEQ ID NO:1408)
(amino acids)
GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO:1409)
Mouse 3C231 light chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
cagcacagtagggagcttcctctcacg (SEQ ID NO:1410)
(amino acids)
QIISRELPLT (SEQ ID NO:141:)
Mouse 3C231 light_ chain variable framework 4 (FW4) sequence:
(DNA)
ttcggtgctgggaccaagctggagctgaaa (SEQ ID NO:1412)
(amino acids)
FGAGTKLELK (SEQ ID NO:14:3)
Mouse 312 heavy chain variable framework 1 (FW1) sequence:
(DNA)
Caggcgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacttgcactgtctct
gggttttcattaacc (SEQ ID NO:1414)
(amino acids)
QAQLKESGPGLVAPSQSLSITCTVSGFSLT (SEQ ID NO: 1415)
Mouse 312 heavy chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
agctatggtgtacac (SEQ ID NO:1416)
(amino acids)
SYGVH (SEQ ID NO:1417)
Mouse 312 heavy chain variable framework 2 (FW2) sequence:
283
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(DNA)
LgggLLcgccagccLccaggaaagggLcLggagLggcLggga (SEQ ID NO:1418)
(amino acids)
WVRQPPGKGLEWLG (SEQ ID N0:1419)
Mouse 312 heavy chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
gtaatatggcctggtggaagcacaaattataattcgactctcatgtccagaatg (SEQ ID NO: 1420)
(amino acids)
VIWPGGSTNYNSTLMSRM (SEQ ID NO:1421)
Mouse 312 heavy chain variable framework 3 (FW3) sequence:
(DNA)
cggatcatcaaagacaactccaagagccaagttttottaaaaatgaacagtotgcaaattgatgacacagccatg
tactactgtgccaga (SEQ ID NO:1422)
(amino acids)
RIIKDNSKSQVFLKMNSLQIDDIAMYYCAR (SEQ ID NO:1423)
Mouse 312 heavy chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
gatcggacacctcgggtgggggcctggtttgcttac (SEQ ID NO:1424)
(amino acids)
DRTPRVGAWFAY (SEQ ID NO::425)
Mouse 312 heavy chain variable framework 4 (FW4) sequence:
(DNA)
tggggccaagggactctggtcactgtctctgcag (SEQ ID NO:L426)
(amino acids)
WGOGILVTVSA (SEQ ID NO:1427)
Mouse 312 light chain variable framework 1 (FR1) sequence:
(DNA)
atcattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatatcctgc (SEQ
ID NO:1428)
(amino acids)
IIVLTOSPASLAVSLGQRATISC (SEC ID NO:1429)
Mouse 312 light chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
agagccagtgagagtgttgctacttatggcaataattttatgcag (SEQ ID NO:1430)
(amino acids)
RASESVATYGNNFMQ (SEQ ID NO:1431)
Mouse E2 heavy chain variable framework 1 (FW1) sequence:
(DNA)
gaagtggtgctggtggagtctgggggaggcttagtggagcctggagggtccctgaaactctcctgtgtagcctct
ggattcgctttcagt (SEQ ID NO:1432)
(amino acids)
EVVLVESGGGLVEPGGSLKLSCVASGFAFS (SEQ ID NO:1433)
Mouse 32 heavy chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
acctttgccatgtct (SEQ ID NO:1434)
(amino acids)
TEAMS (SEQ ID NO:1435)
Mouse 32 heavy chain variable framework 2 (FW2) sequence:
284
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(DNA)
LgyaLLcqccaqacLccggagaaqaqqcLggagLqggLcgca (SEQ ID NO:1436)
(amino acids)
WIRQTPEKRLEWVA (SEQ ID NO:1437)
Mouse 32 heavy chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
qccattaqtaatggtggtggttacacctactatccaqacactctqaagqgg (SEQ ID NO:1438)
(amino acids)
AISNGGGYTYYPDTLKG (SEQ ID NO:1439)
Mouse 32 heavy chain variable framework 3 (FW3) sequence:
(DNA)
cgattctccatctccagagacaatgccaagaataccctgtacctgcaaatgagtagtotgaggtotgaggacacg
gccgtgtattactgtgcaaga (SEQ ID NO:1440)
(amino acids)
RFSISRDNAKNTLYLQMSSLRSED7AVYYCAR (SEQ ID NO:1441)
Mouse 32 heavy chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
cgctactatgatctctactttgactta (SEQ ID NO:1442)
(amino acids)
RYYDLYFDL (SEQ ID NO:1443)
Mouse 32 heavy chain variable framework 4 (FW4) sequence:
(DNA)
Tggqgccgaggcacctctctcatagtctcctca (SEQ ID NO:L444)
(amino acids)
WGRGTSLIVSS (SEQ ID NO:1445)
Mouse 32 light chain variable framework 1 (FR1) sequence:
(DNA)
gatattctgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatttcttgc (SEQ
ID NO:1446)
(amino acids)
DILMTOTPLSLPVSLGDQASISC (SEC ID NO:1447)
Mouse 32 light chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
agatctagtcagaacattgtacatagtaatggaaacacctatttagaa (SEQ ID NO:1448)
(amino acids)
RSSQNIVHSNGNTYLE (SEQ ID N0:1449)
Mouse 82 light chain variable framework 2 (FR2) sequence:
(DNA)
tggtacctgcagaaaccaggccagtctccaaagctcctgatctac (SEQ ID NO:1450)
(amino acids)
WYLQKPGQSPKLLIY (SEQ ID NO:1451)
Mouse 32 light chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
aaagtttccaaccgattttct (SEQ ID NO:1452)
(amino acids)
KVSNRFS (SEQ ID NO:1453)
Mouse 82 light chain variable framework 3 (FR3) sequence:
(DNA)
285
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ggggtccccgacaggttcagtggtagtgggtcagggacagatttcacactcaagacagcagagtggaggctgag
gaLcLgggagLA.LaLLacLgc (SEQ ID N0:1454)
(amino acids)
GVPDRFSGSGSGTD7TLKISRVEAEDLGVYYC (SEQ ID NO:1455)
Mouse 82 light chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
tttcaagattcacatgttcctctcacg (SEQ ID NO:1456)
(amino acids)
FQDSBVPLT (SEQ ID NO:1457)
Mouse 82 light chain variable framework 4 (FR4) sequence:
(DNA)
ttcggtgctgggaccaggctggagctgaaa (SEQ ID NO: 1458)
(amino acids)
FGAGTRLELK (SEQ ID N0:1459)
Mouse B7 heavy chain variable framework 1 (FW1) sequence:
(DNA)
gaggtgcaggtggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctct
ggattcactttcagt (SEQ ID NO:1460)
(amino acids)
EVQVVESGGDLVKPGGSLKLSCAASGFTFS (SEQ ID NO:1461)
Mouse 87 heavy chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
agatatggcatgtct (SEQ ID NO:1462)
(amino acids)
RYGMS (SEQ ID NO:1463)
Mouse 07 heavy chain variable framework 2 (FW2) sequence:
(DNA)
tgggttcgccagactccagacaagaggctggagtgggtcgca (SEQ ID NO:1464)
(amino acids)
WVRQTPDKRLEWVA (SEQ ID NO:1465)
Mouse 87 heavy chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
accattagtagtggtggtacttacatctactatccagacagtgtgaagggg (SEQ ID NO:1466)
(amino acids)
TISSGGTYIYYPDSVKG (SEQ ID NO:1467)
Mouse 07 heavy chain variable framework 3 (FW3) sequence:
(DNA)
cgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacaca
gccatgtattactgtgcaagg (SEQ ID N0:1468)
(amino acids)
RFTISRDNAKNTLYLQMSSLKSEDTAMYYCAR (SEQ ID NO:1469)
Mouse 87 heavy chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
gataactacggtagtagctacgactatgctatggactac (SEQ ID NO:1470)
(amino acids)
DNYGSSYDYAMLY (SEQ ID NO:1471)
Mouse 07 heavy chain variable framework 4 (FW4) sequence:
(DNA)
286
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tqqqqtcaaqqaacctcaqtcaccqtctcctca (SEQ ID N0:1472)
(amino acids)
WGOGTSVTVSS (SEC) ID NO:1473)
Mouse 57 light chain variable framework 1 (F51) sequence:
(DNA)
gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgc (SEQ
ID NO:1474)
(amino acids)
DVLMTOTPLSLPVSLGDQASISC (SEQ ID NO:1475)
Mouse 37 light chain variable complementarity determining regions I (CDRI)
sequence:
(DNA)
agatctagtcagaccattgtacatagtaatggaaacacctatttagaa (SEQ ID NO:1476)
(amino acids)
RSSQTIVHSNGNTYLE (SEQ ID NO:1477)
Mouse B7 light chain variable framework 2 (FR2) sequence:
(DNA)
tggtacctgcaaaaaccaggccagtctccaaagctcctgatctac (SEC ID N0:1478)
(amino acids)
WYLQKPGQSPKLLIY (SEQ ID NO:1479)
Mouse 37 light chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
aaagtttccaaccgattttct (SEC ID NO:1480)
(amino acids)
KVSNRFS (SEQ ID NO:1481)
Mouse 57 light chain variable framework 3 (FR3) sequence:
(DNA)
ggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaaga7.cagcagggtggaggctgag
gatctgggagtttattactgc (SEQ ID NO:1482)
(amino acids)
GVPDRFSGSGSGTDTTLKISRVEAEDLGVYYC (SEQ ID NO:1483)
Mouse 37 light chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
tttcaagattcacatgttcctctcacg (SEQ ID NO:1484)
(amino acids)
FQDSHVELT (SEQ ID NO:1485)
Mouse 57 light chain variable framework 4 (FR4) sequence:
(DNA)
ttcggtgctgggaccaagctggagctgaaa (SEQ ID NO:1486)
(amino acids)
FGAGTKLELK (SEQ ID N0:1487)
Mouse 8C7F3 heavy chain variable framework 1 (FPV1) sequence:
(DNA)
gaagtgatgctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctct
ggattcactttcagt (SEQ ID NO:1488)
(amino acids)
EVMLVESGGGLVKPGGSLKLSCAASGFITS (SEQ ID NO:1489)
Mouse 8C7F3 heavy chain variable complementarity determining regions 1
(CDR1) sequence:
(DNA)
287
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acctatqccatqtct (SEQ ID NO:1490)
(amino acids)
TYAMS (SEQ ID NO:1491)
Mouse 8C7F3 heavy chain variable framework 2 (FW2) sequence:
(DNA)
tgggttcgccagactccggagaagaggctggagtgggtcgca (SEQ ID NO: 1492)
(amino acids)
WVRQTPEKRLEWVA (SEQ ID NO:1493)
Mouse 8C7F3 heavy chain variable complementarity determining regions 2
(CDR2) sequence:
(DNA)
gccattagtaatggtggtggttacacctactatccagacagtctgaagggg (SEQ ID NO:1494)
(amino acids)
AISNGGGYTYYPDSLKG (SEQ ID NO:1495)
Mouse 8C7F3 heavy chain variable framework 3 (FW3) sequence:
(DNA)
cgattcaccatctccagagacaatqccaagaacaccctqtacctqcaaatgagcagtctgaggtotqaggacacq
gccacgtattactgtgcaaga (SEC) ID NO:1496)
(amino acids)
RFTISRDNAKNTLYLQMSSLRSED7ATYYCAR (SEQ ID NO:1497)
Mouse 8C7F3 heavy chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
cgttactatgatcactactttgactac (SEC) ID NO:1498)
(amino acids)
RYYDHYFDY (SEQ ID NO:1499)
Mouse 8C7F3 heavy chain variable framework 4 (FW4) sequence:
(DNA)
tgcmgccaaqqcaccqctctcacqqtctcctca (SEQ ID NO:1500)
(amino acids)
WGQGTALTVSS (SEQ ID NO:1501)
Mouse 8C7F3 light chain variable framework 1 (FR1) sequence:
(DNA)
atcattgtgctgacccaatctccagcttotttggctotqtctctagggcagaqggccaccatatcctgc (SEQ
ID NO:1502)
(amino acids)
IIVLIQSPASLAVSLGQRATISO (SEQ ID NO:1503)
Mouse 8C7F3 light chain variable complementarity determining regions 1
(CDR1) sequence:
(DNA)
agagccagtgagagtgttgctacttatggcaataattttatgcag (SEQ IC NO: 1504)
(amino acids)
RASESVATYGNNFMQ (SEQ ID NO:1505)
Mouse 8C7F3 light chain variable framework 2 (FR2) sequence:
(DNA)
tggtatcagcagaaaccaggacagccacccaaactcctcatctat (SEQ ID NO:1506)
(amino acids)
WYQQKPGQPPKLLIY (SEQ ID NO:1507)
Mouse 8C7F3 light chain variable complementarity determining regions 2
(CDR2) sequence:
(DNA)
cLLgcaLccacccLagaLLcL (SEQ ID NO:1508)
(amino acids)
288
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LASTLDS (SEQ ID NO:1509)
Mouse 8C7F3 light chain variable framework 3 (FR3) sequence:
(DNA)
ggggt=ctgccagghtcagtggcagtgggtctaggaragacttcaccctcaccaTtgatcctgtggaggctgat
gatgctgcaacctattactgt (SEQ ID NO:1510)
(amino acids)
GVPARFSGSGSRIDETLTIDPVEADDAATYYC (SEQ ID NO; 15:1)
Mouse 8C7F3 light chain variable complementarity determining regions 3
(CDR3) sequence:
(DNA)
caqcaaaataatgagqatcctccqacq (SEQ ID N0:1512)
(amino acids)
QQNNEDPPT (SEQ ID NO:1513)
Mouse 8C7F3 lighL chain variable framework 4 (FR4) sequence:
(DNA)
ttcqqtqqaqqcaccaaqctqqaaatcaaq (SEQ ID NO: 1514)
(amino acids)
EGGGIKLEIK (SEQ ID N0:1515)
289
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Mouse Hll heavy chain variable framework 1 (FW1) sequence:
(DNA)
gaagtggtgctggtggagtotgggggaggcttagtggagoctggagggtccctgaaactctoctgtgtagcctct
gqattcgcttttagt (SEQ ID NO:1516)
(amino acids)
EVVLVESGGGLVEPGGSLKLSOVASGFAFS (SEQ ID NO: 1517)
Mousc H11 heavy chain variablc complcmcntarity dctcrmining rcgions 1 (CDR1)
sequence:
(DNA)
acctttgccatgtct (SEQ ID NO:1518)
(amino acids)
TEAMS (SEQ ID NO:1519)
Mouse 1111 heavy chain variable framework 2 (FW2) sequence:
(DNA)
LggaLLcgccagacLccggagaagaggcLggagLgggLcgca (SEQ ID NO:1520)
(amino acids)
WIRQTPEKRLEWVA (SEQ ID NO:1521)
Mouse 1111 heavy chain variabic complcmcntarity determining regions 2 (CDR2)
sequence:
(DNA)
gccattagtaatggtggtggttacacttactatccagacactctgaagggg (SEQ ID NO:1522)
(amino acids)
AISNGGGYTYYPDTLKG (SEQ ID NO:1523)
Mouse H11 heavy chain variable framework 3 (FW3) sequence:
(DNA)
cgattcaccatctccagagacaatgccaagaataccctgtacctgcaaatgagtagtctgaggtctgaggacacg
gccgtgtattactgtgcaaga (SEQ ID NO:1524)
(amino acids)
RFTISRDNAKNTLYLQMSSLRSED7AVYYCAR (SEQ ID NO:1525)
Mouse H11 heavy chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
cgctactatgatctctactttgactta (SEQ ID NO:1526)
(amino acids)
RYYDLYFDL (SEQ ID NO:1527)
Mouse H11 heavy chain variable framework 4 (FW4) sequence:
(DNA)
tggggccaaggcacctctctcatagtctcctca (SEQ ID NO:1528)
(amino acids)
WGQGTSLIVSS (SEQ ID NO:1529)
Mouse H11 light chain variable framework 1 (FR1) sequence:
(DNA)
gatattctgatqacccaaactccactctccctgcctqtcagtcttggagatcaagcctccatttcttgc (SEQ
ID NO:1530)
(amino acids)
DILMTOTPLSEPVSLGDQASISC (SEQ ID N0:1531)
Mouse H11 light chain variable complementarity deLermining regions 1 (CDR1)
sequence:
(DNA)
agatctagtcagaacattgtacatagtaatggaaacacctatttagaa (SEC ID NO:1532)
(amino acids)
RSSQNIVHSNGNTYLE (SEC ID NO:1533)
290
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Mouse H11 light_ chain variable rramewoLA 2 (FR2) sequence:
(DNA)
tggtacctgcagaaaccaggccagtctccaaagctcctgatctac (SEQ ID NO:1534)
(amino acids)
WYLQKPGQSPKLLIY (SEQ ID NO:1535)
Mouse H11 light chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
aaagtttccaaccgattttct (SEQ ID NO:1536)
(amino acids)
KVSNRFS (SEQ ID NO:1537)
Mouse H11 light chain variable framework 3 (FR3) sequence:
(DNA)
ggggtccccgacaggttcagtggtagtgggtcagggacagatttcacactcaagatcagcagagtggaggctgag
gaLcLgggagLLLaLLacLgc (SEQ ID NO:1538)
(amino acids)
GVPDRFSGSGSGTDFILKISRVEAEDLGVYYC (SEQ ID NO:1539)
Mouse H11 light chain variable complcmcntarity dctormining regions 3 (CDR3)
sequence:
(DNA)
tttcaagattcacatgttcctctcacg (SEQ ID NO:1540)
(amino acids)
FQDSHVPLT (SEQ ID NO:1541)
Mouse H11 light chain variable framework 4 (FR4) sequence:
(DNA)
tteggtgctgggaccaggctggagctgaaa (SEC) ID NO:1542)
(amino acids)
FGAGTRLELK (SEQ ID NO:1543)
Mouse 39 heavy chain variable framework 1 (FW1) sequence:
(DNA)
gaggtgcaggtggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctct
ggattcactttcagt (SEQ ID NO:1544)
(amino acids)
EVQVVESGGDLVKPGGSLKLSCAASGFIFS (SEQ ID NO:1545)
Mouse 39 heavy chain variable complcmcntarity determining regions 1 (CDR1)
sequence:
(DNA)
agatatggcatgtct (SEQ ID NO:1546)
(amino acids)
RYGMS (SEQ ID NO:1547)
Mouse 39 heavy chain variable framework 2 (FW2) sequence:
(DNA)
tgggttcgccagactccagacaagaggctggagtgggtcgca (SEQ ID NO: 1548)
(amino acids)
WVRQTPDKRLEWVA (SEQ ID NO:1549)
Mouse 39 heavy chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
accattagtagtggtggtacttacatctactatccagacagtgtgaagggg (SEC) ID NO:1550)
(amino acids)
TISSGGTYIYYPDSVKG (SEQ ID NO:1551)
291
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Mousc 39 hcavy chain variable framcwork 3 (FW3) scqucncc:
(DNA)
cgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacaca
qccatqtattactqtqca (SEQ =D NC:1552)
(amino acids)
RFTISRDNANNTLYLQMSSLKSEDTAMYYCAR (SEQ ID NO:1553)
Mouse 39 heavy chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
agggataactacggtagtagctacgactatgctatggactac (SEQ ID NO:1554)
(amino acids)
DNYGSSYDYAMDY (SEQ ID NO:1555)
Mouse 39 heavy chain variable framework 4 (FW4) sequence:
(DNA)
tggggtcaaggaacctcagtcaccgtctcctct (SEQ ID N0:1556)
(amino acids)
WGQGTSVTVSS (SEQ ID NO:1557)
Mouse 39 light chain variable framework 1 (FR1) sequence:
(DNA)
caaattqttctcacccaqtetccaqcaatcatqtctqcatetccaqgqqaqqaqqucaccctaacctqc (SEQ
ID NO:1558)
(amino acids)
OIVLTOSPAIMSASEGEEVTLIC (SEQ ID N0:1559)
Mouse 39 light chain variable complementarity determining regions 1 (CDR1)
sequence:
(DNA)
agtgccagctcaagtgtaagttacatgcac (SEC) ID NO:1560)
(amino acids)
SASSSVSYMII (SEC ID NO:1561)
Mouse 39 light chain variable framework 2 (FR2) sequence:
(DNA)
tggttccagcagaggccaggcacttctcccaaactctggatttat (SEQ ID NO:1562)
(amino acids)
TAIFQQRFGTSFKLWIY (SEQ ID NO:1563)
Mouse 39 lighL chain variable complemenLariLy deLermining regions 2 (CDR2)
sequence:
(DNA)
accacatccaacctggcttct (SEQ ID NO:1564)
(amino acids)
TTSNLAS (SEQ ID NO:1565)
Mouse 39 light chain variable framework 3 (FR3) sequence:
(DNA)
ggagtecctgctcgcttcagtggcagtggatctgggacctcttactctctcacaatcagccgaatggaggctgaa
gatgctgccacttattactgc (SEQ ID NO:1566)
(amino acids)
GVPARFSGSGSGTSYSLTISRMEAEDAATYYC (SEQ ID NO:1567)
Mouse 39 light chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
cagcaaaggagtaqttacccattc (SEC IC NO:1568)
(amino acids)
QQRSSYPF (SEQ ID NO:1569)
292
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Mousc 39 light chain varLablo framcwork 4 (FR4) scqucncc:
(DNA)
acgttcggctcggggacaaagttggaaataaaa (SEQ ID NO:1570)
(amino acids)
TFGSGTKLEIK (SEQ ID NO:1571)
mu3C2B1 scFNT sequence
(DNA)
Gaagtgatgctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctct
ggaatcactttcagtacctataccatgtcgtgggttcgccagactccggagaagaggctggagtgggtcgcaacc
attagtactggtggtgataaaacctactattcagacagtgtgaagggtcgattcaccatctccagagacaatgcc
aagaacaacctgtacctccaaatgagcagtctgaggtctgaggacacggccttgtattactgtgcaaggggaacc
acggctatgtattactatgctatggactactggggtcaaggaacctcagtcaccgrxtcctcaggtggcggagga
tctggcggaggtggaagcggcggaggcggatccgacattgtgctgacacagtctcctgcttccttagctgtatct
ctggggcagagggccaccatctcatgcagggccagcaaaagtatcagtacatctgactataattatattcactgg
taccaacagaaaccaggacagccacccaaactcctcatctatcttgcatccaaccagaatctggggtccctgcc
aggl_l_cagUggcagUgggLcUgggacagacULcacccUcaacaUccaUccUglggaggaagaagaUgcLgcaacc
tattactgtcagcacagtagggagcttcctctcacgttcggtgctgggaccaagcr.ggagctgaaa (SEQ ID
NO: 1572)
(amino acids)
EVMLVESGGGLVKPGGSLKLSCAASGITFSTYTMSWVRQTPEKRLEWVATISTGGDKTYYSDSVKGRF7ISRDNA
KNNLYLQMSSLRSEDTALYYCARG7TAMYYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVS
LGQRATISCRASKSISTSDYNYIHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAAT
YYCQHSRELPLTFGAGTKLELK (SEQ ID NO:1573)
mu20A10 scFli full sequence
(DNA)
gaagtgatgctggtggaatctggcggcggactggttaagcctggcggatctctgaagctgagctgtgccgccagc
ggcttcacctttagcacatacgccatgagctggatccggcagacccctgagaagagactggaatgggttgccagc
atcggcagagccggcagcacctactacagcgattctgtgaagggcagattcaccacagccgggacaacgtgcgg
aacatcctgtacctgcagatgagcagcctgcggagcgaggataccgccatgtactactgtgccagaggacccatc
tacaacgactacgacgagttcgcctattgqggccagggcacactggttacagtttctqctggtggcqqaqqatct
ggcggaggtggaagcggcggaggcggatccaatatcatgatgacacagagccccagcagcctggctgtgtctgct
ggcgagaaagtgaccatgtcctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcc
tggtatcagcagaagcccggccagtctcctaagctgctgatctactgggccagcaccagagaaagcggcgtgccc
gatagattcacaggcagcggcagcggaaccgacttcaccctgacaatcagctctg7_gcaggccgaagatctggcc
gtgtactattgccaccagtacctgtccagcctgacctttggcgccggaacaaagc7_ggaactgaag (SEQ ID
NO: 1574)
(amino acids)
EVMLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWIRQTPEKRLEWVASIGRAGSTYYSDSVKGRFT:SRDNVR
NILYLQMSSLRSEDTAMYYCARGP:YNDYDEFAYWGQGTLVTVSAGGGGSGGGGSGGGGSNIMMTQSPSSLAVSA
GEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLA
VYYCHQYLSSLTFGAGTKLELK (SEQ ID NO:15/5)
hu20A1OM scFV
(DNA
gaggtgcagctggttgaatctggcggcggacttgtgaagcctggcggatctctgagactgagctgtgccgccagc
ggcttcacctttagcacatacgccatgagctgggtccgacaggcccctggaaaaggccttgaatgggttgcctct
atcggcagagccggcagcacctactacagcgattctgtgaagggcagattcaccar.cagccgggacaacgccaag
aacagcctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgccagaggacccatc
tacaacgactacgacgagttcgcctattggggccagggcacactggtcacagtcagctctggcggtggcggaagc
ggaggcggtggctccggtggcggaggcagcgacatcgtgatgacacagagcccttctagcctggccgtgtctctg
ggagagagagccacaatcagctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcc
tqqtatcagcagaagcccqqacagtctcccaagctqctgatctactgggccagcaccagagaaagcggcgtqccc
gatagattcacaggctctggcageggcaccgacttcaccctgacaattagcagtc7.gcaggccgaggacgtggcc
gtgtactactgtcaccagtacctgagcagcctgacctttggcggcggaacaaagg7_ggaaatcaag (SEQ ID
NO: 1576)
(amino acids)
293
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASIGRAGSTYYSDSVKGRFT:SRDNAK
NSLYLQMNSLRAEDTAVYYCARGP:YNDYDEFAYWGQGTLVIVSSGGGGSGGGGSGGGGSDIVMTQSPSSLAVSL
GERATISCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVA
VYYCHQYLSSLTFGGGTKVEIK (SEQ ID N0:1577)
hu20A10MR scFV
(DNA
gaggl_gcagcUggl_l_gaaLcUggcggcggacLLgUgaagccUggcggaLcUclgagacUgagcUgUgccgccagc
ggcttcacctttagcacatacgccatgagctgggtccgacaggcccctggaaaaggccttgaatgggttgcctct
aUcggcagagccggcagcaccUacLacagcgaULcUgUgaagggcagaLUcacca_cagccgggacaacgccaag
aacagcctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgccagaggacccatc
tacaacgactacgacgagttcgcctattggggccagggcacactggtcacagtcagctctggcggtggcggaagc
ggaggcggtggctccggtggcggaggcagcgacatcgtgatgacacagagcccttctagcctggccgtgtctctg
ggagagagagccacaatcagctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcc
UggLaUcagcagaagcccggacagLcUcccaagcl_gcl_gaUcLacUgggccagcaccagagaaagcggcgUgccc
gatagattcacaggctctggcagcggcaccgacttcaccctgacaattagcagtcr.gcaggccgaggacgtggcc
gtgtactactgtcaccagtacctgagcagcctgacctttggcggcggaacaaagg7.ggaaatcaagaga(SEQ
ID NO:5001)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASIGRAGSTYYSDSVKGRFT:SRDNAK
NSLYLQMNSLRAEDTAVYYCARGP:YNDYDEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSLAVSL
GERATISCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFIGSGSGTDFILTISSLQAEDVA
VYYCHOYLSSLTFGGGTKVEIKR(SEQ ID NO: 5002)
hu20A10MRT scFV
(DNA
ciacicitcicacictcmttgaatctqcicgcfcciciacttgtgaacicctcmccigatctctgagactgagctcitq
ccgccacic
ggcttcacctttagcacatacgccatgagctgggtccgacaggcccctggaaaaggccttgaatgggttgcctct
atcggcagagccggcagcacctactacagcgattctgtgaagggcagattcaccacagccgggacaacgccaag
adcagccUgUaccUgcagaUgaacLcccUtgagagccgagyacaccyccgUgUacUaLUgUyccagagyacccaUc
tacaacgactacgacgagttcgcctattggggccagggcacactggtcacagtcagctctggcggtggcggaagc
ggaggcggtggctccggtggcggaggcagcgacatcgtgatgacacagagcccttctagcctggccgtgtctctg
ggagagagagccacaatcagctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcc
tggtatcagcagaagcccggacagtctcccaagctgctgatctactgggccagcaccagagaaagcggcgtgccc
gaLagaLUcacaggcUcUggcagcggcaccgacLLcacccUgacaaLLagcaglx_gcaggccgaggacgUggcc
gtgtactactgtcaccagtacctgagcagcctgacctttggcggcggaacaaaggr.ggaaatcaagagaacc(SE
Q ID NO:5003)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASIGRAGSTYYSDSVKGRFT:SRDNAK
NSLYLQMNSLRAEDTAVYYCARGP:YNDYDEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSLAVSL
GERATISCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVA
VYYCHQYLSSLTFGGGTKVEIKRT(SEQ ID NO: 5004)
hu20A10C2 scFV
(DNA)
gaggtgcagctggttgaatctggcggcggacttgtgaagcctggcggatctctgagactgagctgtgccgccagc
ggcttcacctttagcacatacgccatgagctgggtccgacaggcccctggaaaaggccttgaatgggttgcctct
atcggcagagccggcagcacctactacagcgattctgtgaagggcagattcaccar.cagccgggacaacgccaag
aacagcctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgccagaggacccatc
tacsacgactscgacgagttcgcctattggggccagggcacactggtcacagtcagctctggcggtggcggsagc
ggaggccigtggctccqqtggcqqaggcagcgacattgtqctgacccagtctccagcctccttcmccgtqtctcca
ggacagagggccaccatcacctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcc
tggtatcagcagaaaccaggacaacctcctaaactcctgatttactgggccagcaccagagaaagcggggtccca
gccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgr.ggaagctaatgatactgca
aattattactgtcaccagtacctgagcagcctgaccttcggcggagggaccaagg7ggagatcaaacga (SEQ
ID NO:1570)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASIGRAGSTYYSDSVKGRFT:SRDNAK
NSLYLOMNSLRAEDTAVYYCARGP:YNDYDEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTOSPASLAVSP
294
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
GQRATITCKSSQSVLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPARFSGSGSGTDFTLTINPVEANDTA
NYYCHQYLSSLTFGGGTKVEIKR (SEQ ID NO:1579)
hu20A10C2-R scFV
(DNA)
Gaggtgcagctggttgaatctggcggcggacttgtgaagcctggcggatctctgagactgagctgtgccgccagc
ggcttcacctttagcacatacgccatgagctgggtccgacaggcccctggaaaaggccttgaatgggttgcctct
atcggcagagccggcagcacctactacagcgattctgtgaagggcagattcaccar.cagccgggacaacgccaag
aacagcctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgccagaggacccatc
tacaacgactacgacgagttcgcctattggggccagggcacactggtcacagtcagctctggcggtggeggaagc
ggaggcggtggctccggtggcggaggcagcgacattgtgctgacccagtctccagcctccttggccgtgtctcca
ggacagagggccaccatcacctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcc
tggtatcagcagaaaccaggacaacctcctaaactcctgatttactgggccagcaccagagaaagcggggtccca
gccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgr.ggaagctaatgatactgca
aattattactgtcaccagtacctgagcagcctgaccttcggcggagggaccaagg7ggagatcaaa (SEQ ID
NO: 5005)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASIGRAGSTYYSDSVKGRFT:SRDNAK
NSLYLQMNSLRAEDTAVYYCARGP:YNDYDEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSP
GQRATITCKSSQSVLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPARFSGSGSGTDFTLTINPVEANDTA
NYYCHQYLSSLTFGGGTKVEIK(SEQ ID NO:5006)
hu20A10C2RT scFV
(DNA)
gaggtgcagctggttgaatctggcggcggacttgtgaagcctggcggatctctgagactgagctgtgccgccagc
ggcttcacctttagcacatacgccatgagctgggtccgacaggcccctggaaaaggccttgaatgggttgcctct
atcggcagagccggcagcacctactacagcgattctgtgaagggcagattcaccacagccgggacaacgccaag
aacagcctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgccagaggacccatc
tacaacgactacgacgagttcgcctattggggccagggcacactggtcacagtcagctctggcggtggcggaagc
ggaggcggtggctccggtggcggaggcagcgacattgtgctgacccagtctccagcctccttggccgtgtctcca
ggacagagggccaccatcacctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcc
tggtatcagcagaaaccaggacaacctcctaaactcctgatttactgggccagcaccagagaaagcggggtccca
gccaggttcagcggcagtgggtctgggaccgatttcaccctcacaattaatcctgggaagctaatgatactgca
aattattactgtcaccagtacctgagcagcctgaccttcggcggagggaccaaggr.ggagatcaaacgaacc(SE
Q ID NO:5007)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASIGRAGSTYYSDSVKGRFT:SRDNAK
NSLYLQMNSLRAEDTAVYYCARGP=YNDYDEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSP
GQRATITCKSSQSVLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPARFSGSGSGTDFTLTINPVEANDTA
NYYCHULSSLTFGGGTKVEIKRT(SEQ ID NO:5008)
hu20A10N scFV
(DNA)
caggtgcagctggttgaatctggcggcggacttgtgaagcctggcggatctctgagactgagctgtgccgccagc
ggcttcacctttagcacatacgccatgagctggatcagacaggcccctggcaaaggcctggaatgggtggcgtct
attggcagagccggcagcacctactacagcgactctgtgaagggcagattcaccar.cagccgggacaacgccaag
aacagcctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgccagaggacccatc
tacaacgactacgacgagttcgcctattggggccagggcacactggtcacagtttctagcggcggtggcggaagc
ggaggcggtggctccggtggcggaggcagcgaaattgtgctgacacagagccccgccacactgtcactttctcca
ggcgaaagagccacactgagctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcc
tggtatcagcagaagcccggccaagctcctcggctgctgatctattgggccagcacaagagagagcggcatccct
gccagattttctggcagcggctctggcaccgatttcaccctgaccataagcagccggaacctgaggacttcgcc
gtgtattactgccaccagtacctgagcagcctgacctttggcggaggcaccaaggggaaatcaagcgg (SEQ
ID NO:1580)
(amino acids)
QVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWIRQAPGKGLEWVASIGRAGSTYYSDSVKGRFT:SRDNAK
NSLYLQMNSLRAEDTAVYYCARGP:YNDYDEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSP
GERATLSCKSSOSVLYSSNOKNYLAWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSGTDFTLTISSLEPEDFA
VYYCHQYLSSLTFGGGTKVEIKR (SEQ ID NO:1581)
295
CA 03187555 2023- 1- 27
VVCO 2022/027039
PCT/US2021/071017
hu20A10N-R scFV
(DNA)
Caggtgcagctggttgaatctggcggcggacttgtgaagcctggcggatctctgagactgagctgtgccgccagc
ggcttcacctttagcacatacgccatgagctggatcagacaggcccctggcaaaggcctggaatgggtggcgtct
attggcagagccggcagcacctactacagcgactctgtgaagggcagattcacca7cagccgggacaacgccaag
aacagcctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgccagaggacccatc
tacaacgactacgacgagttcgcctattggggccagggcacactggtcacagtttctagcggcggtggcggaagc
ggaggcggtggctccggtggcggaggcagcgaaattgtgctgacacagagccccgccacactgtcactttctcca
ggcgaaagagccacactgagctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcc
tggtatcagcagaagcccggccaagctcctcggctgctgatctattgggccagcacaagagagagcggcatccct
gccagattttctggcagcggctctggcaccgatttcaccctgaccataagcagcc7_ggaacctgaggacttcgcc
gtgtattactgccaccagtacctgagcagcctgacctttggcggaggcaccaaggggaaatcaag (SEQ ID
NO: 5009)
(amino acids)
QVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWIRQAPGKGLEWVASIGRAGSTYYSDSVKGRFT:SRDNAK
NSLYLQMNSLRAEDTAVYYCARGP:YNDYDEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSP
GERATLSCKSSQSVLYSSNQKNYLAWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSGTDFTLTISSLEPEDFA
VYYCHQYLSSLTFGGGTKVEIK(SEQ ID NO: 5010)
hu20A10NRT scFV
(DNA)
Caggtgcagctggttgaatctggcggcggacttgtgaagcctggcggatctctgagactgagctgtgccgccagc
ggcttcacctttagcacatacgccatgagctggatcagacaggcccctggcaaaggcctggaatgggtggcgtct
attggcagagccggcagcacctactacagcgactctgtgaagggcagattcaccar.cagccgggacaacgccaag
aacagcctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgccagaggacccatc
tacaacgactacgacgagttcgcctattggggccagggcacactggtcacagtttctagcggcggtggcggaagc
ggaggcggtggctccggtggcggaggcagcgaaattgtgctgacacagagccccgccacactgtcactttctcca
ggcgaaagagccacactgagctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcc
tggtatcagcagaagcccggccaagctcctcggctgctgatctattgggccagcacaagagagagcggcatccct
gccagattttctggcagcggctctggcaccgatttcaccctgaccataagcagccr.ggaacctgaggacttcgcc
gtgtattactgccaccagtacctgagcagcctgacctttggcggaggcaccaaggr.ggaaatcaagcggacc
(SEQ ID NO:5011)
(amino acids)
QVQLVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWIRQAPGKGLEWVASIGRAGSTYYSDSVKGRFT:SRDNAK
NSLYLQMNSLRAEDTAVYYCARGP:YNDYDEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSP
GERATLSCKSSQSVLYSSNQKNYLAWYQQKPGQAPRLLIYWASTRESGIPARFSGSGSGTDFTLTISSLEPEDFA
VYYCHQYLSSLTFGGGTKVEIKRT(SEQ ID NO:5012)
hUMNE6+R marir
(DNA)
Gaggl_gcagcl_gql_qqaqUcUcmgclaggccUggl_caagccUggcmgclUccclgagacUcUccUgUgcagccUc
L
ggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaacc
attagtggcggaggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgcc
aagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataac
tatggccgcaactatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcggcggtggc
ggatccggcggtggcggatccggcggtggcggatccgaaattgtgttgacacagtctccagccaccctgtctttg
tctccaggggaaagagccaccctcacctgcagcgccaccagcagtgttagctacar.ccactggtaccaacagagg
cctggccagagccccaggctcctcatctatagcacctccaacctggccagcggcacccagccaggttcagtggc
agtgggtctgggagcgactacactctcaccatcagcagcctagagcctgaagatttgcagtttattactgtcag
cagcgtagcagctcccctttcacctttggcagcggcaccaaagtggaaattaaaaga (SEQ ID NO :5013)
(amino acids)
EVOLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGGGTYIYYPDSVKGRF7ISRDNA
KNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSL
SPGERATLTCSATSSVSYIHWYOORPGOSPRLLIYSTSNLASGIPARFSGSGSGSDYTLTISSLEPEDFAVYYCO
QRSSSPFTFGSGTKVEIKR (SEQ ID NO:5014)
huMNE6+RT serif
(DNA)
Gaggl_gcagcUggUggagUcUgggggaggccUggl_caagccUggggggUccclgagacUcUccUgUgcagccUcL
ggattcaccttcagtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtgggtctcaacc
296
CA 03187555 2023- 1- 27
LZ -Z0Z GSSL9t0 VO
L6Z
41111ooyollqobboteoL)booblIbllobebllobeyblowllebbobbwobeellqbblloybbubbobblioll
eybbqb
61361P616pP66o366Pooboo6oPooloblobiloo66136336loolobilooboor.616Poopiloo661P
(vNa)
zE-EET-V-S-I HYD-OTYOZnul
(OZOG:ON ai 02S) IA 999
NVIUNVEAdNIMUZaaIOSOSOSaUVdAOSEINSVIAIIIMddOOdMOOAMHNASAOSISASMSYUOMIMVUO0d
SATISVdSOIZAIOSO9DOSOODOSODOOSSAIALION9MACUAaAANOODMIIMAAVIOaVUZSNNOZAUNN
VNGUSI=3119MASOdAXIII9DSSIISAMTI9M9dliOUAMSWX5Sai3DSVVOSWIS95dMAZ959Sa/VIOAa
(sIDT0e ouTwe)
(610 ON
CI 02s)eeeole5e555beeooebbbebbob5oqqeoeoqqqoobqobebbbeqbeoeobeoqbweqqeq
qvve3fr43vqvblvvl3bevbfq.bloolvvqqvvovol000eollqvboovbbblole6blbeobbobvollUm
Dobepoolbbbboeebebbaoqeepoleobblopelllebloowepeloolopeepebbeopeeebeobeolel
bbloeobqeoeqooweqebbobeooeqbeoqbqbebeeqbeopbebeobwoeoqeooeoobbbebeoebbeoo
go.45.4boo.65.4.4pogoobyoogogbp000pbqobgEggpopboogybbobbqbbobboogybbobbqbbobboo
g
eb6o5.6q5bobboo4004o153oe345.6oeooebbbeeeobb564o454ebo4loe4ee5oeqoe44ee4e5555
bbbqqoebebobqtqoeqqeqbqboobboeoebbetoobebebqoobeoeebqeeeobqoqeqbqoeoqoeebee
opboeloolobPbeoololepolopilloboobbbPlobibeololob000pPloeleiPpelopelobbobbibeibe
lle
3ovvoqoqbbbqbe55-43555beebbbeoowbbeoobooqb55335e5qeoobqe-4355-45eoqqooeoqqe55
qoloobyobqbqpoqoqopb26qopoqbbbbbbqpobp2oqbbloobbybbbbbqoqbvbfrabbqobvpbqbbvb
(IMO)
Ados MU-ZONPVIR
(810G:ON (II 02G) UMIaAMM0903MadrIMIGHOOAA
MIONliaAdNILUZJIOLSSSSSSaUlidASSarINSWIAMMddOSdNOOAMONASASSISASMSYUDILIMUOSd
SAVISVdSOIZAIOS0000S0000SOODOSSAMALIONOMACUAZAANOODIUVOAAAVMOZVUISNNOIXISNX
VNGUSIZATIONASOdAXIAIOSSSIISAMTION0dVOUAMSWASSaIaOSVVOSWISOOdMAZOOOMVIOAS
(sIDT0e ouTwe)
(LTOG:ON CI
02s)
e5L)wwwoliebeffy_bbeeL)oubbbebb:A)ZLY4.4wL)eLY111111511L)bebbbellbeLbeL)11511:)
ellItell
qeeeobweqebqeewbee55.4.5.400qeeqqeeoeowooeoqqqebooe555.4o4E65.45eobbobeoqqbbe
pobypooqbbbbotybybecLogyypogyobbqopygggybqopqpypygooqopyypybbypoyvybypfmogyq
bbloeobqeoeqpoweqebbobeooeqbeoqbqbebeeqbeoobebeobwoeoqeooepobbbebeoebbeoo
wlblboobbllooloobvpololbv000vbloblb11232boolvbbobblbbobboolebbobblbbobbool
pbbobbqbbobboowpwaboopoqbboppowbbEeppobbbbloqbqwboqwwqwwbowwwqqwwqwbbbb
bffyll.oebebobqEweqqe.45.4.5oobboe3ebbeEoobe6ebwobe3eebqeee3bwqeqbqovoweebee
L):ffpoweL)ebebwoolloquoliwb:x.)bbbewbqtwoll)elielleL)ellwebbL)bblibelibelille
oovvoqoqbbbqbebbqobbbbeebbbeooqobbeoobooqbbbqobebqeoobqeqobbqbeoqqooeoqqebb
qoloobeablbloololovbebq000lbbbbbbloobveolbbloobbebbbbblolbe5515blobeoblbbeo
(IMO)
Aloe X-Emognm
(9105:ON alOES) IUMIaAMMOSOLLMSSSUO
00XXAVa(12,32USIMTIXOSOSOSOSaUVaIOSWINSISXITIUdSOOdU0OXMHIASAGSIVSOMTIVU20,3S
7S'LLVdS(IVIAI2S0000S00f)0S000f)SSAIATISOOMXaW0XaXNUOXNaU/DXXAVIZ2VUUNIAIMXTINM
YNGUSIZATIONASOdAAIAIDOOSIMSAM3ZUMOdVOUAMSNOAUSeLLIOSVVOSZUZSOOdMAZOOOSaMOA3
(sIDT0e ouTwe)
(GTOG:ON
CI 02s) 332Pfme221-422Pbbqfme23323bb3be3bbqqq33e3qqq3333q3fm3bvqb3b23
be3.1.5.4oeqqeqq152o5qqlebvebqoobebewobeofmoqvooeoqoqoeoewebobebbbw.4555.1.52
o55.1.52o4.4552oobe000eobbobeoobbwoevoogooeofmqvgogeogoogoe.fm0000befmoobbqoo
bbvbel)eyooellbtlloyoLY_Eq)ellobellqbqbeobvooyoobobeobllooyoll000eL)obebeyebbbb
yoolloll
blllolbwooepobeoololbeovoebqqbqbqleeebooqebbobblbbobboolebbobbqbbobboolebb
obblabobbobv3Z25163326-4.6613332obbbeopbE65-41211v6612365121125121322oboobb121
oPP1P6P6PooPiEloPile16-161066oPop66pEoo6P6P61336PoPP6ipPPobiolPibl000PoPPEPP
oobovvoybybyoogogyooyoggyboobbbyybgbyowyby000ygovqvgyoygooyobbybbobbgbyggy
LIOILOITZOZSIVIDd 6COLZO/ZZOZ OM
LZ -Z0Z GSSL9t0 VO
86Z
(vNa)
ze-8Z-8-M UV3-OTVOZnw
(g8g1:ON aiOn) **ImarIvONHavaxmaxmvzsrlsOxasalloNsulluasmwsiasx
vziewmamOrmixasnaN>maNe9wzdausuuNarlAaxmluDrIN=xrIONOsONxvdvaysusaNAurmose
H323d3U0S00(1220.11.0AdUNadOMaIATIMMUOUNOMIAUSTIZASOMOIndliMIXICOliaMOUMHAY00
VV.3110V2dIVIS'IdOSVIMdVaIdaldVdaLLUMISAMM900,11rISSZAOHOAANVICINVaAdNISTIACIOS
OSO
SaUYdAOSEUMSVMAIIIMdd00dMODAMVIANMONSSAIASOSSMOMIMYUDOdSAVISVdSOMIADIS0000S
DOODSOODOSSAIATI9ODMAVaaCIACINAIdOUVOAAAVIaaVUZSNNOZAUNNVMUSILDIOXASCISAAISO
VU9ISVAMTI9M9dInUAMSIANXISLIASSVVOSUUZS99dRAU595SZAZOAZdUliVIVITIVUdrIVIILIATIl
iN
(sIDT0e ouTwe)
('8;T ON ai 02s) 212-4126-
435o400000fy4000bfmobqwovoqq000boubovqoovol2551212oovoob
eoeqbeoqo-4555eooeqqoobbqeboeobbbbeeobbbbebboobobebobbeeebqe555-4-4e5e5-45eoeq
oobbvbbobblvemv1vbevvbeoblovvblevoelblooffmvffmol000vebvve6vebeboobvvvbbbbb
blpbabloppebbepobblboebebeepebb1111bleboelbebbpbabeeboebbelpleeplobeboeelel
oq3be3oeebeo3Ebbeobeeoeqbob00000boe5eobobebfmobeoqqbeebqbebebqoeeblbqebbebb
vvbwvbpvbvvfmooqqqvboob-435v-45-4o55-
4v5vvbfmbvvc)qopqovvvovqfmoopfmbqvqqqpoovvo
eeeo44e4e4e45goo4oeeebeeebeobbbboeeeo54oe44wooeo4e44554oeo464004o4wo45555
qbqqoybaboobbqqoppbobbbqogepegogeoetobqopboqqqybbqoebbebeopepeobgboobebbobb
lobloblooP6Pibilobbeb000bbithipobithioloobPoobepobliPPoPloopobepoopPloolooP6P
00000bw000eeoeeoeeboeeeoqebe55.455eeooebbbebbobboqqooefrwobeobebwoeqbeooeo
qblovqq2-4-4222obw2qpb-4221obvvbbqbwoqp2-11.p2owpwoovoq-
11.2boopbbbqoqbbbqb2obb
obvp-44e6epobeopp-46556:)beyebebeopeobeope654pe-444eb4o4eee4004opeepubbeopeee
be3be3ge45540055400e4oee6eebeooeeobeoo4oe45.4o545obebeoobeobebeeob400eo4eooe
pobbbvb2oebbeo31315-LboobbllooloobvoololbvpooebloblblleovbobPobbebbobblbbool
35535bobbebboEee55355-
465355.43.4obeo.45eoeoqbbloeoeobbbeo35555.4.4eq3353.4.45e5oe5
32132b32232.43123339bbebvoobqbqqwwvqbqbooboovovbbyboob2b2bwooq322b.42b2obqo
pe-
4641,z)beoeebeepz)5:Dee:Debbboobeogeopeoggebyobbbee5454oggebz)bepeqpeqpoeobeobb
oobybeobbogegogoo5q55bgeebgwobbeeeebbw000bbeoeboo-4555-4obebgeooboegeoeobe
qlloovollobboevoobooblblobvb.43262blololvbbobbloob226161132bbobbobblo1P2b1lb
b3be3bqbbebboobbeooboob3eooq35.435.4.43355qo5335.433.4o5.4.433533e5.45e33E-4-
43355.4e
(vNa)
(C8ST:ON aI Os) 44uddrivOwinvaxiaNamasas0ArmaHeNeuuusemweiss
70fEWDRIMMENA1020dNNUEdMSONEdaUOUUMMAGXEHUEOrlIVIENArIONOOOMXifElfaliSUSZNAW120
0
smaadallospeasaainAduwadOma:AriammusumomimsrmAsalevriavmixiapvaarisuamvs
9ViidUOV2dUrindOSVISAVaidaUdVdMMIN=MM9V9aMrISSZXOHOXXAVga2VOASSISTIASISS9S9
JAUCHAOSaUMSVMAIUMLISOOd>100AMVUANXONSSAUASOSSXOSNMAXaDVSAVUSSdSOMMINSOODOS
OSODSDeSeliSALNILSOSMAiidSGACMAIdOUliDAANIVLaaSUUSNMArlINUANGUSILDISMASCISAALSS
krUOISVAMZUUM2dMOUIMSNVAMSZIAOSVVOSUMZSOOdNAZOODSZAZNAZdUknalarriliadarriliMATI
liN
(sTov ouTum)
(zegi:oN ai 02s) peirbloboloopoobl00066P061PoPolloop63PboPloovot.66PPooP
oobeovqbeogogEftheooe-moobbgeboeo555522o6b5525boobobebobbee25-42555-4-42525-
452
oeqoobbebbobblebee4ebeeebeobweebqevoeqb400bbeebbeowooeebeebbeebeboobeeebb
bUbbqebebwooebbboobbaboeDebeeoebbqqqq5qeboeqUebbebebeeboebbew4eeowbeboee
qelolobeooeebeoobbbeobeeoelbob000005oebe3b3bebbeobeollbeeblbebebloyeblblebb
ebbeybP2bevbegbPooq-
Lqeboob.4362.45.4355.4P52266252v3.432.43e2232.452332625.42.4.4.4233
323 22333
b55-45-4-4oebbboo55-4wo3bo555-4oqeoeqoqt.o2b3bwoboqq-4255-
4oebbef2ooe3eobqboobebb
off)qablobwoeteqUqqobbeb000bbebwobebqowobeoobeoobqqeeoew000be0000ewowo
efm00000bl0000es.oevoebeebloeebblobeveoeebboobobblqwoebloobvooqblooelbeooeo
36132.432.45.4boobbq3qebeeb33bbP3bqb.43.43523qev3eb.433323.4.432b33Pebbobvobb3b
v3bb
232 322
523523-42-455-40055-400eqoeebeebeooeeobvooqoe-45.4o5-45obefmoobeof2beeobwo-45-
42ooe
bqbeyebybobbquelquilecLbllobbwobeobeb000bebeoy:)ebIlybIlyollellyeLny_lebbobbebb
obboby
ebblbbebbobblolebbebbobblbbloblolllbeoelqbbloeoeobbbeoobbbbllewobollbeboeb
32132.63223213.42333Pbbeb233blbl3el3elblv33b33elvbb2b3bebb3bl33b2362512623613
3216133123pe6636163pe3266633623123323112623666yR6161311#636e3p13p133pp6e366
oobvbvobboqvobvoobqbbbqvvbbqovbvbvvbvbw000vbvobbooqvbbqobvb-42oobovqvovobt,
LIOILOITZOZSIVIDd
6COLZO/ZZOZ OAA
LZ -Z0Z GSSL9t0 VO
66Z
XXI-ZE-SEET-17-8-I
(68GT:ON UI OSS) 44UddrIVONWPMADDLIVMS700A70aHOMOUUUSONNOISS
AvsviamamOrisNArIssOaNxHEam5swzdaHoHHmarulaxszHEoriNrisNArIONOsOmAvEvaysHsammsH
Av
VaaliddVAdOAHNUMd9allidIVINNAaSH7711SUNSUOATIINIS777A00MOVadVNIXIa0VaariOUMHAVO
0
VValOVSWISadOSViidVdidaidlid:IDINISANI999,117SS7AOHDAANViaNVaAdNiirliaai9S9S9
SaUlidAOSSUMSYMAI7751ddOOdMOOAMWIANNONSSAZASOSSMOMIMIPIOOdSAWISIfdSOM7ADISOODOS
9099SOODOSSAMATI9OOMAVa2GACINAIdOUV3AAAVDMVU7SNNOZA7SNMVNCUSIMallONASCISAAMSO
VUOISVAME70X0dVOUAMSNVAISSISOSVVOS7U7SOOdXA7000SENIOAEdUVVH777V7d777VMAd7VIAT
(epTov ouTum)
(88S1:ON aI Os) eelpblobolooppoblopobbeoblepeollopoboeboelopeoebbeepoe
oobvovqbeowqEbbeooeq-443355.4eboeobb5beeobbbbvbboobobebobbeeebqebbblqebebqbe
ouwo.6512.65o55-412512124125121212512obqousbquvo2-
4540055121255voq000w251225512125125oo512121255
6565-4y5ebq000ebbboobblboybebeeoe55-4-4-4-11.-
4eboeqbybbebebeeboebbeqogevoqobebove
qvlolobvoovvemoobbfmobevovlbob00000bovemobobebbvafmollfmver4bebeoolobolvloob
epboqwebobouppeoppobgewpabeopeggeobeeebeopegoobbbqopebeebegoogoebgepeebge
oewebqbeoeobloo.4355eobebbebeeqbebbeobweqql000eoqeqqbbweoqbwowlwoqbbbb
qbqqov55.600bbqq000bobbbqoqvopqoqvov5obwoboqqqQ55-4ovbbvfmoovovob-45c)obpbbobb
g3bq3bwoebe4E44obbeb0006bebwobe54p400beoo6yoo544eeoew0005e0000ewowoebe
opoopbqopopeepeepeeboeeeogebebbqbbeuppebbbebbobboggopebqopbeobebqopeqbeopeo
ibioPilPiiPPPobioPiebiePiobPPbbibiooiPPiiePopoipooPpyymbooPbbbioibbbibPobb
obv3.4.455e3obe000.45555obevebebeooeobeoobbfrwelqqe5woweeewowoeeovbbeooeee
byobvoqvqbbwobbqoppw226v2.6voovvobvpoqopqblobqba62b2pobvpbpbvpobwovoqvoo2
pobbbebeoebbeop4o45-Lbz)obbqqopqopbepogo4beopowbqp64644epebobeobbebbobbqbboo4
o5645.6obbebbobeebbo554553554o4obeo4beoeo4b5weoeobbbeoo556544ewobo445eboeb
321o2.53223213.42333Pbbebvooblbllewelblboobooeovbb2boob2b2bl000w2261262oblo
pelfrwobeoeebeepoboeeoebbboobeoqeopeoqqebeobbbve5.45.43.4.4e535epeqoewoeobeo55
pobvbvobboqwwwobq-Lbbbqvvbqwobbyvvvbbw000bbvoybooqbbbqobvb.4233bovqvovobv
44400sQ-44obboZepz)5:Dp5-464:Dbeb4oebefy4o4:D4ebboffy400bee5-45-44pebbp55D55-
4o4ee54-45
bqobeo5-4.65ebboo5beooboobovoogobwbqqoob54oboobwogobqqoobooebqbeooyqqoobbge
(VNa)
EIM-OTINZnq
(L9cI:ON GI Os) ,,,uciarivOwNrivaxiaNzvisrisOArisatioNsuuuseNweis
SAvavwxaxOriaNA70sOdmiuudNOONadauOuuxarinaxasuu07NriaNA7ON000xxvdvavSuSaxAuSux
iliaalladNadOAHNUMd9a1UZINNWACISHVIUSUMSUOMINIS777A90.19V7dVMIXICIDVaarMUMHAV9
OVFMEOVEdErlSqd0SVIIAIMIAdUdVd.LIIM7H7M.101f0,117SS7X0HOXXAWMEInASSIZTIZCLIOSOS
O
MalladADSEUMSVMAITIIMSOOdNOOAMV7ANNONSSA7ASOSSNOSNMANSOVSAVaSSdSOMWNINSOODOS
sesesessevsAmATIDOsiuvasaxaNxiasuvoxxviviassurisswOrumNuANausimausNAsasxxiss
VEDISVAMSZEXadMOUIMSKVAMS,1130SVVOS7X7S90dX117000SSAZNASdUVVH777V7d777VMAdrIVN
(slype ouple)
(98ST:ON SI Os) vegebwbow0000bwoobbeob4eoeo4wooboeboewoeoebbee
oovoofmovq6vowlbbfmooellloobbleboeobbbbeeobbffmaboobobvEobbeveblvbbblqvbvb
16roP10066P66o661P6eP1e6rPP6P0610PPE1PP0P1610066PP66P010ooPP6PP66Pe6P6006PP
25555.65-42525wooebbboobbgboebebeeoe55-44-44bgeboy-
4525525ebeeboebbegogeeogobeb
oev42404obeoo2e13eoo1313beofmeoelbob0000013oe13eo13o13e13beo13eo44522545ebeoowb
o424
ooUvaboawebof:oeooe0000bqewoobeooe33eobeee6vooewo5553opebeebewowebaeoee
blvoeweblbeoeobloo-Lobbeobebbebeelbebbeobwellwooeolellbbloeoqbloololloolb
bb636.4.432bbboobbqqopobobbb.43.423eqoqvoP535.1.3353.4.4.4266.4326626Poo23235.4
6336266
obbwbgobwoeEeqbqlobbeb000bbebwo525-4owobeoobeoobqqeeoew000fm0000ewo-wo
efm00000bw000eeoeeoebeebweebbqobeveoeebboobobb-mooebwobvoo4bwoeqbeooeo
of).43e3oe35353o553olebeeboobbeo535333obeoleeovbwooeo3webooeebbobeobbobeoff)
eovollebeleb000blbobbobevebefmooeobvoobbbwelolebloblobeelooloqbeoobb000bee
623523.42.456.43obbqooe.43226P2bPooevobt,33.432.4blobqbab262336236P52236.4o3.46
.42332
5-4522252.6o55-4o5w-4651o55-wobeobeb000bebe3eo25-4e5qeoqeqeeoo-
4256o55255o55352
255-45.6ebbobbw-4855255o55.455-4obwqq-45eoe-4455.4oeoeobbbeoo5555-4-
42400boqqbeboeb
oelloyboyeoellolleoL)oebbebyooblIblloyllowqbqyooboL)eqvbbybobebboblloubeobebIly
beobllo
oeqblooleoeebboblboeeoebbboobeoleooeollebeobbbveblblollebobeoewelooeobeobb
ooembvpbbolvoZvoobq-LbbblvPbb.43252begbPfilopooebvobboolebblobP51233bo21P32362
111popp11p6606po36006151o6p6-136ep61o1o1e6636613.16ep116610e66066066131me6616
b4ob4vb4bpvbboobbvooboobovooqobwbqwobb4oboobwowbqqooboovbqb2oopqqoobbqv
LIOILOITZOZSIVIDd 6COLZO/ZZOZ OM
LZ -Z0Z GSSL9t0 VO
00E
(E6ST:ON aiOSS) 44UddrIVONH7VaZIGNIVZS700370(1HOMOUUUSONNOISSa
VEWINCIMOrlaNXISSOdNMUUdMOONSdaUOUUNCMACIASS2nMWIENAZONOOOMAVdVaVSUSMIAU7S000
3222dEE3S3OGE3Omm0AdEwadOmaiwrimmESEN3Ar1miNISTrIASDmOvridvmiAia3vaarISEmHAvOS
VlicillOVadVISrldOSVIMdVdIdaldVdalMUMIHAMM900,117SSZAOHOAANVICINVaAdNIMTIACIOSO
SO
SZUYdASSSUISVMAIVINdd05dMOOAMIEIANNONSSAZASOSSMOILIMUO9dSAWISlidSOIZADIS9999S
0000SOODOSSAMATIOOOMAILMIXCINAIdOUTiOAANCIJIHIPIUNNOZAUNNYNCUSISAUOXASCISAAMSO
VUOISVAM270M0dVOUAMSFIVAMSZIAOSVVOS7117S90dNAZOODS2A70A2dINVH777V7d777VMAdrIVN
(spToe ouTwe)
(z6s1:ON ai oas) eeleblobol00000bl000bbeobleovoll000boebollooeoebbevoovoob
epelbuololbbbeooqqqapobbqeboeobbbbeeobbbbebboobobebobbeeeblebbbqlebeblbeoll
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(VNa)
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oeqoeboeeoeqole000ebbebeoo5.45.4.4eqoeqbqbooboovoebbeboobebeErwooqoeebqebeobqo
ovlbloobeovebevooboveoebbboobeoleopeollebeobbbeeblblollebobeovqoeloovobeobb
oobebeobboleloloob4-Lbbbleeblqoobbeeeebell0000bbeoeboolbbblobebleooboeleoeobe
111oovoqqobboEeooboob1bqobefrwebeb1010qebbobbwobeebqbqwebbobbobbloqeebqqb
ninbeoninbenbn::)neleonnon::)e-pn-r)511::):Thnionn::)nio-
onlinneninennello::)nnie
(vNa)
XXI-zE-13Z-8Z-I
(9ZOG:ON UIOES)**EddrIVONHaVaLl
ammvisrlsOarmalloNsulauzoNwsizsavzvwmaNOrmNarionaNmalaNsowzaausEuNammuzzuusrIN
rIENX'IONO0C5XXVdVaVSUSaMAUSIallniaallddliXdOXHMILIdOdIRMINNTAIXaSWIMISUMSUAMII
3VAIA
TISADVIADOAAAIAMadMSdOdaldSdOZHNNZEIMMOVO3MUSSUAOHOAAAVaaaVOASSISTIACIOSOSO
IauadAssauIsvmAiriaNdsOodNOOmvaANNONssAriAsOssNpswIANasysAvassdsalmwiNsesses
0000S0000vSALATIO00mAvasaxabuidOuv0AmiviaaSuaSSMaxaiNuANauSizau0xASaSALISO
VUOISVAM2ZUM2dMOUIMSNVXMSZIAOSVVOS'IMZSOOdMAZOODSZNINA2dUVVInggirldriqqVMAdrIVN
(spToe ouTwe)
(szos:oN
a: 02s)eeqebqoboqoopoobapoobbeobqeoeoqqopoboeboqqopeoebbeeopeoobeoeqbeoqoqbbb
e30g44433664e63v36666ee36666e6633636ebo66eee64e66644ebeb46e0443366e660664e6ee4e
6eee
beo64oeeb4eeo44bzoobbee6beo43poeebeebbeebeboobeee5666664e6eb4000e666=6646oebebe
eoe
bb4;44b4eboe4bebbebebeeboebbego4eeo4ofieboev4e4o4obeopeebeoobbbeobeeou4b3b33333
b3ebe
360be6f,e36eo446ee646e6eoo4o6oge433be3b344oe6363e33e3o33b4e43336e33eq4e36eee6e3
3e4o3
b6b400ebee6e4004oeb4eoeeb4eoeuoe64beoeo6wpo4o6beobefteBee46e66e646664o44o4eo4e4
4400
bb4booeb4W5435400beoe4o15400blizob4bebbobbb4151541515415154=4W5154344400beeobe4
opobb00004415
4opoo4o440o4b4b4o0eobeebeebweebbqobeveoeebboobobbqqqooebwobeooqbqooeqbeooeo
obggegoeqbgboo55.4ogebeeboobbeo5.45.4ogobeogeeoefrwooeoggoeboovebbobeobbobeobb
eoeoqqebeqeb000bqUobbobeeebebeooeobeoobbbloeqoqebqobqobeeqooqoqbeoobb000bee
beobeoqelbbloobblooeloeebeebeooeeobeooloeqbloblbobebeoobeobebeeobloolbleooe
bqbevebebobbqobqoqb-Lbqobbqopbeobeb000bebeovoebqebqvoqeqevooqebbobbebbobbobe
255.45.6ebbobbqoqebbebbobb.455.4o5qoqqq5eoeqqbbqoeoeobbbeoo5555.4.4eqooboqqbebo
eb
oegoeboeeoegoge000ebbebe3o5.45.4oegoeqbgeoobooegebbebobebboerwobeobebgebeobqo
oellbIll)ollyoyebtoblIboyez)ebbboobeollyoovollqybeobbbvebqbqoqqybL)beuelloylloo
yobeobb
pobebeobboleobeoobq-Lbbbleebbwebebeebeblopooebeobboolebblobeblepoboeleoeobe
qlloovollobbobroobooblblobebqobeeblololebbobbloobeellbbloebbobbobbloleebblb
61361e616eebboobbeoo6006oRoolo6loblioobbloboobloolobliooboop616pooelloo661e
(vNa)
LIOILOITZOZSIVIDd
6COLZO/ZZOZ OAA
VVCO 2022/027039
PCT/US2021/071017
qaqqtqcaqctqqtqqaqtctqqqqqaqacttaqtqaagoctqqaqqqtccctqaaactctcctqtqcaqcctct
ggLLLcacLLLcagLagLLaLggaaLgLci-LgggLLcgccagacLccagacaagaggcUggaqUgggl_cgcaacc
attagtaatggtggtagacacaccttctatccagacagtgtgaaggggcgattcaccatctccagagacaatgcc
aaqaacaccctqtatctqcaaatqaqcaqtctqaaqtctqaqqacacaqccatqtatttatqtqtaaqacaqact
gggacggagggctggtttgcttactggggccaagggactctggtcactgtctctgcaggtggcggaggatctggc
ggaggtggaagcggcggaggcggatccgatgttgtgatgacccagactccactcactttgtcggttaccattgga
caaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggaaagacatatttgaattggttg
ttacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacagg
ttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttat
tattgctggcaaggtacacattttectcagacgttcggtggaggcaccaagctggaaatcaaa (SEQ ID
NO: 1598)
(amino acids)
EVQLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISNGGRHTFYPDSVKGRF7ISRDNA
KNTLYLQMSSLKSEDTAMYLCVRQ7GTEGWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDVVMTQTPL7LSVTIG
QPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVY
YCWQGTHFPQTFGGGTKLEIK (SEQ ID NO:1599)
hu25E6 full sequence
(DNA)
qaqqtqcaqctqqtqqaatctqqcqqaqqactqqtcaaqcctqqaqqcaqcctqaqactqaqctqcqccqccaqc
ggcttcacattcagcagctacggcatgagctgggtgcggcaggcccctggcaagggcctggaatgggtcagcacc
atcagcaacggcggaagacacaccttctaccccgacagcgtgaagggcagattcaccatctcaagagataacgcc
aagaacagcctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagacagacc
qqcacaqaqqqctqqttcqcctactqqqqccaqqqcaccctqqtqaccqtqtccaqcqqcqqtqqcqqaaqcqqa
ggcggtggctccggtggcggaggcagcgacatcgtgatgacccagacccctctgtctctgagcgtgacccctggc
cagcctgccagcatctcttgtaaaagcagccagagcctgctggacagcgacggcaagacctacctgaactggtac
ctgcagaagcccggccaaagccctcagctgctgatctacctggtgtccaagctggatagcggtgttcctgataga
ttcagcggatctggcagcggcaccgacttcaecctgaagatcagcagagtggaagccgaggacgtgggcgtgtac
tactqctqqcacmcacacacttcccccaqacattcqqccacmcaccaaqqtqqaaatcaaq (SEQ ID
NO: 1600)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSTISNGGRHTFYPDSVKGRF7ISRDNA
KNSLYLQMNSLRAEDTAVYYCARQTGTEGWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPG
QPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY
YCWQGTHFPQTFGQGTKVEIK (SEQ ID NO:1601)
hu25E6R
(DNA)
Gaggtgcagctggtggaatctggcggaggactggtcaagcctggaggcagcctgagactgagctgcgccgccagc
ggcttcacattcagcagctacggcatgagctgggtgcggcaggcccctggcaagggcctggaatgggtcagcacc
aUcaqcaacqqcqqaaqacacaccLUcLaccccqacaqcqUqaaqqqcaqaUlcaccaLcUcaaqaqaLaacqcc
aagaacagcctgtacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagacagacc
ggcacagagggctggttcgcctactggggccagggcaccctggtgaccgtgtccagcggcggtggcggaagcgga
ggcggtggctccggtggcggaggcagcgacatcgtgatgacccagacccctctgtctctgagcgtgacccctggc
cagcctgccagcatctcttgtaaaagcagccagagcctgctggacagcgacggcaagacctacctgaactggtac
ctgcagaagcccggccaaagccctcagctgctgatctacctggtgtccaagctggatagcggtgttcctgataga
ttcagcggatctggcagcggcaccgacttcaccctgaagatcagcagagtggaagccgaggacgtgggcgtgtac
tactgctggcagggcacacacttcccccagacattcggccagggcaccaaggtggaaatcaagcgg (SEQ ID
NO; 5029)
(amino acids)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSTISNGGRHTFYPDSVKGRF7ISRDNA
KNSLYLQMNSLRAEDTAVYYCARQ7GTEGWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPG
QPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY
YCWOGTHFPOTFGQGTKVEIKR (SEQ ID NO:5030)
hu25E6RT full sequence
(DNA)
gaggtgcagctggtggaatctggcggaggactggtcaagcctggaggcagcctgagactgagctgcgccgccagc
ggclAcacal_l_cagcagcLacggcaUgagcUgggl_gcggcaggcccoUggcaagggccUggaaUgggl_cagcacc
atcagcaacggcggaagacacaccttctaccccgacagcgtgaagggcagatteaccatctcaagagataacgcc
304
CA 03187555 2023- 1- 27
LZ -Z0Z GSSL9t0 VD
co
DlbeibblibilloebtbDobbal:DDobobbbloleoeloleoe6obloobollIlebblpebbebeDoeoeoblboo
b
p66o66136136loovbeiblio66e63.0366e6loobebioloobroo6poobilepovi00006p0000ploo
goowfm00000fy40000es.oveoebeeogeeeffy45beeooeobffmoobboggeoefm00000ggoeoeoeobb
beoffyLlobwegoe-4646D6664boebbeboobeeffy4bebeobeogebeebwooeo-44Debooeobbobeobb
qolvlobobvollvb212bqoolqbqbbobvqvbblobvvooqbqbbloovqolvbloblobvowoobvvvoobb
opobeubeoblopulbbloee6qopelopebeeobboebobeoebblobloobebepobeobeeeelbllowle
ofmoobwobeopEbq000pefri.bobebwwqbqow000ebe000ebqebqboqeoebobeobbvbbobbqbb
oogobbqbboffm5bobulabboffy46bobbofmooqbgboovbqffywoovobffmoobbbbqougooboqqffy4
o565ebeoeo55ooe5eoe5eoo5obloeloe1515oo5ooeoe55e5oo555o5loo5eoeeble5eo5looel
bloofmovvemvpobovvqvbefmvoloqvoovollvfmoffAmeblbobvovboopovlolwovovovfmvbb
obboeupbeolepoeobepabbblpebbloobbbeupbbloppobbpobboblbbblobebleobboelobeobe
oqlvovoqqobboEeooboobobqobebwebebwobeobbebbqoobeeoqbbqoebbebbobbloqeebbqb
5.4361206.4.6bubbooffmooboobovoogobwbqqoobbqoboofywogobqqooboolabgEmoowqwoffy41
2
uvo-93sznq
(09T:ON ai 02s) ..uddriliONImaxiam=visrlsOxrisaliemeuuuzemweiz
speavwxamormsurissodmmEudxsowsdausuumazAaxasEuszmaNxrioNosompfdlealesusamAuzso
so2222aaupsosa220mmOnauwadOmaixrrimmusumoxamiArisrirmAsomovrlavmixia3vaarisumun
v
00VVdU3liadUrIS1dOSVI1dVdIddUdVdIIINIarINI0003I0daHIDOM3AXAOrlaaVaAUSINqIaaIOSO
SalaUCHAOSCMISMAIZUNdS09dUOTIMMAINDaSTIUSOSSMOSISVd0DIIASTYMIOINAACIS9999
S0900S0000VSAIATIOOOMAVZMOSIOIOUADZANVICISSYISSNOZATINNIEUDISLIZUOMASCHAZIHUO
ONSLIVAMErIEMadnEAMSGMSS,1130SVIMS'IM'ISOOdNAMMOSENIOAEdEliVITMWDTTYPLIAdqW1
(sIDTD2 ouTwv)
(3091: ON 01 Os) eegefogobowooDobwoobbeabgeoeogwooboeboegooeoebbee
ooeoobeoe4beoqQ4655eooe4.4400654eboeo5565eeo6555ebboobobebo55eeeb4e55544ebeb
qbvovloobbvbbobblvbevlebvP2bPoblove61P232-1.61o36622662313332P522662e62633622
e5555.65qebebwooe5553365153ebebeeoe55qqqqbqebovqbebbebebeeboebbewleeowbe5
ovv32-43-43b2oovvb2oobbb2obvvovqbob00000bovbvpbobvbb2obvoqqb2vbqb2b2bqovvbqbq
ebbebbeebeebeebeebezny444efoobwbegbwbbgebeebbebeeDwegoeeeDegbeoowbebge-444
eooveoeeeoqqeqeqeq5ooweeebeeebeo5555oeeeobweqqwooeoqe.4.455.4oeoqbwoqoqqo
31666.&461132beb33bb-Llop3b3abbl3lv3el31232636133631-4126613266262332323blb33b
ebbobbqobqobwoebeq5-4-4355eb000bbebwobebqowobvoobeoobqqeeoew000fm0000ewo
wovbv00000bw000vvoveovvvvoqvvvbbqobvvooeobbvbbqbboqqbovb2owoqqq32ovovqbb
esQffy4D64-4e4-4e44-4e66644-
4ebbefyLlobbeffy4bebeobwogeeeebweoezYmebeoebbbeogebb
qbeobbwe3qqbEe3ebwooqbebbqowebbweeew.45.455.4oqeqoqeewobobeeeooqoqbe3obb
yooffmfmoyqqbqqbeiggyy6qqqvgyoybyvybbqwerifmgybyggogoobybypqbweogbyypbqqogogy
poloobeooeeoeffy4qeopeqq5boqbqqweoweooqoebe000efraebqbqq5qebooqe55355e55355
obvvbbqbbvbbobblolvbbebbobblbb2oblowlbloeolbblolovbbbvvoobbbbw2113b111bbq
obbbebbowbbbqopbpowbeeqbqbqpqqqpqbqvpobwopowbbebwqbewbwqbwobebqwwwobwqwq
bq000voee5eeoobqee3ebebeo3qoqeooe3qqebobbbbev5.45.4.6e3e5e3oqewq400e3e3ebe.455
4ber4eelibeqqeDoeez)bDabbbqbebbliobbebeeoebeoDqDebeoobDqqbbbqqoqbqeebbqeqqbeqbe
oqqqovoqqqbbqoqoobeobqbqooqoqoeeebwooqbbbebbqoobeebqbeqqoebebbbbbqoqbebbqb
blobeoblbbebboobbeopboobotooqobloblloobbwboobloolobllooboovbqbeooelloobble
(liNa)
zE-EE1-6-8-M UNE0-9ECZnw
(ZEOC:ON (II 02S) mumizAmmoosamodausompx
xmmasysAusixruacuosososauadAosarimsmxirrinasnoammxmzximoasarrinssmnsisvan
OdIAS'IS'IdIaLWAICISOODOS0000SDOOOSSAMATIOOOMAV0IMO3ManUliOAAAV=VUZSINIKOZAZSNM
WIGUSIZAUOMASOdAaIHUOSNSIISAMagOMOdliCAIAMSWOASSaLaOSIDiOSqU'ISOOdMMOSOSENIOAa
(p-ç3 e ouTwe)
(TEOG:ON ai
s) 33282332
3t-46.4.63bbbgb3vb5eb33beebbqbebe3be3g2f2eb33323q3eb33e3bb3f23bbq3gebb3be3gq
ebvlieblioollqbqtbobellebbllobeeoollblIbbllooellollebllobllobeoll000beeeoubb000
beebeobllo
pelbbweeblopelooebeeobboebobeoebblobloobebeoobeobeeeelbllowleobeoobloobeo
obbl000p26163Z2blowlbqolopoovbeopot,6126qboleovbab236626bobblbboolobblbbobb
p66361e66366166:36636pool616ope6166l000po666e336666loploo6o116610666e6eopo66
ooybyoybyoobobwygovqbgbooboovoybbyboobbbobwobvoyybqybyobwoy4bwobyoyybyy
LIOLLORZOZSahlad 6C0LZ0/ZZ0Z OM
LZ -Z0Z GSSL9t0 VO
90C
DlbeibblibilloebtbDobbalz)DobobbbloleoeloleoebobloobolqlebbwebbebeDoeoeoblboob
p66o66136136loovbeiblio66e63.0366e6loobebioloobroo6poobilepovi00006p0000ploo
goowfm00000fy40000es.oveoebeeogeeeffy45beeooeobffmoobboggeoefm00000ggoeoeoeobb
beoffyLlobwegoe-4646D6664boebbeboobeeffy4bebeobeogebeebwooeo-44Debooeobbobeobb
qolvlobobvollvb212bqoolqbqbbobvqvbblobvvooqbqbbloovqolvbloblobvowoobvvvoobb
opobeubeoblopulbbloee6qopelopebeeobboebobeoebblobloobebepobeobeeeelbllowle
ofmoobwobeopEbq000pefri.bobebwwqbqow000ebe000ebqebqboqeoebobeobbvbbobbqbb
oogobbqbboffm5bobulabboffy46bobbofmooqbgboovbqffywoovobffmoobbbbqougooboqqffy4
o565ebeoeo55ooe5eoe5eoo5obloeloe1515oo5ooeoe55e5oo555o5loo5eoeeble5eo5looel
bloofmovvemvpobovvqvbebvvoloqvoovollvfmobbbveblbobvovboopovlolwovovovfmvbb
obboeupbeolepoeobepabbblpebbloobbbeupbbloppobbpobboblbbblobebleobboelobeobe
oqlvovoqqobboEeooboobobqobebwebebwobeobbebbqoobeeoqbbqoebbebbobbloqeebbqb
5.4361206.4.6bubbooffmooboobovoogobwbqqoobbqoboofywogobqqooboolabgEmoowgwoffy41
2
uv0-9ESZnq
(LO9T:ON ai Os) 44UddrIVONWIliGADDLIVISZSOArMall5M9UUUSSMNSI
HSXVEYIAMICDIMENX'10E0dNMEEdX0ONEdaUSEUXMACRESUEOrlIVIENX'ION00051X1fdlialfSES,
DIAUSE
AliVaaUadVAdOAHMUIdOcTUUdIVINNAUSIVIZUSUMSUOIJIMINISTIZASOMOVadVMIAICIOVaMOUSHA
V
00VVdU3liadUrIS1dOSVI1dVdIddUdVdIIIXIarIXI0003I0d3HIDOM3AAAOrlaaVEAUSIXaIaclIOS
O
SalaUCHAOSarDISAZAMIXdS09dUOTIMMAIMaSTIUSOSSX3SISVd0DIMAS1YMMOINAACIS9999
S0900S0000VSAIAZIOOOMAVaMOSIOIOUADZANVICISSYISSNOZATINNYNCIUSLIAUOMASCHALIHUO
ONSLIVAMErniXadnEAMSGMSS,1130SVIMS=SOOdWMOOSENIOAEdUltVWITIWIdT=AdqW1
(sIDTD2 ouTwv)
(9091:0N cu Os) eegebwbow0000fywoobbeabgeoeogwooboeboegooeoeb
beeoovoobeoe4beow4556eooe44400554eboeobbbbevo555.6ebboobobe5obbeee54e55544e
b261bvovloobbgbbobb-L25221Pb2P2beoblovEtilveovlbloobb22bbvolopovvb22662Pb2boo
bet,v5.655bbqebe5.4333e5563355-
453ebebeeoebbqqq1bqvboeqbebbebebeeboebbvqoqeeoqo
bybovvq2qowbv3322b2oobbbvobvvovqbob00000bovb2obobvbbvobvoqqb22bqbvb2ooqobo
4ewobeaboggoebDboeDoeDo:DobgewoobeooeggeDbewebeooegoobffy4oDebeebeqoogoebge
oeebqeoewebqEeoeobooqobbeobebbebeeqbebbeobweqqwooeoqeqq5bweoqbwoqoqqo
olbbabqb1132beb33bb-Llop3b3bbbl3lv3el3lv32b3bl33b3lq12bblovbbebvp32323blb33b
ebbobbqobqobwoebeq5-4-4355eb000bbebwobebqoqoofmoobeoobqqeeoew000fm0000ewo
wovbv00000bw000vvoveovvvvoqvvvbbqobvvooeobbvbbqbboqqbovb2owoqqq32ovovqbb
esQffy4D64-4e4-4e444bebb644-
4ebbefyLlobbeffy4bebeobwogeeeefyLpeoezYmebeoebbbeogebb
qbeobbwe3qqbEe3ebwooqbebbqowebbweeew.45.455.4oqeqoqeewobobeeeooqoqbe3obb
yooffmfmoyqqbqqbeiggyy6qqqvgyoybyvybbqwerifmgybyggogoobybypqbweogbyypbqqogogy
poloobeooeeoeffy4qeopeqq5boqbqqqoeoqoeooqoebe000efraebqbqq5qebooqe55355e55355
obvvbbqbbvbbobblolvbbebbobblbb2oblowlbloeolbblolovbbbvvoobbbbw2113b111bbq
obbbebbowbbbqopbpowbeeqbqbqpqqqpqbqvpobwopowbbebqoqbewbwqbwobebqwwwobqoqwq
bq000voee5eeoobqee3ebebeo3qoqeooe3qqebobbbbev5.45.4.6e3e5e3oqewq400e3e3ebe.455
4bbgeelibeggepoeez)bpabbbqbebbliobbebeeoebeopqpwbeoobpqqbbbqqoqbgeebbgeqqbegbe
oqqqovoqqqbbqoqoobeobqbqooqoqoeeebwooqbbbebbqoobeebqbeqqoebebbbbbqoqbebbqb
5135e051.6bebboobbeopboobotooqobloblloobbwboobloolobllooboov515tooelloobble
7-8Z-8-1. UVO-92CZnw
(g091:ON ai Os) **uddayOwsavaxIax=mIsasOxasassxsuuuasxwoia
sAvavwxmlOriaNxassOdismuudxsowadausuuxarmaxasuusamaNArION000xxvdvaysusaaimao
902222,33110S00a220.1.10AdUNadOMaIXTIMMUOUMWJIMINISTIZASOMOVadVMIXICOVaMOUMHAV
oolevaunvaawislaOsvimavaid,padvaizimisAmmo0oamOdsfunOmpxxmmaavansimmlacuoso
sosauadAssaamsAruizzOasOodmOzAmmAmmeasarlasOssmosisvdOodmAszsadmaimuas0000
SOSSOSOOOOSSAIMIDOOMX1i3M0aIOLOUYOXAMICWIWISNWOrlArISN1IiNaUSII,DIOXASadAaIHUO
ONSIISAMTIOX0dVOUAMSNDASS,1130SVVOSUUZS90dXAUDOOMMAadUVVIIMVUTITIVIATIVN
(gPT02 0u-cum)
(1709T:ON ai ryas) ve.425.4353.4333335q33355235.42323.4.4333b3eb3eq33e3ebbee
oovoobeoeqbeogoge6beooeqqqoobbgeboeobbffmeobbbbebboobobebobbeeeb.42555.4.42525
4bvoelloobbebbobbllebeellebveebeobweebIleeoe4bwobbeebbeollooL)eebeebbeybeboobee
ebbbbbblebebl000ebbboobblboebebeeoebblqqqblebovlbebbebebeeboebbewleeolobeb
ovv1213135233v25233566236P2oPlbob000pobovfmobobPbbvpbeollb2P515262513P26161
p66e6Eme6ep6ev6ve6eooliir6336136e1613661e6pe66r6peoloeloppeoel6poop6e6leill
voovvoyvvoqqvgyqvgboogovvybyvybyobbbboyvvobwyggwooyogyqqbbqovoqbwogoggo
LIOLLORZOZSahLad 6C0LZ0/ZZ0Z OM
LZ -Z0Z GSSL9t0 VO
LOC
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byobbloblovlovlblbobbbqbovbbvboobvvbblbvbeobvolvb22bl000vollovboovobbobvobb
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(131,Toe oupme)
(0I91:oN ai Os) ee4e543534333335w3obbe354e3e3443335oeb34433e3e55ee
33vo3bP321b23.431bbbe33.4.111336bleb3e3bbbbve3bbbbPabo36362b366222612bbb112626
53355553333353::33355f,335
ebbbbbb.42b2bwoovbbboobblbovb2bvvovbbqqqqb.42bovqb2bb2b2b22boebb2.433223.43b2b
peygew4obeopeebeoz)55beobeeoegbobopoo:Dboebepbobebbeobeogqbeebqbebebqoee5454
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o.4555&45.4.43ebE53355-
Lqoo3535.65.43.4eoewqeoeboblooboqqqebbwebbebeooeoe35.45335
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4=ebez)0000fr_looDzmeDeeDeveeollees,55-4obeeozmobbeffpgbbogqfpoebezyLloo444-
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lmoobb
e0055v5e0e115115bllee&411eqeoebeeeb5qebqbeqebeqwwobebeoqbeeoqbeeobqwwqe
ooloobvoovvovbb1123321.4.bbolbqqlovowvooqovbv000vblvbqbqqb12booqvbbobbvbbobb
obewbbqbbpbboEbwqwbbebbobbqbbwobwwqbqopoqbbqowebbbeepobbbbqopqwbqqqbbq
obbbebboe555-43e5eoebee-4545-4e-megbgeoobeoeoebbebqoqbeebwqbeobebgeveobwgeg
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olllovolllbblowobvoblblooloweeebl000lbbbebbloobeebqbeqwebebbbbblolbebblb
61p6P06166P66=66Pooboo6pPooloblobiloobbloboobloolobiloo6poP616eopelloo661P
XX7-zE-EE1-T7-8-.1 UNF3-9acznw
(6091:0N al Os) **UddrIVONE'lliGASCUIVISMOArlOaHOMOUUUSOMNOI
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xlivaaudavxdOximumasauudimmxasimusumsuomuirisqqqAsomovuoimixiaovaarisumilAv
ooleydunv2dwisrldOsvizdvdidddvdizimi2Ammo0oandstunOmpxxmma2v2Asimruacuoso
sosauadAssarimsAruirrnaneamOrummAmmeasazasOssmosisvaCeamAszszdmaimuas0000
SOSSOSOODOSSAIMIDOOMXIiaMOSIOLOUYOXAMICWIWISNWOrlArISNXIiNaUSII,DIONASadAaIHUO
ONSIISAMTION0dVOUAMSNDASS,1130SVVOSZUZS90dXMOODMVIOAadUVVIIMVUTITIVIATIVN
(gp102 0u-cum)
(8091:0N ai n2s) et-425.4obow0000bwoobbeobqeoeoqwooboeboewoeoeb
bev33233be3eq5e33qbffm33e-m33bbqe5323b6bbev3555.625b33b3emb3b6eee5-42555-4-42
beblibeoelloobbvbbL)bb-
LebeellybeyebyoblloyebIleeoeliblloobbyebbeolloouyebyebbeybyboo
bevebbbbbblebebl000ebbboobblboebebeeoebbqq1lblvboeqbebbebebeeboebbeloleeolo
bybovv.421olobt,33225e3obbbPobP2oelbob00000bo252363.62bbeob2311622blbebvoolobo
leloo6p36olloy6o6oepopp0006ipl0006eooelleo6pee6pooploo666-poe6ep6ploolop6le
ovvfoqvovqovbqbvovobooqobbvobvbbvbvvqbvbb2obwv-m000voqvqqbbqovoqbwoqoqqo
LIOILOITZOZSIVIDd 6COLZO/ZZOZ OM
LZ -Z0Z GSSL9t0 VO
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oerbleoeloe616eoeo6-ooio66eobebbebeelbe66eo6lovill000eole11661oeolblooloilo
o-4555.6-45-4-4oe555oof000bobbbqoqeoegogeoebobgooboqqqebbqoebbebeooeoeobgboob
ebbLIbbllobqobwoebv-454-4obbeb000bbvbwobv&LID4Dobeoobvoofy44vvoeg0000be0000vwo
qooelov00000bl0000veoveoebevoqvvebblbbevooeobbbeoobbolleoebe00000llovovovobb
beobblobweloulblbobbbqboebbeboobeebblbebeobeolebeebloopeolloebopeobbobeobb
qolebbobeoqqeEeqebq33-44515b3beqebbqobee33qblbb33eq3qebq3bq3be3333beee33bb
000fmebeobwovqbEr4o12125.400egooebseob5opbobeoubbqobwobsbuoobeobeevegbqqogoge
o5eoobqoobe3oE5q0000e5q53bebqolo151ol0000e5eoo3e51e5lbole3e5o5eo55e55o55155
oolobbqabobbe5bobeebbobblbbobboemoolblbooeblbbl000vobbbepobbbbweloobollbbq
obbbebepeobbooebeoebeopbobwelpelblboobopeoebbeboobbbobloobepeeblebeoblopel
bqoobeoeebeepoboeeqebebevoqoqeooeoqqebeobbbeebqbobeoeboopoeqoqqooeoeoebeebb
obboveofmoquoovofmobbbqe125.6.400bbbseobbq0000bbeobbobqbbbqoblabgeobbougobeobe
olqeovollobboEeoobooboblobebqoebebloobeobbebbloobeeolbbloebbebbobbqolee5515
blobeoblbbebbooffmooboobovooqobloblloobbqobooblooloblloobooeblbeooelloobble
Uy3-92gznq
(gT9T:ON ai02S) **UddrIVONWIVCLIDIMMVMSZOO,n9aHOMOUUU2ONNOI
aSZVaVIADIGNMENEIOndisDIUUdNODNadaUOUUXCIAGAaaUUD'INriaNArIONOOOXAVdVaVSUS,DIAU
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AVV3aUddVAdOAHMUIdDalUdIVINNACISHTIUSUNSUDAZIINISTIZADDIDIEMVMIAIMVaTIDUIHAV
90iiVciU0liSaVIS'IdOSYLIciiidIdaldliaLLIMITIMI009,110daHIOOMOIAAOrlaTiaAUSINTLE
LIOSO
salAuadAnsarimsArixiqumdsonduorrimisruzmnasarmossmnsisynnizAsruridizakmAasnnnn
S9999539900VGAZATIOOOMXVIMOMMOMOUAD'UNVDIMSWISSNOZXTINNVNaUSIMIUOMASCHXIMUU9
DNSIJMAMarDIXadIOUAMSNDASS,11.30SWIOSUXaSDOd>WIGODSaNIOAadUliliWITIVridarlalLIA
dqVIA1
(spToe ouTwe)
(17T9T:oN ai oss) velebloboloopoobq000bbeobleovoll000boebolloovoeb
bev33vo3be3eqEe3q3q55be33qqqq33bbqe53e3bbbbee3bbbbebb33b3beb3bbeeebqe555qqe
beblbeoqqoobbebbobb-Lebeelebevebeobweebqveollbwobbeebbeoq000vebeebbeebeboo
beeebbbabbgebeb400ebbbooffy4boebebeeoeffy444-464eboe-4bebbebebeeb3ebbeg3geeogo
beboevqeqoqobeooeebeoobbbeobeeoeq5o500000boebeobobebbeobeoqqbeebqbebeooqobo
qvloobeobolloeboboeopeopooblel000beopelleobeeebeopeloobbblooebeebelooloeble
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oqbbbbqbqwebbboobb-Lq000bobbbqoqeoeqoqeoeboblooboqqq2bbwebbebeoovoyobqboob
ebbobb4obqobqooebe454-
43bbeb000bbefy:toobefy43400beoobeoob44eeoe43000be0000e43o
qooebe00000bq0000eeoeeoeveeoqeeebbqobeeooeobbebbqbboqqboebeoqooqqqqeoeoe.455
eyobbqobqqvggeqqqbebb6qqqyaoybqobbebfritiebeobeogyvvybqoyoyoqqqybeoebbbeogebb
qbeobbqoeoqqbEeoebq000qbebbqowebbqoeeeqoq5155qoqeqoqeewobobeeeooloqbeoo55
voobbebeoellbqqbblq22bqlleleoebevebbleblbelebelloloobebeolbeeoqbevobllolole
poqoobeoopeopEbqqeopeqqbboqbqqqopoqopoowebeopoebqebqbqqbqebooqebbobbebbobb
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qbblevqbellepoeeobo-Lbbblbebbqobbebegoebeooloebeoobollbbbllolbleebblellbelbe
olllovo111661oloobeo61613ololoeeebl0001666e6613obee616elloebe666661316e6616
bqobeo5.4.65ebboo5beoobooboeooqobqobqqoobbqoboobwoqobqqooboo25.46eooeqqoobbqe
U110-93SZnw
(E191:ON GI OES) 44UddrIVONEIVallaM:VISZOOXIOCIHOMOUUUSONNOIE
SZY2VIAINCIMOZ2NAZ020dMMUUdM00142daUOUUMarlAUX22UUVINZ2N7JIONOOOMXVOialiSUSZMAU
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002222dAUDS300220.1.10AdUWAdOMAIX=MUMMUTIINISTTIA03.101fIdVMIXIMVACIOUZHAV
0011rYdUOV3dUrISIdOSVIIdVdMdaldNidMIMMIaAMI000.1.10delUMDOMOAAAOACMIMAUSINTLICL
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sosauadAssamismAirrioasoodxorumNammasarlasossxosislidoodIAsasadIcaNniassoss
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ONSIZSAM2q0M0dVOUAMSPDXSSAZASSVVOSUU'ISOOdMAUDOSS2M0A2dUVVIVITIV=VIVIAdqVG4
(spToe ouTwe)
(zI91:oN al oas) veqefoqobow0000foq000fobeofoqeoeoqq000boeboqqooeoefobee
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ebbebbeebeeberbeebeooqqqeboobqobeqbqobbqebeebbebevoqoeqovevoeqbeooebebqeqqq.
LIOILOITZOZSIVIDd 6COLZO/ZZOZ OM
LZ -Z0Z GSSL9t0 VO
60C
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06bubbobb.4(thuu4uf)
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4e.vo4obeboee4e4o4obeopeebepobbbeobeepe4boboopoobo
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4eobeeebeo:Je
43obb5433v6vvbv43343vb4e.ovvb4vov4ovb4bvovob43343b5vobv6bvbvv4bvbbgbgbbb434434v
34v 44
439f.b4b33e6466405433be3e4ob400bb4ob4J5ebbobbb465461546643046654044
400BEeobe4000bb0000
1461opoololloo1616loopoBeebevogyeebb15bevooeobbbyoobbolleoveiv00000llovovovobb
beobblobloeloe15153555q5oebbeboobeeffrabebeobeolebeebl000eolloebooeobbobeo55
gogebbobvoggebegebqooqqbqbbobegebbqobevooqbqbbqoovqogebqobqobvog000bvevoobb
poobeebeobqooeqbbqoee6qopeqooebeeobboebobeoebbqobwobebeoobeobeeeelbqqoqoqe
obeoobqoobeoobbqoopoefq.bobebqoqoqbqoq0000ebe000ebqebqboqeoebobeobbebbobbqbb
oolobbqbbobbeZbobeebbobblbbobbobeoolblbooeblbbloopeobbbepobbbbqoeloobollbbq
obbbebeoeobbooebeoebeopbobqoeqoeq5-45oobooeoebbeboobbbobwobeoeebqebeobqooeq
bwobeovebeepoboveqpbebevoqogeopeoqq2b2obbbewbqbobeoeb000peqoqqopeovoybeebb
obboepobeoqeopeobeoabbbqee&Erwobbbeeobbqopoobbeobbobqbbbqobebqeobboeqobeobe
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blobeoblbbebboobeivooboobovooqoblobllootibqobooblooloblloobooeblbeooelloobble
zE-8Z-8Z-M UNE0-93gZnq
(17cog:oN ai 02s) .11ddrIvONHqvax
maxmvzszo0ArmaHoxouuuaoxvioiasAvaypolaxOzaNxzondiuuudxoopladauouuxarmaxaauuoz
WIZNArIONOOONAVdVaVSUSIMAUSUAVVICIUddVAdOMINUIdOdUUdasINKACISUTIESUMSUAMIIIIVAM
ATISAOVTAOSAARIAMIdXSdOdarldSdalliMMIS'IMI00031.0daHLOOMOAXAOrlaSliZAUSINTIIGIO
SO
SOMZUCHAOSTINSAZAIMINdSOOdUOTIMMAMMOaSarrISOSSMOSISIMODIMASTYMMOMKAAaS0000
S0990S0000VGAZATLOOOMXV.IMOMIOMOUAOrIANVMUMFISSNOZATINNVNaUSIMZUOMASadkIMUU0
ONSLIVAMEaUXU&IOUAMSIAMASSaIAOSVVDS=SOOdMArlannSENTOAEdUVVIVITTWIdarlaViAdaVVI
(epToe ouTwe)
(U:og:ON
ai Os) ee4e64o6o40003o6403obbeob4eoeo4;000boe6oe400eoebbvepoeoobeoe4beo4o4
35 3 :L355
wwpfiwobqoppliqwpop4b4onfibEwfifipoqpnowpfimpfifimpliwboofivewpfififififibqwfip
liqopopfifiboofifiqflopfimfim
vo-
ebb444464vboe4bebbebeBEebovbbe4o4evo4obeboee4e4o4obeooevbeoobbbvo6Eeoe46ob00000
bo
ubpobobubbeobuoqqbuubqbefmooqoboqu400buoboqqoubobouoou0000bquq000fmocuqquofmuub
uocu
403034oeb4eoevb4eoe4oeb4beoeofq.004obbeobebbebee4bebbgb45554oq4o4eo4e 44
400bb4booe6466406400beoe4ob400bb4o646ebbobbb4bb4664664004bbb4o 44
400beeobeq000bbooclo
1461peoplolloo1616100e0beeepeoqevebbqobevoovobbebbqbboqqboveivoqooTmeopoeqbb
eeo55-4o5qqeqqeqqq5e5554-41e5bebqobbeffrabebeobeoqeeeebqoeoeoqqqebeoebbbeoqe55
qbeobbqovollbbeoebq000qbebblowebblovveloqbqbbloleqolveloobobeevoololbeoobb
epobbebeopqqbqqbbqqeefrmeqeDebweebEqpbqbeqebeqwwobebeoqbeepqbeepbqwwqe
ooqoobeooeeoeffy4qeooeqq5boqbqqqoeoweooqoebe000ebqe5.45.4.45.4ebooqebbobbebbobb
obeebblibbebbotbqoqwbbebbobbqbbwobqoqoqbqoeoqbbqoqowbbbewoobbbbqowqqobqqqbbq
obbbebboebbbwebeoebeeqbqbqelqqeqbqeoobeoeoebbebqolbeebqoqbeobebqeveobqoqeq
bl000voeebeepobleeoebebeooloqeooeollebobbbbeeblblbeoebeooleloqqooeoeoebelbb
1661evibelleooevobo-66616e661066e6eeoebeooloebroo6o1166611016leebbielibeibe
oqqqovoqq.455.4o400beobqbqooqoqoeeebwoo.4566255.400bee5.45eqqoebe56555.1.o.4525
5.45
bqobeobqbbebboobbeoobooboeoogobqobqqoobb4oboobwogobqqoobooebqbeooeqqoobbge
zE-8Z-8Z-I UNM-9SCZnw
(L191:ON (II 02S) **UddrIVONEFIVUZMUMMVMSZOOZZOUHOMOUUU2OMNOI
assysywmamOrIEN,ariosOcuoDDIamofmsdamarinaxamDlornstrisNxriONOoOmxvdvavmlszmmim
l
xvvolauadvxdOmmixdocluudimmxasuzzusumsuomuizszammmovadvmmaDvamoumunv
001ilidUOliSdUriSrldOSIiIldVdIddUdlidIIIMIZAMI00031.0daHLOOMOAXADAGSVEAUSIYILIG
IOSO
SOSaUCHAOSarDISMAITIOdSOOdMOZAMMAINOCISTIUSOSSMOSISVelOdIASUrldIOINAICIS0000
SOOOSSOSOOSSASNIISOOMXV3M02.10.10UVOAXAVIU2VUUSNWOrlkISNMVNUUSIZZUOMASUdX,IIHUS
ONSLISAMTIOMOdVOUAMSWASSZIAOSVIMSqU'ISOOdX/V1000SENTOAEdUVVH=V7=IVIAETIE4
(epToe ouTwe)
(9191:0N GI Os) yellybIloboll00000bll000bbyobllyoyoqq000boybollqooyoyb
bevoovoobeoelbeoloqbbbeoolllwobbleboeobbbbeeobbbbebboobobebobbeeeblebbblle
beblbeolloobbebbobb-Lebeelebeeebeoblovefilveollbloobbeebbeol000vebeebbeebeboo
bere6666661e6e6l000e666=6616oe6e6evoe66111161r6oel6e66e6e6ee6oe66eloleeolo
byboyegegogobeooyebyoobbbeobeyoyqbob00000boybeobobebbeobeogqbeebqbebyoogobo
LIOLLORZOZSallad
6COLZO/ZZOZ OM
W02022/027039
Per/lUS2021/071017
tctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctgataa (SEQ ID
NO: 5035)
(amino acids)
MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSTISNG
GRHTFYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQTGTEGWFAYWGQGTLVTVSSGGGGSGGGGS
GGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGS
GSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGQGTKVEIKKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLV
TVAFTIFWVRSKRSRLLHSDYMNM7PRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELN
LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT
YDALHMQALPPR** (SEQ ID NO:5036)
mu25E6-CAR T-28-28-3z-1XX
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgcagctg
gtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggtttcactttc
agtagttatggaatgtcttgggttcgccagactccagacaagaggctggagtggg7cgcaaccattagtaatggt
ggtagacacaccttctatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctg
tatctgcaaatgagcagtctgaagtctgaggacacagccatgtatttatgtgtaagacagactgggacggagggc
Uggl_l_l_gcl_LacUggggccaagggacUoUggl.cacUgUcUcl_gcaggUggcggaggaLcUggcggaggLggaa
gc
ggcggaggcggatccgatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctcc
atctcttgcaagtcaagtcagagcctcttagatagtgatggaaagacatatttgaattggttgttacagaggcca
ggccagtotocasagogootastotatotggtgtotasactggactotggagtccotgacaggttcactggcagt
ggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaa
ggtacacattttcctcagacgttcggtggaggcaccaagctggaaatcaaaaagcacctgtgtccttctccactgtt
ccccggccctagcaagcctttctgggtcctggtggtggtgggcggagtgctggcctgctacagcctgctggtgaccgtg
gcct
ttatcatcttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactcctagaagacctgg
gcct
accagaaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcg
caga
cgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttg
gaca
agagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgttcaatgaactgca
gaaa
gataagatggcggaggccttcagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggccttttcc
aggg
tctcagtacagccaccaaggacaccttcgacgcccttcacatgcaggccctgccccctcgctgataa (SEQ ID
NO: 5037)
(amino acids)
MALPVTALLLPLALLLHAARPEVQLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISNG
GRHTFYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYLEVRQTGTEGWFAYWGQGTLVTVSAGGGGSGGGGS
GGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGS
GSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIKKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLV
TVAFTIFWVRSKRSRLLHSDYMNM7PRRPGPTRKEYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELN
LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDT
FDALHMQALPPR** (SEQ ID NO:5038)
hu2bE6-CAR T-28-28-3z-1XX
aUggceLLaccagLgaccgccLLgcUccUgccgcUggccUl_gcl_gcLccacgccgccaggccggaggl_gcagcl_g
gtggaatctggcggaggactggtcaagcctggaggcagcctgagactgagctgcgccgccagcggcttcacattc
agcagctacggcatgagctgggtgcggcaggcccctggcaagggcctggaatggg7cagcaccatcagcaacggc
ggaagacacaccttctaccccgacagcgtgaagggcagattcaccatctcaagagataacgccaagaacagcctg
tacctgcagatgaacagcctgcgggccgaggacaccgccgtgtactactgcgccagacagaccggcacagagggc
tggttcgcctactggggccagggcaccctggtgaccgtgtccagcggcggtggcggaagcggaggcggtggctcc
ggtggcggaggcagcgacatcgtgatgacccagacccctctgtctctgagcgtgacccctggccagcctgccagc
atctcttgtaaaagcagccagagcctgctggacagcgacggcaagacctacctgaactggtacctgcagaagccc
ggccaaagccctcagctgctgatctacctggtgtccaagctggatagcggtgttcctgatagattcagcggatct
ggcagcggcaccgacttcaccctgaagatcagcagagtggaagccgaggacgtgggcgtgtactactgctggcag
ggcacacacttcccccagacattcggccagggcaccaaggtggaaatcaagaagcacctgtgtccttctccactgtt
ccccggccctagcaagcctttctgggtcctggtggtggtgggcggagtgctggcctgctacagcctgctggtgaccgtg
gcct
LLaLcaLciAcLyyyLyayyayLaayayydycayycLocLyeacayLyacLacaLyaacaLyacLceLayadyaccLyy
yocl
accagaaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcg
caga
cgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttg
gaca
agagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgttcaatgaactgca
gaaa
gataagatggcggaggccttcagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggccttttcc
aggg
tctcagtacagccaccaaggacaccttcgacgcccttcacatgcaggccctgccccctcgctgataa (SEQ ID
NO: 5039)
(amino acids)
310
CA 03187555 2023- 1- 27
VVCO 2022/027039
PCT/US2021/071017
MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSTISNG
GRHTFYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQTGTEGWFAYWGQGTLVTVSSGGGGSGGGGS
GGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWYLQKPGQSPQLLIYLVSKLDSGVPDRFSGS
GSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGQGTKVEIKKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLV
TVAFTIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELN
LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGIIDGLFQGLSTATKDT
FDALHMQALPPR** (SEQ ID NO:5040)
MNC2-1XX
muMNC2-CAR T-8-4-1BB-3z-LXX
atggccttaccagtgaccgccttgetcctgccgctggccttgctgctccacgccgccaggccggaggtccagctg
gaggagtcagggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttc
agtggctatgccatgtcttgggttcgccagactccggagaagaggctggagtgggcgcaaccattagtagtggt
ggtacttatatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctg
tacctgcaaatgagcagtctgaggtctgaggacacggccatgtattactgtgcaagacttgggggggataattac
tacgaatacttcgatgtctggggcgcagggaccacggtcaccgtctectccgccaaaacgacacccccatctgtc
tatggcggtggcggatccggcggtggcggatccggcggtggcggatccgacattg7.gatcacacagtctacagct
tccttaggtgtatctctggggcagagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggctat
agULaLaUgcacUggLaccaacagagaccaggacagccacccaaacLccUcalcUaLcUl_gcaUccaaccUagaa
tctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggag
gaggatqctqcaacctattactqtcagcacagtagggagcttccgttcacgttcqqacmgcmaccaagctqqaq
ataaaacgggctgatgotgoaccaactgtatecacaacaaccoctgoccocagacctoctaccocagoccotaca
attgccagccagcctctgagcctgaggcccgaggcttgtagacctgctgctggcggagccgtgcacaccagagga
ctggatttcgcctgcgacatctacatctgggcgcccttggccgggacttgtgggg7.ccttctcctgtcactggtt
atcaccctttactqcaaacqqqgcagaaagaaactcctqtatatattcaaacaaccatttatgagaccaqtacaa
actactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaag
ttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctotataacgagctcaatctaggacga
agagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaac
cotcaggaaggcctgttcaatgaactgcagsaagataagatggoggaggoottcagtgagattgggatgaaaggc
gagcgccqqacmgcaaggcmcacgatcmccttttccagggtctcagtacagccaccaaggacaccttcgacqcc
cttcacatgcaggccctgccccctcgctgataa (SEQ ID NO:1618)
(amino acids)
MALPVTALLLPLALLLHAARPEVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVATISSG
GTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARLGGDNYYEYFDVWGAGTTVTVSSAKTTPPSV
YGGGGSGGGGSGGGGSDIVITQSTASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQRPGQPPKLLIYLASNLE
SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGGGTKLEIKRADAAPTVSITTPAPRPPTPAPT
IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQ
TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN
PQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR** (SEQ ID
NO: 1619)
huMNC2-CAR T-8-4-1BB-3z-LXX
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgcagctg
gtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttc
agtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggctcaaccattagtagtggc
ggaacctacatatactaccccgactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactg
tatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttgggggggataattac
tacgaatacttcgatgtctggggcaaagggaccacggtcaccgtctcctccggcggtggcggatccggcggtggc
ggatccggcggtggcggatccgacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagg
gccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacagcactggtatcagcagaaa
ccaggacaacctcctaaactcctgatttacctggcatccaatctggagagcggggr.cccagccaggttcagcggc
agtgggtctgggaccgatttcaccctcacaattaatcdtgtggaagctaatgatactgcaaattattactgtcag
cacagtagggagctgcctttcacattcggcggagggaccaaggtggagatcaaacgaactacaacaacccctgcc
cccagacctcctaccccagcccctacaattgccagccagcctctgagcctgaggcccgaggcttgtagacctgct
gctggcggagccgtgcacaccagaggactggatttcgcctgcgacatctacatctgggcgcccttggccgggact
tgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattc
aaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaa
claaqqaqqatqtqaactqacracitqaaqttcadcaqqacicgcacracqcccccqcqtacaagcagcmccagaacca
q
etetataacgagetcaatetaggacgaagagaggagtacgatgttttggacaagagacgtggeogggaccetgag
atggggggaaagccgagaaggaagaaccctcaggaaggcctgttcaatgaactgcagaaagataagatggcggag
gccULcagUgagaLUgggaUgaaaggcgagcgccggaggggcaaggggcacgaUggcoLLLLccagggLeLcagL
311
CA 03187555 2023- 1- 27
WC)2022/027039
PCT/US2021/071017
acaqccaccaaqqacaccttcqacqcccttcacatqcaqgccctqccccctcqctqataa (SEQ ID
NO: 1620)
(amino acids)
MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSG
GTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGMTVSSGGGGSGGG
GSGGGGSDIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSG
SGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRT=PAPRPPTPAPTIASULSLRPEACRPA
AGGAVEITRGLDFACDIYIWAPLAG7CGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE
EGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAE
AFSEIGMKGERRRGNGHDGLFQGLSTATKDTFDALHMQALPPR** (SEQ ID NO:1621)
muMNC2-CAR T-8-28-3z-1XX
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtccagctg
gaggagtcagggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttc
agtggctatgccatgtcttgggttcgccagactccggagaagaggctggagtggg7cgcaaccattagtagtggt
ggtacttatatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctg
tacctgcaaatgagcagtctgaggtctgaggacacggccatgtattactgtgcaagacttgggggggataattac
LacgaaLacLUcgaUgUcUggggcgcagggaccacggl_caccgUcUccUccgccaaaacgacacccccaUcUgUc
tatggcggtggcggatccggcggtggcggatccggcggtggcggatccgacattgr.gatcacacagtctacagct
tccttaqqtqtatctctqqqqcaqaqqqccaccatctcatqcaqqqccaqcaaaaqtqtcaqtacatctqqctat
agttatatgcactggtaccaacagagaccaggacagccacccaaactcctcatctatcttgcatccaacctagaa
tctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggag
gaggatgctgcaacctattactgtcagcacagtagggagcttccgttcacgttcggaggggggaccaagctggag
ataaaacqqqctqatqctqcaccaactqtatccacaacaacccctqcccccaqacctcctaccccaqcccctaca
attgccagccagcctctgagcctgaggcccgaggcttgtagacctgctgctggcggagccgtgcacaccagagga
ctggatttcgcctgcgacatctacatctgggcgcccttggccgggacttgtgggg7.ccttctcctgtcactggtt
atcaccctttactgcaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactcctagaagacct
gggcctaccagaaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttc
aqcaqqaqcqcaqacqcccccqcqtacaaqcaqqqccaqaaccaqctctataacqaqctcaatctaqqacqaaqa
gaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccct
caggaaggcctgttcaatgaactgcagaaagataagatggcggaggccttcagtgagattgggatgaaaggcgag
cgccggaggggcaaggggcacgatggccttttccagggtctcagtacagccaccaaggacaccttcgacgccctt
cacatgcaggccctgccccctcgctgataa (SEQ ID NO:1622)
(amino acids)
MALPVTALLLPLALLLHAARPEVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVATISSG
GTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARLGGDNYYEYFDVWGAGMTVSSAKTTPPSV
YGGGGSGGGGSGGGGSDIVITQSTASLGVSLGQRATISCRASKSVS7SGYSYMHWYQQRPGQPPKLLIYLASNLE
SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGGG7KLEIKRADAAPTVSITTPAPRPPTPAPT
IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRP
GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP
QEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR** (SEQ ID
NO: 1623)
huMNC2-CAR T-8-28-3z-1XX
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgcagctg
gtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttc
agtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggrxtcaaccattagtagtggc
ggaacctacatatactaccccgactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactg
tatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttgggggggataattac
tacgaatacttcgatgtctggggcaaagggaccacggtcaccgtctcctccggcggtggcggatccggcggtggc
ggatccggcggtggcggatccgacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagg
gccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacar.gcactggtatcagcagaaa
ccaggacaacctcctaaactcctgatttacctggcatccaatctggagagcgggg7.cccagccaggttcagcggc
agtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgtcag
cacagtagggagctgcctttcacattcggcggagggaccaaggtggagatcaaacgaactacaacaacccctgcc
cccagacctcctaccccagcccctacaattgccagccagcctctgagcctgaggcccgaggcttgtagacctgct
qctqqcqqaqccqtqcacaccaqaqqactqqatttcqcctqcqacatctacatctqqqcqcccttqqccqqqact
tgtggggtccttctcctgtcactggttatcaccctttactgcaggagtaagaggagcaggctcctgcacagtgac
tacatgaacatgactcctagaagacctgggcctaccagaaagcattaccagccctatgccccaccacgcgacttc
gcagccUaLcgcUccagagUgaagLUcagcaggagcgcagacgcccecgcgUacaagcagggccagaaccagcl_c
tataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatg
312
CA 03187555 2023- 1- 27
WC)2022/027039
PCT/US2021/071017
gggggaaagccgagaaggaagaaccctcaggaaggcctgttcaatgaactgcagaaagataagatqgcggaggcc
LUcagUgagaLlgggaUgaaaggegagegccggaggggcaaggggeacgaUggcc_ULUccagggLeUcagLaca
gccaccaaggacaccttcgacgcccttcacatgcaggccctgccccctcgctgataa (SEQ ID NO:1624)
(amino acids)
MALPVTALLLPLALLLHAARPEVIOLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSTISSG
GTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGSGGG
GSGGGGSDIVLTQSPASLAVSPWRATITCRASKSVSTSGYSYMHWYWKPGQPPKLLIYLASNLESGVPARFSG
SGSGTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRTT7TPAPRPPTPAPTIASQPLSLRPEACRPA
AGGAVHTRGLDFACDIYIWAPLAG7CGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF
AAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEA
FSEIGMKGERRRGKGIIDGLFQGLS7ATKDTFDALIIMQALPPR** (SEQ ID NO:1625)
muMNC2-CAR T-28-28-3z-1XX
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtccagctg
gaggagtcagggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttc
agtggctatgccatgtcttgggttcgccagactccggagaagaggctggagtggg7.cgcaaccattagtagtggt
ggtacttatatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctg
LaccUgcaaaUgagcagUcUgaggLcUgaggacacggccaUgUaLLacUgUgcaagacLUgggggggaLaaLLac
tacgaatacttcgatgtctggggcgcagggaccacggtcaccgtctcctccgccaaaacgacacccccatctgtc
tatggcggtggcggatccggccigtggcggatccggcggtgqcggatecgacattg7.qatcacacacitctacagct
tccttaggtgtatctctggggcagagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggctat
agttatatgcactggtaccaacagagaccaggacagccacccaaactcctcatctatcttgcatccaacctagaa
tctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggag
gaggatgctgcaacctattactgtcagcacagtagggagcttccgttcacgttcggaggggggaccaagctggag
ataaaacgggctgatgctgcaccaactgtatccaagcacctgtgtccttctccacegttccccggccctagcaag
cctttctgggtcctggtggtggtgggcggagtgctggcctgctacagcctgctggegaccgtggcctttatcatc
ttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactcctagaagacctgggcct
accagaaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcagg
agcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggag
tacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaa
ggcctgttcaatgaactgcagaaagataagatggcggaggccttcagtgagattgggatgaaaggcgagcgccgg
aggggcaaggggcacgatggccttttccagggtctcagtacagccaccaaggacaccttcgacgcccttcacatg
caggccctgccccctcgctgataa (SEQ ID NO:5041)
(amino acids)
MALPVTALLLPLALLLHAARPEVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVATISSG
GTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARLGGDNYYEYFDVWGAGTTVTVSSAKTTPPSV
YGGGGSGGGGSGGGGSDIVITQSTASLGVSLGQRATISCRASKSVS7SGYSYMHWYQQRPGQPPKLLIYLASNLE
SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFOGG7KLETKRADAAPTVSKHLCPSPLFPGPSK
PFWVLVVVGGVLACYSLLVTVA=FWVRSKRSRLLIISDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR
SADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERR
RGKGHDGLFQGLSTATKDTFDALHMQALPPR** (SEQ ID NO:5042)
huMNC2-CAR T-28-28-3z-1XX
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgcagctg
gtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttc
agtggctatgccatgagctgggtccgccaggctecagggaaggggctggagtggg7.ctcaaccattagtagtgge
ggaacctacatatactaccccgactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactg
tatctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgcgagacttgggggggataattac
tacgaatacttcgatgtctggggcaaagggaccacggtcaccgtctcctccggcggtggcggatccggcggtggc
ggatccggcggtggcggatccgacattgtgctgacccagtctccagcctccttggccgtgtctccaggacagagg
gccaccatcacctgcagagccagtaagagtgtcagtaccageggatactectaca7.gcactggtatcagcagaaa
ccaggacaacctcctaaactcctgatttacctggcatccaatctggagagcggggecccagccaggttcagcggc
agtgggtctgggaccgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgtcag
cacagtagggagctgcctttcacattcggcggagggaccaaggtggagatcaaacgaactaagcacctgtgtcct
tctccactgttccccggccctagcaagcctttctgggtcctggtggtggtgggeggagtgctggcctgctacagc
ctgctggtgaccgtggcctttatcatettctgggtgaggagtaagaggagcagg=ctgcacagtgactacatg
aacatqactcctagaagacctqcmcctaccagaaagcattaccagccctatqccccaccacqcqacttcqcagcc
tatcgctecagagtgaagttcagcaggagegcagacgcceccgcgtacaagcagggccagaaccagetctataac
gagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatgggggga
aagccgagaaggaagaacccUcaggaaggccUgLUcaaUgaacUgcagaaagaLaagaUggcggaggccULcagL
313
CA 03187555 2023- 1- 27
LZ -Z0Z GSSL9t0 VO
VIE
ebeboobeeebbbtbblebebqznx)ebbboobblboebebeeoebb1111bleboelfiebbebebeyboebbelo
1evolobeboppielolo6pooep6Roo666eobeeov1bob00000boebeobobebbyobeolibee616e6e
5-432e.6-
45.4ebbe5beebeebeebvebeooqqqeboobqobeqbqobbqebeebbebeeoqoeqoeweoeqbeoo
ebefogeteooveovvvoage4E-4e4.6400qovvebvvveleoffobbovvvobwv-444oDovo4v44e64ovo4
bl33l31loolbbbblblqp2bbboobbql000bobbbloqeovlolvoybobloobo1112bblovbbvb2332
Depeaboabebboebloblobqoppbelbllobbeboopbbebwobeblowobepobepobgleppeloppob
e0000t,wowoeEe00000bw000eeoeeoeeeeqqeeebblbeveooeobbobeobfrmooeoqqw000q
ofmobvq.bobuobvioqbqouq-412-44-
4buobqqqqs51225wobubuqoofmobsoquoopowqouovqoubobv
b56w155.615eoE5gbeol5beoo5e000leo55o5eoob5woeeoogooeo5elelogeoloolo55e000
obve,voobbloob5vemoevooelbblovoolvoelobvqqbqbeofmoovoobobvoblooeol000voobvfm
eebbbbepologbqqlogbapopepobepologbepepebllbgblleeeboolebbobblbbobboolebbobb
b35 55
3uvo5335.6-412-4o1212-41252512oovq5qovqqw45-45-4obbovouffmboo5125125-
4po52312126-4121212o5qoquq.
bl000eoeebeeooboeeoebebeoologeooeolleboobbbeyblbeowebe000eloegeleoelooeobb
ebbobbqfmllvoovvolo-Lbbblfmabwbembevbbemooloffmooboolbbblobvbleobblvlbfmlfm
plqopuollebblolopbeobgbloolowebeblopolbbbbbblopbeeplbbloobbebbbbblolbebblb
6.435e35.16bebboobbeoobooboeooqobwbqwobbqoboobwowbqwobooebqbeoovq.wobbqe
XX7-7E-GGT-6-8,1 UV3-92NNnq
(LZ91:ON Os)
4*UddrIVONIVIVaaDDLIVMSZDOZZDaHDXDUUUaDX14
9I2SavalaimamOriaNaripaOdNmuudmOONadaueuumarinaxammOrunaNAri0m000mAvdvaliSuSamA
u
rrannnaaaadaunspnaaa0=0AduwadOxaixTmlufmmumArisrmAnaloviElemixianyaarinal
nAveslivallovadurisriaOsvimavamaaualiamiamiarimissallizasssu003xxmlivaavapausim
risxsm
DADSOSaaAdADSWINSMSAINIXdSMOdU003MHIASASSIliSaVIMAaaDdSliSNIlidSOIrlAIOSOODOSOD
OOSSODOSSAIASZOOOMAGNOACIANUDAMMIOHANVICESWISSNOZATINXVNQUSLIdUOXASadAAIA.10
DOSIMVAMEZUMOdMOUAMSNSAUSLIAOSAAOSZYISOOdXATIOSSEAAMAEdUVVHMVUdrIVIVMATIVN
(sp-pe ouTwe)
(9z91:ON (II 02S) PE0-42b3bo33333bw33bb23bqv323q333b32b3ll
Doeoebbeeooepobez)e4be:D4o-455beoo-444-
400bbgeboep5555eeobbbbebb:Dpbobebpbbeeebge
656-11.ebe5qbe3ggoobbebbobbge5eege5eeebeobwee5gee3gqbwobbeebbe3g000ee5ee55e
ebvb33bvvebbbe5blvbeblo33Pabb335blbovb2b2e32661111612632162ff2bebveb32bb213
qevoq3beboeeqwww5e3oeebe33555eobeeoeqbobopoo35oebeo53bebbeobeoqqbee5.45e5e
bqovvbqbqvbbvbbv2b92bevbvvb233.4.4.42boobwbeqfplobbqvb22bbvb223.432.4.32vvovqb2
33
ebeb-421144eo3e2oee23a4e4e4e4b400wee25eee5eo55553eeeo5we444z)DoeD4e-445543234
643343-4-4334555545-4q32555335.6-
433353555433e3e434e3e535433534qqe5543e55e5e33e
3235.4b33owb5355y436336-
43326w46.4.4366vb000bbv&433626.43.43362336233b.4.42232.433336
e3333vq334338Ee333335r43333ee3ee3eeeeeqeee651352ee325555343553.4q632344233334
.1.5v152552v23b2335132112113233b33blvb2g4)13552551v253352312232313.4.3132113133
2
556qpqw5Erq.523554623aq353qqbqopoqbebEqoqqobbqp32233-qp3235eqpqqqwbbqoqoppeopo
goggoeobbeoobEe5eofmooqq5bweoegeoeqqbeeqbgbyeogooeoobqbeobwoeewooeogbbeb
bebbbbeooqo4e3b4z4baezy4eeobeDoqo4beooz)eoq34qbqqeeeDooqebb3bb4bbDbbDoqebb3bb
45535533qe55355y455355e3q33433533834583433eebbev3qbbbb3e3e554eqbb3e3eb3eq
3ev55mq553el3vele555ee3e1513231e35lv33523832552513.45es.513152352612823513321
61333#3PP6P23361PPoe626P331312332311P636666Pe61616P326P33121321312321132166
35535.6352-4-4233283535553525.6335525e23552333325833.63-4-4555-4433532355-
423252352
3q-443833qeff)33333686-4b3333333eeeb33333b5b2553336226362qq326265566433626636
5455eebI513855335583353353833135435qq3355335335433435qq33533854be33844335548
XX7-zE-SET-P-8-.1 UVD-9aNNnw
XXT-92N14
(t7T7os:oN ai Os) .EadavONurriaamax
IliIS'1003'10(1HOMOUUUa0XWOISSZliallUADMOrlaN3q0a0dINDIUUdX0014adaUSUUHarlAaAaa
UUOriNga
NAZONOOONAVdVaVSUS,DIAUSUAVVaaUddVAdOAIDIUMdOdUUdIENNACISHTIUSUYSHAMII3VAMATI
SXOWIASSAANIAM,RdMSdOdIrldSdOrIHMZUMI2AM.1000,111=USHOOXANVIAINVEAdNIZTIACISSOS
ossulednosarmsvrixirrixddOodmOnxwmasxnsisAsmsyunmmunodsAvrisvEsarinias0000so
osessesessAuumememAalAaAANasszulipAAAymaavuzsmOrarisNmyNausimausmAsadAmme
t)ssiIsAmaritmayammswixt)saLat)slitrisravistyckimArisamOnaduAiliwirrivadrirrivI
AdriliN
(sp-ç38 ouTwe)
(E170g:oN ai 02s) rel#613631333336133366#361#3#31133360#631133#3#66e#
oovoobvovqbvoqoqbbbvooqqqqoobbqvbovobbbbvvobbbbvbboobobvbobbvvvbqvbbbqqvbvb
LIOLLORZOZSahlad
6COLZO/ZZOZ OM
WC)2022/027039
PCT/US2021/071017
aqqaaqaaccctcaqqaaqqcctqttcaatqaactqcaqaaaqataaqatqqcqqaqqccttcaqtqaqattqqq
aUgaaaggcgagcgccggaggggcaaggggcacgaUggccULLUccagggl-cicagUacagccaccaaggacacc
ttcgacgcccttcacatgcaggccctgccccctcgctgataa (SEQ ID NO: 1628)
(amino acids)
MALPVTALLLPLALLLHAARPEVOLVESGGGLVKPGGSLRLSCAASGFTFSRYGMSWVRQAPGKRLEWVSTISGG
GTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGG
GGSGGGGSEIVLTQSPATLSLSPGERATLTCSATSSVSYIHWYWRPGQSPRLLIYSTSNLASGIPARFSGSGSG
SDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKITTPAPRPPTPAPTIASULSLRPEACRPAAGGAVH
TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
RVKFSRSADAPAYKOGONOLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPIDEGLFNELQKDKMAEAFSEIG
MKGERRRGKGIIDGLTQGLSTATKUTFDALIIMQALPPR** (SEQ ID NO:1629)
muMNE6-CAR T-8-4-28-3z-1XX
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgaaggtg
gtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtg7agtctctggattcactttc
agtagatatggcatgtcttgggttcgccagactccaggcaagaggctggagtggg7.cgcaaccattagtggtggc
ggtacttacatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctg
LaccUgcaaaUgagcagUcUgaagLcUgaggacacagccaUgUaLcacUgUacaagggaLaacLacggLaggaac
tacgactacggtatggactactggggtcaaggaacctcagtcaccgtctcctcaggcggtggcggatccggcggt
qqcqqatccqqcqqtqqcqqatcccaaattqttctcacccaqtctccaqcaatca7qtctqcatctccaqqqqaq
gaggtcaccctaacctgcagtgccacctcaagtgtaagttacatacactggttccagcagaggccaggcacttct
cccaaactctggatttatagcacatccaacctggcttctggagtccctgttcgct7_cagtggcagtggatatggg
acctcttactctctcacaatcagccgaatggaggctgaagatgctgccacttattactgccagcaaaggagtagt
tccccattcacqttcqqctcqqqqacaaaqttqqaaataaaaacaacaacccctqcccccagacctcctacccca
gcccctacaattgccagccagcctctgagcctgaggcccgaggcttgtagacctgctgctggcggagccgtgcac
accagaggactggatttcgcctgcgacatctacatctgggcgcccttggccgggacttgtggggtccttctcctg
tcactggttatcaccctttactgcaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactcct
agaagacctgggcctaccagaaagcattaccagccctatgccccaccacgcgact7.cgcagcctatcgctccaga
qtqaaqttcaqcaqqaqcqcaqacqcccccqcqtacaaqcaqqqccaqaaccaqcr.ctataacqaqctcaatcta
ggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagar.ggggggaaagccgagaagg
aagaaccctcaggaaggcctgttcaatgaactgcagaaagataagatggcggaggccttcagtgagattgggatg
aaaggcgagcgccggaggggcaaggggcacgatggccttttccagggtctcagtacagccaccaaggacaccttc
gacgcccttcacatgcaggccctgccccctcgctgataa (SEQ ID NO: 1630)
(amino acids)
MALPVTALLLPLALLLHAARPEVKVVESGGDLVKPGGSLKLSCVVSGFTFSRYGMSWVRQTPGKRLEWVATISGG
GTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYHCTRDNYGRNYDYGMDYWGQGTSVTVSSGGGGSGG
GGSGGGGSQIVLTQSPAIMSASPGEEVTLTCSATSSVSYIHWFQQRPGTSPKLWIYSTSNLASGVPVRFSGSGYG
TSYSLTISRMEAEDAATYYCQQRSSSPFTFGSGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH
TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLIISDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR
VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGM
KGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR** (SEQ ID NO: 1631)
huMNE6-CAR T-8-4-28-3z-1XX
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgcagctg
gtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttc
agtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtggg7.ctcaaccattagtggcgga
ggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgccaagaacaccctg
tatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataactatggccgcaac
tatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcggcggtggcggatccggcggt
ggcggatccggcggtggcggatccgaaattgtgttgacacagtctccagccacccr.gtctttgtctccaggggaa
agagccaccctcacctgcagcgccaccagcagtgttagctacatccactggtaccaacagaggcctggccagagc
cccaggctcctcatctatagcacctccaacctggccagcggcatcccagccaggtr.cagtggcagtgggtctggg
agcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcagc
tcccctttcacctttggcagcggcaccaaagtggaaattaaaacaacaacccctgcccccagacctcctacccca
gcccctacaattgccagccagcctctgagcctgaggcccgaggcttgtagacctgctgctggcggagccgtgcac
accagaggactggatttcgcctgcgacatctacatctgggcgcccttggccgggacttgtggggtccttctcctg
tcactqqttatcaccctttactqcaqqaqtaaqaqqaqcaqqctcctqcacaqtqactacatqaacatqactcct
agaagacctgggcctaccagaaagcattaccagccctatgccccaccacgcgact7.cgcagcctatcgctccaga
gtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagc7_ctataacgagctcaatcta
ggacgaagagaggagUacgaUgULLUggacaagagacgUggccgggacccUgaga_ggggggaaagccgagaagg
aagaaccctcaggaaggcctgttcaatgaactgcagaaagataagatggcggaggccttcagtgagattgggatg
315
CA 03187555 2023- 1- 27
WC)2022/027039
PCT/US2021/071017
aaacmcgagcgccqqaqqqgcaacmgcacgatqqccttttccacmtctcaqtacagccaccaaqqacaccttc
gacgcccULcacaUgcaggcccUgcccccUcgcl_gaLaa (SEQ ID NO:1632)
(amino acids)
MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSLRLSCAASGFTESRYGMSWVRQAPGKRLEWVSTISGG
GTYIYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRDNYGRNYDYGMDYWGQGTLVTVSSGGGGSGG
GGSGGGGSEIVLTQSPATL5L5PGERATLTCSATSSVSYTHWYQQRPGQSPRLLIYSTSNLASGIPARFSGSGSG
SDYTLTISSLEPEDFAVYYCQQRSSSPFTFGSGTKVEIKITTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH
TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR
VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLENELQKDKMAEAFSEIGM
KGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR** (SEQ ID NO: 1633)
muMNE6-CAR T-28-28-3z-1XX
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgaaggtg
gtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgr.agtctctggattcactttc
agtagatatggcatgtcttgggttcgccagactccaggcaagaggctggagtgggr.cgcaaccattagtggtggc
ggtacttacatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctg
tacctgcaaatgagcagtctgaagtctgaggacacagccatgtatcactgtacaagggataactacggtaggaac
tacgactacggtatggactactggggtcaaggaacctcagtcaccgtctcctcaggcggtggcggatccggcggt
ggcggetUccggcggUggcggaUcccaaal_l_gl-
LcUcacccagUcUccagcaalca_gUcl_gcaUcUccaggggag
gaggtcaccctaacctgcagtgccacctcaagtgtaagttacatacactggttccagcagaggccaggcacttct
cccaaactctqqatttatagcacatccaacctggcttctggagtccctqttcgct7cagtggcagtggatatcmg
acctcttactctctcacaatcagccgaatggaggctgaagatgctgccacttattactgccagcaaaggagtagt
tccccattcacgttcggctcggggacaaagttggaaataaaaaagcacctgtgtccttctccactgttccccggc
cctagcaagcctttctgggtcctggtggtggtgggcggagtgctggcctgctacagcctgctggtgaccgtggcc
tttatcatcttctqcmtgaggagtaagaggagcaggctcctqcacagtqactacar.gaacatgactcctagaaga
cctgggcctaccagaaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaag
ttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacga
agagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaac
cctcaggaaggcctgttcaatgaactgcagaaagataagatggcggaggccttcagtgagattgggatgaaaggc
gagcgccqqacmgcaaggcmcacgatcmccttttccagggtctcagtacagccaccaaggacaccttcgacqcc
cttcacatgcaggccctgccccctcgctgataa (SEQ ID NO:5045)
(amino acids)
MALPVTALLLPLALLLHAARPEVKVVESGGDLVKPGGSLKLSCVVSGFTFSRYGMSWVRQTPGKRLEWVATISGG
GTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYHCTRDNYGRNYDYGMDYWGQGTSVTVSSGGGGSGG
GGSGGGGSQIVLTQSPAIMSASPGEEVTLTCSATSSVSYTHWFQQRPGTSPKLWIYSTSNLASGVPVRFSGSGYG
TSYSLTISRMEAEDAATYYCQQRSSSPFTEGSGTKLEIKKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA
FIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGR
REEYDVLDKRRGRDPEMGGKPRRKNPQEGLENELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTA7KDTFDA
LHMQALPPR** (SEQ ID NO:5046)
huMNE6-CAR T-28-28-3z-1XX
aUggccULaccaqUqaccgccUl_gcUccUgccgcUggccUl_gcl_gcLccacgccgccaggccgclaggLycagcl_
g
gtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttc
agtaggtatggcatgagctgggtccgccaggctccagggaagaggctggagtggg7ctcaaccattagtggcgga
ggcacctacatatactacccagactcagtgaagggccgattcaccatctccagagacaacgccaagaacaccctg
tatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtaccagagataactatggccgcaac
tatgattatggcatggattattggggccagggcaccctggtgaccgtgagcagcggcggtggcggatccggcggt
ggcggatccggcggtggcggatccgaaattgtgttgacacagtctccagccacccr.gtctttgtctccaggggaa
agagccaccctcacctgcagcgccaccagcagtgttagctacatccactggtaccaacagaggcctggccagagc
cccaggctcctcatctatagcacctccaacctggccagcggcatcccagccaggt7_cagtggcagtgggtctggg
agcgactacactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcagc
tcccctttcacctttggcagcggcaccaaagtggaaattaaaaagcacctgtgtccttctccactgttccccggc
cctagcaagcctttctgggtcctggtggtggtgggcggagtgctggcctgctacagcctgctggtgaccgtggcc
tttatcatcttctgggtgaggagtaagaggagcaggctcctgcacagtgactacaT.gaacatgactcctagaaga
cctgggcctaccagaaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaag
ttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacga
agagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaac
cctcaqqaacmcctqttcaatqaactqcacraaagataagatqqcqqaqqccttcaqtqagattqqqatqaaacmc
gagcgccggaggggcaaggggcacgatggccttttccagggtetcagtacagccaccaaggacaccttcgacgcc
cttcacatgcaggccctgccccctcgctgataa (SEQ ID N0:5047)
(amino acids)
316
CA 03187555 2023- 1- 27
LZ -Z0Z GSSL9t0 VO
LIE
eobeoleoleoqqoquopebbagoopaboeepebeEeobloebobeoebooebleovbbeboqqblooppebepe
eobeZvoleboleollbloblffmooeboeeblooeebbooqeblbobebwbeeobeeebbwbevobbolloe
4boobfmooboobovoolob-Lobqloobbqoboobloolob4-Looboo26162332113obbleoot.00.6p-
405
4yy000p6Q6106oloo6Poollobool066600pol0612106101Poolool06111161o6oPooll0006op610
66loo6
.D4eDe4DeeD4De44454c)DeDeDee4e4e4DDeDDC)4DDoe4D4DDDe44Dv4oeeDvD4e444
eoollqqlleollobbebeobloellleo3111111eollobbebeobloellleo311111qeollobbebeob
wv111P331111-41Polqobbebv3613211123o1111qq231136626235132111233-4.11111Pollob
(VNG)
81 'II 9X ToMVeIN EcixoeI
(6E9T:ON GI OS) eelebloebbeboeebe0616
33vollblvolvoZvobooebobbbl32Pb3ebbeZ2P2b2eblo3lPblobve3llfiloo25663526P22626
obloobbloolloelobb5eboelobeobeobebeElllbeoblvfmeoeeoeboeoobb000frabobebbobeo
oggoggyquoquovibobubuyoovoybbyvoquouvopbboob000vvbqububuvuoggobvoquogybuyouv
bybob4obeb4000yobeLY_ebeybeboblIbeyb-
4511ollolleooybIlboobbIlyobbybelloobeoobeoebbey
oelblvobeoleoqeollo-Leooebbol0000boeeoebebeoblovbabeoebooebleoebbebollbl0000
efmovvobbemolvbolvo-L16-4.3616fmoovbovvbloovebboolvbqbobvbweivvo62226613622366
3lq3eqbo3bbe33b33b3e33q3bwbqq335Eq3b33bq333bqqo3b33efrabe33eqq33bbqe00E.00
beloblee000ebebebo400fmoogebooebbbooeoebeebegeoogooe54.4445.4oboeo3g23oboebeb
bqooboqebeoqbooeebqbeqqq.boqobebeobeuqeqeqoobbebbbqbboeqfrilobbeqqbeqoeebeobq
oe111103311111-poollobbebeobloPillepoiliiilloollobbebeobloellieopliiilleollobb
(vNa)
81-rn-cx-1omvaN-Edx0,11
(8E9T:ON
ai Os) ..aaN0AmawisuaormaaNylirmarlausmzovriaxsaAsssaaOwNNaNsansuagai
IGSMIGMINGddNNEMaSIINNEDS'IISIME3MASLIAI/NOUdOSGMXNSII3IZUdYNG
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(PT L)w oupme)
(LE9T:ON GI OES) ee4e540ebbeboee
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(vNa)
paqeATlow 81-'II
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(vNa)
dTuTw laolowoad TEuTuTN
(cE9T:ON GI OES) 05400402204002245e020402402042244404040
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(dzaiw) aaaowoad zai Tew-cuTN
(17c9T:oN ai 02s) bpooq263326563323262ebvq2oowoebqlqqfywbov
ooqvooboebebblooboqebeoqbooee5.452.4.4.45owbebeobveqeqewo552555.46boeq5.45o552.
4
(vNa)
(Awow) aaaowoad AND Tew-cuTN
(81713;:oN ai Os) ..uddrivOmpi
vaamaxzvisasOormausNouuuzsmNsiasavaviziamOzaNazondmmudxsowaausuumaulaxami
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00S0000SSAIAUSOOOMXGNOXGXNUOXNGUIOXXAVIGaVUUSNNOWLINMVNGUSIZZUOMASGWILIO
DOSIMSAMaZUMOdVOTIAMSNDAUSZIAOSVVOSUUZSOOdXAZOODSaNIOAadUVVIVIZZWIdggrIVIAdrIVN
LIOILOITZOZSIIMOd 6COLZO/ZZOZ OAA
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4eboeq6e55ebe5e
eb3vabv131223.43626322121313.Emoovvfmoo6562362vov1636333336326236362662362311
beebqbebebweebqbqebbebbeebeebeebeebeooqqaeboobwbeqbqobbqebeebbebveowewe
333 33323
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2.4.462
qeqobbqoqeoeqEeoqbqbeeeeobeoobbbeobqeowqeoovoo55.6252o5555-wwqe-45-455eqqooq
4o1323vw4beoe3eo4ebbqqeoeb3o4ebb3bb4bb3bb3o4ebb313b4bb3bboo4ebb3bb4bb3bb424
3-45--b-4-45-4553555535555-43-45-43-4-4-45384
peqqeeqe55bbbEbl13ebee3b153ellelble33bb3e3ebbvbl3qbbebl3l5e3bebleee35l33el
b3oovo225223o6-
42232beb233.43.423323.4.425365562y6.46.162325233.42.432q3.4232.4.432.466
-455-452-452-4-423o2835555-45e5.6-4obbebevbebbooqovfmooboqq555-4-4o-45-
42oobqww55-452
oqqqovoqqebbqowobeobqbqooqoqovvebq000qbbbvbbwobvvbqbeqqobbebbbbbvoqbebbeb
blobepolbbeMpobelepobooboepoloblobqqopbbloboobqopqoblloobooeblbeopelloobble
(vNa)
8T-7I-DLEN-Edx0a-zg-SST-17-8-ZONNnw
ZONN
eqez-Ea3 jo WILLI UT sucrmeqnw XXT -/+ 81-71 aTcuonpuT 14-4-Fm eaTpoqTque wag
(9T :Q ai oss) eele5we55e5ovebe3bqbooe3qq5qeo4e35vo53oe535
bblovveovelemelvvveovvelqoowelqabvvo4q5133255bobvEyevvfmbobloobbqoo4q3213555253
2
lo6po6RoEmbebillEmoblebeeapeoebapooEb000616o6e66o6eooliolleleoleoe6o6e6eeoo
vorbbreoqeorroy5boob000rybqybrEmyroqlobroqeoqebeeovebebobw5rbwoovobeoqebe
e6e6o616ee6i13lo1312332616336612366ebe1336233623266ee3el6leo6poleoleolioleoo
ebbol0000boeeoebebeobloebobeoebooebleoebbebollbl0000ebeoeeobbbeoleboleollbq
oble,fmoo2532yZ1332955331y51.53525135y23622256135y236531132153355233533532331
obaobaa006620500ba0010541.3obooebabeooeaaoobbavnnvonnp-r)nippmembeboaobeo
oqebooebbbooeoebeebeqeoowoebqq-4-45-4oboeooqeooboebebbwoboqebeoqbooeebqbeqqq.
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qlbloveeeebbeEblbobbeebeoeleolllbweeeeebbebblbobbeebeoeleo.4115.4oeeeeebbebb
(vNa)
8T-r1I-9X-T3ivaN-Z-7I
(11791:ON aiOas) eplabwebbeboeebeoblbopeollble
olvobvobooeboEbbweeboebbebeeebeebloolebwbevollblooebbbobebeeebebobloobblo
p.4132.4obbemboy.43623523626y6.4.4.46yobqyby23223253yoobb000bqbobybbob233.4.43.
4.42.42
olvoebobebeeooeoebbeeo4eoeeoebboob000eebqebebeveolwbeoleolebeeoeebeboblobe
bq000vobeoqebeebebobqbee6-45wwqeooebqboobbqvobbebewobeoobeoebbeeoeqbqeobe
3123123113123326631333363223262623613#636#326332612326626311613333262022366
beoleboleollb.4351b5e3oeboeebwoeebboolebqbobvblobeeobeeebblobeeobbolloelboo
5b23363363233.4obqobq-Loobbqoboobwo.435.4-
43353325.462332.4.43366.420000bploblppo
00v5ebebowobeopqebooebbboopoebuebeqpooqopeblqqqbwbopooqpooboebebbwoboqpb
eogbooeebqbeqqqbowbe6eoUeeqeqewobbebbbqbboyqUqUobbeqqbyweqUobbeybeoeqeoq
qlbloveeeebbeEblbobbeebeoeleolllbloveveebbebblbobbeebeoeleoqq15.4oeeeeebbebb
(VN(1)
8T-qI-EX-T3ivaN-z-rn
(0D,91:0N GI Os) yellybIloebbyboyebyoblIbooyoll
gbleoleobeobooebobbbloeeboebbebeeebeebloogeblobeeoglblooebbbobebeeebebobloo
bbloolgovlobbZyboygobeobyob26261115goblybevoevopbovoobboopbqbobebbobvpollog
leleolpop6o6eEevooeop66proleopeop66o3600pee6le6p6pepolio6poleole6peopp6e636
qobvbwoovobvoqvbvvbvbobqbvvbqbqoqoqvoovbqboobbqvobbvbvqoobvoobvovbbvvovqbq
LIOILOITZOZSIIMOd
6COLZO/ZZOZ OAA
LZ -Z0Z GSSL9t0 VO
61E
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ebbvfmooeoeoblboobebbobbqobqobqooebeqbqqobbeb000bbebqoobebqoqoofmoofmoobqqe
eovl3333be3333eq33q33e5m33333b3333ge3ee3e33leqb3ee33e35.435.4e5.4.35bboeeeeqe
65106ppoop6E6666p66pii6opoll600lio6p666p16povo6pol6lopliplooppo6lo6ip66p6
bebbe.65-4.6-400qeooqeoeeowooeoqqoe5eoe5bEr4o-4565-45ea65-45eoqqbbeoobwoo-
45555q3q
evfml3ovvoolvoblloq213.4.23133.432vv000v33b232562332.526232233216bw23612121162
lewbbloqeoeqEeoqbqbeeeeobeoobbbeobqeoqoqeoovoobbbebeobbbbqoqoqeqbqbbeqqooq
35e3v3qbe3eoe3qe5-Lbqqe3eb33qebb355.45b35533lebb35bqbb3bb33qebb35blbb355qeq
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(VNa)
8T-ZI-Z,VaN-Edx0a-zE-8Z-8-Z3NNnw
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efmobvv121213o66266616bovlblbobb21162132262oblow41.423311111123qqoffmfmoblov
aqqvoolqqqqqeoqqobbebeobweqqqeooqqqqqqeolqobbooeq6boobbqoeeqoobbqoqoqqqeoe
85233.63eeeqq15eeqv5.43631333335.433355835qe383.4.433353853e.433838568833833583
8
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62613332bbb33tb163962622386.6.4111blvbov16866868682.6386621312v3l3b26322121313
5833E.E.5833555835883eqb3b33333b3ebe363bebbe3b23qqbeebq58585q388646qvbbebbeeb
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beolbwellelleeeobwelebleelobeebbl6looleelleeoeol000eolllebooebbblolbbblbe
366o6voll66poo6v000-6666o6p6p66-pippoolpoffyloovilip6looloppplooiooppop66poo
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bbebeoebbeoo4o4b4boobb4400400beoo4o4be000ebqob4b44eoeboo4ebbobb4bbobboo4ebb
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45
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(vNa)
8T-qI-I.V.RN-Edx03-zg-SST-17-8-ZONIAIng
(69T:ON aI OaS) eele5l3e55e53e85e054500
epqlbleoleobeobooebobbblpeeboebbebeuebeeblooleblobeeollblopebbbobebeeebebob
wobbloolloelobbbeboelobvobeobebeb1115e35.4efmeoeeoeboeoobb000bqbobvbbobeool
.1.3132.4yoqyoybobvfmvooe3265223.423223vbboob000y26.42.6262y23.4.43623q23.4vfmy
32262
bobqobebl000eobeolebeebe6351522515lowleooebqboobbqe35525213352335voebbeeoe
-45.4vobeoqeoqeoqqoqeooebbow000boeeoebebeobwebobeoebooebqeoebbeboqqbw000eb
eoepo666eole6olvoll61.36166eooe6oee61008865031851506851058805888551058805501
welboobbeoobooboeoo-Lobwbgloobbloboobloo4oblloobooeblbeooelgoobble00y0abe
1061ep000p6ebebowobeooqvbooebbbooeoebeebeqeoowoebqqqqbwboeooqeooboebebbq
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blobvoolbbvbboobbvooboobovooqobloblloobbqoboobloolobllooboovbqb2oovqloobbly
(vNa)
8T-7I-.11/3N-Edx03-XXTzE-SET-P-8-ZONGinw
(91,91:01,1 ai cos) eeeEE,E,E.4
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bbveoleoeeoebEoob000eeblv5ebeeeollo5eoleo4ebeeoeebeboblobebl000eobeolebeebe
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436p5pobvp4v4p43355v5554653Q-4545355p445v4ovv5v3543v444v33444444v344355v5v35
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61o6po6166p660066poo6006opoolo6lo6iloo6510600bloolo6iloo600p616poopiloo661p
(vNa)
8T-UI-Zvaisi-Edxoa-zE-8Z-8-ZONIAIng
(GD,9T:ON alOES) eelebloeb5e60eebe051
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ovfmovvobbb231263.493.4-
46.435.1662332bovvb.433226533.42&463526.4obv23522256.4362236
bollovlboo5beoobooboeooqoblobqloobblobooblooloblloo5ooeblbwooelloobbleoopo
obvloblee000ezebeboloobeoo4ebooebbbooeoe5ee6v4eoo400e5444454oboeoo4eooboebe
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000wobpoqvfmvEv5o545vebqbqoqoqvoopbqboobbqvoffmfmwobvoofmovffmvov-45-4pobvoq
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(vNa)
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(vNa)
8T-r1I-mvam-cdx0a-xxize-OZ-8Z-9Nwnq
(Tsos:0N ai Oas) eelebloebbeboevbe054.600
1?o1161rolPo6PobooP6o66613PP6oP66P6PPP6PPo1oolP6106PPoliblooP66636P6PPP6P6o6
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366per6le66611e6e616poiloo66p663661y6ep1e6pee6ro613pe61ep31161oo66pe66p3133
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bobqbvvbqbqoqoqgoopbqboobbquobbsbuqoobvoobuovbbvuovqbquobvoqvo4voqqoqvooubb
pqoppoboevoebefmobwebobypebooebgeoebbebo446-4poppebeoveobbbvp4vbogyo-4454p54
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pnoopfinnoopoenppnpipopioopnillinionopopipponopnpfinioonolpnpoinooppninpilino
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523523.421bbloobblope13226P2bPopevobvpolowl.bloblbob252335235P622obloo2312332
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obbqbbobbebbobeebbobbqbbobbqowbeoqbeoeo4bbweoeobbbeooWAADqqewoboqqbeboeb
ovq.ovbovvovxo4v000eDDebezYoD444e4oe4D4DooboowoeDDebooDeDeD40004oeeD4eDeoD4o
pelbloobeoeebeeooboeeoebbboobeoleopeollebeobbbveblblollebobeoewelooeobeobb
pobebyobbolvlogoobg-LbbblvP51.433552evvbfiloppo5bvp2boolbbblobP61233bovqP32352
111opyolloBBDEepoBooBlfrapBefrapeBefraololebboBErappBeefrafralpeBBDB6DBfrapleef
ralB
fy4olovobqbbvbboobbvooboobovooqobqobqqoobb4oboobwowbqqoof000vbqbvoovqqoobbqv
(Na)
8T-UI-IVabl-Edxoa-z E-EST-17-8-0TVOZn4
(6g91:oN ai 02s) eele5loebbebovebeafrabooeollbleoaeobvobooe5o5
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eovUmeoleoeeoebbooboopet,blebebeeeollobeoqeolebeeoeebeboblobebwoovobeolebe
efmeob.452v515.431319poeblboob512355v52133523352ov5.6223v15123523q2olvollolvoo
ebboa0000boeeoebebeobloebobeoebooebaeoebbeboaaba0000ebeoeeobbbeoaeboaeoaaba
35.455yooybovvEgoovy5boogy535y5-435pyo5yyp56-435yy3553-4-43Q-
453356yoo5oo5oyoog
3613611.0366136o36133-361looboop616poopiloo661e00-6.005e405-4220002526e6oloo6e
ooqvUooebbbopeoebeebe4eoo400e5444454oboeoo4eooboebe55400bo4ebeolLooee545e44
qb0l0bebe0beeqele10055e5551.65oelblbobbelqbeloevbeobloellleoollqqqleollo55e5
eoblovqqqeop.41.4-4.4.4P3.4-
4356P5235.432.4.4.4233.4q1:41323.4.4obboovqbboobbqp2233355.43.43
-4-41.2ovebeoobweeeqqbeeqvbqobow0000bq000bbeobqeoeoqq000boeboewoeoebbeeooe
oobeovqbeow4E552ooeqqq=55-4eboeo555522o555525boobobebobbee25-42555-4-42525-452
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ebvc0000blopoo2P32P326226132Pbblobet,23225boobobblqqopebloobPooqbloo21523323
obileipel6160066131p6pe6o066p361610136poleepe6l000pollop600ee6606e366.16e366
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1.5olobebeobeeleqeqopbbe555.1653e.45.4bobbeqqbeloevbeobqoeqqqeooqqq4.4.4v3.4.43
55e5
eoblovq.4.4v33.41.4-4.4.493.4-
4obbvb2ob.432.4.4.4233.4qq.41323.4.4obboovqbboobbqp2233obb.43.43
qqqwpwabupobwwwwqqabeuqubwbowoopobqopobbwobqwpwoqqopobowbowqopwowbbwwoow
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bbbbqvbebq000ebbboobbqbovbebeeoebbqqqqbqeboeqbvbbebebeeboebbeqo4eeoqobeboee
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ponollopnonoenoRp000nipl000npooplienneppnpooRioonnnioopnppnelooiopnipoppnip
oegoebqbeoeobwogobbeobebbebeeqbebbeobweggwooeogeqq5bweogbwogoggoo-45555
gel4qovEibboobblwoobobbb434eoe4o4eoe5obwobo444ebbwebbebeooeoeob4boobebbobb
wElqaellooebeqZqqobbeb000bbebloobebqoqoofmoobvoobqqeeoeq0000fm0000eqooqooebe
00000bw000veovvoveboeevolvfmbblbbevoovbbbvbbobbolwoebloobvobebloovlbvoovo
qbloellelleepobloelebleelobeebblblopleelleepeoloppeolllebopebbblolbbblbeobb
ofmo44552oofm00045555obeyebebeooeo52=5564o2444e5woweeewowoeeoebbeooeee
beobeo-4245643o65400e4oeebeebeooeeofmoo4o246.4ob4bobefmoobeobebeeobwoeo4eooe
pobbbvbeoebbeooqoqb-Lboobbqqooqoofmooqoqbe000vbqobqbqqeoebobeobbebbobbqbbooq
obblbbobbebboEeebbobblbbobblolobeolEeoeoqbbloeoeobbbeoobbbbllewobollbeboeb
oP1oP60PP0P101PoopP66e6Poo61611P1oP1616poboopoP66P6006P6PEll000loPP61P6Po610
ot-45400beoeefmeooboeeoebBboobeo4eoovo4426eobbbye5454o4425ofmoe4oewoeobeobb
oobefmobbo4e4o4=5455b4ve54-400bbeevebbw000bbvoeboo45554o52542ooboe4eoeobe
aqqooeoqqobboteooboobabqoDebloebebqoqoqebbobbqoobeebqbqqoebbobbobbqoqeebqqb
blobeoblbbebboobbeopbooboeooqobloblloobbwboobloolobllooboovbqbeooelloobble
(Vtla)
8T-7I-ile3N-gdwoz-7 C-8Z-8-OTVOZnq
(L99L:oN ui oas) ee4eD4oebbefweebeoD4Do.oeo444eo4woDeobooe
bobbbweeboebbebeeebeeblooleblobeeollblooebbbobebeeebebobloobbwolloelobbbe
bovlobPobeobeZ2511qfmoblvb2232232bovoobboop616362bbobepollollvqeoleo2636262
eppepyBBeepleoeepeBBooboopeebleBeBeeepllobeoleplebeepeeBeEpoBlobefrapopeoBeol
vbvpbvbob4bvvb4b4o4o4poovb4boobb4vobbvbv400bpoobvovbbvvov4b42obvo4po4vo44o4
eooebbog0000boeeoebebeobwebobeoebooebgeoebbybo44.61t0000ebeoeeobbbeogebogeog
4bloblbbeooeboeeblopeebbooleblbobeblobeeobeevbblobeeobbolloelboobbvoobooboe
oolobloblloobbwboob-Loo4361133633261523324-Loobb1200v00b210b122000vb2b2bolo
obeopqwboopbbEoppowbeebeqppoqoppbqqqqbqpbowooqppabowbebbqopboqpbwolbooppbqb
e4.445ogobebeoEee4e4e400bbefx65455oe4E4bobbe4gbeweebeobwe444e=444444eo44o5
bwbwobliow-mwooqqqqaqwDqqobbebwobqowqqqwznymqqqwoliqobboowqbboobbqDwwqoobbq
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povoofmovlbvowlbbfmooellwobbleboeobbbbeeobbbbvbboobobvbobbeveblvbbblqvbvb
16P3P10066P66o661P6ePle6PPP6PobloPPE1PPoP1610066PP66P010ooPP6PP66Pe6P6006PP
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35545.6obbe553E2855355-455355ww5m3q5eoeo455-weoeo.6552oo5555qqe400b3qqbeboeb
oeweboeeoewle000ebbefmoobqbqqeweqbqfpooboovovbbeboobebeepwooweebqebeobqo
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(visla)
8T-qI-IVaN-Edx03-XXIzE-EST-17-8-0TVozn4
(s99T:oN ai oss) eeqe&webbebovebeobqbooeo.4.45.423qeofmobooeb3b
65.4o225o255252285285-woqv5qobeeo-4-45-woe5563525veebebofywobbwoqqoeqo55525oe
l3U23be3bebebq336eobaebeeoeeoeboeopbb000babobebbobeoo33o33e3eoleoebobebeeoo
eovffmeoleoeeoebbooboopev5lebebeeeollobeoqeolebeeoeebeboblobebl000eobeolebe
25253&452e515.43131Popeblboobblvobbe521335e33523P5.622pelblvobeoleoleollolvoo
p66ol00006opeop6e6eo6lop6o6pop600p6leop66e6o116l0000p6popeo666eole6oleoll61
obqbbvooebovebwoeebbooqvbqbobebqobveobeeebbqobeeobboqweqboobbeoobooboeooq
.354:)54400DD4ofy.DoD400Lo544zrobooeD4Dwooe44o.3554e00-
poobploblpp300262DeboxooDe
oolvbooebbbooeoebeebeqeoowoe5.4.4.4.45.4353eooqeooboebe55.43353.4ebeoqbooee5.45
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qbolobvb2obvv12-
42.43obbvbbbqbbovqbqbobbvqqb2lovvb2obwvqq.4233.4.4q4.4.4voqqobbvb
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1.4.4y32252335432224qbeeqe54353433333543336523542323443335325343332325522332
335232.4523.43.4t5fmopaT433355.4253835555223555625533.635253552225.42655.4.4252
5.452
oqloobbebbobblebeeqebeeebeafrweebqeeoqq5qpobbet,55eowooeebeebbeebeboobeee55
bbbblvb2bl000vbbboobblbovb2bvvovbb1111b12bovlbvbb2b2bv2bovbbeloqvvolobvb322
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#6#3333361333322322326#26132P6613622232266336366111332613362331613321623323
obggegoe-45-45o355-4ogebeeboobbeobqbqogobeogeeoefywooeoggoeboovebbobeobbobeobb
e323qqe5e3e533353535535e2e5e5e33e3523355543e333e5335335ee3333345e33553335ee
523523123553335533323322622.6233823523333235.435353.625e335835e5e2353333532332
blbeevbebobbloblo15-Lblobbloobeobeb000bebepeoeblebleoleleeoolebbobbebbobbobe
25535.625.635543.4255255365155.435.43.4.4.45232.4q55132323.6552335555.4.4233353
.4.4525325
ot-weboeeoewle000ebbebeoo5-45-wwwe-45-42ooboovqybbebobebbob-wobeo525-425eobqo
3235333323225535353223255533523323323-4425235552253533-4425352323323332352355
33625235.6332312335315553225.61325ebeebe63333325235.63338553358538335383838358
qlloovollobboEeoobooblblobefrwbeeblowlebbobbloobeellbbloebbobbobbloleebblb
5.435.425.162255335523353353233335435.4.43355335335.433.435.4.43353326.452332.4
.43355.42
(vNa)
8T-r1I-IILIN-Edx001-XXTzE-EET-V-8-0ilioznw
(g99T:ON alOES) eeqebqoebbeboyebeobqbooeollbleoleobeobooebob
bblovvboebbvfmvveivvbloolvblobvvoqqbqoorbbbobebvP2b2bobloofiblool.432135552532
loemobpo6e6e61116pobleberopeopboppobboop6i6o6e66o6poogioiipieogeop6o6p6epoo
govfobvvoqvovvovbboob000vvbqvbvbvvvoqqobvogvoqvbvvovvbs,bobqobvbwoovobvoqvbv
LIOILOITZOZSIILIOd 6COLZO/ZZOZ OM
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io6-136ioov6viET-1066106poo66126-1=6106-
loipobloop6voobiielooPipopoblopoopeippipolobe
000006i0000PpoPlooPe6oPPPoiP6P66-166pPooP666P66o66o-noop6ioo6Po6P6iooPi6PooPo
-1.5.4ovqqvqqvvvobwv4v5-42v4obvt,55-45-400qvt-442vovoq000voqqqvboov555-4o-4555-
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.DDe.D44DDeDe.D.D4D5DDDeeeDeDeeDeDD51.-444eD4.4eee4.4oeeyDDeeee
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offrabbobbebboEeeb53551653551olobeolEeoeoabbloeoeobbbe33555511ea=53115e5oe5
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qlloovollobboZvoobooblblob2.6.4325261o1312563651336226151132bbobbobblo1P26116
bl3be3blbbebboobbeooboob3e=q35135113obbaobooblooloblloobooeblbeoovlwobbae
(vNa)
8I-UI-Iliam-Edx03-XXIzE-Elz-8-0-Peoznq
(g991:ON ai pas) vvqvbqovaEyebovvfmoblboovollbqvolvaftaboae
bob55.43eeboebEebeeebeebqooqebqofteo44.64opebbbobebeeebe535.43355woqweqoabele
boygobyafmobvEy5-44q5yobqybyvoyvoyb3yoobboo36-4535y.6535yooggoggygyogyoybobyby
popeovbbepoqepeeoebboob000eebqebebeeeoqqobeoleoqebeeoeebebobwbebwooeobeoq
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lleolilloll
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qbqobqbbeooeboeebwoeebbooqebqbobebqobeeobeevbbqobeeobboqweqboobbvoobooboe
33.435.436.11.336bl3bo35-L33.1.35.11.33633wbqft33vq-L33bbqv00.6.00.62-405-
42200025eft63w
oftooqebooebbEooeoebeebeqeoowoe54-44-45-4oboeooqvooboebebbwoboqebeo-45=225-45
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bebeobwellleoollqq-Lleollobbebeobloellle=111111eoqlobbooelbboobbloeeloobbq
313111P3veftoobl32PellqbvP126.43b3loo3336q33366v36.4232311333bo25311=2326622
3oPoo62oP16Po1316662oo1111=661263po66662Po6666266=636263662PP612666112626
qbvoqwobbebbobbqebeeqebveebeobweebqeeo4qbwoktheektheowooeebeebbewbeboobee
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33be3boll32bo63233Po3ooblPl333be33ellP3bvevbe33Pl33bb6l3ovfteft.4.33132612322
blypeloefraBepeofrappobbeoBebbebeelbeBBeofrapellloopeolellBErapeo2bloololloolb
bbbqbqqovbbboobbqwoobobbbqoqvovqoqvovbobwoboqqqvbbqovbbvbvoovovobqboobvbb
3ffy4ob-43b4oyZe4b-443bbybopobbyb4obeb-43-400fmooftoob-4-4eyoe-43000fm0000e-
400-400
eft00000bl0000eeoeeoebeebweebblobeeeoeeb633636611.4332blooftooqblooelbeooeo
3fy.4121o2151boobbloqvbegb3obbv36161313523q223v61333231132b3ovebboftobboftobb
e323llebele533351535536evebe523323523355513el3leblobl3beel33l3a5e33553335ee
332332
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o3be6p3563gy3Evoo5456bqyy5.6-43y5y5yp5y53333p5m35633gy55-435e5gy33532-4y32352
11130P3113663EP33633616136P6136pe610131P6636613.06PP116613P66366366131PP6616
bq35ge5qbeebboobbeopbooboepogobwbqq33.6.6goboobqopqobqqopbopebqbeopeggpobbge
(vNa)
6T-'1I-DiIN-Edx0I-XXIzE-8Z-8-0-PiOznw
(T799T:oN ai 02s) pequbwebbebopebeobqboopoqqbqpoqeobeoboopbobbbq
322532552522252254334e54obee34454=e55635e5e2e5e5354=554=44oe4o555e53e435
2362362626111Z23bl2f2e32v32b3233bb333blb3b2bb3b233.4131121231232b3bvb2233232
552234e3ee3e5E335333ee54ebe6eee3443Ee34233efte3ee6e535435e54333e35v34e5ee5e
boblftebqbqoqoqeoovb.453355.4eobbebewobeoobeoebbeeoeqbqeobeoqeoqeoqloqeooebb
ow000bovpovfm5v3543v5ofmovboov5-4vopbbv53-4-45-1.333ovftovvobbftoqvboqpoq-45-
435-4
bbyooybovebqoovebbooqebqbobebqofteoEeeebbqofteobboqqoeqboobbeoobooboeooqobq
oblloobbloboobwolob-Llooboovbqb23321133664200.E.00beloblee000ebeembolooftool
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3
io6e6pobeeipivioo66e666-1663P-16-1636610ii6PioPPEPo6-
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(vNa)
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bobwbvpoosoubbuvoqpouwoubboob000vvbqububvpuoqqobuoquoqubuvolapbpbobwbvbwoo
eobeogebvelovbobgbveb-
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eollogeooebbow000boeeoebebeobloebobeoebooebleoebbebollbl0000ebeoeyobbbeole
bolvolgbloblbZ2332532ebloo226633125163525.1.36y236222661362235631.4.32163366233
boofoeo3.4351351.43355-
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bebowobeooqeEooebbbooeovbeebeqeoowo25-4q1.45-4oboeooqeooboe5255woboqebeoqbo
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epqqabbebeobwegggeopqqqqqqeoggobbebeobqoeqqqeoogglqqqeoggobbooeqbboobbqpee
w3bblolollleoeebe33b3evellqbeeleb1353133333.6.433obbv35le3e3ll33353ebollooe
op66pypopoo6popiElpo-01666poolilloo6ElpElopp6666rpo6666p6630606p6366epp6ip666
qqvbebqbeoqq=55255o55-4.ebeeqebeeebeobweebqevoqqbwobbeebbeoq000eebeebbeebe
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(vNa)
8T -ZI-MVal-Edx03-XXTzE-8Z-8Z-01VOznw
(9991:0N ai 02s) eeae6aoebbeb3eebeobqbooeoaab2e02e0be0b00eb3b
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(vNa)
8T-qI-MV3N-Edx0a-zE-SEIT-D-8-93GZnw
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(T7gog:oN ai 02s) eelebloebbeboeebeoblbooeoalfrae01e05e05
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bwobeoeebeeoobovegybebeyollogeooeoggebeobbbeybgbobeoeb0000elloggooeoeoebeebb
obboevobeolepoeobeo-Lbbblvebbwobbbeeobblopoobbvobboblbbblobebleobboelobeobe
ollvovollobboZvoobooboblobvb.43252bloobvpbbvbbwobvvoqbbqovbbebbobblo1P26616
6136e36166e66o366pop6opEopoolo6136113066106306loolobliooboop616poopiloo661e
(vNa)
LIOILOITZOZSIILIOd
6COLZO/ZZOZ OAA
LZ -Z0Z GSSL9t0 VO
La
oovoobeoeqbeoloqb55e3o4.41.43355.4e5oeobbbbeeobbbbebboobobebobbeeebqeb55.4.4e5e
5
Ilmoqqoa55255o55.42522-
425vvvfm35.432v5.4vvoqq513355225523.43332v622552v52533522
e5655.654ebebl000e555o355.4boebebeeoe65444q54e5oe4bebbebebyeboebbe4o4evowbeb
oeegegogobeooeebeoobbbeobeeoegbob00000boebeobobebbeobeogqbeebqbebebqoee5-45-4
ebbebbP2bevbegbP2bPoolqqvbooblobvlblobblvb22bbvb2voloelo222321beopeb2612111
eooveoeeeoqqeleqe.45-Looqoveebeeebeob5553eeeoblovqqwooeoqeqq5bweoqbwowqqo
pqbbbbqb.4.432bbboobb-Lwoobobbb.43-423Q-
43.4232boblooboqqq2bb.432bbeb233232obqboob
ebbobbqabqobwowbwqbqqobbwboopbbwbwobwbqowobpoobwoobqqwwowqoppobwoopowqop
gooybe00000bg0000eeoeeoeveeogeeebbqobeeooeobbe55-4.65oggboebeogooqqqqeoeoe-455
ewaecoqobqqwqqwqqqfpwbbecrnebbefoqoffowbfoqfpwbeorpeoqewwwfoqowowzrmwbwowbbbeoq
wbb
IlmobbloeoqqbEeoebwooqbvbbqowebbweeeqoqbqbbqoqeloqeewobobeeeooqoqbeoobb
eoobbvb2ovqqbqqbblq22b.4.11v1232bv22bblvbqbelvb211oloobvbvolbveoqb223b1131312
poloonpooepoennlipopplinnoinillopolopoolopnp000pnipninlinipnoolpfinonnpfinonn
obey5.6-45.6ebbobbqogebbebbobbqbbeobwwqbweo.455-4owebbbeeoobbbbweggobqq-455-4
obbElebboebbbqoebeoebeeq5-45qeqqqeqbqt.00beoeoebbvbqoqbeebqoqbeobebqeweobqoqeq
bwoovoeebeeoobqeeoebebeoowleooeoqqebobbbbevbqbqbeoefmooqeqoqqooeoeoebeq55
qbblvvqfmllvoovvobo-Lbbblfmbbqobbvbevovbvooloebvoobollbbbllolbleebblvq1bvlbv
olllouolllbblolopbeoblbloololoeueblopolbbbebblopbeeblbelloebebbbbblolbebblb
bqobeo5-4.652553o5fmoobooboeoogobwbgwobbwboobwogobqwoboo25-462oovqqoobbge
(vNa)
8T-ZI-IliaN-Edx02-XXTzE-SST-17-8-93gZnw
(0L91:ON (II 02S) PP1P6101966P6oPP6P061600P011611901P06P06
ooybobbbqoeeboe5bebeeebeybgooge5gobveo-446-4=2555obebeeebebobqoobbwoqwegob
bbv5o2qobeobeo52525-4-45235-4ebeeoeeovboeoobb0005-45obebbobeooqqoqqeqvoqeoebob
eUevooeoeff)eeoqeoeeoe6booD000ee6qe6eUeeeolqoUeoqeoleUeeoveUebo64o6v6wooeof)
eolebvebeboblEeeb15-Lowlvooeblboobbleobbebeloobeoobeoebbeeovlbqeobeoleoleol
qplepoPbbolopoobo2P32625vobloybobvogboo25.1.23ebbP53-4151333325232235552312531
poll6lo6166poop6opebioopy6600le616o6e6lo6peoberp661o6epo66olloel6=66poo600
bovoowfoqa6.4.40055.4oboofoqooqa6.4.400bootToqfmoovqqooffoqt.00.6.005e4054ee000
ebefotTo
oloobvpoleboovbbbopeoebvvb21Poolopeb1111bwbovoolvoobovb2bblopbolvb23163322
bqbvqqq53.435eEeobee-
Leqewobbe555.45boeqbqbobbvqqbeweebeobweqqqe333.4.4.4.4.4e3.4
qobbybyob.432.41.4yooq-
Lqqqqyoqqobbvb2ob.432.4q1.233.4.4.4.4.4q23.4.4obbooeqbboobb.4322.433
55-4o41,11-44wows,Zwpz)bwwww4-4-45wegwbgoboqp:xxx,bwooffmQ54wowo-
44ppobps,bps,goowowb
bevoovoobeoegEeowq555eooe-moobbge5oeobbbbeyobbbbebboobobebobbeeebgebbbqqe
bEfrifmovqopbbubbobbav6vvqpbvpvbwobqovEtiqvvovqbqopbbvvbbvpqpoovvbvvbbvpbvboo
bevv5.655.65.4ebefywooebbboo55.453ebebeeoebbqq.415.4E,53eqbebbebebeeboebbvwqeeo
w
bybovvq2lolobv3322b2oobbbvobvvovlbob00000bovb2obobvbbvobvollb22b1bvb2oolobo
qpqoabeaboqqou.bobopopeoppobqwwoobwoopqqwobewebwoopwobbbqopebeebeloowebqw
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Dqfpbbblibqqowbtbzn,b5aq=obobbbqoqwowqoqwowbobqooboqqqwbbqowbbebs,Dowzmobqboob
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400egogebwEr4obeog000beeeoobb
000bevbeobwovq5bweebwoewoebeeob5oebobeoebbqobwobebeoobeobeeee45qwwqe
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polobbqbbobbeEbobeebbobbl5bobbobeoplblbooeblbbl000eobbbepobbbbweloobollbbq
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bwobvoeebeeooboeeqebebevoqogeooeoqqebeo655225-45obeoeb0000eqoqqooeoeoebeebb
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6
blobeoblbbebboobbeopbooboeooqobloblloobbwboobloolobllooboovbqbeooelloobble
(vNa)
8T-rn-zvaN-Edx03-zE-9z-9-92gznq
(6991:0N ai Os) yellybIloebbyboyebyoblIbooyoqqbqyollyobeob
povbobbbweeboebbebeeebevblooleblobeeollbwoebbbobebeeebebobloobbloolloelob
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pErpoopop66peolyopeop66=600pee6le6y6epeolio6poleole6epope6e6o6lo6p6l000po6
voqvbvvbvbobqbvvbqboqoqvoovbqboobbqvobbvbvqoobvoobvovbbvvovqbqvobvoqvoqvoq
LIOLLORZOZSflaad
6COLZO/ZZOZ OM
LZ -Z0Z GSSL9t0 VO
8CE
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blob2361.652bboobbvpobooboPpoqobloblloofibwboobloolobllopbooP5152opelloobbqt,
(win)
st-qi-mvaN-cdx0a-xxize-ez-e-9asznw
(7L91:oN ai Das) eelebloebbvboeebeoblbopeollbqeolvobeobooe
b3bbbl3225325Z2522952ebl33l2b13522311513326653bv6222fmb3bl33bbq33ll3vl3bbfm
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ofmoogebooebbEooeoebeebegeoogooe54-4445-4o6oeoogyooboebebbqoobogebeogbooe25-45
eqqq.boqobebeoE22-424eqoobbefx65-455o2-45-45o552-4.452-4oeebeobqoeqqqeooqqq-
meoqqob
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olollleoeebepobloevellqbveleblobol00000bwoobbvobleoeoll000boebollooeoebbee
33v33bP321623.431fibbe33qqql33bbleb3e3bbfibve3bbffmab33b3b26365222612566112626
16rolloo66e66o661e6pele6rpe6po6lopeElepoll6loo66pe66pol000pe6pe66pe6e6006pe
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oee4e4o4oDeo.oeeDeooDDDeobeeoe4DoDoznDooDoebeoboDebbeoDeo44Dee645eDeD4oeeD4D4
ebbebbeebeebeebeebepolqqvbooblobelblobblebeebbvbeeoloeweeeoelbeooebeble111
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3l6BEEra5ral3eBEBo3l33363Bbfra3le3el3le3eb3fra3353l-
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vbbobbqobwbqoovbv4bqqobbvb000bbvbqoobvb4owobvoobvoobqqvvovw000bp0000vqoo
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6136e36166p66=66epo6006opoolo61361loo66136336loolo6iloo6poe616poopiloo661e
(vNa)
8T-rn-mvaN-Edx0a-xxi7c-set-t7-8-9Esznu
(1L91: ON ai 02s) evqe6q3pB6eB3pvbvob4boovoqqBqeoqvobvpboot.
bobbbweeboebEebeeebeebwoqebwbepoqqb-woebbbobebeeebubobwobbqooqqouwbbbe
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ebevbebob4beeE4b4oqo4eooeb4boobb4eobbebeqoobvoobeoebbeeoe4b4eobeo4vo4eo44o4
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45v55-45
54oDeoD4bbeanDobbeoob.7c&oeoo4o54o54400554o5.7.D54004o54400booeD45eooy4400bb4e
(vNa)
8t-rn-amaN-Edx0a-xxi7c-8Z-9-9acznw
(T7L9T:ONaiOac) 212-412b4owbbpbovubsobqboopo4qbqwoqpobuob
opybobbb4peeboebbebeeebeyb4pogeb4obeep-4454opybbbobebeeebebobqopbbwoqwegob
bbveovqobeobeobebefrLqqbeoblebeeoeeoeboeoobb000blbobebbobeoolloqqeleoleoebob
ebveooPovbbveolPo2P3266335333225125v6P223.1.13523123.42be23226Pbobqobebl000vob
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boepoqobqobqqoobbqoboobqooqobqqoobooebqbeooeqqoobbqe00.E.00.6e106-4ee000ebebeb
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blobeoblbbebboobbeopbooboeooq351351133b5a353351331351133533eblbeooelloobble
(vNa)
8T-ZI-Mliati-sdx03-XXIzE-8Z-8-9gsznq
(sL9T:ON ai Ogs) v21261326626322623el533e3qq61231236236
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23.4454354b5e33eb3ee54332255334#5453beb43bee35eee5.613582355344324.63355233533
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(xers) sox Apra mod :euoqApIrq 4on14suo0
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(9g0g:oN ai 03S) ee2v520e55e5DeeBe3Babooe02252e02
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poo6006opooio6io6Tioo66-p6006looio6Tioo600p6i6poopiloo66-1e0ovoz,be4054ve000
pbvbeboqoobepoqebooebbbooeoebeebeqeooqooebqqqqbqoboeooqepoboebebbqooboqebeo
lbooev5lbelllEolobebeobevleleloobbe555155oelblbobbellbeloeebeofraoellleoolll
-4-11.woqqobbubsobqouqqwooqq-44-4-1voqqobb9buob-4312-11.-Ivoo-11.44-
11.12oqqobboowqbboobbq
ovvqooffogo4oql4eovebv:Dob4ovve44qbve4vb4bo400000b400bbwob4eovo4400bovfoo-44
oovoebbeeooeoobeoeqbeoqoqbabeoollqloobbleboeobbbbeeobbbbebboobobebobbeeeble
566.41v62&4623.41336526636612.6v2lvfmvvfmobw2251vvolqbloobb225623.4.33322622662
efmboobeee555E55.4e5e5qpooebbboobbqboebebeeoebbqqq.45.4eboeqbebbebebev5oebbew
gyvogobyboyyqp-43-435yoovy6y33555pobypoyqbob00003boybyobobybbyobyoqqbyybgbyby
powboqewobeoboqwebobovooe0000bqewoobepoeqqvobeeebeooewobbbqooebeebewo
4ovfx4vovver4voeqDv5-4.6vzmobqoollobbvobebbvbeeqbebbefx4555-
4oqqoqeoqeqqqoobbqfpoov
blbblobloobvoglobloobbqoblb2bbobbblbblbbqbbloolbbb.4311133b2vobvq000bb0000ll
bqovooqoqqooqEqbqooeobeebeeoqeeebbqebeeooeobbbvoobboqqeoebe00000qqoeoeoeobb
byoBB-4DB-4pyqpy-45-4BoBB6-4BoeBbeBooBveEB-
4ByBpoBeoqeByyfrappoyoqqpyBopyoBBoByoBB
lole66o6polipEpip61331-1616636pip661o6ppool61661oopiolp61361o6poi0006pppoo66
000beebeobwoeq5bwee5gooegooebeeo55oebobeoebbqobwobebeoobeobeeee45qqogoge
obvoabwobvooZbloo3325.4.bobvbqololblow000ebv000vb12b1b31232153b2obbvbbobblbb
ooqobblbbobbeEbobeebbobbqbbobbobeopqbqbooebqbbq000eobbbepobbbbqoeqooboqqbbq
obb5ebeoeobbooebeovbeoobobweqoeqbqEoobooeoebbvboobbbobqoobvoeebqebeobwoeq
6-1.3.-
D5vovv5vvoobovvqvbebvvoqoqvoovoqqvbvobbbvwfyl.bobvovboopovqoqqoovc)vopbvv55
obboeeobeoqeooeobeobbbqeebbwobbbeeobbw000bbeobbo5-4555gobebgeobboegobeobe
ollvovollobbotvoobooboblob2.6.432b2bloofmobb26613362231bblovbbebbobblolvvbblb
bqobeobqbbebboobbeoobooboeoolobqobqqoobbqoboobqooqobqqoobooebqbeoovqqoobbqe
(11Na)
8t-r1I-mvarl-ON0a-xxi 7E-8z-8-9cznq
(ssos:cm ai pas) wwqwbqowbbwbowwbs,Dbqboowoqqbqwoq
eobeaboowbobbtwww5DwbbwbewwbwwbloolwblobewDillbloowbbbobwbweebwbobwobblool
wvlobbbeboelobeobeobebeblqqbeoblebeeoeeoeboeoobb000blbobebbobeoollolleleol
pov6o6p6ppoopop66ppoleopvop66006000pp61p6p6pppolio6polpolp6peopp6p6o6lo6p61
000yobeoqebeeEe5obqbeebqbqoqoqeooebqboobbqeobbebeqoobeoobeoebbeeoe-45-4eobeoq
eoqwo4qo4eooebbo40000boveoebebeob4ovbobeoebooeb4eoebbebo44b40000ebwoveobbbe
oqpboqeoqqbweqbbeopeboepbqopeebbooqebqbobebwbeepbeeebbwbeeobboTweqboobb
eoobooboeoowbqoblloobbqoboobwoloblloobooeblbeooelwobbleoovoz..be2062E.E.000
ebybybogoofm3ogybooy55bo3voybyvfmgyoogooybqq-445-43.63yoogy335325vbbwobogyby3
4booevbqbeqqqEowbebeobevqeqewobbe5bbqbboeqbqbobbeqqbeweebeobqoe4qqeooqqq.
aggyoglobbebeoUgoeqlgeooqqqqlgeoggobbebeobqpygggeooqqqqqqeoqqobboovq5boobbq
33 13 13 3b33 3E, 33333 3335 35 33 3335353
33vo2.66223323o6P3vq523.4.3156.6P33qqqqo3bb.42632obbbb22365662fib33bo52bobbP226
.42
555-4-4252.6-45eolwobbebbobbqebeeqebee252obweebqyeoqqbwobbeebbeog000eebeebbe
25v5oobeee555555-42525wooebbboo55-45oebef2eoe55-4-11.45-42532-
11.255252522532552qo
leeoqobeboeeqeq3qo5eo3eeUeoof)55e35223eqUob00000boe5eobobvbbeobe3qqbeebqUebe
3335323333853233233333335333333335225233
qovbqvo225.423t,4326q523236.433qobbyobt,65262eqbebbPbqbbb.43.4.43.4Po.424.4.400
bb.46332
5-455-435-400beovqobqoo55-4o5-45255o555-455-11.5qbbwo-
455.6qoqqqoobevobeg000bb0003qq.
bwvooqoqqoo-45-45-400eobeeveeoqeeeffy4ofmeooeo55255-4.650-4-45oebeowoqqqqeoeoe-
455
eyobblloblIgeggyqqqbebb611114ebbebllobbebbIlbebyobeolleyeybqoyoyLrmybeoebbbyoll
ybb
qbvobbqoeoqqbbeoebq000qbvbblowebbloeeeloqbqbblolewleeloobobeeeoololbeoobb
Poobbvbrovqqbqqbblqe26.4.11P1232be22551Pfilbe125211oloobeb2316Peoqb2vob1131312
ooloo6pooppoeffylippopii660-16-mopoiopooipe6p000e6ip6-16ii6ip600ie66366e66366
obvs,bbqbbvbbobbqoqvbbebbobbqbbvobwqoqbqovoqbbqoqovbbbvvoobbbbqovqqobqqqbbq
LIOILOITZOZSIILIOd
6COLZO/ZZOZ OM
WO 2022/027039
PCT/US2021/071017
Promoter MSCV
Leader sequence: Human CD8 alpha (1- 63)
scPv Name: Humanized 20A10 -0 (64 - 807)
Hinge region: Human CD8 alpha (808 - 942)
Transmembrane Domain: Human CD8 alpha (943 - 1014)
Costimulatory Domains: Human 41BB (1015 - 1140) and CD3 zeta
(1141 -
1476)
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgcagctg
gt-LgaaLci-ggt=ggcggaLL031-gaAgccLOYci.ggaLLcii.gagdcl-gdOGL
bLgcPgcdgcgOcLcac0-1.4.
ageacatacgcatgagcAgggtcc4acaggco'cotgciaaaggc:lottgat4gg=t4ctota;tc4goaq9cP
ggcageacctatacagc9attotgt9aagggc4gatcaccat6agcc943gacaaqgc4aagaca.gcct9ta
ctqcagatgaa6tocctq4qagccg4ggace040cgigtactatigagg4cc6atct'acacgata-6
gacgagttegctattggggccagggcacatggtcao g agica 6t
LeCggLggcg9499ca-9,-.4a,.L.,
ccg9cggtg-ggg4alcg4aqgc,10-gtic
(-91,9a got-,11-.219c19LL4A-9aL4dgeeiggcc9
' ' ' '
accatcaactg6aagag-c4gccagagcgtg6t iAct: --(--
9gg4ig"dg549" g. cagcaaccagaAga4ctadctggectggtatcageag
aagcceggccagcetcet4agetgctgatet..aegggqca-gcacagagaag-cggegtgcce :atai
tc:t att*-
cpcagcggct. a
..ctggcacc4acttcaccctg4ca.:ttagctcctgeaggecgq 9: g
n8.r7mIgtRct4.40 ta
cAtg,lcctttggcgn;g9 ,0,
9gt(4041RRI'a 7q'qqccgtltctattqt
agacctcctaceccagcccctacaattgccagccagcCtctgagocZgaggcc7-1''
tgCCeee
'''''87:
'
.
agattt
iE(:). ID 146:1675-i'
CDR leader segJence
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGC7CCACGCCGCCAGGCCG (SEQ ID
NO: 1676)
11201I0 -0 SCtV
GAGGTGCAGCTG(-7-7-GAATCTSGCGOCGGATT&GAAGCTGGCOGAT0T&TGAG4CTOArrtrT.
GGCTTCACCTTTAGCACATACGCCAtGAGCte-G6TCC6ACAGGOCtCTGAAAAG0CCT4GArAG;!7-44-JCT(74
ATCGGCAGAGCGGCAGC*CCIAC2A.CAGCOATtCTGtGAAGGGc;AGATtCA4-CATAGOCGGGA,CACGCC:AM-4
AACAOCCT-GTAcCTOCAQATOAAC:CCCTOAC-AOCCOGi.3ACA00QC 2
47i6.7QTATTGTOCCAPACIACC:CATc
TACAACGACTACGACGAG/TCGCC-ATTGGGCCAGGdCA0ACT6G
CTCGGCG'GTdGCGGiag
GGAGGc-
GGTGI*tcct4GTO0GCGGAGdCaqc6ACATCGtGAirAckt/kaPs:4Cffit.AC4CTSGCOTGTCTCTO
GGAGAGAGAQC0Ac0ATCAACTGCAAQAGQAQCCAGACTQCTGTACTCC;k,90AACCAqAAGAAC7*CCTOGCC
TGGTATCAGCAOAAGCCCGCCAGCdTeCTAAC,tTGCtG1s, CTAT ,C GCACCAGIOAAAC,COGCGTCCC
GATAGATTTTC:TGGCAGCOGCTCT-CidCACCACTCAOCCtGACA:TA9CTOCCTCAdGCC-GAGOA7GT4GC
GTGTACTACTGtCACCA2GtACCTGA6CAGCaTGACCTtrGCGGdGGAACAAAGGItGAAArCA:.1w6ga (SEQ:
ID NO:1677)
CD8 hinge region
ACAACAACCCCTGCCCCCAGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTCTGAGCCTGAGQ0=PAQ
QTT.-0,TQQQfp.G.GTCICT-6GCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGAC(SEQ ID NO:
1678)
"
(SE
02 ID NO:1675)
(SEQ ID NO:1676)
A9ARTRApTTPKPAPK.WOMCAGACGOCOMGOGTACAAGOACIrrA
iiiiiiii!!!!!!!!!11111111i1g/i11111141111111111/1111111111
v.WAX4eTGATAA (SEQ ID NO: 1677) ----
341
CA 03187555 2023-1-27
WO 2022/027039
PCT/US2021/071017
342
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Construct Name: pCDH MSCV h20A10-N CAR 4128
Construct backbone: pCDH CMV MCS (SST)
Promoter MSCV
Leader sequence: Human CD8 alpha (1- 63)
sell,. Name: Humanized 20A10-N (64 - 807)
Hinge region: Human CD8 alpha (808 - 942)
Transmembrane Domain: Human CD8 alpha (943 - 1014)
Costimulatory Domains: Human 41BB (1015 - 1140) and CD3 zeta
(1141 -
1476)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGC7CCACGCCGCCAGGC,..:GcAGQ.TQQA(4CT
GTTGAATCTGGtGGCGGATTGTGAGCCTGCGATaTCtGAGAC=GAGCTTGCCGCdAGCGGCTTCACCTTt
AGcAcArAcGcATGA,GctGGATcAd.AcAG4ccOcTGOcA4aGG6.c7ciAATPGGTG90qT0TATTPGcAGAGc
GGCAGCACCTATACAGCOACTCTGtGAAGE;GCGATTCAtCATdAGCCGGO.CAACGCCAAGAACGCCW-TA
CTGCAGATGAA6TCCCTG4GAGCCGii.GGACACCOCCG*GTACTAiTGTGCCAOAGGACCdATCTACACGACTA
GACGAGTTCGCbTATTGGGGCCAGGCACAtIGGTCACAG TTCtAGCGGCGGTGGCGOia4cGGAGGCGGTGGt
. ...................................... . . . .
tccGGTGGCGGAGGCagc0AAATTGIIGCTGACKAGAOCCtCGCeACACTGTACTTTCtCCAGGCOAAAGAGCt
ACACTGAGCTGbAAGAGCAGCCAGAdCGTGT,GtACTCA-6CAA&AGAAGAkeTACCTdGCCTGatATCAGCA6
AAGCCCGGCCA4GCTCCTOGGCTGCtiGATCtATtGGGOCACAC4AGAGAGAOCGGCAlt.eCTGCCGATTITet
GGCAGCGGCTCtGGCACCATTICAOCCTG.SCC.P,TAACACCTOGAACCTGi,GGAQTTGCCGTGtATTACTG
CACCAGTACCT4AGCAGCTGACC711TGGCWAGCACAAGGTOGAAATCAC-C-G-GACAACAACCdC7GCCCCC
AGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTCTGAGCC7GAGGCCCGAGGCTT3TAGACCTGCTGCT
GGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGAC =
= ,=,. . ...õ .
=
TATMWAOCTOMMTAWAOW00.04.0i0at-itUO.'!",'21-
r:Tr*c+A.V.s.A040A007,00tt4041tti0000
66660,WAA46d6A:AXi*44AA*eed,tdkgr4iXO6i-
']eff,,T(4.kiiii:e4Xiftf*J6kkko;AtAi:e4Xtdi4664kddde,
(SEQ ID NO: 1678)
CD8 leader sequence
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGC7CCACGCCGCCAGGCCG (SEQ ID NO:
1679)
h2OATON scry
CAGGTGCAGCTbGTTGAATCTGGCGdCGGAtIT6TGAhGCOTGGdGGATCTCTGAGACT6AGCTGT6CCGCCAGO
GGCTTCACCTTTAGCACATACGCCATGAGCTGGATCAGACJWGCQCCTGGCAAAGGCCTQGAATGGGTGgegICT
ATTGGCAGAGCGGCAGCACCTAC:*CAGCOACtCTGi.GA*GGGOAGATTCA6CATCAGOCGGGACACGCCAA0
AACAGCCTGTACTGCAGATnAACTdCCTG4GAOCCG*GGCACd.GCCGTGTiCTATTGTGCCAGAdGACCCAT
TACAACGACTAGACGAGi"reGCDIATTGGO.CCAGeOCAADTWXACAGiTTCTAGG$COGTC/GeGGA09
QGA0OCQGTGGtOCG-QT6OCQQAGOCagoAAiiTTOI.QOtGACACA-QACCCCQCOACACEGTOAdT7TOTOC
GCGAAAGAGccACACTGAGCTGcA4GAG04-QtAGAC.74tGcTPTACTCCACAACcAOAAGAActACCT-Q-Qcc
TGGZATCA-C,CApAAQCCO6GCCAAGC.TCCTCQQTGC1,GATCTATTQQQCCACAOAA,GAQAGAGC6GCATCCCT
GCCAGATTTTCTGGCAGCGCTCTGG4ACC4ATtTCAdCCTGACdA7AAGCACCTGGAACCTGAGdACTTCGC
GIGTATTACTGCACCAGTACCTGAOCAGCTG:KCTTTGOCGGGGCACCAiµGGTGGAAArCAAGOGG (SEQ
ID NO: 1680)
CD8 hinge region
ACAACAACCCCTGCCCCCAGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTCTQAW4TG4GGCCCGAG
GCTTGTAGACCTGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGA0 (SEQ ID NO:
1681)
(SEC) :D NO: 1632)
' ' ' '
s = ':
(SEQ :D ND:
Z7AP!
.4PAPWAWMPAPPMPAPPgq4P4PqM;;POPPPWAAPPAWACCAPAAPPAPPTPTNOWPAPP;q44T
TAaahMAAgAgAMMTAttAMT=TOMAAaAtAtITOC-W6dAettnIWATag=6AAA6MA6A
*00440**000t0A0040004taX0WOOt0040W#0*M0000a04WetA0000.4*ft400
343
CA 03187555 2023- 1- 27
W02022/027039
PCT/US2021/071017
ATOAAAACOCe00A0OCAAW8OCAe0ATOOCCTTUCCA000TcrtAaTACAWZACAAOGACACC
TACL+Auc,t.cccTTLA-CATCALccc-P$CC-C-CCTCGOTGATAA (SEQ ID NO: 1684)
344
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Construct Name: pCDH NSCV h20A10 -C2 CAR 41BB
Construct backbone: pCDH CMV MCS (SBI)
Promoter MSCV
Leader sequence: Human CD8 alpha (1- 63)
scPv Name: Humanized 20A10 -C2 (64 - 807)
Hinge region: Human CD8 alpha (808 - 942)
Transmembrane Domain: Human CD8 alpha (943 - 1014)
Costimulatory Domains: Human 41BB (1015 - 1140) and CD3 zeta
(1141 -
1476)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGC7CCACGCCGCCAGGCGGAG(.4.T(.4cAt4CT
GTTGAATCTGGtGGCGGATTGTGAGCCTGCGATaTCtGAGAC=GAGCTTGCCGCdAGCGGCTTCACCTTt
AGCACATACGCATGAGCTGGGrCed.ACAG4CCPCTGPAA4aGG6.C7TGAATPGGTTGCTOTATCPGCAGAGC
GGCAGCACCTATACAGCOATTCT-GtGAAGEriGCGATTCAtCATdAGCCGGO.CAACGCdAAGAACGCCW-TA
cTGeAGATGAA6TcccTG4GAGecGiaGAGiccOccG*GricrAirGT-GccAOAGGAccdArcTAcAcGAcTA
GACGAGTTCGCbTATTGGGGCCAGGCACAtIGGTCACA.GtCAGdTCTGGCGGTGGCGOia4cGGAGGCGGTGGt
tccGGTGGCGOWGCagc4acattgtcletgaccagttcagc6tccttggcg.:,gtcccaggadAgagggc
accatcacctg6AAGAGCAGCCAGAdCGTG&GtACTCA-6CAA&AGAAGAkeTACCTdGCCtggtcatcagcaig
ataaccaggacaa.cotcatAaactcctigattacGGGCACACd.AGAGAAApCggggtccagamEiggttcag
ggcagtgggtar-gggacclatttcaqcctcOcattaatctgtggaagctatgatacgcaaattattaOtg
CACCAGTACCT4AGCAGCcTGACC711CGGCWAGGACAAGTOGAGATCAAC-GAACAACAACCdC7GCCCCC
AGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTCTGAGCCTGAGGCCCGAGGCTT3TAGACCTGCTGCT
GGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGAC
= =
\.µ`' =
µ*".ZSA's".:`,.µ"N.
=
ft0 =Q*,.,4:Q..7`.5.:TaQ:U:V.W.Q;AA=040MQ:ks'4(_-=
TATKAMAOCTOMMTAWAOONMA55MIT4,40.'!",'21-
.T.Tr*c+AVW,*OA007,000M:",40.A.Mittt:A000
Oot*i*A*4666A4AafwAkAeeditkogOpnmff-TAOOAAOt0008AAOAtAAvAtOo004800ft
OgClAccANAPAP'-2.1-AcG4qqcgcTT.OAtAt,Wir.rrir.C.CqtqCO:gg'01000TGATAA (SEQ ID
NO:1685)
CD8 _oader segJon.7o
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO:
1686)
1120,A10 -C2 sEV
GAGGTGCAGCT4GTTGAATCTGGCGOCGGATTGTGAGCTGGOGGATCTCTGAGACTOASCTGTGCCGCCAG
GGCTTCACCTTtAGCACATACGCCAtGAGOte-G6TCCaACAGGCdCCTGGAAAAGGCCriGAATGG61-.7GCCTOt
ATCGGCAGAGCQGGCAGCACCTAC:CAGC9ATTCTGTGA/WGGC.AGATTCACCATCAGQCGGGACAACGCCAA
AACAGCCTGTACTGCAGATGAACTO:CCTO,GA6CCGAGG*CACciGCCGTGTkrATTGtGCCAGAdGACCCATO
TACAACGACTAGACGAG TCGCCTTTGGGCAGGOCAACTOGTCACAGtCAGCTCTGGCGGTOGCGGAag
GOAGGCGGTGGO..ccOOTOGeGGA-Q0Ca9cojac41.99490ccicdgt,<;LQ6019eQL*:LL9gcc41.91-
QLet*
ggaCAgaggg0 aCCatcC,C,tgOA/114AQ0,-QCAO.A6Cc4GCTOTACTCCACAACCAOAAGAACTACCTQQQ0
tggtatcagcagaaacca4gacaacqtcctOaa:ctccgattac.TGC-GCCACAcCAG4GAAAC,049ggtccc4
qacacicittcaqPqgcaqt4qqtctqcOaccOati.tcacQ-eacaAttaatctcrIqqa4gctaatciatactge0
aattattactgteACCAGtACCTGAG4AGCTGACCTItG(XGGGGGACCAAGGTGGA6A7CAAAdGA (SEQ
ID NO: 1687)
CD8 hinge region
ACAACAACCCCTGCCCCCAGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTCTGAGCCTGAGGCCCGAQ
GCTTGTAGACCTGCTGCTGGCGGAGCCGTGCACACCAGAGOACTGGATTTCGCCTGCGAC (SEQ ID NO:
1688)
(SEQ ill NO: 1689)
(SEQ ID No: 1690)
....... ....... ........
04#0 10#0000t#00#0000#44000I000000t#0##00#000g0WKOWOOtOtAtX400#001tAAt
.PTAPPAPPA4PAMOPPTAWATATXWM4P4APAAMPX144PgAPPW4P4TWg*PP;444PP;PO
AddiUWAAtCnA=AAdattlantAMPAAAtTatAt'AAAOAIAMATaatt4OgCTMATMWAtittg
345
CA 03187555 2023- 1- 27
W02022/027039
PCT/US2021/071017
ATOAAAACOCe00A0OCAAW8OCAe0ATOOCCTTUCCA000TcrtAaTACAWZACAAOGACACC
TACL+Auc,t.cccTTLA-CATCALccc-P$CC-C-CCTCGOTGATAA (SEQ ID NO: 1691)
346
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Construct Name: pCDH MSCV h20A10-0 CAR CD28 1XX
Construct backbone: pCDR CMV MCS (SRI)
Promoter MSCV
Leader sequence: Human CD8 alpha (1- 63)
sell,. Name: Humanized 20A10-0 (64 - 807)
Hinge region: Human CD8 alpha (808 - 942)
Transmembrane Domain: Human CD8 alpha (943 - 1014)
Costimulatory Domains: Human CD28 (1015 - 1137) and CD3 zeta 1XX
(1138
- 1473)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGC7CCACGCCGCCAGGCGGAGQT(4cAt4CT
GTTGAATCTGGtGGCGGATTGTGAGCCTGCGATaTCtGAGAC=GAGCTTGCCGCdAGCGGCTTCACCTTt
AGCACATACGCATGAGCTGGGrCed.ACAG4CCPCTGPAA4aGG6g7TGAATPGGTTGCTOTATCPGCAGAGC
GGCAGcACCTA&ACAGCOATTCT-GtGAAGEGATTCAtCATdAGCCGGO.CAACGCdAAGAACGCCT-G-TA
omeAGATGAIN6TccoTG4GAGecGiaGnicoOom*GricmirGmocAOAGGAccdArcTAAcGACTA
GACGAGTTCGCbTATTGGGGCCAGGCACACIGGTCACAGtCAG&CTGGCGGTGGCGOiagoGGAGGCGGTGGC
tccGGTGGCGGAGGCagcOACATCGIIGATGACKAGAOCCtTGAtAGCCTGGCGTGICtCrGGGAGAGAGAGCt
ACCATCAACTGCAAGAGCAGCCAGAdCGTGCSGtACTCCA-6CAA&AGAAGAkCTACCTdGCCTGatATCAGCA6
AAGCCCGGCCP4CCTCCT4AGCTGCtiGATCtACtGGGOCACACd.AGAGAAAOCGGCGTCCCGATP1GATTITCt
GGCAGCGGCTCtGGCACCACTICAOCCTG.SCATTACTCCTOCAGGCCGi,GGATGTGCCGTGtACTACIGt
CACCAGTACCT4AGCAGCTGACC711TWCWCGAAAAAGTOGAAATCAC-ogaACAACAACCdC7GCCCCC
AGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTCTGAGCC7GAGGCCCGAGGCTTGTAGACCTGCTGCT
GGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGACw,,,,,,,
.\\\
,t',,,v(wkr'======;-= =;-= \\N
'zZz `,` ==z -=== \S":`,`,
\\\ NN\
ONs
CJ f'A2 S 'Ac GCAC CCC SAC CC 'CCC AC ACCA 'CC WAAC C2 SC 'C AT
AAWX04tOgAtOM-10A0004WOOAMOONMMTWAONWA040M000000hMOTOA5AM000
0000W0A044**000PPOOtOtkAMOAAttOOONOAtAgAt00.00.004tONt
4VOW=44P4MMAA,APPPP04044PAPPAWAAPPOPT.P40g4Wigg.
A00**OOMMMOk0A00qqccActiA00000tiOftglacOOTGATAA (SEQ ID NO; 1692)
CD8 leader sequence
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGC7CCACGCCGCCAGGCCG (SEQ ID NO:
1693)
finA10-0 scry
GAGGTGCAGCTbGTTGAATCTGGCGdCGGACTT6TGAhGCOTGGdGGATCTCTGAGACT6AGCTGT6CCGCCAGO
GGCTTCACCTTTAGCACATACGCCATGAGCTGGGTCCGAC/WGCQCCTGGAAAAGGCCTTGAATGGGT7GCCICT
ATCGGCAGAGOGGCAGCACCTAO:*OAGCOATtCTGi.GA*GGGOAGATTCA6CATOAGOCGGGACACGCOAA0
AACAGCCTGTP4CTGCAGATnAACTOCCTG4GAOCCG*GGCACC.GCCGTGTiCTATTGTGCCAGAOGACCCAT
TAGAACGACT4GACOAG*CGC=ATTGGO.CCAGeOCAACTOGCACAG;MAGCTCTO$COGTC/GeGGA09
QGA0OCQGTGGtoc-G-QT6OCQQAGOCagOOkCiaC01.QAtGACCA-QACCCOTQATAGOTG-QCOdTGTCTCTO
GAGAGAGAGccACCATCAACTGcA4GAGC*,G-CtAGAC177tGcTPTACTCCACAACcAOAAGAActACCTcc
TGGTATCAC,CApAAQCCO6GCCAC=CC7CCT45,AQCTGO=GATCTACTQOQCCACACCKGAGAAAGC6GCGTC=CCc
GATAGATTTTCTGGCAGCGCTCTGG;CACC4ACtTCACCCTGACAA7TAGCTCCCTGCA6GCCGAGdA7GTGGCC
GIGTACTAZT#CACCAGTACCTGAOCAG#TG:KCTTTGOCGGOGGAACAAGGTGGA4ArCAAGOga (SEQ
ID NO: 1694) .
CD8 hinge region
ACAACAACCCCTGCCCCCAGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTCTQAZ=G4GGCCCGAG
GCTTGTAGACCTGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCG40 (SEQ ID NO:
1695)
(3E0 ID NO: 1696)
---------
(SEC) ID NO: 1697)
ZMTTANK
.4PAWOMMIXPAM4PAPP-W4P4MP;;POPPMPAAPPAK*CCAPAAPOPPTPWANWPAP4MAT
TAaahMAAgAgAMMTAttAMTTt=4AeAAMMtITOC-CWAACten:OAT==AAA6MA6A
*00000**0000000400004NOWWW0040WWW0000#00000t000004(#000
347
CA 03187555 2023- 1- 27
W02022/027039
PCT/US2021/071017
ATOAAAACOCe00A0OCAAW8OCAe0ATOOCCITTvCeA000TcrtAaTACAWZACAAOGACACC
Tt.C.L+Auc,t.cccTTLA-CATCALccc-P$CC-C-CCTCGOTGATAA (sEQ ID NO: 1698)
348
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Construct Name: pCDH MSCV h20A10-N CAR CD28 1XX
Construct backbone: pCDH CMV MCS (SRI)
Promoter MSCV
Leader sequence: Human CD8 alpha (1- 63)
sell,. Name: Humanized 20A10-N (64 - 807)
Hinge region: Human CD8 alpha (808 - 942)
Transmembrane Domain: Human CD8 alpha (943 - 1014)
Costimulatory Domains: Human CD28 (1015 - 1137) and CD3 zeta 1XX
(1138
- 1473)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGC7CCACGCCGCCAGGCCGQAGQT(4cAt4CTG
GTTGAATCTGGCGGCGGACTTGTGAAGCCTGGCGGATCTCTGAGAC -GAGCTGTGCCGCCAGCGGCTTCACCTTT'
AGCACATACGCCATGAGCTGGATCAGACAGGCC6CTGGC GG CCTGGAATGGGTG909.TOTATTGGCAGAGCC
GGCAGCACCTACTACAQ;CGACTCTGTGAAGGC-CAGATTCACCATCA
aCCGGT:FACAACGCCAAGA&CAGCCTGTAC
CTGCAGATGAA6TCCC TGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCAGAGGA666ATCTACAACGACTA6
GACGAGTTCGCbTAT TGGGGCCAGGGCACAC TGGTCACAG TT TCTAGCGG6GGTGGCGGAa
4CGGAGGCGGTGGC
tcoGGTGGCGGAGGCago4AAATTGIIGCTGAC=kAGAdCCCCGCCACACTGTACTTTCtCCAGGC4AAAGAGCC
ACACTGAGCTGbAAGAGCAGCCAGAdCGTG&T,GtACTCCA-6CAACAGAAG4CTACCTdGCCTGarATCAGCA6
AAGcccGGccA4GcTecTOGGcTGctiGATA#GGGOckc,4cAelkAGAGAGAPcGGCATecTGcciGATTITet
GGCAGCGGCTCtGGCACCATTICAqCCTGKCikTAACAkCTdGAACCTGi,GGAQTTGCCGTGT'ATTACTG
CAOCAGTAOCT4AGCAGOTGACO711TWCWAGCAoCAAGGTGAAATCAC-CC,-GACAACAACCdC7GCCCCC
AGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTrTCAGCC7G?iGGCCCGAGGCTTGTAGACCTGCTGCT
GGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCI-Ac
;=N = = = = . , =
= "µk.
= === = , '
' - "'"`"µ
,.;:x!;g4Q7Z";XAGt;.AGC GCA -A:GC .. SAC CC C , CC
AC
C*G.T CAG
' GGCCAGAALCAGC,'C-TAT
4etrad,VORitiq,,,AMT9WOOP.ftATWARiPM:P4&:,
' 'N799P1W129ENWPW E1 Th:CANOVTO.Wki44.4
....õõ7,7,-!10.?ip!p0NOTcOOTGATAA (SEQ ID-NO:.1.6.'8'4')."--
CD8 leader sequence
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGC7CCACGCCGCCAGGCCG (SEQ ID NO:
1700)
h217AIIIN scrV
CAGGTGCAGCTbGTTGAATCTGGCGdCGGACTT6TGAhGCOTGGCGGATCTCTGAGACT6AGCTGT6CCGCCAGO
GGCTTCACCTTTAGCACATACGCCATGAGCTGGATCAGAr'AGGC qCCTGGCAAAGGCCTWAATGGGTGgegICT
ATTGGCAGAGCOGGCAGCACCTACCAGC6ACtCTGi.GAiGGGC:AGATTCACCATCAGOCGGGACACGCCAA0
AACAGCCTGTACCTGCAGATMIACTOCCTGA.GAdCCGAGPACAOGCCGTGT*CTATTGTGccAGAOGAcceAT
TACAACGAOTACGAOGAGi.TOGOO-ATTGGCCAGe4CACACACAOTTTOTAGG$O0GTCIGeGGA09
QGA0OCQGTGGtoc-G-QT6OCQQAGOagcAPkiiTTGIv"tGAOAicA-QACCCO.OWCAOACEGTCAdT7TOTOO
GCGAAAGAGcPACACT'GAGCTGcAAGAGCAtAGAGcTPTACTCCACAACCAOAAG.AActACCTCc
TGGTATCA-C,CApAAQCCO6GCCAAGCTCCTQCQTGC
TATTQOQCCACACAlvGAGAGAGO6GCATCCCT
GCCAGATTTTCTGGCAGCGCTCTGG;CACC4ATtTCACCCACCA7AAGCACCTGGAACCTGAGdACTTCGCC
GIGTATTACTGCACCAGTACCTGACAGCCTQsCCTTTG-pCGG*GGCACCA:tv-GTGGAAATCAAGOGG (SEQ
ID NO: 1701)
CD8 hinge region
ACAACAACCCCTGCCCCCAGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTCTQAZZCTG4GGCCCGAG
GCTTGTAGACCTGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGAd (SEQ ID NO:
1702)
(3E0 ID NO: 1703)
mrxm
ONg:g040TTPAM4P;wmcAGAmem=ocaniqAAGgiAKHPA
PAPPAKTPAZMPPAWM.
__ir7,r7=v.tp..ps.;qT9pcicGGGActttstmAtag=wiAA6ttGAgA
P*p_Awpgzmpicmpqq,r.044:MWT0000#00AA0000004)000000000.440**.00
349
CA 03187555 2023- 1- 27
W02022/027039
PCT/US2021/071017
ATOAAAACOCe00A0OCAAW8OCAe0ATOOCCITTvCeA000TcrtAaTACAWZACAAOGACACC
Tt.C.L+Auc,t.cccTTLA-CATCALccc-P$CC-C-CCTCGOTGATAA (SEQ ID NO: 1705)
350
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Construct Name: pCDH MSCV h20A10-C2 CAR CD28 1XX
Construct backbone: pCDH CMV MCS (SRI)
Promoter MSCV
Leader sequence: Human CD8 alpha (1- 63)
sell,. Name: Humanized 20A10 -C2 (64 - 807)
Hinge region: Human CD8 alpha (808 - 942)
Transraembrane Domain: Human CD8 alpha (943 - 1014)
Costimulatory Domains: Human CD28 (1015 - 1137) and CD3 zeta
1XX (1138
- 1473)
ATGGCCTTACC2.kGTGACCGCCTTGCTCCTG(..:CGCTGGCCITGCTGCTCCACGCCGCCAGGCCGWWQTWAtiCTG
5.77QvacTGGCGGeGGAOTTGTGAAGccTGGctGATdv7tGAGAt77GA6cTOTGcgp0x4c4QQTicAQ-6TT
AGCACATACGCATGAGCTGGGTCC6ACAGI pg7p4A44qpiTp.41.TOpT:Tpqz.:74.T.ppppAG4Gc
GGcAGcAccTAtTAcAsiUTATTcTGiGmo0q774TVgpTOAKptigOcAAOGetAAGAackpecT4TA
CTGCAGATGAA6TCCC;G4GAGCOGGAGACC6CCGtGrACT4TVqTpOpOSOCC6AreTix4lieGA6TA6
rC::;;:nATTGGGCGAGGd.CACAqGOTCA4G4ZAGtICTGCGGTGGCGOilia4cG.GAGGCGGTGGt
0.4:9:c.9:40gXXg10.:t4AggiVOX4.6*10tgOgt)4gtgit:CA44aC:AvagKe
accatcacCgOAAGAGGIACCAGAdeGTG&GtACTftA'6CAACbAGAiGAACTAtCTdW'Et
.............. 3
ffi.c.cggOW:#0.0:Ø:Ø***00..00.:.!q*gt...P.PõP.OPASACt.AGAG4AAOCgS,64tddA:41
tii=:!:'!
qgc44t.gggct.gggacc4attta0actctAttAttbtqt4444.540.444g*4g0:440g-:
pAggAgwpTOAppww.TAwq7ligg0pA0pG4cAAGpTOGAQATcAiawAAcAAcAAccq.c7Gcccc
AGACCTCCTACQCCAGCCCCTACAATTGCCAGCCAGCCTCTGAGCC7GAGGCCCGA6GCTTGTAGACCTGCTGCT
GGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGEGAC'
==µµ µqN,;==, \`'`,,, = V = \\\ k's
= ,`= s. ,Z,Z= `
Ss'
õ =,=.µ =
N\ '
p:A: G:cAGGAG,CA: piNg.,,,,-teeedGCGTACAAGCAGGGC%C.AGNAC\C:
......................
moo _ _
rAA.ii
:
(SEQ ID NO:1706)
CD8 leader sequence
ATGGECTTACCAGTGACCGCCTTGETECTGCCGCTGGCCTTGETGC7CCACGCCGCCAGGCCG (SEQ ID NO:
1707)
h2OAI0C26.EV
p4p0Toow_qmoTTQAATgTQ0cGacqw.T:ToTQAAGrxTGGQGGATclzTGAGAcTGAwroTocgcowc
pGeTTcAccTtrAGcAcKiAcGccAtGAGctc-G4Tpp0A4Apppg.cgTgOv44ppplipmtppOT7qcTct
ATcGwAGAGcOGGcpaciiccTAcTAcAGC4ATTCTMtOpppOWATTCAOCATOAWCGGGAcAcGcAA0
AAckaccToTAmTocAG4ywq7OppywppppApqApApgpgqpTpT4pTAxzwmmA004cecnc
TAc.41caN-Q:u4GacQ>TcaQQ74TTQQtac0iPPOOAP;WH7V4APtcMTqM,7POTOPPP*0
pGAGGQQTGGptTGcQ::GAGe4gc44..p#xx90qggApp.pAgpp09,:q.44-mmggpc.4pgr.c.,4
ii44644:4444Cei4-44*4WOApqipcqiwOpqTqcTppicTpcAdcAAcAGAAGAAcTAccT4occ
tqqtAttAlbAO.A.OrjacatItcct*aa,6teil4ftt,A6TGGGCCACACtAGAP,,.:6CY t
gpgaggtgagOggp*gt4qtg444dd4Atttd4OdtitAd4attA4td6tV444440tA4tgg4;::
44t:-at4.4c4=APtIAMTAWA.Occ.W4=Ttco.4.w'ChiwP:T.O.G44.ArAAA0.4 (SEQ.
:D N(: 1708)
CD8 hinge region
ACAACAACCECTGCCECCAGACCTECTACCQCAGECCETACAATTGCCAGCp.GCCTETGAGCCTGAGGCCCGAQ
GCTTGTAGACCTGOTGCTQGCGGAGCCGTGOACACCAQAGGACTGGATTTCGQCTGOGAC (SEQ ID NO:
1709)
= \ = \ Ass,. \
(SEQ ID NO: 1710) "
351
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
00130
40.4P#WTTWWONPPWNP4gqqgMWO;A4AAQOWAOOAOXn 4AVATCTATAAMAMmCAAT
q?W0F90q0FOKFI*00077WQ*2a*GAgM4Tit=4,KA4kCCTiGiNgAt4.4Aiii6.6i6i
.9W110555IMPWOMOMOOMMOT00404#0i0AAtiAtWaW,IdddtedOW#440tt(0
iqpp.t.3:ppjGAGWiGMAG.GØ0tAtbdtdttttttikkikifebibbiaidai8kibieb
tgRqggg4g;40004.44**400M;tg&:GA:AA (SLQ :12 NC: --2)
Amino Acids
RVICFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDICRRGRDPEMGGKPRRKNPQ
EGLFNELQICDICMAEAFSEIGMKGERRRGKGHDOLFQGLSTATKDTFDALHMQALPP
R--
352
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
Construct Name: pCDH mSCV hC2 CAR 41Bs 6xNFATFoxP3 IL18
Construct backbone: pCDH CMV MCS (SEX)
Promoter MSCV
tul:4111..tr= Iltur= 1111 .401.=
scFv Name: Humanized C2 (64 - 8010)
Hinge region: Human CD8 alpha (811 - 945)
Transmembrane Domain: Human CD8 alpha (946 - 1017)
Costimulatozy Domains: Human 418B (1018 - 1143) and CD3 zeta
(1144 -
1479)
NFAT response element: Human FoxP3 NFAT (6x) response element
(1530 -
1691)
Minimal promoter: mCMV (1698 - 1815)
Leader sequence: Human CD8 alpha (1841- 1903)
C tokine: Human IL18(1904 - 2374)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGC7CCACGCCGCCAGGCOGGAGGTOCAOCTG
GTGGAGTCIGGOGGAGGOOTGGICOGCCTOGG6GGTftCTGAGACTCTCCT6TGCAGCOTCTGGAtTCACCITO
AGTGGC'7ATGCOATGAGeTGGGTCCOCCAC*Ii-,CAG(if;AAC4C4C-nn-AnGTCTCAA=ATTAO:"AGTGGi:
GGAACCTACATATACTACOCCGACI"CAGTGAAGOGCCGATTCACCATCTOCA6AGACAAOGCCAAGAACTCACT
TATOTQcAAATAAQAGO(:TQAQA(.4.i.CGA(.40ACAO(.4(,4cC,IG2A2:AC.:(..;,_GO(TACiACTtQc
=(;(44.G(JA=AATTAO
TACGAATACTTtGATGTCTGGGGCAAGGGACCCGGTCACCGTCTCCTCCGOCGGTGGdGGATCCOGCGGTGG
GGATCCG-GCGGtGGC-C-GACCGACAtTGTGT-CCCAGITCC*GCCTCCTtGGCCGTOTCITCCAc4GACAGAG6
GCCACCATCACTGCAGMiCCAGTAGAGTEriTCGTAOCACGGA ACTCCVsCATGeAdTaGTATCAGCAGAAA
CCAGGACAACerCOTAAAOTOCTGATITACOIGGCATOCAATOTdeAGAGOGeGTOCCAGOCAGli.TCAGCGGO
AGTGGGTCTGGACCGATTTCACCCItACAATT:VerCTG GGAGCTAATQcrACTGCAAATTATTACTGTCA
CACAGTAGGGA4CTGCCTTTCACAGGC4GAPGGACA-OGTGAGATCAMCGAACtACAACAACCCCTG&C
CCCAGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTCTGAGCCTGAGGCCCGAGGOTTGTAGACCTGCT
O'''µ''.::\'''''I'''µ':.:PAC47q:WPTPWq?NqPAC.MiP4P4WPqqqqqq4Ac44PWW/GGCCaQAACCR
Q
OVTAW4gAgggiMUTA004qA4PAPWPAOTAOPAT01;474P4g46006 4Q444
.4.Tggg4PA44Pg.P.:PAWNWPWWTWAPNW.40=QTAMMIWTkggiaaii*AeAt000:Ag4
6qqW.0?1IGAGA=OhtdiliAMGCKatkAMW,A4t4WAG.a:wthWAttbilTTAMAW4tXtftiGi
04m4AA;pgimpOip**TKOOpp47,7gAAtOW4WØ0f0atOT.GATAAGrTTAAAc76CcAGA
ACATTTCTCTGGCOTAACTGGCCGGTACCakaAwv-Waagia:
agcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgtttgacctccatagaagacac
cgggaccgatccagcCTCGAGAGACCCAATGCTAGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCT
GGCCTTGCTGCTCCACGCCGCCAGGCC
GAtaa (SEQ ID NO::713)
CD8 leader seciaence
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (SEQ ID NO:
1714)
C2 .5cFV
GAGGTGCAGCTGTGGAGTCTGGGGOAGGC>CA*GCTGGGGGICCCTGAGACT6TCCIGTCAGCCTet
GGATTCACCITAGT-GCCTATGCCPaiGhGCt-C-CPTCCPCC4GGC1;:CCACGGAAGGGGCTGAGTGGGTCTCAAC
ATTAGTAGTGG6KAACCTACATATCTACtCCOACTON3tGAA4sGCCGATTCACCATdICCACAORCAACC4C
AAGAACTOACTeTATCTGPAAATGAACAGGOTGAGAGPCGAGGACACGGCCGTGTATTAOTGTGCGAGACTTGG6
GGGGATAATTATACGAATACTTCGOGTetGGOGCAUGOGACOACGGTCACGTCTCdTCCGGCOGTGGCGGili
TCCGGCGGTGGGGATCCOGCGGTGOCGGATCCOACAtTGTGCTOACCCAGTTCCAGCTCCTTGdCCGTGTCT
CCAGGACAGAG6GCCACCATCAC=dCAGAbCCAGTAAGA6TGTdAGTACCACGGATAdTCCTACkT3CACTGb
353
CA 03187555 2023- 1- 27
W02022/027039
PCT/US2021/071017
TATCAGCAGAAACCAGGACAACCTCCTAAACTCCTGATTTACCTGGCATCCAATCTGGAGASCGGOGTCCCAGCC
AGGTTCAC/CGGCAGTGGGTCTGGGACCGATTTCACCCTCAOAATTAATCCTGTGGAAGCTAATGATAC=GCAAAT
PATTACTGTCAGCACAGTAGGGAGCTGCCTTTCACATTOGGCGGAGGGACCAAGGTGGAGATCAAACGAACT
(SEQ ID NO: 1715)
CDS hinge region
ACAACAACCCCTGC-
CCCCAGAQQTC(TiTACCIXAOCCCqTAcAATTUCCAGCC*4CCTCTqAWCTGAGGUCCGA4.3
GCTTGTAGACCTGCTGCTOGCSGAGdCGTGCACCCAOAZACTGGATTTCGOCTGCGAQ (SEQ ID NO:
1716)
(SEQ ID NO: 1717)
NN,
N
MaGrGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACMGCAGGGCCAGPaCC4WCTC7A:AACGAGC=7CAAT
CTAGGACGAAGAGAWAGTAC6ATGTTTTWACAAGAGACGTGGCOWGACCCTGAGATGGGGGAAAGCCGAGA
AGGAACAACCCTCAGGAAGGCC:G7ACAACGAACTGCAGAAAGATAAGATGGCCGAGGCCTACAGTGNGA:TGGG
ATGAAPit-Gt'GAGOGCOGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAW4ACCIAAGGACACC
TACGAC=CCTITACATGCAGG-CMTMCCCCTCGC (SEQ ID NO: 1/19)
plasmid
TGATAAGTTTAAACTGCCAGAACATTTCTCTGGCCTAACTGGCCGGTACC (SEQ ID NO: 1720)
=
ac ,q (SEQ =E, N( : 1721)
mCMV
Taggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatcc
acgctgttttgacctccatagsagacaccgggaccgatccagc (SEQ ID NO: 1722)
plasmid
CTCGAGAGACCCAATGCTAGCCACC (SEQ ID NO: 1723)
CDR leader sequence
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCITGCTGCTCCACGCCGCCAGGCOG (SEQ ID NO:
1724)
GAtaa (SEQ ID NO: 1725)
354
CA 03187555 2023- 1- 27
VIM) 2022/027039
PCT/US2021/071017
Construct Name: pCDH MSCV hC2 CAR CD28 130C
Construct backbone: pCDH CMV MCS (SRI)
Promoter MSCV
Leader sequence: Human CD8 alpha (1- 63)
sell,. Name: Humanized C2 (64 - 8010)
Hinge region: Human CD8 alpha (811 - 945)
Transraembrane Domain: Human CD8 alpha (946 - 1017)
Costimulatory Domains: Human CD28 (1018 - 1140) and CD3 zeta 1XX
(1141
- 1476)
ATGGCOTTACCAGTGACCGOOTTGOTOCTGCCGOTGGCOTTGOTGOTOCACGCCGCCAGGCGGAG(.4T(4cAt4CT
GTGGAGTCTGG6GGAGGCTGGTCAkGCCTGGGGTaCCtGAGAt2CTCCTTGCAGCdTCTGGATTCACCTT
AGTGGCTATGCATGAGCTGGGrCed.CCAG4CTPCAGPGA4GGG6.C7GGAGTPGGTCTCACICATTG7AGTGG
GGAACCTACATATACTACCCGACTdAGTGAAGOGCCOATtCAedA7CTCCAOAGACAAdGCCAAGVkCTCACT
TATCTGCAAAT6AACAGCOTGAGAGdCGAGeA4CGGOCG4GTAiTACTGTGGAGACTiGaGGGGOATAATTA
TACGAATACTTtGATGTCTGGGGCAAGGGACCACGGTCAtCGTdTCCTCCGGCGGTGGdGGATCCGGCGGTGGt
GGATCCGGCGGTGGOGGAtCCGACAIIGTGTGkCCAGItTCCGOCTCCTtGGCCGTTCTCCA4GACAGAG
GCCACCATCAC6TGCAGAOCCAGTAkGAGT6TC11.GTACA-
6CGGkTACTCCT41,CATGCAdTSGTATdAGCAGAAA
CCAGGACAACCteCTAAAOTCCTGAtiTTACt-TGOCATOCA.0aCTd0AGAGCGOGGTeCCAGCCAGGtTCAGeGG
AGTGGGTCTGGACCGATI7CACCC7iCACATTATCTIStGGAGCTAATGiiTACTGC*AATTATtACTGTCA
CACAGTAGGGA4CTGCCTtTCACA711CGGCWAGGACAAGGTOGAGATCA*'AC-GAACTACAACAACCCCTGCC
CCCAGACCTCCTACCCCAGCCCCTACAATTGCCAGCCAGCCTCTGAGCCIGAGG=CGAGGCTTGTAGAMTGGE
GC TGGC GGAG CCG T GCAC4C CAGAGGACTG GATT TC C IGCGA
tAtKOMMOttOOMTAMMONAMggl'-,7' 'A-'''2TTIVOOMAA040MOT00604Add4t-WATO
66660***466-4AXi;*i****eedri",:dia=c2AA--;.( .7'27
(4,kiit:iikfte366i4MAtAi:e4t66664kddde,
(SEQ ID NO: 1726)
CD8 leader sequence
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGC7CCACGCCGCCAGGCCG (SEQ ID NO:
1727)
C2 soFV
GAGGTGCAGCTGTGGAGTCTGGGGQAGGecTGGTCAAGOQTGGQGGGTCCCTGAGACTQTCCTGTGCAGCCICT
GGATTCACCTTAGTGGCiATGCCAIIGAGCtGG6TCCdCC*GGClICCAGGGAGGGGCTOGAGTGGdTCTCAACO
ATTAGTAGTGGGGAACC ACATATP4TACCCOACTAGTGAAGGCCGATCACCATTCCAGAOACAACGC
AAGAACTCACTOTATCTGAAATGAACAGCMWAeC-0.14GGACPXOGCCO;reTATTAT$TOCGP4ACTT-GG
QGGQATAATTATACGAA ACTTQC14Grct-Q-QGCAitAGACdAC-Q-QTCACQTQTCT;C-QQCdQ7QQQQW>.
TCCGGCG-GTGGPGGATCCGCGGT-GPCGGAtCCACAtTc..7tGCTPACCCAQTtTCChGCTC.CTTGCCGT-QTC
CCAGGACAAQpGQQACCATCACC7QcAQApCCAGTAAQApTTcAQTACCACOATAcT:CTACTGCACTQP
TATCAGCAGAAACCAGGAAACCTCTAAATCtTGAtTrACCT(.74CATCCAATCTGGA(1,AGCGGGdTCCCAGCC
AGGITCAGCGGtAGTGGGTCIGGGAdeGArtre:KCCTCA-dAATtAATCCIGTGGAAGCtAATGATC7GCAAAt
TATTACTGTCACACAGTAGGGAGei,GCCT"'UCACATtCSCGG4.GGGACCAAGGTGGA4ArCAAAdGAACT
(SEQ ID NO: 1728)
CD8 hinge region
ACAACAACCOCTGCCOCCAGACCTOCTACCOCAGOCCOTACAATTGCCAGCCAGCCTOTGAGCCTGAGG=GRO
GCTTG7:1,GACCTGCTGCT(4GCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGAC (SEQ ID NO:
172',)
(SEQ ID NO: 1736
. . (SEQ ID NO: 1731)
AGAGTGAAITITMMW6dAatOMMC-affde&taTAMOCAM4MAUAAUMTCTNIWWWWintAAT
0100.00A#0#0400400.00tOtttt00004004W000.00#000.000t00#*1000404040040#
355
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
WOMAAOCOTOMAA00=0=WAZOMOTOMMA402ATAMATOMMOOCCUMOTWOMT000
W4440000WW0400000WOMA000000VM4A0OUVOOM60444440.4040
too=oommoottooroommT000TGArAA (SEQ ID NO: 1732)
Mouse 312 light chain variable framework 2 (FR2) sequence:
(DNA)
tggtatcagcagaaaccaggacagccacccaaactcctcatctat (SEQ ID NO: 1733)
(amino acids)
WYQQKPGQPPKLLIY (SEQ ID NO: 1734)
Mouse 312 light chain variable complementarity determining regions 2 (CDR2)
sequence:
(DNA)
cttgcatccaccctagattct (SEQ ID NO: 1735)
(amino acids)
LASTLDS (SEQ ID NO: 1736)
Mouse B12 light_ chain variable framework 3 (FR3) sequence:
(DNA)
qqqqtccctqccaqqttcaqtqqcaqtqqqtctaqqacaqacttcaccctcacca7.tqatcctqtqqaqqctqat
gatgctgcaacctattactgt (SEQ ID NO: 1737)
(amino acids)
GVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID NO: 1738)
Mouse 312 light chain variable complementarity determining regions 3 (CDR3)
sequence:
(DNA)
cagcaaaataatgaggatcctccgacg (SEQ ID NO: 1739)
(amino acids)
QQNNEDPPT (SEQ ID NO: 1740)
Mouse B12 light chain variable framework 4 (FR4) sequence:
(DNA)
ttcqqtqqaqqcaccaaqctqqaaatcaaqq (SEQ ID NO: 1741)
(amino acids)
FGGGTKLEIK (SEQ ID NO: 1742)
opitopo to which NME1 and NME7AB bind part of the 10 membrane proximal
amino acids
PFPFSAQSGA (SEQ ID NO:1743)
SNIKFRPGSVV (SEQ ID NO:1744)
ASRYNLT (SEQ ID NO:1745)
Fragment of PSMGFR
GTINVHDVET (SEQ ID NO:1746)
Fragment of PSMGFR
FPFS (SEQ ID NO:1747)
Fragment of PSMGFR
SNIKFRPGSVVVQLTLAFRE (SEQ ID NO:1748)
Fragment of PSMGFR
QFNQYKTEA (SEQ ID NO:1749)
Fragment of PSMGFR
VQLTLAFRE (SEQ ID NO:1750)
Fragment ot PSMGFR
356
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
SVSDV (SEQ ID NO:1751)
[00912] IL18 sequence
[00913] (DNA)
[00914] Atagaagacaccgggaccgatccagcctcgagagacccaatgctagccaccatggccttaccagt
gaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgtacttcggcaagctggaaagcaagct
gagcgtgatccggaacctgaacgaccaggtgctgttcatcgatcagggcaacagacccctgttcgaggacatgac
cgacagcgactgcagagacaacgcccctcggaccatcttcatcatcagcatgtacaaggacagccagcctagagg
catggccgtgaccatctctgtgaagtgcgagaagatcagoaccctgagctgcgagaacaagatcatcagcttcaa
agagatgaacccgccggacaacatcaaggacaccaagagcgacatcatattcttccagcggagcgtgcccggcca
cgacaacaagatgcagtttgagagcagcagctacgagggctacttcctggcctgcgagaaagagcgggacctgtt
caagctgatcctgaagaaagaggacgaactgggcgaccgcagcatcatgttcaccgtgcagaacgaggactgata
aaagoLLggcaaLccggLacLgLLggLaaagccacca (SEQ ID NO:1752)
[00915] (amino acids)
IEDIGTDPASRDPMLATMALPVTALLLPLALLLNAARPYFOKLESKLSVIRNLNDQVLFIDQGNRPLFEDMIDSD
CRDNAPRTIFIISMYKDSOPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGMDNK
MWESSSYEGYFLACEKERDLEKLILKKEDELGDRSIMFIVQNED*'KLGNPVLLVKPP (SEQ ID
NO: 1153)
primer
5f- agggagacccaagctggctagttaagcttggatggccttaccagtgaccgccttgc-3' (SEQ ID
NO: 1754)
primer
5'- taggccagagaaatgttctggcattatcagcgagggggcagggcctgc-3' (SEQ ID NO:1755).
5'- tgccagaacatttctctgg-3' (SEQ ID NO:1756)
5f- acagtcgaggctqatcagcgggtttaaacttatcagtcctcgttctgcacgg-3' (SEQ ID NO:
1757)
5'-atgcaggccctgccccctcgctgataagtttaaactgccagaacatttctctggcctaac-3' (SEQ ID
NO :1758)
5'- accggagcgatcgcagatccttcgcggccgcttatcagtcctcgttctgcacggtgaac-3' (SEQ ID
NO: 1759)
5'- attgcactagttgaaagaccccacctgtagg-3' (SED ID NO:1760)
5'- aaLgeLcLagaaLaegggLaLccagg-3' (SEQ ID NO:1761)
D' atagegaattegtaccgagggeeaccatgg-3' (SEQ ID NO:1762)
5'- taggcctcccaccgtacacgcctaggtaccacgccttctgtatg-3' (SEQ ID NO:1763)
5f- taggcctcccaccgtacacgcctaggtacctctgcagtaaatgq-3' (SEQ ID NO:1764)
5f- taaggccatggtggctagc-3' (SEQ ID NO:1765)
5'- aataagtttaaactgccagaacatttotctgg-3' (SEQ ID NO:1766)
5'- atatagcggccgcttatcagtcctcgttctgcacgg-3' (SEQ ID NO:176/)
[00916] 6x FoxP3NFAT mCMV
[00917] (DNA)
357
CA 03187555 2023- 1- 27
WC) 202/1127039
PCT/US2021/071017
[00918] Agaacatttctctggcctaactggccggtaccggcttcattttttccatttactgcagaggcttca
ttttttccatttactgcagaggcttcattttttccatttactgcagaggcttcatttttccatttactgcagag
gcttcattttttccatttactgcagaggcttcattttttccatttactgcagaac-tagttaggcgtgtacggtgg
gaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctc
catagaagacaccgggaccgatccagcctcgagagacccaaLgcLagccaccatgg (SEQ ID NO:1768)
[00919] (amino acids)
[00920] RTFLWPNWPVPASFFPFTAEASFFPFTAEASFFPFTAEASFFPFTAEASFFPFTAEASFFPFTAEL
VRRVRWEAYISRARLVNRQIAWRRHPRCFDLHRRHRDRSSLERPNASHHG (SEQ ID NO:1769)
[00921] 6x FoxP3NFAT mIL2P
[00922] (DNA)
[00923] AgaacaLLLcLcLggccbaacLggccggLaccggcLLcaLLA_LALccabbbacLgcagaggcLLca
ttttttccatttactgcagaggcttcattttttccatttactgcagaggcttcatttttccatttactgcagag
gcttcattttttccatttactgcagaggcttcattttttccatttactgcagaactagtcattttgacaccccca
taatatttttccagaattaacagtataaattgcatctcttgttcaagagttccctatcactctctttaatcacta
ctcacagtaacctcaactcctgcctcgagagacccaatgctagccaccatgg (SEQ ID NO:1770)
[00924] (amino acids)
[00925] RTFLWPNWPVPASFFPFTAEASFFPFTAEASFFPFTAEASFFPFTAEASFFPFTAEASFFPFTAEL
VILIPP*YESRINSINCISCSRVPYHSL*SLLTVISTPASRDPMLATMX (SEQ ID NO:1771)
[00926] 6x FoxP3NFAT miniP
[00927] (DNA)
[00928] Agaacatttctctggcctaactggccggtaccggcttcattttttccatttactgcagaggcttca
ttttttccatttactgcagaggcttcattttttccatttactgcagaggcttcatttttccatttactgcagag
gcttcattttttccatttactgcagaggcttcattttttccatttactgcagaacagtagagggtatataatgg
aagctcgacttccagctcgagagacccaatgctagccaccatgg (SEQ ID NO:1772)
[00929] (amino acids)
[00930] RTELWPNWPVPASEEPETAEASEEPETAEASEEPFTAEASEFPFTAEASEEPFTAEASEEPETAEL
VEGI*WKLDFQLERPNASHHG (SEQ ID NO:1773)
[00931] 6x IL2NFAT mCMV
[00932] (DNA)
[00933] Agaacatttctotggcctaactggccggtaccggaggaaaaactgttcatacagaaggcgtggag
gaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcat
acagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgt7_tcatacagaaggcgtacta
gttaggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccat
ccacgctgttttgacctccatagaagacaccgggaccgatccagcctcgagagacccaatgctagccaccatgg
(SEQ ID N0:1774)
[00934] (amino acids)
[00935] RTFLW2NWPVPEEKLEHTEGVEEKLEHTEGVEEKLF=EGVEEKLEHTEGVEEKLEHTEGVEEKLF
HTEGVLVARVRWEAYISRARLVNRQIAWRRHPRCEDLHARHADRSSLERPNASHHG (SEQ ID NO: :775)
358
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
[00936] 6x IL2NFAT mIL2P
[00937] (DNA)
[00938] Agaacatttctctggcctaactggccggtaccggaggaaaaactgtttcatacagaaggcgtggag
gaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcat
acagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtacta
gtcattttgacacccccataatatttttccagaattaacagtataaattgcatctcttgttcaagagttccctat
cactctotttaatcactactcacagtaacctcaactcctgcctcgagagacccaa-tgctagccaccatgg
(SEQ ID NO:1776)
[00939] (amino acids)
[00940] RTFLWPNWPVPEEKLFHTEGVEEKLFHTEGVEEKLFHTEGVEEKLFHTEGVEEKLFHTEGVEEKLF
HTEGVLVILIPP*YYSRINSINCISCSRVPYHSL*SLLTVISTPASRDPMLATMX (SEQ ID NO:1777)
[00941] 6x IL2NFAT miniP
[00942] (DNA)
[00943] Agaacatttctctggcctaactggccggtaccggaggaaaaactgttcatacagaaggcgtggag
gaaaaacLgLLLcaLacagaaggcgAgaggaaaaacLgLLLcaLacagaaggcg_ggaggaaaaacLgLLLcaL
acagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtacta
gLagagggLabaLaaLggaagcLcgacLLccagcLcgagagacccaatgctagccaccaLgg (SEQ ID
NO:1778)
[00944] (amino acids)
[00945] RTELWPNWPVPEEKLEHTEGVEEKLEHTEGVEEKLEHTEGVEEKLEHTEGVEEKLEHTEGVEEKLF
HTEGVLVEGI*WKLDFQLERPNASHHG (SEQ ID NO:1779)
Human NME1
(DNA)
atggccaactgtgagcgtaccttcattgcgatcaaaccagatggggtccagcggggtcttgtgggagagattatc
aagcgttttgagcagaaaggattccgccttgttggtctgaaattcatgcaagettccgaagatcttctcaaggaa
cactacgttgacctgaaggaccgtccattctttgccggcctggtgaaatacatgcactcagggccggtagttgcc
atggtctgggaggggctgaatgtggtgaagacgggccgagtcatgctcggggagaccaaccctgcagactccaag
cctgggaccatccgtggagacttctgcatacaagttggcaggaacattatacatggcagtgattctgtggagagt
gcagagaaggagatcggcttgtggtttcaccctgaggaactggtagattacacgagctgtgctcagaactggatc
tatgaatga (SEQ ID NO:1780)
(amino acids)
MANCERTFIAIKPDGVORGLVGEITKRFPOKGFRLVGLKFMOASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVA
MVWEGLNVVKTGRVMLGETNPADSKPGTIRGDECIQVGRNIIHGSDSVESAEKEIGLWEHPEELVDYTSCAQNWI
YE- (SEQ ID NO:1781)
Human NME7
(DNA)
atgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgottcacttcttcgacgttatgag
cttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggacc
aaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtatta
aLLgacLaLggggaLcaaLaLacagcLcgccagcLgggcagLaggaaagaaaaaacgcLagcccLaaLLaaacca
gatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatg
atgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatc
cagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactg
ctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggc
ataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagt
ggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgtaaaccccatgctgtcagt
359
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
claaggactgttgggaaagatcctgatggctatccgagatgcaggttttqaaatctcagctatgcagatgttcaat
aLggaLcgggLLaaLgLLgaggaaLLcLaLgaagLLLaLaaaggagLagLgaccgaaLaLcaLgacaLggLgaca
gaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgt
ggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaa7.ctttgqtaaaactaagatc
cagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggat
aattag (SEQ ID N0:1782)
[00946] (amino acids)
MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVL
IDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELI
WITTGPIIAMEILDDAIMEWKALLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSS
GGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVT
EMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRATFGKTKIQNAVHCTDLPEDGLLEVQYFFKILD
N- (SEQ ID NO:1783)
[00947] hC2 CD28 1XX (CD28 h/tm)
[00948] (DNA)
[00949]
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgcagctggt
ggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccacagtggct
atgccatg
agctgggtccgccaggctccaggg aaggggctgg agtgggtctc aacc attagtagtggcgg aacctac
atatactacccc g actc a
gtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgagg
ac acggc
cgtgtattactgtgcgagacttgggggggataattactacgaatacttcgatgtctggggcaaagggaccacggtc
accgtctcctccg
gcggtggcgg atccggcggtggcggatccggcggtggcgg atccgac attgtgctgaccc agtctcc
agcctccaggccgtgtctc
caggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagcggatactcctacatgcactggtatca
gcagaaa
cc agg ac aac ctcctaaactcctg atttacctggcatcc aatctgg agagcggggtccc agccaggttc
agcggcagtgggtctggg
accgatttcaccctcacaattaatcctgtggaagctaatgatactgcaaattattactgtcagcacagtagggagctgc
catcacattegg
cggagggaccaaggtggagaLcaaacgaactaagcacctgtglccactccactgaccccggccctagcaagccatctgg
gtcctg
gtggtggtgggcggagtgctggcctgctacagcctgctggtgaccgtggcattatcatcttctgggtgaggagtaagag
gagcaggc
tectgcacagtgactacatgaacatgactectagaagacctgggcctaccagaaagcattaccagccctatgccccacc
acgcgactt
cgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctat
aacgag
ctcaatctagg acg aag ag aggagtacg atgttttgg acaag ag acgtggccggg accctgag
atggggggaaagccgag aagg a
agaaccctcaggaaggcctgacaatgaactgcagaaagataagatggcggaggcatcagtgagattgggatgaaaggcg
agcgc
cggaggggcaaggggcacgatggccttttccagggtctc agtacagcc
accaaggacaccttcgacgcccttcacatgc aggccct
gccccctcgctgataa (SEQ ID NO: 1784)
[00950] (amino acids)
[00951] MALPV TALLLPLALLLHAARPE V QL V ES GGGLV KPGGSLRLS CAA S GFTF
S GYAMS WVRQAPGKGLEWVSTIS S GGTYIYYPDSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCARLGGDNYYEYFDVWGKGTTVTVSSGGGGSGGGGSGGGGSDIVL
TQS PAS LAYS PGQRATITCRAS KS VS TS GYSYMHAVYQQKPGQPPKWYLASNLES
360
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
VPARFS GS GS GTDFTLTINPVEANDTANYYCQHSRELPFTFGGGTKVEIKRTKHLCPS
PLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG
PTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQN QLYNELNLGRREEYDVLD
KRRGRDPEMGGKPRRKNPQEGLENELQKDKMAEAFSEIGMKGERRRGKGHDGLFQ
GLSTATKDTFDALHMQALPPR-- (SEQ ID NO:1785)
[00952] CD8 leader sequence
[00953] (DNA)
[00954] Atggccttaccagtgaccgccttgacctgccgctggccttgctgctccacgccgccaggccg (SEQ ID
NO:1786)
[00955] (amino acids)
[00956] MALPVTALLLPLALLLHAARP(SEQ ID NO:1787)
[00957] hMNC2 scFV
[00958] (DNA)
[00959]
gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctg
gattcaccttcagtggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaccattag
tagtggcg
gaacctacatatactaccccgactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatct
gcaaatgaa
cagcctgagagccgaggacacggccgtgtattactgtgcgagacttgggggggataattactacgaatacttcgatgtc
tggggcaaa
gggaccacggtcaccgtctcctccggcggtggcggatccggcggtggcggatccggcggtggcggatccgacattgtgc
tgaccc
agtctccagcctccaggccgtgtctccaggacagagggccaccatcacctgcagagccagtaagagtgtcagtaccagc
ggatact
cctacatgcactggtatcagcagaaaccaggacaacctcctaaactcctgatttacctggcatccaatctggagagcgg
ggtcccagc
c aggttc agcggc agtgggtctggg accg atttc accctc ac aattaatcc tgtgg
aagctaatgatactgc aaattattactgtcagc ac
agtagggagctgcctttcacattcggcggagggaccaaggtggagatcaaacgaact(SEQ ID NO:1788)
[00960] (amino acids)
[00961] EV QLVESGGGLVKPGGSLRLSCAASGFTFSGYAMS WVRQAPGKGLEW VS
TIS S GGTYIYYPDS VKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARLGGDNYYE
YFDVWGKGTTVTVSSGGGGS GGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASK
SVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFS GS GS GTDFTLTINPVEAN
DTANYYCQHSRELPFTFGGGTKVEIKRT (SEQ ID NO:1789)
[00962] CD28 hinge region (811-855)
[00963] (DNA)
[00964] Aagcacctgtgtccttctccactgttccccggccctagcaagcct (SEQ ID NO:1790)
[00965] (amino acids)
[00966] KHLCPSPLFPGPSKP (SEQ ID NO:1791)
[00967] CD28 transmembrane domain (856-936)
361
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
[00968] (DNA)
[00969] Ttctgggtcctggtggtggtgggcggagtgctggcctgctac
agcctgctggtgaccgtggcctttatcatcttctggg
tg (SEQ ID NO:1792)
[00970] (amino acids)
[00971] FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:1793)
[00972] CD28 (937-1059)
[00973] (DNA)
[00974]
Aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactcctagaagacctgggcctaccagaaag
cattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc (SEQ ID NO:1794)
[00975] (amino acids)
[00976] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID
NO:1795)
[00977] CD3 zeta 1XX
[00978] (DNA)
[00979]
Agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaa
tctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatgggggg
aaagccgagaaggaagaa
ccctcaggaaggcctgacaatgaactgcagaaagataagatggeggaggcatcagtgagattgggatgaaaggcgagcg
ccgg a
ggggcaaggggcacgatggccattccagggtctcagtacagccaccaaggac acc ttcgacgccc ttc ac
atgc aggc cc tgccc
cctcgctgataa (SEQ ID NO:1796)
[00980] (amino acids)
[00981] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK
PRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDAL
HMQALPPR--
[00982] (SEQ ID NO:1797)
CD28 hinge region
...
CD3z 1XX
............. .................. ........
CD28 transmembrane
[00983]
[00984] h20A1ON CD28 1XX (CD28 h/tm)
[00985] (DNA)
[00986] atggccttac
cagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgcaggtgcagctggtt
gaatctggcggcggacttgtgaagcctggcggatctctgagactgagctgtgccgccagcggcttcacctttagcacat
acgccatga
362
CA 03187555 2023- 1- 27
WO 2022/027039
PCT/US2021/071017
gctggatcagacaggcccctggcaaaggcctggaatgggtggcgtctattggcagagccggcagcacctactacagcga
ctctgtg
aagggcagattcaccatcagccgggacaacgccaagaacagcctgtacctgcagatgaactccctgagagccgaggaca
ccgccg
tgtactattgtgccagaggacccatctacaacgactacgacgagttcgcctattggggccagggcacactggtcacagt
ttctagcggc
ggtggcggaagcggaggcggtggctccggtggcggaggcagcgaaattgtgctgacacagagccccgccacactgtcac
tttctc
caggcgaaagagccacactgagctgcaagagcagccagagcgtgctgtactccagcaaccagaagaactacctggcctg
gtatca
gcagaagcceggccaagctcctcggctgctgatctattgggccagcacaagagagagcggcatccctgccagattnctg
gcagcg
gctctggcaccgatttcaccctgaccataagc agcctggaacctgaggacttcgccgtgtattactgccacc
agtacctgagc agcctg
acctttggcggaggcaccaaggtggaaatcaagcggaagcacctgtgtccttctccactgttccccggccctagcaagc
catctgggt
cctggtggtggtgggeggagtgctggcctgctacagcctgctggtgaccgtggccatatcatcactgggtgaggagtaa
gaggagc
aggctcctgcacagtgactacatgaacatgactcctagaagacctgggcctaccagaaagcattaccagccctatgccc
caccacgc
gacttcgcagcctatcgctccagagtgaagttcagcagg
agcgcagacgcccccgcgtacaagcagggccagaaccagctctataa
cgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaag
ccgaga
aggaagaaccctcaggaaggcctgttcaatgaactgcagaaagataagatggeggaggccttcagtgagattgggatga
aaggcga
gcgccggaggggcaaggggcacgatggccnticcaggglctcagtacagccaccaaggacaccncgacgcccncacatg
cagg
ccctgccccctcgctgataa (SEQ ID NO:1798)
[00987] (amino acids)
[00988] MALPVTALLLPLALLLHAARPQVQLVESGGGLVKPGGSLRLSCAASGFTF
S TYAMS WIRQAPGKGLEWVAS IGRAGS TYYSD S VKGRFTIS RDNAKNSLYLQMNSL
RAEDTAVYYCARGPIYNDYDEFAYWGQGTLVTVSSGGGGSGGGGS GGGGSEIVLTQ
SPATLSLSPGERNILSCKSSQS VLY SSN QKN YLAW Y QQKPGQAPRLLIY W ASTRESGI
PARFS GS GS GTDFTLTIS SLEPEDFAVYYCHQYLS SLTEGGGTKVEIKRKHLCPSPLFP
GPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRS KRSRLLHSDYMNMTPRRPGPTR
KHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKR
RGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGL
STATKDTFDALHMQALPPR-- (SEQ ID NO:1799)
[00989] CD8 leader sequence
[00990] (DNA)
[00991] Atggccttaccagtgaccgccttgctectgccgctggccttgctgctccacgccgccaggccg (SEQ
ID
NO:1800)
[00992] (amino acids)
[00993] MALPVTALLLPLALLLHAARP (SEQ ID NO:1801)
[00994] h20A10N scFV
[00995] (DNA)
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[00996] Caggtgc agctggttg aatctggcggcgg acttgtgaagcctggcggatctctgagactg ag
ctgtgccgcc agcg
gcttcacctttagcacatacgccatgagctggatcagacaggcccctggcaaaggcctggaatgggtggcgtctattgg
cagagccg
gcagcacctactacagcgactctgtgaagggcagattcaccatcagccgggacaacgccaagaacagcctgtacctgca
gatgaac
tccctgagagccgaggacaccgccgtgtactattgtgccagaggacccatctacaacgactacgacgagttcgcctatt
ggggccag
ggcacactggtcacagthctagcggcggtggcggaageggaggcggtggctccggtggeggaggcagcgaaattgtgct
gacac
agagccccgccacactgtcactttctccaggcgaaagagccacactgagctgcaagagcagccagagcgtgctgtactc
cagcaac
cagaagaactacctggcctggtatcagcagaagcccggccaagctcctcggctgctgatctattgggccagcacaagag
agagcgg
catccctgccagattactggcagcggctctggcaccgatttcaccctgaccataagcagcctggaacctgaggacttcg
ccgtgtatta
ctgccaccagtacctgagcagcctgacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO:1802)
[00997] (amino acids)
[00998] EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVS
TISSGGTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGGDNYYE
YFDVWGKGTTVTVSSGGGGS GGGGS GGGGSDIVLTQS PA SLAVSPGQRATITCRAS K
S VS TS GYS YMHWYQQKPGQPPKLLIYLASNLES GVPARFS GS GS GTDFTLTEN PVEAN
DTANYYCQHSRELPFTFGGGTKVEIKRT (SEQ ID NO:1803)
[00999] CD28 hinge region (811-855)
[001000] (DNA)
[001001] Aagcacctgtgtccttctccactgttccccggccctagcaagcct (SEQ ID NO:1804)
[001002] (amino acids)
[001003] KHLCPSPLEPGPSKP (SEQ IDN 0:1805)
[001004] CD28 transmembrane domain (856-936)
[001005] (DNA)
[001006]
Ttctgggtcctggtggtggtgggcggagtgctggcctgctacagcctgctggtgaccgtggcctttatcatcttctggg
tg (SEQ ID NO:1806)
[001007] (amino acids)
[001008] FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:1807)
[001009] CD28 (937-1059)
[001010] (DNA)
[001011]
Aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactcctagaagacctgggcctaccagaaag
cattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc (SEQ ID NO:1808)
[001012] (amino acids)
[001013] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID
NO:1809)
[001014] CD3 zeta 1XX
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[001015] (DNA)
[001016]
Agagtgaagttcagcaggagcgcagacgcceccgcgtacaagcagggccagaaccagctctataacgagetcaa
tctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaagg
aagaa
ccetcaggaaggcctgttcaatgaactgcagaaagataagatggeggaggectteagtgagattgggatgaaaggcgag
cgcegga
ggggcaaggggcacgatggccuttccagggtctcagtacagccaccaaggacaccucgacgcccttcacatgcaggccc
tgccc
cctcgctgataa (SEQ ID NO:1810)
[001017] (amino acids)
[001018] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK
PRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDAL
HMQALPPR--
[001019] (SEQ ID NO:1811)
[001020]
CD28 hinge region
NEEMINISZEM
CD3z 1XX
L\\\\
[001021] CD28 transmembrane
[001022]
[001023] Humanized 3C2B1 scFv sequence
[001024] (DNA)
[001025]
Gaggtgcagctggtggaatccggeggaggactcgtgaagcccggcggcagcctgeggctgagctgcgccgcttc
tggcatcaccttcagcacctacaccatgagctgggtcagacaggcccctggcaagggcctggaatgggtggccacaatc
tctacagg
eggagataagacctactacagegacagegtgaaaggcagattcaccatctetagagataatgccaagaacaccctgtac
ctgcaaat
gaacagcctgegggccgaggacaccgccgtgtactattgtgctagaggeacaacagccatgtactactacgccatggac
tactgggg
ccagggcacaaccgtgaccgtgtccagcggcggtggcggaagcggaggcggtggctccggtggcggaggcagcgatatc
gtgct
gacccagagccctgcttctctggccgtgtccccaggccagcgggccacaatcacctgtagagccagcaagtccatcagc
accagcg
actacaactacatccactggtatcagcagaaacctggccaacctcctaagctgctcatctacctggccagcaacctgga
aagcggcgt
gcccgccagattcageggatctggctctggcaccgacttcaccctgacaattagccccgtcgaggccgaggacgccgcc
acctacta
ctgccagcacagcagagagctgcctctgacctttggcggcggaacaaaggtggaaatcaag (SEQ ID NO:1812)
[001026] (amino acids)
[001027] EVQLVESGGGLVKPGGSLRLSCAASGITFSTYTMSWVRQAPGKGLEWVA
TISTGGDKTYYSDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARGTTAMYY
YAMDYWGQGTTVTVS SGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRA
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S KSIS TS DYNYIHWYQQKPGQPPKLLIYLAS NLES GVPARFS GS GS GTDFTLTISPVEA
EDAATYYCQHSRELPLTEGGGTKVEIK (SEQ ID NO:1813)
[001028] Humanized 5C6F3 scEv sequence
[001029] (DNA)
[001030]
Gaggtgcagctggtggaaagcggcggcggactggtgaaacccggeggcagcctgagactctcttgtgccgccag
cggatttacattcagcacctacgccatgagctgggtgcggcaggcccctggcaagggcctggaatgggtcgccgctatc
tccaacgg
cggaggctacacctactatcctgattctctgaagggcaggacaccatcagcagagataacagcaagaataccctgtacc
tgcaaatga
actctctgcgggccgaggacaccgccgtgtactactgcgctagaagatactacgaccactacttcgactactggggcca
gggcacac
tggtgacagtgtccagcggcggtggeggaageggaggeggtggctccggtggeggaggcagcgatatcgtgatgaccca
atctcc
tagcagcctgagcgtgtccccaggcgagcgggccagcatcagctgtagaagcteccagaccatcgtgcacagcaacggc
aacacc
tacctggaatggtatcagcagaaacctggccagagccccaagctgctgatctacaaggtgtctaatagattcagcggcg
tgcctgata
gatttagcggatctggctccggcacagacttcaccctgacaatactggagttgaggccgaggacgtgggcgtctactac
tgcaccag
gacagccacgtgcccctgaccacggeggeggaacaaaggtggaaatcaag (SEQ ID NO:1814)
[001031] (amino acids)
[001032] EVQLVESGGGLVKPGGSLRLSCAASGFIFSTYAMSWVRQAPGKGLEWVA
AISNGGGYTYYPDSLKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCARRYYDHYF
DYWGQGTLVTVS SGGGGSGGGGS GGGGSDIVMTQSPSSLSVSPGERASISCRS SQTIV
HSNGNTYLEWYQQKPGQSPKLLIYKVSNRFS GVPDRFS GS GS GTDFTLTISGVEAED
VGVYYCFQDSHVPLTEGGGTKVEIK (SEQ ID NO:1815)
[001033] Those skilled in the art will recognize, or be able to ascertain
using no more than
routine experimentation, many equivalents to the specific embodiments of the
invention
specifically described herein.
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