Note: Descriptions are shown in the official language in which they were submitted.
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CONSTRUCTS TARGETING HPV16-E7 PEPTIDE/MHC COMPLEXES AND USES
THEREOF
CROSS-REREFENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No.
62/158,735,
filed on May 8, 2015, and U.S. Provisional Application No. 62/197,480, filed
on July 27,
2015, all of which are hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] This invention pertains to antibody constructs that specifically bind
MHC molecules
complexed with HPV16-E7 peptides, and uses thereof including treating and
diagnosing
diseases.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0003] The content of the following submission on ASCII text file is
incorporated herein
by reference in its entirety: a computer readable form (CRF) of the Sequence
Listing (file
name: 7500420002405EQLI5T.txt, date recorded: May 6, 2016, size: 125 KB).
BACKGROUND OF THE INVENTION
[0004] Human papillomaviruses (HPVs) are small, non-enveloped DNA viruses that
cause
warts or benign papillomas upon infection in epithelial cells. Persistent
infections with high-
risk HPV types can lead to more serious cytological abnormalities or lesions
that, if
untreated, may progress to cancer. Annual incidence of HPV-associated cancers
is >26,000 in
the US (according to CDC) and >600,000 worldwide (Forman D. et al., Vaccine,
30: Suppl
5:F12-23, 2012). Different types of HPV are responsible for causing almost all
cases of
cervical cancer (Doorbar J., (lin. Sci., 110:525-541, 2006), the third leading
cause of cancer-
related deaths in women worldwide. In addition to cervical cancers, HPV is
also associated
with anogenital cancers (anus, penis, vagina and vulva) and head/neck cancers
(oropharynx:
back of the throat, including the base of the tongue and tonsils). In a meta-
analysis of 5,046
head and neck squamous cell carcinomas (HNSCC) cancer specimens from 60
studies, the
overall HPV prevalence was found to be 25.9%. Among HPV-positive HNSCC, HPV-16
was
found in 86.7% of the HPV-positive oropharyngeal SCCs, 68.2% of oral SCCs and
69.2% of
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laryngeal SCCs. (Kreimer A.R. et al., Cancer Epidemiol Biomarkers Prey. 14:467-
475,
2005). In a more recent study (The Cancer Genome Atlas Network, Nature,
517:576-582,
2015) that classified HPV status by presence of >1,000 mapped RNA sequencing
(RNA-Seq)
reads of HPV E6 and E7 proteins, 36 out of 279 (12.9%) were found to be HPV-
positive.
[0005] Cancer development upon persistent infection with high risk HPV subtype
is mainly
attributable to the expression of two potential oncogenes, E6 and E7, which
have been
documented to degrade p53 and Rb in a proteasome-dependent manner, thereby
promoting
genomic instability and cellular transformation (Doorbar, supra). The HPV E6
and E7
oncoproteins are continuously expressed in the lesions, while tumor arises
only several years
after the initial cellular immortalizing events. In fact, the continuous
expression of E6 and E7
is required for maintenance of the transformed phenotype, and prevention of
cell growth
arrest and/or apoptosis (McLaughlin-Drubin M.E. & Munger K., Virology, 384:335-
344,
2009). Since established lesions do not express Li, there is an unmet need for
treatment of
HPV cancers despite the availability of approved prophylactic vaccines
directed against the
Li protein of HPV-6, 11, 16 and 18 subtypes.
[0006] Proteins expressed by oncogenic viruses such as HPV represent excellent
targets for
immunotherapy due to absence of expression in normal cells. E6 and E7 are
intracellular
proteins that have not been targeted by traditional drug development efforts
using low
molecular weight compounds or antibody approaches against cell surface
proteins. Recent
studies in HPV16-positive high-grade vulvar intraepithelial neoplasia have
demonstrated T
cell and clinical responses in 50% of patients treated with vaccination
approaches that
utilized E6/E7 peptides (Kenter G.G. et al., N. Engl. J. Med. 361:1838-1847,
2009; Welters
M.J. et al., N. Engl. J. Med. 361:1838-1847, 2010; Daayana S. et al., Br. J.
Cancer
102:1129-1136, 2010). Among 13 distinct predicted HLA-A*02:01-binding E7
peptides,
only E711-19 (YMLDLQPET), was found to be naturally processed and displayed on
HPV-16
transformed HLA-A*02:01 expressing cell lines in vitro (Riemer A.B. et al., J.
Biol. Chem.
285(38):29608-29622, 2010). This peptide is conserved in 16 out of 17 HPV-16
variants in
the HPV database (Zhang G.L. et al., Database, 1-12, 2014).
[0007] Recent advances in using phage display to generate mAbs have made it
possible to
select agents with exquisite specificity against defined epitopes from large
antibody
repertoires. A number of such mAbs specific for solid tumor antigens, in the
context of HLA-
A01 and HLA-A02, have been successfully selected from phage display libraries
(Noy et al.,
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Expert Rev. Anticancer Ther. 5(3):523-536, 2005; Chames et al., Proc. Natl.
Acad. Sci. USA
97:7969-7974, 2000; Held et al., Eur. J. Immunol. 34:2919-2929, 2004; Lev et
al., Cancer
Res. 62:3184-3194, 2002; Klechevsky et al., Cancer Res. 68(15):6360-6367,
2008). More
recently, a human mAb specific for human WT1/HLA-A02 complex, a well-described
T cell
epitope, has been shown to inhibit multiple cancer cell lines and primary
cancer cells via Fc-
mediated effector cell function (Dao et al., Sci. Transl. Med. 5:176ra33,
2013; Veomett et al.,
Clin. Cancer Res. doi:10.1158/1078-0432, 2014) in cellular assays and in in
vivo models.
[0008] The disclosures of all publications, patents, patent applications and
published patent
applications referred to herein are hereby incorporated herein by reference in
their entirety.
BRIEF SUMMARY OF THE INVENTION
[0009] The present application in one aspect provides constructs (such as
isolated constructs)
that bind to a complex comprising an HPV16-E7 peptide and an MHC class I
protein
(referred to herein as an "HPV16-E7/MHC class I complex," or "E7MC"). In some
embodiments, the constructs ("anti-E7MC constructs") comprise an antibody
moiety
(referred to herein as an "anti-E7MC antibody moiety") that specifically binds
to a complex
comprising an HPV16-E7 peptide and an MHC class I protein.
[0010] Thus, in some embodiments, there is provided an anti-E7MC construct
(such as an
isolated anti-E7MC construct) comprising an antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein. In some
embodiments, the HPV16-E7/MHC class I complex is present on a cell surface. In
some
embodiments, the HPV16-E7/MHC class I complex is present on the surface of a
cancer cell.
[0011] In some embodiments, the anti-E7MC construct comprises an antibody
moiety that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the MHC class I protein is HLA-A. In some embodiments, the
MHC class I
protein is HLA-A02. In some embodiments, the MHC class I protein is the HLA-
A*02:01
subtype of the HLA-A02 allele.
[0012] In some embodiments, according to any of the anti-E7MC constructs (such
as isolated
anti-E7MC constructs) described above, the anti-E7MC construct comprises an
antibody
moiety that specifically binds to a complex comprising an HPV16-E7 peptide and
an MHC
class I protein, wherein the antibody moiety cross-reacts with a complex
comprising the
HPV16-E7 peptide and a second MHC class I protein having a different HLA
allele than the
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MHC class I protein. In some embodiments, the antibody moiety cross-reacts
with a complex
comprising a variant of the HPV16-E7 peptide comprising one amino acid
substitution (such
as a conservative amino acid substitution) and the MHC class I protein.
[0013] In some embodiments, according to any of the anti-E7MC constructs (such
as
isolated anti-E7MC construct) described above, the anti-E7MC construct
comprises an
antibody moiety that specifically binds to a complex comprising an HPV16-E7
peptide and
an MHC class I protein, wherein the HPV16-E7 peptide is about 8 to about 12
(such as about
any of 8, 9, 10, 11, or 12) amino acids in length. In some embodiments, the
HPV16-E7
peptide has an amino acid sequence selected from the group consisting of SEQ
ID NOs: 3-14.
In some embodiments, the HPV16-E7 peptide has the amino acid sequence
YMLDLQPET
(SEQ ID NO: 4).
[0014] In some embodiments, according to any of the anti-E7MC constructs (such
as isolated
anti-E7MC constructs) described above, the anti-E7MC construct comprises an
antibody
moiety that specifically binds to a complex comprising an HPV16-E7 peptide and
an MHC
class I protein, wherein the antibody moiety is a full-length antibody, a Fab,
a Fab', a (Fab')2,
an Fv, or a single chain Fv (scFv). In some embodiments, the antibody moiety
is fully human,
semi-synthetic with human antibody framework regions, or humanized.
[0015] In some embodiments, according to any of the anti-E7MC constructs (such
as isolated
anti-E7MC constructs) described above, the anti-E7MC construct comprises an
antibody
moiety that specifically binds to a complex comprising an HPV16-E7 peptide and
an MHC
class I protein, wherein the antibody moiety binds to the HPV16-E7/MHC class I
complex
with an equilibrium dissociation constant (Kd) between about 0.1 pM to about
500 nM (such
as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50
nM, 100
nM, or 500 nM, including any ranges between these values). In some
embodiments, the
isolated anti-E7MC construct binds to the HPV16-E7/MHC class I complex with a
Kd
between about 0.1 pM to about 500 nM (such as about any of 0.1 pM, 1.0 pM, 10
pM, 50
pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM, including any
ranges
between these values).
[0016] In some embodiments, the anti-E7MC construct comprises an antibody
moiety that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the antibody moiety comprises: i) a heavy chain variable
domain comprising
a heavy chain complementarity determining region (HC-CDR) 1 comprising the
amino acid
sequence of G-F/G/Y-S/T-F-S/T-S-Y-A/G (SEQ ID NO: 183), or a variant thereof
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comprising up to about 3 (such as about any of 1, 2, or 3) amino acid
substitutions, an HC-
CDR2 comprising the amino acid sequence of I-N/I PX X G G/T/I-T/A/P or I-S-X-
S/D-
G/N-G/S-N-T/I/K (SEQ ID NO: 184 or 185), or a variant thereof comprising up to
about 3
(such as about any of 1, 2, or 3) amino acid substitutions, and an HC-CDR3
comprising the
amino acid sequence of any one of A-R-S/R-Y/S/G-Y/V Y/W G X Y D, AR G X XX Y
Y/G/S, or AR G X X Y Q/W-W-S-X-D-D (SEQ ID NOs: 186-188), or a variant
thereof
comprising up to about 3 (such as about any of 1, 2, or 3) amino acid
substitutions; and ii) a
light chain variable domain comprising a light chain complementarity
determining region
(LC-CDR) 1 comprising the amino acid sequence of N-I-G-S-N/K or L-R-S/N-X-Y
(SEQ ID
NO: 189 or 190), or a variant thereof comprising up to about 3 (such as about
any of 1, 2, or
3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence
of A/Q/N-
S/A/V-W/Y/R-D-S/D-S-L/S/G-X-X-X-V (SEQ ID NO: 191), or a variant thereof
comprising
up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions,
wherein X can be any
amino acid.
[0017] In some embodiments, the anti-E7MC construct comprises an antibody
moiety that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the antibody moiety comprises: i) a heavy chain variable
domain comprising
an HC-CDR1 comprising (and in some embodiments consisting of) the amino acid
sequence
of any one of SEQ ID NOs: 57-77, or a variant thereof comprising up to about 5
(such as
about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising
(and in some
embodiments consisting of) the amino acid sequence of any one of SEQ ID NOs:
78-98, or a
variant thereof comprising up to about 5 (such as about any of 1,2, 3,4, or 5)
amino acid
substitutions, and an HC-CDR3 comprising (and in some embodiments consisting
of) the
amino acid sequence of any one of SEQ ID NOs: 99-119, 244, and 245, or a
variant thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions; and
ii) a light chain variable domain comprising an LC-CDR1 comprising (and in
some
embodiments consisting of) the amino acid sequence of any one of SEQ ID NOs:
120-140
and 246, or a variant thereof comprising up to about 5 (such as about any of
1, 2, 3, 4, or 5)
amino acid substitutions, an LC-CDR2 comprising (and in some embodiments
consisting of)
the amino acid sequence of any one of SEQ ID NOs: 141-161, or a variant
thereof
comprising up to about 3 (such as about any of 1, 2, or 3) amino acid
substitutions, and an
LC-CDR3 comprising (and in some embodiments consisting of) the amino acid
sequence of
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any one of SEQ ID NOs: 162-182 and 247-250, or a variant thereof comprising up
to about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0018] In some embodiments, the anti-E7MC construct comprises an antibody
moiety that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the antibody moiety comprises: i) a heavy chain variable
domain comprising
an HC-CDR1 comprising (and in some embodiments consisting of) the amino acid
sequence
of any one of SEQ ID NOs: 57-77, an HC-CDR2 comprising (and in some
embodiments
consisting of) the amino acid sequence of any one of SEQ ID NOs: 78-98, and an
HC-CDR3
comprising (and in some embodiments consisting of) the amino acid sequence of
any one of
SEQ ID NOs: 99-119, 244, and 245; or a variant thereof comprising up to about
5 (such as
about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR regions;
and ii) a light
chain variable domain comprising an LC-CDR1 comprising (and in some
embodiments
consisting of) the amino acid sequence of any one of SEQ ID NOs: 120-140 and
246, an LC-
CDR2 comprising (and in some embodiments consisting of) the amino acid
sequence of any
one of SEQ ID NOs: 141-161, and an LC-CDR3 comprising (and in some embodiments
consisting of) the amino acid sequence of any one of SEQ ID NOs: 162-182 and
247-250; or
a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4,
or 5) amino acid
substitutions in the LC-CDR regions.
[0019] In some embodiments, the anti-E7MC construct comprises an antibody
moiety that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the antibody moiety comprises a) a heavy chain variable
domain comprising
(and in some embodiments consisting of) the amino acid sequence of any one of
SEQ ID
NOs: 15-35 and 233-237 or a variant thereof having at least about 95% (such as
at least about
any of 95%, 96%, 97%, 98%, or 99%) sequence identify to any one of SEQ ID NOs:
15-35
and 233-237; and b) a light chain variable domain comprising (and in some
embodiments
consisting of) the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-
243 or a
variant thereof having at least about 95% (such as at least about any of 95%,
96%, 97%, 98%,
or 99%) sequence identity to any one of SEQ ID NOs: 36-56 and 238-243. In some
embodiments, the antibody moiety comprises a heavy chain variable domain
comprising (and
in some embodiments consisting of) the amino acid sequence of any one of SEQ
ID NOs: 15-
35 and 233-237 and a light chain variable domain comprising (and in some
embodiments
consisting of) the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-
243.
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[0020] In some embodiments, the anti-E7MC construct comprises a first antibody
moiety that
competes for binding to a target HPV16-E7/MHC class I complex with a second
antibody
moiety according to any of the antibody moieties described above. In some
embodiments, the
first antibody moiety binds to the same, or substantially the same, epitope as
the second
antibody moiety. In some embodiments, binding of the first antibody moiety to
the target
HPV16-E7/MHC class I complex inhibits binding of the second antibody moiety to
the target
HPV16-E7/MHC class I complex by at least about 70% (such as by at least about
any of
75%, 80%, 85%, 90%, 95%, 98% or 99%), or vice versa. In some embodiments, the
first
antibody moiety and the second antibody moiety cross-compete for binding to
the target
HPV16-E7/MHC class I complex, i.e., each of the first and second antibody
moieties
competes with the other for binding to the target HPV16-E7/MHC class I
complex.
[0021] In some embodiments, according to any of the anti-E7MC constructs
described above
(such as isolated anti-E7MC constructs), the isolated anti-E7MC construct is a
full-length
antibody. In some embodiments, the isolated anti-E7MC construct is
monospecific. In some
embodiments, the isolated anti-E7MC construct is multi-specific. In some
embodiments, the
isolated anti-E7MC construct is bispecific. In some embodiments, the isolated
anti-E7MC
molecule is a tandem scFv, a diabody (Db), a single chain diabody (scDb), a
dual-affinity
retargeting (DART) antibody, a dual variable domain (DVD) antibody, a knob-
into-hole
(KiH) antibody, a dock and lock (DNL) antibody, a chemically cross-linked
antibody, a
heteromultimeric antibody, or a heteroconjugate antibody. In some embodiments,
the isolated
anti-E7MC construct is a tandem scFv comprising two scFvs linked by a peptide
linker. In
some embodiments, the peptide linker comprises (and in some embodiments
consists of) the
amino acid sequence GGGGS.
[0022] In some embodiments, according to any of the anti-E7MC constructs
described above
(such as isolated anti-E7MC constructs), the anti-E7MC construct comprises an
antibody
moiety that specifically binds to a complex comprising an HPV16-E7 peptide and
an MHC
class I protein, wherein the isolated anti-E7MC construct further comprises a
second antigen-
binding moiety that specifically binds to a second antigen. In some
embodiments, the second
antigen-binding moiety is an antibody moiety. In some embodiments, the second
antigen is
an antigen on the surface of a T cell. In some embodiments, the T cell is
selected from the
group consisting of a cytotoxic T cell, a helper T cell, and a natural killer
T cell. In some
embodiments, the second antigen is selected from the group consisting of CD3y,
CD36,
CD3c, CD3; CD28, 0X40, GITR, CD137, CD27, CD4OL, and HVEM. In some
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embodiments, the second antigen is CD3E, and the isolated anti-E7MC construct
is a tandem
scFv comprising an N-terminal scFv specific for the HPV16-E7/MHC class I
complex and a
C-terminal scFv specific for CD3E. In some embodiments, the second antigen is
an antigen
on the surface of a natural killer cell, a neutrophil, a monocyte, a
macrophage, or a dendritic
cell.
[0023] In some embodiments, according to any of the anti-E7MC constructs (such
as isolated
anti-E7MC constructs) described above, the anti-E7MC construct comprises an
antibody
moiety that specifically binds to a complex comprising an HPV16-E7 peptide and
an MHC
class I protein, wherein the isolated anti-E7MC construct is a chimeric
antigen receptor
(CAR). In some embodiments, the chimeric antigen receptor comprises an
extracellular
domain comprising the antibody moiety, a transmembrane domain, and an
intracellular
signaling domain. In some embodiments, the intracellular signaling domain
comprises a
CD3 intracellular signaling sequence and a co-stimulatory signaling sequence.
In some
embodiments, the co-stimulatory signaling sequence is a CD28 intracellular
signaling
sequence.
[0024] In some embodiments, according to any of the anti-E7MC constructs (such
as isolated
anti-E7MC constructs) described above, the anti-E7MC construct comprises an
antibody
moiety that specifically binds to a complex comprising an HPV16-E7 peptide and
an MHC
class I protein, wherein the isolated anti-E7MC construct is an
immunoconjugate comprising
the antibody moiety and an effector molecule. In some embodiments, the
effector molecule is
a therapeutic agent selected from the group consisting of a drug, a toxin, a
radioisotope, a
protein, a peptide, and a nucleic acid. In some embodiments, the therapeutic
agent is a drug or
a toxin. In some embodiments, the effector molecule is a label.
[0025] In yet other embodiments, there is provided a pharmaceutical
composition comprising
an anti-E7MC construct (such as an isolated anti-E7MC construct) according to
any of the
embodiments described above. In some embodiments, the pharmaceutical
composition
further comprises a cell (such as an effector cell) associated with the anti-
E7MC construct. In
some embodiments, there is provided a host cell expressing or associated with
an anti-E7MC
construct, or polypeptide component thereof. In some embodiments, there is
provided a
nucleic acid encoding an anti-E7MC construct, or polypeptide component
thereof. In some
embodiments, there is provided a vector comprising the nucleic acid. In some
embodiments,
there is provided an effector cell expressing or associated with an anti-E7MC
construct. In
some embodiments, the effector cell is a T cell.
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[0026] In some embodiments, there is provided a method of detecting a cell
presenting a
complex comprising an HPV16-E7 peptide and an MHC class I protein on its
surface,
comprising contacting the cell with an anti-E7MC construct (such as an
isolated anti-E7MC
construct) according to any of the embodiments described above comprising a)
an antibody
moiety that specifically binds to a complex comprising the HPV16-E7 peptide
and the MHC
class I protein and b) a label, and detecting the presence of the label on the
cell.
[0027] In some embodiments, there is provided a method of treating an
individual having an
HPV16-E7-positive disease, comprising administering to the individual an
effective amount
of a pharmaceutical composition comprising an anti-E7MC construct (such as an
isolated
anti-E7MC construct) according to any of the embodiments described above. In
some
embodiments, the pharmaceutical composition further comprises a cell (such as
an effector
cell) associated with the isolated anti-E7MC construct. In some embodiments,
there is
provided a method of treating an individual having an HPV16-E7-positive
disease,
comprising administering to the individual an effective amount of an effector
cell expressing
any of the anti-E7MC CARs described above. In some embodiments, the effector
cell is a T
cell. In some embodiments, the HPV16-E7-positive disease is cancer. In some
embodiments,
the cancer is squamous cell carcinoma, cervical cancer, anal cancer, vaginal
cancer, vulvar
cancer, penile cancer, head and neck cancer, or oropharyngeal cancer. In some
embodiments,
the cancer is an HPV16-E7-positive squamous cell carcinoma (SCC).
[0028] In some embodiments, there is provided a method of diagnosing an
individual having
an HPV16-E7-positive disease, comprising: a) administering an effective amount
of an
isolated anti-E7MC construct comprising a label according to any of the
embodiments
described above to the individual; and b) determining the level of the label
in the individual,
wherein a level of the label above a threshold level indicates that the
individual has the
HPV16-E7-positive disease. In some embodiments, there is provided a method of
diagnosing
an individual having an HPV16-E7-positive disease, comprising: a) contacting a
sample
derived from the individual with an isolated anti-E7MC construct comprising a
label
according to any of the embodiments described above; and b) determining the
number of
cells bound with the isolated anti-E7MC construct in the sample, wherein a
value for the
number of cells bound with the isolated anti-E7MC construct above a threshold
level
indicates that the individual has the HPV16-E7-positive disease. In some
embodiments, the
HPV16-E7-positive disease is cancer. In some embodiments, the cancer is
squamous cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
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and neck cancer, or oropharyngeal cancer. In some embodiments, the cancer is
an HPV16-
E7-positive squamous cell carcinoma (SCC).
[0029] Also provided are methods of making any of the constructs described
herein, articles
of manufacture, and kits that are suitable for the methods described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows the size exclusion chromatography (SEC) chromatogram of
HPV16-E7
11-19 peptide/HLA-A*02:01 complex following concentration by ultrafiltration.
Properly
folded peptide/MHC complex monomers: 212 mL; misfolded aggregates: 111 mL;
free 132M:
267 mL.
[0031] FIG. 2 shows the results of phage clone ELISA for specific binding of
biotinylated
HPV16-E7 11-19 peptide/HLA-A*02:01 versus biotinylated C3 control peptide/HLA-
A*02:01.
[0032] FIG. 3 shows the results of phage clone FACS binding assays for binding
of HPV16-
E7 11-19 peptide-loaded T2 cells versus C3 control peptide-loaded T2 cells. 1:
Cell only
negative control; 2: 2 antibody only control; 3: HPV16-E7 11-19 peptide/HLA-
A*02:01-
specific antibody phage clone.
[0033] FIG. 4 shows the results of phage clone #11 FACS binding assays for T2
cells loaded
with HPV16-E7 11-19 peptides having a single alanine substitution at position
1,5, or 8. 1:
Cell only negative control; 2: 2 antibody only control; 3: HPV16-E7 11-19
peptide/HLA-
A*02:01-specific antibody phage clone #11.
[0034] FIG. 5 shows the results of phage clone FACS binding assays for binding
of HPV16-
E7 11-19 peptide-loaded T2 cells versus T2 cells loaded with peptides derived
from normally
expressed endogenous proteins. For each phage clone, the peptides loaded from
left to right
are HPV16-E7 11-19, A2E1, A2E2, A2E3, A2E4, A2E5, A2E6, A2E7, A2E8, A2E9,
A2E11,
and A2E17.
[0035] FIG. 6 shows SDS-PAGE analysis for determining purity of anti-HPV16-E7
11-
19/MHC bispecific antibodies.
[0036] FIG. 7 shows the T-cell killing of HPV16-E7 11-19 peptide-loaded T2
cells mediated
by anti-HPV16-E7 11-19/HLA-A*02:01 bispecific antibodies prepared from various
phage
clones at 1 t.g/m1 and 0.2 .t.g/ml. Negative controls include T2 cells loaded
with AFP158
peptide and bispecific antibody specific for AFP158/HLA-A*02:01. For each
phage clone,
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the peptides loaded from left to right are 1 iig/m1HPV16-E7 11-19, 1
iig/m1AFP158, 0.2
iig/m1HPV16-E7 11-19, and 0.2 t.g/m1 AFP158. NC peptide: AFP158 peptide; NC
Antibody: anti-AFP158/HLA-A*02:01 bispecific antibody.
[0037] FIG. 8A shows the T-cell killing of two cancer cell lines (CaSki and MS-
751)
mediated by anti-HPV16-E7 11-19/MHC bispecific antibodies (BsAb).
[0038] FIG. 8B shows the dose-dependence for T-cell killing of two cancer cell
lines (CaSki
and MS-751) mediated by anti-HPV16-E7 11-19/MHC bispecific antibodies (BsAb)
at
varying BsAb concentrations.
[0039] FIG. 9 shows a schematic representation of a chimeric antigen receptor
construct.
[0040] FIGS. 10A and 10B show the results of BsAb FACS binding assays for T2
cells
loaded with HPV16-E7 11-19 peptides having a WT sequence or a single alanine
substitution
at positions 1-9. FIG. 10A shows results for BsAbs based on clones US-7, 7-1,
7-3, and 7-6.
FIG. 10B shows results for BsAbs based on clones 7-7 and 7-8.
[0041] FIGS. 11A-11C show flow cytometry analysis of T cells transduced with
various
CARs having an anti-HPV16-E7 affinity matured or parental scFv; cells were
stained with
HPV16-E7 11-19 peptide /HLA-A*02:01 tetramers and co-stained with anti-CD4
antibody
and anti-CD8 antibody. FIG. 11A shows flow cytometry analysis for US-7 4-1BB,
7-1 4-
1BB, 7-3 4-1BB, 7-6 4-1BB, 7-7 4-1BB, and 7-8 4-1BB CAR-T cells. FIG. 11B
shows flow
cytometry analysis for 7-9 4-1BB, US-7 CD28, 7-1 CD28, 7-3 CD28, 7-6 CD28, and
7-7
CD28 CAR-T cells. FIG. 11C show flow cytometry analysis for 7-8 CD28 and 7-9
CD28
CAR-T cells, and mock-transduced T cells.
[0042] FIG. 12 shows the killing of cancer cell lines positive for HLA-A*02:01
and either
positive or negative for HPV16-E7, mediated by T cells expressing an anti-
HPV16-E7/HLA-
A*02:01 CAR having an affinity matured or parental scFv. Mock-transduced cells
were
included as controls.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present application provides isolated constructs (referred to
herein as "anti-E7MC
constructs") that comprise an antibody moiety (referred to herein as an "anti-
E7MC antibody
moiety") that specifically binds to a complex comprising an HPV16-E7 peptide
and an MHC
class I protein (referred to herein as an "HPV16-E7/MHC class I complex," or
"E7MC"). The
anti-E7MC constructs specifically recognize HPV16-E7/MHC class I complexes,
such as
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MHC-presented HPV16-E7 peptides on the surface of cells expressing HPV16-E7.
Anti-
E7MC constructs may specifically bind to the N-terminal portion, the C-
terminal portion, or
the middle portion of the HPV16-E7 peptide in the complex, and/or cross-react
with at least
one complex comprising the HPV16-E7 peptide and a different subtype of the MHC
class I
protein (e.g., the anti-E7MC construct binds to both an HPV16-E7 peptide/HLA-
A*02:01
complex and an HPV16-E7 peptide/HLA-A*02:02 complex). The anti-E7MC constructs
allow for specific targeting of E7MC-presenting cells (i.e., cells presenting
on their surface an
HPV16-E7 peptide bound to an MHC molecule), such as disease cells expressing
HPV16-E7.
This strategy provides a significant technical advantage over using antibodies
directed against
the HPV16-E7 protein, which cannot specifically target E7MC-presenting cells.
Furthermore,
when fused to a detectable moiety, the anti-E7MC antibody moiety allows for
diagnosis and
prognosis of HPV16-E7-positive diseases or disorders with high sensitivity to
changes in the
number and distribution of E7MC-presenting cells, a potentially more relevant
measure of
disease progression than circulating HPV16-E7 levels.
[0044] Using phage display technology, we generated multiple monoclonal
antibodies that
are specific and high affinity against HPV16-E7 11-19 peptide/HLA-A*02:01
complex. Flow
cytometry and T-cell mediated cytotoxicity assays demonstrated that the
antibodies
recognized HPV16-E7 peptide-pulsed T2 cells in an HPV16-E7- and HLA-A*02:01-
restricted manner. When armed as anti-CD3 bispecific antibodies, the
antibodies re-directed
human T cells to kill HPV16-E7-positive and HLA-A*02:01-positive target cells.
The data
presented herein demonstrate that antibodies against HPV16-E7 peptides in the
context of an
HLA complex can be effective therapeutic agents for cancer indications, such
as solid tumor
indications.
[0045] The present application thus provides constructs (such as isolated
constructs)
comprising an antibody moiety that specifically binds to a complex comprising
an HPV16-E7
peptide and an MHC class I protein. The construct can be, for example, a full-
length anti-
E7MC antibody, a multi-specific anti-E7MC molecule (such as a bispecific anti-
E7MC
antibody), an anti-E7MC chimeric antigen receptor ("CAR"), or an anti-E7MC
immunoconjugate.
[0046] In another aspect, there are provided nucleic acids encoding the anti-
E7MC constructs
or the anti-E7MC antibody moiety portion of the constructs.
[0047] In another aspect, there are provided compositions comprising an anti-
E7MC
construct comprising an antibody moiety that specifically binds to a complex
comprising an
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HPV16-E7-peptide and an MHC class I protein. The composition can be a
pharmaceutical
composition comprising an anti-E7MC construct or an effector cell expressing
or associated
with the anti-E7MC construct (for example a T cell expressing an anti-E7MC
CAR).
[0048] Also provided are methods of making and using the anti-E7MC constructs
(or cells
expressing or associated with the anti-E7MC constructs) for treatment or
diagnostic purposes,
as well as kits and articles of manufacture useful for such methods.
Definitions
[0049] As used herein, "treatment" or "treating" is an approach for obtaining
beneficial or
desired results, including clinical results. For purposes of this invention,
beneficial or desired
clinical results include, but are not limited to, one or more of the
following: alleviating one or
more symptoms resulting from the disease, diminishing the extent of the
disease, stabilizing
the disease (e.g., preventing or delaying the worsening of the disease),
preventing or delaying
the spread (e.g., metastasis) of the disease, preventing or delaying the
recurrence of the
disease, delay or slowing the progression of the disease, ameliorating the
disease state,
providing a remission (partial or total) of the disease, decreasing the dose
of one or more
other medications required to treat the disease, delaying the progression of
the disease,
increasing or improving the quality of life, increasing weight gain, and/or
prolonging
survival. Also encompassed by "treatment" is a reduction of pathological
consequence of
cancer (such as, for example, tumor volume). The methods of the invention
contemplate any
one or more of these aspects of treatment.
[0050] The terms "recurrence," "relapse" or "relapsed" refers to the return of
a cancer or
disease after clinical assessment of the disappearance of disease. A diagnosis
of distant
metastasis or local recurrence can be considered a relapse.
[0051] The term "refractory" or "resistant" refers to a cancer or disease that
has not
responded to treatment.
[0052] "Activation", as used herein in relation to T cells, refers to the
state of a T cell that has
been sufficiently stimulated to induce detectable cellular proliferation.
Activation can also be
associated with induced cytokine production, and detectable effector
functions.
[0053] The term "antibody moiety" includes full-length antibodies and antigen-
binding
fragments thereof. A full-length antibody comprises two heavy chains and two
light chains.
The variable regions of the light and heavy chains are responsible for antigen
binding. The
variables region in both chains generally contain three highly variable loops
called the
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complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-
CDR1,
LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and
HC-CDR3). CDR boundaries for the antibodies and antigen-binding fragments
disclosed
herein may be defined or identified by the conventions of Kabat, Chothia, or
Al-Lazikani (Al-
Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat
1991). The
three CDRs of the heavy or light chains are interposed between flanking
stretches known as
framework regions (FRs), which are more highly conserved than the CDRs and
form a
scaffold to support the hypervariable loops. The constant regions of the heavy
and light
chains are not involved in antigen binding, but exhibit various effector
functions. Antibodies
are assigned to classes based on the amino acid sequence of the constant
region of their heavy
chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE,
IgG, and IgM,
which are characterized by the presence of a, 6, , y, and 11 heavy chains,
respectively.
Several of the major antibody classes are divided into subclasses such as lgG1
(y1 heavy
chain), lgG2 (y2 heavy chain), lgG3 (y3 heavy chain), lgG4 (y4 heavy chain),
lgA 1 (al heavy
chain), or lgA2 (a2 heavy chain).
[0054] The term "antigen-binding fragment" as used herein refers to an
antibody fragment
including, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a
disulfide
stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFy (dsFv-dsFv'), a
disulfide stabilized
diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer
(bivalent
diabody), a multispecific antibody formed from a portion of an antibody
comprising one or
more CDRs, a camelized single domain antibody, a nanobody, a domain antibody,
a bivalent
domain antibody, or any other antibody fragment that binds to an antigen but
does not
comprise a complete antibody structure. An antigen-binding fragment is capable
of binding to
the same antigen to which the parent antibody or a parent antibody fragment
(e.g., a parent
scFv) binds. In some embodiments, an antigen-binding fragment may comprise one
or more
CDRs from a particular human antibody grafted to a framework region from one
or more
different human antibodies.
[0055] The term "epitope" as used herein refers to the specific group of atoms
or amino acids
on an antigen to which an antibody or antibody moiety binds. Two antibodies or
antibody
moieties may bind the same epitope within an antigen if they exhibit
competitive binding for
the antigen.
[0056] As used herein, a first antibody moiety "competes" for binding to a
target E7MC with
a second antibody moiety when the first antibody moiety inhibits target E7MC
binding of the
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second antibody moiety by at least about 50% (such as at least about any of
55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) in the presence of an equimolar
concentration of the first antibody moiety, or vice versa. A high throughput
process for
"binning" antibodies based upon their cross-competition is described in PCT
Publication No.
WO 03/48731.
[0057] As use herein, the term "specifically binds" or "is specific for"
refers to measurable
and reproducible interactions, such as binding between a target and an
antibody or antibody
moiety, that is determinative of the presence of the target in the presence of
a heterogeneous
population of molecules, including biological molecules. For example, an
antibody or
antibody moiety that specifically binds to a target (which can be an epitope)
is an antibody or
antibody moiety that binds this target with greater affinity, avidity, more
readily, and/or with
greater duration than its bindings to other targets. In some embodiments, an
antibody or
antibody moiety that specifically binds to an antigen reacts with one or more
antigenic
determinants of the antigen (for example an HPV16-E7 peptide/MHC class I
protein
complex) with a binding affinity that is at least about 10 times its binding
affinity for other
targets.
[0058] An "isolated" anti-E7MC construct as used herein refers to an anti-E7MC
construct
that (1) is not associated with proteins found in nature, (2) is free of other
proteins from the
same source, (3) is expressed by a cell from a different species, or, (4) does
not occur in
nature.
[0059] The term "isolated nucleic acid" as used herein is intended to mean a
nucleic acid of
genomic, cDNA, or synthetic origin or some combination thereof, which by
virtue of its
origin the "isolated nucleic acid" (1) is not associated with all or a portion
of a polynucleotide
in which the "isolated nucleic acid" is found in nature, (2) is operably
linked to a
polynucleotide which it is not linked to in nature, or (3) does not occur in
nature as part of a
larger sequence.
[0060] As used herein, the term "CDR" or "complementarity determining region"
is intended
to mean the non-contiguous antigen combining sites found within the variable
region of both
heavy and light chain polypeptides. These particular regions have been
described by Kabat et
al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health
and Human
Services, "Sequences of proteins of immunological interest" (1991); by Chothia
et al., J. Mol.
Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745
(1996), where
the definitions include overlapping or subsets of amino acid residues when
compared against
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each other. Nevertheless, application of either definition to refer to a CDR
of an antibody or
grafted antibodies or variants thereof is intended to be within the scope of
the term as defined
and used herein. The amino acid residues which encompass the CDRs as defined
by each of
the above cited references are set forth below in Table 1 as a comparison.
TABLE 1: CDR DEFINITIONS
Kabatl Chothia2 MacCallum3
VH CDR1 31-35 26-32 30-35
VH CDR2 50-65 53-55 47-58
VH CDR3 95-102 96-101 93-101
VL CDR1 24-34 26-32 30-36
VL CDR2 50-56 50-52 46-55
VL CDR3 89-97 91-96 89-96
1Residue numbering follows the nomenclature of Kabat et al., supra
2Residue numbering follows the nomenclature of Chothia et al., supra
3Residue numbering follows the nomenclature of MacCallum et al., supra
[0061] The term "chimeric antibodies" refer to antibodies in which a portion
of the heavy
and/or light chain is identical with or homologous to corresponding sequences
in antibodies
derived from a particular species or belonging to a particular antibody class
or subclass, while
the remainder of the chain(s) is identical with or homologous to corresponding
sequences in
antibodies derived from another species or belonging to another antibody class
or subclass, as
well as fragments of such antibodies, so long as they exhibit a biological
activity of this
invention (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl.
Acad. Sci. USA,
81:6851-6855 (1984)).
[0062] The term "semi-synthetic" in reference to an antibody or antibody
moiety means that
the antibody or antibody moiety has one or more naturally occurring sequences
and one or
more non-naturally occurring (i.e., synthetic) sequences.
[0063] "Fv" is the minimum antibody fragment which contains a complete antigen-
recognition and -binding site. This fragment consists of a dimer of one heavy-
and one light-
chain variable region domain in tight, non-covalent association. From the
folding of these
two domains emanate six hypervariable loops (3 loops each from the heavy and
light chain)
that contribute the amino acid residues for antigen binding and confer antigen
binding
specificity to the antibody. However, even a single variable domain (or half
of an Fv
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comprising only three CDRs specific for an antigen) has the ability to
recognize and bind
antigen, although at a lower affinity than the entire binding site.
[0064] "Single-chain Fv," also abbreviated as "sFv" or "scFv," are antibody
fragments that
comprise the VH and VL antibody domains connected into a single polypeptide
chain. In
some embodiments, the scFv polypeptide further comprises a polypeptide linker
between the
VH and VL domains which enables the scFv to form the desired structure for
antigen
binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.
269-315
(1994).
[0065] The term "diabodies" refers to small antibody fragments prepared by
constructing
scFv fragments (see preceding paragraph) typically with short linkers (such as
about 5 to
about 10 residues) between the VH and VL domains such that inter-chain but not
intra-chain
pairing of the V domains is achieved, resulting in a bivalent fragment, i.e.,
fragment having
two antigen-binding sites. Bispecific diabodies are heterodimers of two
"crossover" scFv
fragments in which the VH and VL domains of the two antibodies are present on
different
polypeptide chains. Diabodies are described more fully in, for example, EP
404,097; WO
93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448
(1993).
[0066] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric
antibodies
that contain minimal sequence derived from the non-human antibody. For the
most part,
humanized antibodies are human immunoglobulins (recipient antibody) in which
residues
from a hypervariable region (HVR) of the recipient are replaced by residues
from a
hypervariable region of a non-human species (donor antibody) such as mouse,
rat, rabbit or
non-human primate having the desired antibody specificity, affinity, and
capability. In some
instances, framework region (FR) residues of the human immunoglobulin are
replaced by
corresponding non-human residues. Furthermore, humanized antibodies can
comprise
residues that are not found in the recipient antibody or in the donor
antibody. These
modifications are made to further refine antibody performance. In general, the
humanized
antibody will comprise substantially all of at least one, and typically two,
variable domains,
in which all or substantially all of the hypervariable loops correspond to
those of a non-
human immunoglobulin and all or substantially all of the FRs are those of a
human
immunoglobulin sequence. The humanized antibody optionally also will comprise
at least a
portion of an immunoglobulin constant region (Fc), typically that of a human
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immunoglobulin. For further details, see Jones et al., Nature 321:522-525
(1986); Riechmann
et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-
596 (1992).
[0067] "Percent (%) amino acid sequence identity" or "homology" with respect
to the
polypeptide and antibody sequences identified herein is defined as the
percentage of amino
acid residues in a candidate sequence that are identical with the amino acid
residues in the
polypeptide being compared, after aligning the sequences considering any
conservative
substitutions as part of the sequence identity. Alignment for purposes of
determining percent
amino acid sequence identity can be achieved in various ways that are within
the skill in the
art, for instance, using publicly available computer software such as BLAST,
BLAST-2,
ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including any
algorithms needed
to achieve maximal alignment over the full-length of the sequences being
compared. For
purposes herein, however, % amino acid sequence identity values are generated
using the
sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids
Research
32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).
[0068] The terms "Fc receptor" or "FcR" are used to describe a receptor that
binds to the Fc
region of an antibody. In some embodiments, an FcR of this invention is one
that binds an
IgG antibody (a y receptor) and includes receptors of the FcyRI, FcyRII, and
FcyRIII
subclasses, including allelic variants and alternatively spliced forms of
these receptors.
FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an
"inhibiting
receptor"), which have similar amino acid sequences that differ primarily in
the cytoplasmic
domains thereof. Activating receptor FcyRIIA contains an immunoreceptor
tyrosine-based
activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB
contains an
immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic
domain (see
review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). The term includes
allotypes,
such as FcyRIIIA allotypes: FcyRIIIA-Phe158, FcyRIIIA-Va1158, FcyRIIA-R131
and/or
FcyRIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-
92
(1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J.
Lab. Clin. Med.
126:330-41 (1995). Other FcRs, including those to be identified in the future,
are
encompassed by the term "FcR" herein. The term also includes the neonatal
receptor, FcRn,
which is responsible for the transfer of maternal IgGs to the fetus (Guyer et
al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol . 24:249 (1994)).
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[0069] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is
structurally
similar to major histocompatibility complex (MHC) and consists of an a-chain
noncovalently
bound to 32-microglobulin. The multiple functions of the neonatal Fc receptor
FcRn are
reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays
a role in
the passive delivery of immunoglobulin IgGs from mother to young and the
regulation of
serum IgG levels. FcRn can act as a salvage receptor, binding and transporting
pinocytosed
IgGs in intact form both within and across cells, and rescuing them from a
default
degradative pathway.
[0070] The "CH1 domain" of a human IgG Fc region (also referred to as "Cl" of
"Hl"
domain) usually extends from about amino acid 118 to about amino acid 215 (EU
numbering
system).
[0071] "Hinge region" is generally defined as stretching from G1u216 to Pro230
of human
IgG1 (Burton, Molec. Immuno1.22:161-206 (1985)). Hinge regions of other IgG
isotypes may
be aligned with the IgG1 sequence by placing the first and last cysteine
residues forming
inter-heavy chain S-S bonds in the same positions.
[0072] The "CH2 domain" of a human IgG Fc region (also referred to as "C2" of
"H2"
domain) usually extends from about amino acid 231 to about amino acid 340. The
CH2
domain is unique in that it is not closely paired with another domain. Rather,
two N-linked
branched carbohydrate chains are interposed between the two CH2 domains of an
intact
native IgG molecule. It has been speculated that the carbohydrate may provide
a substitute
for the domain-domain pairing and help stabilize the CH2 domain. Burton, Molec
Immunol.
22:161-206 (1985).
[0073] The "CH3 domain" (also referred to as "C2" or "H3" domain) comprises
the stretch
of residues C-terminal to a CH2 domain in an Fc region (i.e. from about amino
acid residue
341 to the C-terminal end of an antibody sequence, typically at amino acid
residue 446 or 447
of an IgG).
[0074] A "functional Fc fragment" possesses an "effector function" of a native
sequence Fc
region. Exemplary "effector functions" include C lq binding; complement
dependent
cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell
receptor; BCR),
etc. Such effector functions generally require the Fc region to be combined
with a binding
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domain (e.g. an antibody variable domain) and can be assessed using various
assays known in
the art.
[0075] An antibody with a variant IgG Fc with "altered" FcR binding affinity
or ADCC
activity is one which has either enhanced or diminished FcR binding activity
(e.g., FcyR or
FcRn) and/or ADCC activity compared to a parent polypeptide or to a
polypeptide
comprising a native sequence Fc region. The variant Fc which "exhibits
increased binding" to
an FcR binds at least one FcR with higher affinity (e.g., lower apparent Kd or
IC50 value) than
the parent polypeptide or a native sequence IgG Fc. According to some
embodiments, the
improvement in binding compared to a parent polypeptide is about 3 fold, such
as about any
of 5, 10, 25, 50, 60, 100, 150, 200, or up to 500 fold, or about 25% to 1000%
improvement in
binding. The polypeptide variant which "exhibits decreased binding" to an FcR,
binds at least
one FcR with lower affinity (e.g., higher apparent Kd or higher IC50 value)
than a parent
polypeptide. The decrease in binding compared to a parent polypeptide may be
about 40% or
more decrease in binding.
[0076] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a
form of
cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on
certain cytotoxic
cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable
these cytotoxic
effector cells to bind specifically to an antigen-bearing target cell and
subsequently kill the
target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are
absolutely
required for such killing. The primary cells for mediating ADCC, NK cells,
express FcyRIII
only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet,
Annu. Rev.
Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an
in vitro
ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may
be
performed. Useful effector cells for such assays include peripheral blood
mononuclear cells
(PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the
molecule of interest may be assessed in vivo, e.g., in an animal model such as
that disclosed
in Clynes et al. PNAS (USA) 95:652-656 (1998).
[0077] The polypeptide comprising a variant Fc region which "exhibits
increased ADCC" or
mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence
of human
effector cells more effectively than a polypeptide having wild type IgG Fc or
a parent
polypeptide is one which in vitro or in vivo is substantially more effective
at mediating
ADCC, when the amounts of polypeptide with variant Fc region and the
polypeptide with
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wild type Fc region (or the parent polypeptide) in the assay are essentially
the same.
Generally, such variants will be identified using any in vitro ADCC assay
known in the art,
such as assays or methods for determining ADCC activity, e.g. in an animal
model etc. In
some embodiments, the variant is from about 5 fold to about 100 fold, e.g.
from about 25 to
about 50 fold, more effective at mediating ADCC than the wild type Fc (or
parent
polypeptide) .
[0078] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a
target cell in
the presence of complement. Activation of the classical complement pathway is
initiated by
the binding of the first component of the complement system (C lq) to
antibodies (of the
appropriate subclass) which are bound to their cognate antigen. To assess
complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.
Immunol. Methods
202:163 (1996), may be performed. Polypeptide variants with altered Fc region
amino acid
sequences and increased or decreased Clq binding capability are described in
US patent No.
6,194,551B1 and W099/51642. The contents of those patent publications are
specifically
incorporated herein by reference. See, also, Idusogie et al. J. Immunol. 164:
4178-4184
(2000).
[0079] Unless otherwise specified, a "nucleotide sequence encoding an amino
acid sequence"
includes all nucleotide sequences that are degenerate versions of each other
and that encode
the same amino acid sequence. The phrase nucleotide sequence that encodes a
protein or an
RNA may also include introns to the extent that the nucleotide sequence
encoding the protein
may in some version contain an intron(s).
[0080] The term "operably linked" refers to functional linkage between a
regulatory
sequence and a heterologous nucleic acid sequence resulting in expression of
the latter. For
example, a first nucleic acid sequence is operably linked with a second
nucleic acid sequence
when the first nucleic acid sequence is placed in a functional relationship
with the second
nucleic acid sequence. For instance, a promoter is operably linked to a coding
sequence if the
promoter affects the transcription or expression of the coding sequence.
Generally, operably
linked DNA sequences are contiguous and, where necessary to join two protein
coding
regions, in the same reading frame.
[0081] "Homologous" refers to the sequence similarity or sequence identity
between two
polypeptides or between two nucleic acid molecules. When a position in both of
the two
compared sequences is occupied by the same base or amino acid monomer subunit,
e.g., if a
position in each of two DNA molecules is occupied by adenine, then the
molecules are
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homologous at that position. The percent of homology between two sequences is
a function
of the number of matching or homologous positions shared by the two sequences
divided by
the number of positions compared times 100. For example, if 6 of 10 of the
positions in two
sequences are matched or homologous then the two sequences are 60% homologous.
By way
of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally,
a
comparison is made when two sequences are aligned to give maximum homology.
[0082] An "effective amount" of an anti-E7MC construct or composition as
disclosed herein,
is an amount sufficient to carry out a specifically stated purpose. An
"effective amount" can
be determined empirically and by known methods relating to the stated purpose.
[0083] The term "therapeutically effective amount" refers to an amount of an
anti-E7MC
construct or composition as disclosed herein, effective to "treat" a disease
or disorder in an
individual. In the case of cancer, the therapeutically effective amount of the
anti-E7MC
construct or composition as disclosed herein can reduce the number of cancer
cells; reduce
the tumor size or weight; inhibit (i.e., slow to some extent and preferably
stop) cancer cell
infiltration into peripheral organs; inhibit (i.e., slow to some extent and
preferably stop)
tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to
some extent one or
more of the symptoms associated with the cancer. To the extent the anti-E7MC
construct or
composition as disclosed herein can prevent growth and/or kill existing cancer
cells, it can be
cytostatic and/or cytotoxic. In some embodiments, the therapeutically
effective amount is a
growth inhibitory amount. In some embodiments, the therapeutically effective
amount is an
amount that extends the survival of a patient. In some embodiments, the
therapeutically
effective amount is an amount that improves progression free survival of a
patient.
[0084] As used herein, by "pharmaceutically acceptable" or "pharmacologically
compatible"
is meant a material that is not biologically or otherwise undesirable, e.g.,
the material may be
incorporated into a pharmaceutical composition administered to a patient
without causing any
significant undesirable biological effects or interacting in a deleterious
manner with any of
the other components of the composition in which it is contained.
Pharmaceutically
acceptable carriers or excipients have preferably met the required standards
of toxicological
and manufacturing testing and/or are included on the Inactive Ingredient Guide
prepared by
the U.S. Food and Drug administration.
[0085] The term "label" when used herein refers to a detectable compound or
composition
which can be conjugated directly or indirectly to the anti-E7MC antibody
moiety. The label
may be detectable by itself (e.g., radioisotope labels or fluorescent labels)
or, in the case of an
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enzymatic label, may catalyze chemical alteration of a substrate compound or
composition
which is detectable.
[0086] It is understood that embodiments of the invention described herein
include
"consisting" and/or "consisting essentially of' embodiments.
[0087] Reference to "about" a value or parameter herein includes (and
describes) variations
that are directed to that value or parameter per se. For example, description
referring to
"about X" includes description of "X".
[0088] As used herein, reference to "not" a value or parameter generally means
and describes
"other than" a value or parameter. For example, the method is not used to
treat cancer of type
X means the method is used to treat cancer of types other than X.
[0089] As used herein and in the appended claims, the singular forms "a,"
"or," and "the"
include plural referents unless the context clearly dictates otherwise.
Anti-E7MC constructs
[0090] In one aspect, the present invention provides HPV16-E7/MHC class I
complex-
specific constructs (anti-E7MC constructs) that comprise an antibody moiety
that specifically
binds to a complex comprising an HPV16-E7 peptide and an MHC class I protein
("HPV16-
E7/MHC class I complex," or "E7MC"). The specificity of the anti-E7MC
construct derives
from an anti-E7MC antibody moiety, such as a full-length antibody or antigen-
binding
fragment thereof, that specifically binds to the E7MC. In some embodiments,
reference to a
moiety (such as an antibody moiety) that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein means that the moiety binds to the
E7MC
with a) an affinity that is at least about 10 (including for example at least
about any of 10, 20,
30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more) times its binding
affinity for each
of full-length HPV16-E7, free HPV16-E7 peptide, MHC class I protein not bound
to a
peptide, and MHC class I protein bound to a non-HPV16-E7 peptide; or b) a Kd
no more than
about 1/10 (such as no more than about any of 1/10, 1/20, 1/30, 1/40, 1/50,
1/75, 1/100,
1/200, 1/300, 1/400, 1/500, 1/750, 1/1000 or less) times its Kd for binding to
each of full-
length HPV16-E7, free HPV16-E7 peptide, MHC class I protein not bound to a
peptide, and
MHC class I protein bound to a non-HPV16-E7 peptide. Binding affinity can be
determined
by methods known in the art, such as ELISA, fluorescence activated cell
sorting (FACS)
analysis, or radioimmunoprecipitation assay (RIA). Kd can be determined by
methods known
in the art, such as surface plasmon resonance (SPR) assay utilizing, for
example, Biacore
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instruments, or kinetic exclusion assay (KinExA) utilizing, for example,
Sapidyne
instruments.
[0091] Contemplated anti-E7MC constructs include, for example, full-length
anti-E7MC
antibodies, multi-specific (such as bispecific) anti-E7MC molecules, anti-E7MC
chimeric
antigen receptors (CARs), and anti-E7MC immunoconjugates.
[0092] For example, in some embodiments, there is provided an anti-E7MC
construct (such
as an isolated anti-E7MC construct) comprising an anti-E7MC antibody moiety
that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID
NO: 4).
In some embodiments, the MHC class I protein is HLA-A02. In some embodiments,
the
MHC class I protein is HLA-A*02:01 (GenBank Accession No.: AA020853). In some
embodiments, the anti-E7MC construct is non-naturally occurring. In some
embodiments, the
anti-E7MC construct is a full-length antibody. In some embodiments, the anti-
E7MC
construct is a multi-specific (such as bispecific) molecule. In some
embodiments, the anti-
E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-
E7MC
construct is an immunoconjugate. In some embodiments, the anti-E7MC construct
binds the
E7MC with a Kd between about 0.1 pM to about 500 nM (such as about any of 0.1
pM, 1.0
pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM,
including
any ranges between these values). In some embodiments, the anti-E7MC construct
cross-
reacts with at least one (such as at least any of 2, 3, 4, 5, or 6) complex
comprising the MHC
class I protein and a variant of the HPV16-E7 peptide having one amino acid
substitution
(such as a conservative amino acid substitution). In some embodiments, the
anti-E7MC
construct cross-reacts with at least one (such as at least any of 2, 3, 4, or
5) complex
comprising the HPV16-E7 peptide and a different subtype of the MHC class I
protein.
[0093] In some embodiments, there is provided an anti-E7MC construct
comprising an anti-
E7MC antibody moiety that specifically binds to a complex comprising an HPV16-
E7 11-19
peptide (SEQ ID NO: 4) and HLA-A*02:01. In some embodiments, the anti-E7MC
construct
is non-naturally occurring. In some embodiments, the anti-E7MC construct is a
full-length
antibody. In some embodiments, the anti-E7MC construct is a multi-specific
(such as
bispecific) molecule. In some embodiments, the anti-E7MC construct is a
chimeric antigen
receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate.
In some
embodiments, the anti-E7MC construct binds the E7MC with a Kd between about
0.1 pM to
about 500 nM (such as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500
pM, 1 nM,
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nM, 50 nM, 100 nM, or 500 nM, including any ranges between these values). In
some
embodiments, the anti-E7MC construct cross-reacts with at least one (such as
at least any of
2, 3, 4, 5, or 6) complex comprising the MHC class I protein and a variant of
the HPV16-E7
peptide having one amino acid substitution (such as a conservative amino acid
substitution).
In some embodiments, the anti-E7MC construct cross-reacts with at least one
(such as at least
any of 2, 3, 4, or 5) complex comprising the HPV16-E7 peptide and a different
subtype of the
MHC class I protein.
[0094] In some embodiments, there is provided an anti-E7MC construct
comprising an anti-
E7MC antibody moiety that specifically binds to a complex comprising an HPV16-
E7
peptide and an MHC class I protein, wherein the anti-E7MC antibody moiety
comprises: i) a
heavy chain variable domain sequence comprising an HC-CDR1 comprising the
amino acid
sequence of SEQ ID NO: 183, or a variant thereof comprising up to about 3 (for
example
about any of 1, 2, or 3) amino acid substitutions, an HC-CDR2 comprising the
amino acid
sequence of SEQ ID NO: 184 or 185, or a variant thereof comprising up to about
3 (for
example about any of 1, 2, or 3) amino acid substitutions, and an HC-CDR3
comprising the
amino acid sequence of any one of SEQ ID NOs: 186-188; or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
ii) a light chain
variable domain comprising an LC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 189 or 190, or a variant thereof comprising up to about 3 (for example
about any of 1, 2,
or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid
sequence of SEQ
ID NO: 191, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or
3) amino acid substitutions. In some embodiments, the anti-E7MC construct is
non-naturally
occurring. In some embodiments, the anti-E7MC construct is a full-length
antibody. In some
embodiments, the anti-E7MC construct is a multi-specific (such as bispecific)
molecule. In
some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In
some
embodiments, the anti-E7MC construct is an immunoconjugate. In some
embodiments, the
anti-E7MC construct binds the E7MC with a Kd between about 0.1 pM to about 500
nM
(such as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10
nM, 50
nM, 100 nM, or 500 nM, including any ranges between these values). In some
embodiments,
the anti-E7MC construct cross-reacts with at least one (such as at least any
of 2, 3, 4, 5, or 6)
complex comprising the MHC class I protein and a variant of the HPV16-E7
peptide having
one amino acid substitution (such as a conservative amino acid substitution).
In some
embodiments, the anti-E7MC construct cross-reacts with at least one (such as
at least any of
CA 02988397 2017-12-05
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2, 3, 4, or 5) complex comprising the HPV16-E7 peptide and a different subtype
of the MHC
class I protein.
[0095] In some embodiments, there is provided an anti-E7MC construct
comprising an anti-
E7MC antibody moiety that specifically binds to a complex comprising an HPV16-
E7
peptide and an MHC class I protein, wherein the anti-E7MC antibody moiety
comprises: i) a
heavy chain variable domain sequence comprising an HC-CDR1 comprising (and in
some
embodiments consisting of) the amino acid sequence of any one of SEQ ID NOs:
57-77; or a
variant thereof comprising up to about 5 (for example about any of 1, 2, 3, 4,
or 5) amino acid
substitutions; an HC-CDR2 comprising (and in some embodiments consisting of)
the amino
acid sequence of any one of SEQ ID NOs: 78-98; or a variant thereof comprising
up to about
(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; and an
HC-CDR3
comprising (and in some embodiments consisting of) the amino acid sequence of
any one of
SEQ ID NOs: 99-119, 244, and 245; or a variant thereof comprising up to about
5 (for
example about any of 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a
light chain variable
domain sequence comprising an LC-CDR1 comprising (and in some embodiments
consisting
of) the amino acid sequence of any one of SEQ ID NOs: 120-140 and 246; or a
variant
thereof comprising up to about 5 (for example about any of 1, 2, 3, 4, or 5)
amino acid
substitutions; an LC-CDR2 comprising (and in some embodiments consisting of)
the amino
acid sequence of any one of SEQ ID NOs: 141-161; or a variant thereof
comprising up to
about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and an
LC-CDR3
comprising (and in some embodiments consisting of) the amino acid sequence of
any one of
SEQ ID NOs: 162-182 and 247-250; or a variant thereof comprising up to about 5
(for
example about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some
embodiments, the
anti-E7MC construct is non-naturally occurring. In some embodiments, the anti-
E7MC
construct is a full-length antibody. In some embodiments, the anti-E7MC
construct is a multi-
specific (such as bispecific) molecule. In some embodiments, the anti-E7MC
construct is a
chimeric antigen receptor. In some embodiments, the anti-E7MC construct is an
immunoconjugate. In some embodiments, the anti-E7MC construct binds the E7MC
with a
Kd between about 0.1 pM to about 500 nM (such as about any of 0.1 pM, 1.0 pM,
10 pM, 50
pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM, including any
ranges
between these values). In some embodiments, the anti-E7MC construct cross-
reacts with at
least one (such as at least any of 2, 3, 4, 5, or 6) complex comprising the
MHC class I protein
and a variant of the HPV16-E7 peptide having one amino acid substitution (such
as a
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conservative amino acid substitution). In some embodiments, the anti-E7MC
construct cross-
reacts with at least one (such as at least any of 2, 3, 4, or 5) complex
comprising the HPV16-
E7 peptide and a different subtype of the MHC class I protein.
[0096] In some embodiments, there is provided an anti-E7MC construct
comprising an anti-
E7MC antibody moiety that specifically binds to a complex comprising an HPV16-
E7
peptide and an MHC class I protein, wherein the anti-E7MC antibody moiety
comprises: i) a
heavy chain variable domain sequence comprising an HC-CDR1 comprising (and in
some
embodiments consisting of) the amino acid sequence of any one of SEQ ID NOs:
57-77; an
HC-CDR2 comprising (and in some embodiments consisting of) the amino acid
sequence of
any one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising (and in some
embodiments
consisting of) the amino acid sequence of any one of SEQ ID NOs: 99-119, 244,
and 245; or
a variant thereof comprising up to about 5 (for example about any of 1, 2, 3,
4, or 5) amino
acid substitutions in the HC-CDR sequences; and ii) a light chain variable
domain sequence
comprising an LC-CDR1 comprising (and in some embodiments consisting of) the
amino
acid sequence of any one of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising
(and
in some embodiments consisting of) the amino acid sequence of any one of SEQ
ID NOs:
141-161; and an LC-CDR3 comprising (and in some embodiments consisting of) the
amino
acid sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a variant
thereof
comprising up to about 5 (for example about any of 1, 2, 3, 4, or 5) amino
acid substitutions
in the LC-CDR sequences. In some embodiments, the anti-E7MC construct is non-
naturally
occurring. In some embodiments, the anti-E7MC construct is a full-length
antibody. In some
embodiments, the anti-E7MC construct is a multi-specific (such as bispecific)
molecule. In
some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In
some
embodiments, the anti-E7MC construct is an immunoconjugate. In some
embodiments, the
anti-E7MC construct binds the E7MC with a Kd between about 0.1 pM to about 500
nM
(such as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10
nM, 50
nM, 100 nM, or 500 nM, including any ranges between these values). In some
embodiments,
the anti-E7MC construct cross-reacts with at least one (such as at least any
of 2, 3, 4, 5, or 6)
complex comprising the MHC class I protein and a variant of the HPV16-E7
peptide having
one amino acid substitution (such as a conservative amino acid substitution).
In some
embodiments, the anti-E7MC construct cross-reacts with at least one (such as
at least any of
2, 3, 4, or 5) complex comprising the HPV16-E7 peptide and a different subtype
of the MHC
class I protein.
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[0097] In some embodiments, there is provided an anti-E7MC construct
comprising an anti-
E7MC antibody moiety that specifically binds to a complex comprising an HPV16-
E7
peptide and an MHC class I protein, wherein the anti-E7MC antibody moiety
comprises a
heavy chain variable domain comprising (and in some embodiments consisting of)
the amino
acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or a variant
thereof having at
least about 95% (for example at least about any of 96%, 97%, 98%, or 99%)
sequence
identity, and a light chain variable domain comprising (and in some
embodiments consisting
of) the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, or a
variant
thereof having at least about 95% (for example at least about any of 96%, 97%,
98%, or 99%)
sequence identity. In some embodiments, the anti-E7MC construct is non-
naturally occurring.
In some embodiments, the anti-E7MC construct is a full-length antibody. In
some
embodiments, the anti-E7MC construct is a multi-specific (such as bispecific)
molecule. In
some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In
some
embodiments, the anti-E7MC construct is an immunoconjugate. In some
embodiments, the
anti-E7MC construct binds the E7MC with a Kd between about 0.1 pM to about 500
nM
(such as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10
nM, 50
nM, 100 nM, or 500 nM, including any ranges between these values). In some
embodiments,
the anti-E7MC construct cross-reacts with at least one (such as at least any
of 2, 3, 4, 5, or 6)
complex comprising the MHC class I protein and a variant of the HPV16-E7
peptide having
one amino acid substitution (such as a conservative amino acid substitution).
In some
embodiments, the anti-E7MC construct cross-reacts with at least one (such as
at least any of
2, 3, 4, or 5) complex comprising the HPV16-E7 peptide and a different subtype
of the MHC
class I protein.
[0098] In some embodiments, there is provided an anti-E7MC construct
comprising a first
anti-E7MC antibody moiety that competes for binding to a target HPV16-E7/MHC
class I
complex with a second anti-E7MC antibody moiety according to any of the anti-
E7MC
antibody moieties described herein. In some embodiments, the first anti-E7MC
antibody
moiety binds to the same, or substantially the same, epitope as the second
anti-E7MC
antibody moiety. In some embodiments, binding of the first anti-E7MC antibody
moiety to
the target HPV16-E7/MHC class I complex inhibits binding of the second anti-
E7MC
antibody moiety to the target HPV16-E7/MHC class I complex by at least about
70% (such
as by at least about any of 75%, 80%, 85%, 90%, 95%, 98% or 99%), or vice
versa. In some
embodiments, the first anti-E7MC antibody moiety and the second anti-E7MC
antibody
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moiety cross-compete for binding to the target HPV16-E7/MHC class I complex,
i.e., each of
the first and second antibody moieties competes with the other for binding to
the target
HPV16-E7/MHC class I complex.
[0099] For example, in some embodiments, there is provided an anti-E7MC
construct
comprising an anti-E7MC antibody moiety that competes for binding to a target
HPV16-
E7/MHC class I complex with an antibody moiety comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 183, or a variant thereof comprising up to about 3 (for example about any
of 1, 2, or 3)
amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ
ID NO:
184 or 185, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or 3)
amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of
any one
of SEQ ID NOs: 186-188; or a variant thereof comprising up to about 3 (for
example about
any of 1, 2, or 3) amino acid substitutions; and ii) a light chain variable
domain comprising
an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, or a
variant
thereof comprising up to about 3 (for example about any of 1, 2, or 3) amino
acid
substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:
191, or
a variant thereof comprising up to about 3 (for example about any of 1, 2, or
3) amino acid
substitutions.
[0100] In some embodiments, there is provided an anti-E7MC construct
comprising an
anti-E7MC antibody moiety that competes for binding to a target HPV16-E7/MHC
class I
complex with an antibody moiety comprising i) a heavy chain variable domain
sequence
comprising an HC-CDR1 comprising (and in some embodiments consisting of) the
amino
acid sequence of any one of SEQ ID NOs: 57-77; or a variant thereof comprising
up to about
(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; an HC-
CDR2
comprising (and in some embodiments consisting of) the amino acid sequence of
any one of
SEQ ID NOs: 78-98; or a variant thereof comprising up to about 5 (for example
about any of
1, 2, 3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising (and in
some
embodiments consisting of) the amino acid sequence of any one of SEQ ID NOs:
99-119,
244, and 245; or a variant thereof comprising up to about 5 (for example about
any of 1, 2, 3,
4, or 5) amino acid substitutions; and ii) a light chain variable domain
sequence comprising
an LC-CDR1 comprising (and in some embodiments consisting of) the amino acid
sequence
of any one of SEQ ID NOs: 120-140 and 246; or a variant thereof comprising up
to about 5
(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; an LC-
CDR2 comprising
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(and in some embodiments consisting of) the amino acid sequence of any one of
SEQ ID
NOs: 141-161; or a variant thereof comprising up to about 3 (for example about
any of 1,2,
or 3) amino acid substitutions; and an LC-CDR3 comprising (and in some
embodiments
consisting of) the amino acid sequence of any one of SEQ ID NOs: 162-182 and
247-250; or
a variant thereof comprising up to about 5 (for example about any of 1, 2, 3,
4, or 5) amino
acid substitutions.
[0101] In some embodiments, there is provided an anti-E7MC construct
comprising an
anti-E7MC antibody moiety that competes for binding to a target HPV16-E7/MHC
class I
complex with an antibody moiety comprising i) a heavy chain variable domain
sequence
comprising an HC-CDR1 comprising (and in some embodiments consisting of) the
amino
acid sequence of any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising (and in
some
embodiments consisting of) the amino acid sequence of any one of SEQ ID NOs:
78-98; and
an HC-CDR3 comprising (and in some embodiments consisting of) the amino acid
sequence
of any one of SEQ ID NOs: 99-119, 244, and 245; or a variant thereof
comprising up to about
(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-
CDR
sequences; and ii) a light chain variable domain sequence comprising an LC-
CDR1
comprising (and in some embodiments consisting of) the amino acid sequence of
any one of
SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising (and in some embodiments
consisting of) the amino acid sequence of any one of SEQ ID NOs: 141-161; and
an LC-
CDR3 comprising (and in some embodiments consisting of) the amino acid
sequence of any
one of SEQ ID NOs: 162-182 and 247-250; or a variant thereof comprising up to
about 5 (for
example about any of 1, 2, 3, 4, or 5) amino acid substitutions in the LC-CDR
sequences.
[0102] In some embodiments, there is provided an anti-E7MC construct
comprising an
anti-E7MC antibody moiety that competes for binding to a target HPV16-E7/MHC
class I
complex with an antibody moiety comprising a heavy chain variable domain
comprising (and
in some embodiments consisting of) the amino acid sequence of any one of SEQ
ID NOs: 15-
35 and 233-237, or a variant thereof having at least about 95% (for example at
least about any
of 96%, 97%, 98%, or 99%) sequence identity, and a light chain variable domain
comprising
(and in some embodiments consisting of) the amino acid sequence of any one of
SEQ ID
NOs: 36-56 and 238-243, or a variant thereof having at least about 95% (for
example at least
about any of 96%, 97%, 98%, or 99%) sequence identity.
[0103] The different aspects are discussed in various sections below in
further detail.
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Anti-E7MC antibody moiety
[0104] The anti-E7MC constructs comprise an anti-E7MC antibody moiety that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein.
[0105] In some embodiments, the anti-E7MC antibody moiety specifically binds
to an
E7MC present on the surface of a cell. In some embodiments, the cell is a
cancer cell. In
some embodiments, the cancer cell is in a solid tumor. In some embodiments,
the cancer cell
is a metastatic cancer cell.
[0106] In some embodiments, the HPV16-E7 peptide is an MHC class I-restricted
peptide.
In some embodiments, the HPV16-E7 peptide is from about 8 to about 12 (such as
about any
of 8, 9, 10, 11, or 12) amino acids in length.
[0107] In some embodiments, the HPV16-E7 peptide comprises (and in some
embodiments
consists of) the sequence of amino acids 7-15 of HPV16-E7 (TLHEYMLDL, SEQ ID
NO:
3), amino acids 11-19 of HPV16-E7 (YMLDLQPET, SEQ ID NO: 4, also referred to
herein
as "HPV16-E7 11-19"), amino acids 16-25 of HPV16-E7 (QPETTDLYCY, SEQ ID NO:
5),
amino acids 44-52 of HPV16-E7 (QAEPDRAHY, SEQ ID NO: 6), amino acids 46-55 of
HPV16-E7 (EPDRAHYNIV, SEQ ID NO: 7), amino acids 49-57 of HPV16-E7
(RAHYNIVTF, SEQ ID NO: 8), amino acids 82-90 of HPV16-E7 (LLMGTLGIV, SEQ ID
NO: 9), or amino acids 86-93 of HPV16-E7 (TLGIVCPI, SEQ ID NO: 10).
[0108] In some embodiments, the MHC class I protein is HLA-A, HLA-B, HLA-C,
HLA-
E, HLA-F, or HLA-G. In some embodiments, the MHC class I protein is HLA-A. In
some
embodiments, the HLA-A is HLA-A02. In some embodiments, the HLA-A02 is HLA-
A*02:01.
[0109] In some embodiments, the anti-E7MC antibody moiety is a full-length
antibody. In
some embodiments, the anti-E7MC antibody moiety is an antigen-binding
fragment, for
example an antigen-binding fragment selected from the group consisting of a
Fab, a Fab', a
F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), and a
single-chain
antibody molecule (scFv). In some embodiments, the anti-E7MC antibody moiety
is an scFv.
In some embodiments, the anti-E7MC antibody moiety is human, humanized, or
semi-
synthetic.
[0110] In some embodiments, the anti-E7MC antibody moiety specifically binds
to the N-
terminal portion of the HPV16-E7 peptide in the complex. In some embodiments,
the anti-
E7MC antibody moiety specifically binds to the C-terminal portion of the HPV16-
E7 peptide
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in the complex. In some embodiments, the anti-E7MC antibody moiety
specifically binds to
the middle portion of the HPV16-E7 peptide in the complex.
[0111] In some embodiments, the anti-E7MC antibody moiety (or the anti-E7MC
construct
comprising the anti-E7MC antibody moiety) binds to the complex comprising the
HPV16-E7
peptide and the MHC class I protein with an affinity that is at least about 10
(including for
example at least about any of 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500,
750, 1000 or
more) times its binding affinity for each of full-length HPV16-E7, free HPV16-
E7 peptide,
MHC class I protein not bound to a peptide, and MHC class I protein bound to a
non-HPV16-
E7 peptide. In some embodiments, the anti-E7MC antibody moiety (or the anti-
E7MC
construct comprising the anti-E7MC antibody moiety) binds to the complex
comprising the
HPV16-E7 peptide and the MHC class I protein with a Kd no more than about 1/10
(such as
no more than about any of 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200,
1/300, 1/400,
1/500, 1/750, 1/1000 or less) times its Kd for binding to each of full-length
HPV16-E7, free
HPV16-E7 peptide, MHC class I protein not bound to a peptide, and MHC class I
protein
bound to a non-HPV16-E7 peptide.
[0112] In some embodiments, the anti-E7MC antibody moiety (or the anti-E7MC
construct
comprising the anti-E7MC antibody moiety) binds to the complex comprising the
HPV16-E7
peptide and the MHC class I protein with a Kd between about 0.1 pM to about
500 nM (such
as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50
nM, 100
nM, or 500 nM, including any ranges between these values). In some
embodiments, the anti-
E7MC antibody moiety (or the anti-E7MC construct comprising the anti-E7MC
antibody
moiety) binds to the complex comprising the HPV16-E7 peptide and the MHC class
I protein
with a Kd between about 1 pM to about 250 pM (such as about any of 1, 10, 25,
50, 75, 100,
150, 200, or 250 pM, including any ranges between these values). In some
embodiments, the
anti-E7MC antibody moiety (or the anti-E7MC construct comprising the anti-E7MC
antibody
moiety) binds to the complex comprising the HPV16-E7 peptide and the MHC class
I protein
with a Kd between about 1 nM to about 500 nM (such as about any of 1, 10, 25,
50, 75, 100,
150, 200, 250, 300, 350, 400, 450, or 500 nM, including any ranges between
these values).
[0113] In some embodiments, the anti-E7MC antibody moiety specifically binds
to a
complex comprising an HPV16-E7 peptide and an MHC class I protein, wherein the
anti-
E7MC antibody moiety cross-reacts with at least one complex comprising the
HPV16-E7
peptide and an allelic variant of the MHC class I protein. In some
embodiments, the allelic
variant has up to about 10 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10) amino acid
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substitutions when compared to the MHC class I protein. In some embodiments,
the allelic
variant is the same serotype as the MHC class I protein. In some embodiments,
the allelic
variant is a different serotype than the MHC class I protein. In some
embodiments, the anti-
E7MC antibody moiety does not cross-react with any complex comprising the
HPV16-E7
peptide and an allelic variant of the MHC class I protein. In some
embodiments, the anti-
E7MC antibody moiety cross-reacts with at least one (such as at least any of
2, 3, 4, or 5)
complex comprising the HPV16-E7 peptide and a different subtype of the MHC
class I
protein.
[0114] In some embodiments, the anti-E7MC antibody moiety specifically binds
to a
complex comprising an HPV16-E7 peptide and an MHC class I protein, wherein the
anti-
E7MC antibody moiety cross-reacts with at least one complex comprising the MHC
class I
protein and a variant of the HPV16-E7 peptide having one amino acid
substitution (such as a
conservative amino acid substitution). In some embodiments, the anti-E7MC
antibody moiety
does not cross-react with any complex comprising the MHC class I protein and a
variant of
the HPV16-E7 peptide.
[0115] For example, in some embodiments, the anti-E7MC antibody moiety
specifically
binds to a complex comprising HPV16-E7 11-19 (SEQ ID NO: 4) and an MHC class I
protein (such as HLA-A02, for example HLA-A*02:01). In some embodiments, the
anti-
E7MC antibody moiety further binds to at least one (including at least about
any of 2 or 3) of:
a complex comprising an alanine-substituted HPV16-E7 peptide of SEQ ID NO: 12
and an
MHC class I protein (such as HLA-A02, for example HLA-A*02:01); a complex
comprising
an alanine-substituted HPV16-E7 peptide of SEQ ID NO: 13 and an MHC class I
protein
(such as HLA-A02, for example HLA-A*02:01); and a complex comprising an
alanine-
substituted HPV16-E7 peptide of SEQ ID NO: 14 and an MHC class I protein (such
as HLA-
A02, for example HLA-A*02:01).
[0116] In some embodiments, the anti-E7MC antibody moiety specifically binds
to: a
complex comprising an HPV16-E7 peptide of SEQ ID NO: 4 and an MHC class I
protein
(such as HLA-A02, for example HLA-A*02:01); a complex comprising an alanine-
substituted HPV16-E7 peptide of SEQ ID NO: 12 and an MHC class I protein (such
as HLA-
A02, for example HLA-A*02:01); and a complex comprising an alanine-substituted
HPV16-
E7 peptide of SEQ ID NO: 13 and an MHC class I protein (such as HLA-A02, for
example
HLA-A*02:01).
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[0117] In some embodiments, the anti-E7MC antibody moiety specifically binds
to: a
complex comprising an HPV16-E7 peptide of SEQ ID NO: 4 and an MHC class I
protein
(such as HLA-A02, for example HLA-A*02:01); a complex comprising an alanine-
substituted HPV16-E7 peptide of SEQ ID NO: 12 and an MHC class I protein (such
as HLA-
A02, for example HLA-A*02:01); and a complex comprising an alanine-substituted
HPV16-
E7 peptide of SEQ ID NO: 14 and an MHC class I protein (such as HLA-A02, for
example
HLA-A*02:01).
[0118] In some embodiments, the anti-E7MC antibody moiety specifically binds
to: a
complex comprising an HPV16-E7 peptide of SEQ ID NO: 4 and an MHC class I
protein
(such as HLA-A02, for example HLA-A*02:01); and a complex comprising an
alanine-
substituted HPV16-E7 peptide of SEQ ID NO: 12 and an MHC class I protein (such
as HLA-
A02, for example HLA-A*02:01).
[0119] In some embodiments, the anti-E7MC antibody moiety specifically binds
to: a
complex comprising an HPV16-E7 peptide of SEQ ID NO: 4 and an MHC class I
protein
(such as HLA-A02, for example HLA-A*02:01); and a complex comprising an
alanine-
substituted HPV16-E7 peptide of SEQ ID NO: 14 and an MHC class I protein (such
as HLA-
A02, for example HLA-A*02:01).
[0120] In some embodiments, the anti-E7MC antibody moiety specifically binds
to a
complex comprising HPV16-E7 11-19 (SEQ ID NO: 4) and HLA-A*02:01. In some
embodiments, the anti-E7MC antibody moiety cross-reacts with at least one
(including at
least about any of 2, 3,4, 5, or 6) of: a complex comprising HPV16-E7 11-19
(SEQ ID NO:
4) and HLA-A*02:02 (GenBank Accession No.: AFL91480), a complex comprising
HPV16-
E7 11-19 (SEQ ID NO: 4) and HLA-A*02:03 (GenBank Accession No.: AAA03604), a
complex comprising HPV16-E7 11-19 (SEQ ID NO: 4) and HLA-A*02:05 (GenBank
Accession No.: AAA03603), a complex comprising HPV16-E7 11-19 (SEQ ID NO: 4)
and
HLA-A*02:06 (GenBank Accession No.: CCB78868), a complex comprising HPV16-E7
11-
19 (SEQ ID NO: 4) and HLA-A*02:07 (GenBank Accession No.: ACR55712), and a
complex comprising HPV16-E7 11-19 (SEQ ID NO: 4) and HLA-A*02:11 (GenBank
Accession No.: CAB56609).
[0121] In some embodiments, the anti-E7MC antibody moiety specifically binds
to: a
complex comprising HPV16-E7 11-19 (SEQ ID NO: 4) and an MHC class I protein
(such as
HLA-A02, for example HLA-A*02:01); and a complex comprising an HPV16-E7 11-19
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variant having the amino acid sequence of YMLDVQPET (SEQ ID NO: 11) and an MHC
class I protein (such as HLA-A02, for example HLA-A*02:01).
[0122] In some embodiments, the anti-E7MC antibody moiety is a semi-synthetic
antibody
moiety comprising fully human sequences and one or more synthetic regions. In
some
embodiments, the anti-E7MC antibody moiety is a semi-synthetic antibody moiety
comprising a fully human light chain variable domain and a semi-synthetic
heavy chain
variable domain comprising fully human FR1, HC-CDR1, FR2, HC-CDR2, FR3, and
FR4
regions and a synthetic HC-CDR3. In some embodiments, the semi-synthetic heavy
chain
variable domain comprises a fully synthetic HC-CDR3 having a sequence from
about 5 to
about 25 (such as about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22,
23, 24, or 25) amino acids in length. In some embodiments, the semi-synthetic
heavy chain
variable domain or the synthetic HC-CDR3 is obtained from a semi-synthetic
library (such as
a semi-synthetic human library) comprising fully synthetic HC-CDR3s having a
sequence
from about 5 to about 25 (such as about any of 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25) amino acids in length, wherein each amino acid
in the sequence
is randomly selected from the standard human amino acids, minus cysteine. In
some
embodiments, the synthetic HC-CDR3 is from about 7 to about 15 (such as about
any of 7, 8,
9, 10, 11, 12, 13, 14, or 15) amino acids in length.
[0123] The anti-E7MC antibody moieties in some embodiments comprise specific
sequences or certain variants of such sequences. In some embodiments, the
amino acid
substitutions in the variant sequences do not substantially reduce the ability
of the anti-E7MC
antibody moiety to bind the E7MC. For example, alterations that do not
substantially reduce
E7MC binding affinity may be made. Alterations that substantially improve E7MC
binding
affinity or affect some other property, such as specificity and/or cross-
reactivity with related
variants of the E7MC, are also contemplated.
[0124] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain comprising an HC-CDR3 comprising the amino acid sequence of
any one of
SEQ ID NOs: 186-188, or a variant thereof comprising up to about 3 (for
example about any
of 1, 2, or 3) amino acid substitutions; and ii) a light chain variable domain
comprising an
LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or a variant
thereof
comprising up to about 3 (for example about any of 1, 2, or 3) amino acid
substitutions.
[0125] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain comprising an HC-CDR3 comprising the amino acid sequence of
any one of
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SEQ ID NOs: 186-188; and ii) a light chain variable domain comprising an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 191.
[0126] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 183, or a variant thereof comprising up to about 3 (for example about any
of 1, 2, or 3)
amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ
ID NO:
184 or 185, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or 3)
amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of
any one
of SEQ ID NOs: 186-188, or a variant thereof comprising up to about 3 (for
example about
any of 1, 2, or 3) amino acid substitutions; and ii) a light chain variable
domain comprising
an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, or a
variant
thereof comprising up to about 3 (for example about any of 1, 2, or 3) amino
acid
substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:
191, or
a variant thereof comprising up to about 3 (for example about any of 1, 2, or
3) amino acid
substitutions.
[0127] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 183, or a variant thereof comprising up to about 3 (for example about any
of 1, 2, or 3)
amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ
ID NO:
184 or 185, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or 3)
amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of
any one
of SEQ ID NOs: 186-188; and ii) a light chain variable domain comprising an LC-
CDR1
comprising the amino acid sequence of SEQ ID NO: 189 or 190, or a variant
thereof
comprising up to about 3 (for example about any of 1, 2, or 3) amino acid
substitutions, and
an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191.
[0128] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 183, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or
185, and
an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 186-
188; or a
variant thereof comprising up to about 3 (such as about any of 1, 2, or 3)
amino acid
substitutions in the HC-CDR sequences; and ii) a light chain variable domain
comprising an
LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 191; or a variant thereof
comprising up
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to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the
LC-CDR
sequences.
[0129] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 183, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or
185, and
an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 186-
188; and
ii) a light chain variable domain comprising an LC-CDR1 comprising the amino
acid
sequence of SEQ ID NO: 189 or 190, and an LC-CDR3 comprising the amino acid
sequence
of SEQ ID NO: 191. The sequences of the CDRs noted herein are provided in
Table 2 below.
TABLE 2
HC-CDR1 consensus SEQ ID NO:
183 G-F/G/Y-S/T-F-S/T-S-Y-A/G
HC-CDR2 consensus SEQ ID NO:
1 1
8
4 I-N/I-P-X-X-G-G/T/I-T/A/P
HC-CDR2 consensus SEQ ID NO:
2 185 I-S-X-S/D-G/N-G/S-N-T/I/K
HC-CDR3 consensus SEQ ID NO:
1 186 A-R-S/R-Y/S/G-Y/V-Y/W-G-X-Y-D
HC-CDR3 consensus SEQ ID NO:
2 187 A-R-G-X-X-X-Y-Y/G/S
HC-CDR3 consensus SEQ ID NO:
3 188 A-R-G-X-X-Y-Q/W-W-S-X-D-D
LC-CDR1 consensus SEQ ID NO:
1 189 N-I-G-S-N/K
LC-CDR1 consensus SEQ ID NO:
2 190 L-R-S/N-X-Y
LC-CDR3 consensus SEQ ID NO:
191 A/Q/N-S/A/V-W/Y/R-D-S/D-S-L/S/G-X-X-X-V
[0130] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain comprising an HC-CDR3 comprising the amino acid sequence of
any one of
SEQ ID NOs: 99-119, 244, and 245, or a variant thereof comprising up to about
5 (such as
about any of 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a light chain
variable domain
comprising an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID
NOs:
162-182 and 247-250, or a variant thereof comprising up to about 5 (such as
about any of 1,
2, 3, 4, or 5) amino acid substitutions.
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[0131] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain comprising an HC-CDR3 comprising the amino acid sequence of
any one of
SEQ ID NOs: 99-119, 244, and 245; and ii) a light chain variable domain
comprising an LC-
CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 162-182 and
247-
250.
[0132] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
any one of
SEQ ID NOs: 57-77, or a variant thereof comprising up to about 5 (such as
about any of 1,2,
3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid
sequence of any
one of SEQ ID NOs: 78-98, or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the
amino acid
sequence of any one of SEQ ID NOs: 99-119, 244, and 245, or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions; and ii) a light
chain variable domain comprising an LC-CDR1 comprising the amino acid sequence
of any
one of SEQ ID NOs: 120-140 and 246, or a variant thereof comprising up to
about 5 (such as
about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising
the amino
acid sequence of any one of SEQ ID NOs: 141-161, or a variant thereof
comprising up to
about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC-
CDR3
comprising the amino acid sequence of any one of SEQ ID NOs: 162-182 and 247-
250, or a
variant thereof comprising up to about 5 (such as about any of 1,2, 3,4, or 5)
amino acid
substitutions.
[0133] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
any one of
SEQ ID NOs: 57-77, or a variant thereof comprising up to about 5 (such as
about any of 1,2,
3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid
sequence of any
one of SEQ ID NOs: 78-98, or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the
amino acid
sequence of any one of SEQ ID NOs: 99-119, 244, and 245; and ii) a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of any one of
SEQ ID
NOs: 120-140 and 246, or a variant thereof comprising up to about 5 (such as
about any of 1,
2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid
sequence of
any one of SEQ ID NOs: 141-161, or a variant thereof comprising up to about 3
(such as
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about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising
the amino acid
sequence of any one of SEQ ID NOs: 162-182 and 247-250.
[0134] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of
any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of
any
one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of
any
one of SEQ ID NOs: 99-119, 244, and 245; or a variant thereof comprising up to
about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR
sequences; and
ii) a light chain variable domain sequence comprising an LC-CDR1 comprising
the amino
acid sequence of any one of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising
the
amino acid sequence of any one of SEQ ID NOs: 141-161; and an LC-CDR3
comprising the
amino acid sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a
variant thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
LC-CDR sequences.
[0135] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of
any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of
any
one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of
any
one of SEQ ID NOs: 99-119, 244, and 245; or a variant thereof comprising up to
about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, wherein the
amino acid
substitutions are in HC-CDR1 or HC-CDR2; and ii) a light chain variable domain
sequence
comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID
NOs:
120-140 and 246; an LC-CDR2 comprising the amino acid sequence of any one of
SEQ ID
NOs: 141-161; and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 162-182 and 247-250; or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions, wherein the amino acid
substitutions are in HC-
CDR1 or HC-CDR2.
[0136] In some embodiments, the anti-E7MC antibody moiety comprises i) a heavy
chain
variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of
any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of
any
one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of
any
one of SEQ ID NOs: 99-119, 244, and 245; and ii) alight chain variable domain
sequence
comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID
NOs:
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120-140 and 246; an LC-CDR2 comprising the amino acid sequence of any one of
SEQ ID
NOs: 141-161; and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 162-182 and 247-250. The sequences of the HC-CDRs noted herein are
provided in
Table 3 below and the LC-CDRs noted herein are provided in Table 4 below.
TABLE 3
SEQ ID NO: 57 SEQ ID NO: 78 SEQ ID NO: 99
HC-CDR1 1 GYTFIDYY HC-CDR2 1 INPKSGGT HC-CDR3 1 ARSYYYGGVKDY
SEQ ID NO: 58 SEQ ID NO: 79 SEQ ID NO: 100
HC-CDR1 2 GGTFSSYA HC-CDR2 2 IIPIFGTA HC-CDR3 2
ARGVYGYSGSAPYDE
SEQ ID NO: 59 SEQ ID NO: 80 SEQ ID NO: 101
HC-CDR1 3 GGTFSMAA HC-CDR2 3 IIPMIGIP HC-CDR3 3 ARGPLYWWHSDS
SEQ ID NO: 60 SEQ ID NO: 81 SEQ ID NO: 102
HC-CDR1 4 GFTFSSYA HC-CDR2 4 ISYDGTNK HC-CDR3 4 ARM HGSYGSYFDK
SEQ ID NO: 61 SEQ ID NO: 82 SEQ ID NO: 103
HC-CDR1 5 GGTFRSYA HC-CDR2 5 IIPIFGTP HC-CDR3 5 ARSSMYQYSHDD
SEQ ID NO: 62 SEQ ID NO: 83 SEQ ID NO: 104
HC-CDR1 8 GYTFANYG HC-CDR2 8 ISASNGNT HC-CDR3 8 ARSYYAGYYMFMDF
SEQ ID NO: 63 SEQ ID NO: 84 SEQ ID NO: 105
HC-CDR1 9 GFTFSSYW HC-CDR2 9 INQDGSEK HC-CDR3 9 ARYSVWGMMDE
SEQ ID NO: 64 SEQ ID NO: 85 SEQ ID NO: 106
HC-CDR1 10 GFPFSSYS HC-CDR2 10 ISSSSSTI HC-CDR3 10
ARQNYYADYDY
SEQ ID NO: 65 SEQ ID NO: 86 SEQ ID NO: 107
HC-CDR1 11 GYTFSDYG HC-CDR2 11 ISTYNGNT HC-CDR3 11 ARGYTGSYYATGDE
SEQ ID NO: 66 SEQ ID NO: 87 SEQ ID NO: 108
HC-CDR1 13 GYTFTSYG HC-CDR2 13 ISPYNGNT HC-CDR3 13
ARGYKDY
SEQ ID NO: 67 SEQ ID NO: 88 SEQ ID NO: 109
HC-CDR1 17 GDIFRKFA HC-CDR2 17 IIPNLDIT HC-CDR3 17
ARGSIYQWSGWDS
SEQ ID NO: 68 SEQ ID NO: 89 SEQ ID NO: 110
HC-CDR1 22 GFNFSDYY HC-CDR2 22 ISPGGTT HC-CDR3 22 ARGHSFSYGEDY
SEQ ID NO: 69 SEQ ID NO: 90 SEQ ID NO: 111
HC-CDR1 26 GYTFTGYY HC-CDR2 26 INPNSGGT HC-CDR3 26 ARGWHQYGIMDY
SEQ ID NO: 70 SEQ ID NO: 91 SEQ ID NO: 112
HC-CDR1 27 GFTFSSYA HC-CDR2 27 ISGSGGST HC-CDR3 27
ARRSVWSAYDS
SEQ ID NO: 71 SEQ ID NO: 92 SEQ ID NO: 113
HC-CDR1 31 GFTFSSYW HC-CDR2 31 IKEDGSVK HC-CDR3 31 ARRGVWGHIDF
SEQ ID NO: 72 SEQ ID NO: 93 SEQ ID NO: 114
HC-CDR1 32 GGTFSSYA HC-CDR2 32 IIPILGIA HC-CDR3 32
ARGTYTWGSDT
SEQ ID NO: 73 SEQ ID NO: 94 SEQ ID NO: 115
HC-CDR1 39 GFSFSRHA HC-CDR2 39 ISGSGGNT HC-CDR3 39 ARYWGSYDK
SEQ ID NO: 74 SEQ ID NO: 95 SEQ ID NO: 116
HC-CDR1 40 GYSFSDYY HC-CDR2 40 INPNTGVT HC-CDR3 40
ARNVYRYSMMHDS
SEQ ID NO: 75 SEQ ID NO: 96 SEQ ID NO: 117
HC-CDR1 41 GFTFSSYS HC-CDR2 41 ISSSSSYI HC-CDR3 41
ARRGVWGYFDS
SEQ ID NO: 76 SEQ ID NO: 97 SEQ ID NO: 118
HC-CDR1 US-5 GGTFSSYG HC-CDR2 US-5 IIPIFGTT HC-CDR3 US-5
ARVYGSYFYDD
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SEQ ID NO: 77 SEQ ID NO: 98 SEQ ID NO: 119
HC-CDR1 US-7 GGSFSGYY HC-CDR2 US-7 INHSGST HC-CDR3 US-7 ARAPQSWYVGDV
TABLE 4
SEQ ID NO: SEQ ID NO:
120 141 SEQ ID NO: 162
LC-CDR1 1 SSNIGTNP LC-CDR2 1 GNY LC-CDR3 1 AAWDDSLTGYV
SEQ ID NO: SEQ ID NO:
121 142 SEQ ID NO: 163
LC-CDR1 2 NIGMKS LC-CDR2 2 YDS LC-CDR3 2 QVWDTTGDHPGYV
SEQ ID NO: SEQ ID NO:
122 143 SEQ ID NO: 164
LC-CDR1 3 SSNIGNNY LC-CDR2 3 DNY LC-CDR3 3 GTWDSSLSAYV
SEQ ID NO: SEQ ID NO:
123 144 SEQ ID NO: 165
LC-CDR1 4 NIGSKS LC-CDR2 4 YNS LC-CDR3 4 QVWDSSSDHWV
SEQ ID NO: SEQ ID NO:
124 145 SEQ ID NO: 166
LC-CDR1 5 QGISSW LC-CDR2 5 AAS LC-CDR3 5 QQANSFPLT
SEQ ID NO: SEQ ID NO:
125 146 SEQ ID NO: 167
LC-CDR1 8 NSNIETNP LC-CDR2 8 SYY LC-CDR3 8 ASWDDSVQGYV
SEQ ID NO: SEQ ID NO:
126 147 SEQ ID NO: 168
LC-CDR1 9 SLRSYY LC-CDR2 9 GKN LC-CDR3 9 NSRDSSGNHQV
SEQ ID NO: SEQ ID NO:
127 148 SEQ ID NO: 169
LC-CDR1 10 NIGSKN LC-CDR2 10 DDH LC-CDR3 10 QVWDSSSDHVV
SEQ ID NO: SEQ ID NO:
128 149 SEQ ID NO: 170
LC-CDR1 11 SSNIGINT LC-CDR2 11 GNS LC-CDR3 11
QSYDTSLTVI
SEQ ID NO: SEQ ID NO:
129 150 SEQ ID NO: 171
LC-CDR1 13 TGAVTSGYY LC-CDR2 13 STN LC-CDR3 13 LLYYGGAQLWV
SEQ ID NO: SEQ ID NO:
130 151 SEQ ID NO: 172
LC-CDR1 17 QSISSY LC-CDR2 17 GAS LC-CDR3 17 QQSYSAPLT
SEQ ID NO: SEQ ID NO:
131 152 SEQ ID NO: 173
LC-CDR1 22 SSNIGSNS LC-CDR2 22 SNN LC-CDR3 22
AAWDDSLNGYV
SEQ ID NO: SEQ ID NO:
132 153 SEQ ID NO: 174
LC-CDR1 26 SSNIGAGYD LC-CDR2 26 ESN LC-CDR3 26 QSYDTSLKGV
SEQ ID NO: SEQ ID NO:
133 154 SEQ ID NO: 175
LC-CDR1 27 ILRNYY LC-CDR2 27 GQN LC-CDR3 27 DSRDSSGNHWV
SEQ ID NO: SEQ ID NO: SEQ ID NO: 176
134 NLRNSY 155 GKN LC-CDR3 31 NSRDSSGFPPYV
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LC-CDR1 31 LC-CDR2 31
SEQ ID NO: SEQ ID NO:
135 156 SEQ ID NO: 177
LC-CDR1 32 KLGEKY LC-CDR2 32 QND LC-CDR3 32 QSWASSSVYV
SEQ ID NO: SEQ ID NO:
136 157 SEQ ID NO: 178
LC-CDR1 39 SLRNYY LC-CDR2 39 GKD LC-CDR3 39 NSRDSSGNHRV
SEQ ID NO: SEQ ID NO:
137 158 SEQ ID NO: 179
LC-CDR1 40 SSNIGSNH LC-CDR2 40 NNN LC-CDR3 40
AAWDDSLNVPV
SEQ ID NO: SEQ ID NO:
138 159 SEQ ID NO: 180
LC-CDR1 41 TLRSFY LC-CDR2 41 GKN LC-CDR3 41 NSRDSGGDPVV
SEQ ID NO: SEQ ID NO:
139 160 SEQ ID NO: 181
LC-CDR1 US-5 TGAVTSSYY LC-CDR2 US-5 STS LC-CDR3 US-5 LLYYGGARV
SEQ ID NO: SEQ ID NO:
140 161 SEQ ID NO: 182
LC-CDR1 US-7 SSNIGTRM LC-CDR2 US-7 NNN LC-CDR3 US-7 AAWDDNLKSYV
[0137] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence of any one of SEQ ID NOs:
15-35 and
233-237, or a variant thereof having at least about 95% (including for example
at least about
any of 96%, 97%, 98%, or 99%) sequence identity, and a light chain variable
domain
comprising the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-
243, or a
variant thereof having at least about 95% (including for example at least any
of 96%, 97%,
98%, or 99%) sequence identity.
[0138] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence of any one of SEQ ID NOs:
15-35 and
233-237 and a light chain variable domain comprising the amino acid sequence
of any one of
SEQ ID NOs: 36-56 and 238-243.
[0139] The heavy and light chain variable domains can be combined in various
pair-wise
combinations to generate a number of anti-E7MC antibody moieties.
[0140] For example, in some embodiments, the anti-E7MC antibody moiety
comprises a
heavy chain variable domain comprising an HC-CDR1 comprising the amino acid
sequence
of SEQ ID NO: 57, or a variant thereof comprising up to about 5 (for example
about any of 1,
2, 3, 4, or 5) amino acid substitutions; an HC-CDR2 comprising the amino acid
sequence of
SEQ ID NO: 78, or a variant thereof comprising up to about 5 (for example
about any of 1, 2,
3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising the amino acid
sequence of
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SEQ ID NO: 99, or a variant thereof comprising up to about 5 (such as about
any of 1, 2, 3, 4,
or 5) amino acid substitutions; and a light chain variable domain comprising
an LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 120, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 141, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 162, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0141] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 57, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 99, or a variant thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 120, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 141, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 162, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0142] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 57, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 78, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 99; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
120,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 141, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 162.
[0143] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 58, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 79, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 100, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
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comprising the amino acid sequence of SEQ ID NO: 121, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 142, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 163, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0144] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 58, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 79, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 100, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 121, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 142, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 163, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0145] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 58, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 79, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 100; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
121,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 142, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 163.
[0146] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 59, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 80, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 101, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 122, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
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comprising the amino acid sequence of SEQ ID NO: 143, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0147] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 59, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 101, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 122, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 143, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 164, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0148] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 59, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 101; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
122,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 143, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 164.
[0149] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 60, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 81, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 102, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 123, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 144, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
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comprising the amino acid sequence of SEQ ID NO: 165, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0150] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 60, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 81, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 102, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 123, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 144, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 165, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0151] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 60, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 81, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 102; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
123,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 144, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 165.
[0152] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 61, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 82, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 103, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 124, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 145, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 166, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
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[0153] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 61, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 82, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 103, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 124, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 145, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 166, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0154] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 61, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 82, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 103; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
124,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 145, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 166.
[0155] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 62, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 83, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 104, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 125, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 146, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 167, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0156] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
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NO: 62, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 104, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 125, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 146, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 167, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0157] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 62, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 104; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
125,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 146, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 167.
[0158] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 63, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 84, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 105, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 126, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 147, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 168, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0159] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 63, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 84, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 105, or a variant
thereof
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comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 126, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 147, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 168, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0160] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 63, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 84, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 105; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
126,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 147, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 168.
[0161] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 64, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 85, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 106, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 127, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 148, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 169, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0162] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 64, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 85, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 106, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
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the amino acid sequence of SEQ ID NO: 127, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 148, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 169, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0163] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 64, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 85, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 106; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
127,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 148, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 169.
[0164] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 65, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 86, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 107, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 128, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 149, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 170, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0165] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 65, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 107, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 128, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 149, and an LC-CDR3 comprising the amino acid sequence
of SEQ
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ID NO: 170, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0166] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 65, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 107; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
128,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 149, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 170.
[0167] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 66, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 87, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 108, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 129, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 150, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 171, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0168] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 66, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 87, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 108, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 129, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 150, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 171, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
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[0169] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 66, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 87, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 108; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
129,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 150, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 171.
[0170] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 67, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 88, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 109, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 130, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 151, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 172, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0171] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 67, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 109, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 130, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 151, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 172, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0172] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
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NO: 67, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 109; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
130,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 151, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 172.
[0173] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 68, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 89, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 110, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 131, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 152, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 173, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0174] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 68, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 110, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 131, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 152, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 173, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0175] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 68, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 110; and a light chain
variable
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domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
131,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 152, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 173.
[0176] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 69, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 90, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 111, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 132, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 153, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 174, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0177] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 69, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 111, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 132, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 153, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 174, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0178] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 69, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 90, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 111; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
132,
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an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 174.
[0179] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 70, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 91, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 112, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 133, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 154, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 175, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0180] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 70, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 91, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 112, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 133, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 154, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 175, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0181] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 70, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 91, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 112; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
133,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 154, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 175.
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[0182] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 71, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 92, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 113, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 134, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 155, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 176, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0183] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 71, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 113, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 134, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 155, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 176, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0184] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 71, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 113; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
134,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 155, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 176.
[0185] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
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NO: 72, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 93, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 114, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 135, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 156, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 177, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0186] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 72, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 114, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 135, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 156, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 177, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0187] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 72, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 114; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
135,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 156, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 177.
[0188] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 73, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
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NO: 94, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 115, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 136, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 157, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 178, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0189] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 73, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 94, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 115, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 136, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 157, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 178, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0190] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 73, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 94, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 115; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
136,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 157, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 178.
[0191] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 74, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 95, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
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ID NO: 116, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 137, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 158, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 179, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0192] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 74, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 116, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 137, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 158, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 179, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0193] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 74, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 116; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
137,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 158, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 179.
[0194] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 75, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 96, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 117, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
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comprising the amino acid sequence of SEQ ID NO: 138, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 159, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 180, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0195] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 75, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 117, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 138, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 159, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 180, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0196] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 75, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 117; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
138,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 159, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 180.
[0197] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 76, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 97, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 118, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 139, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
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comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 181, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0198] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 76, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 97, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 118, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 139, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 160, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 181, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0199] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 76, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 97, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 118; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
139,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 160, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 181.
[0200] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 98, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 119, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 140, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 161, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
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comprising the amino acid sequence of SEQ ID NO: 182, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0201] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 182, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0202] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
140,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 182.
[0203] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 98, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 244, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 140, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 161, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 182, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
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[0204] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 244, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 182, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0205] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 244; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
140,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 182.
[0206] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 98, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 245, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 140, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 161, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 182, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0207] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
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NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 245, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 182, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0208] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 245; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
140,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 182.
[0209] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 98, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 119, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 140, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 161, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 247, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0210] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119, or a variant
thereof
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comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 247, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0211] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
140,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 247.
[0212] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 98, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 119, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 140, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 161, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 248, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0213] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
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the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 248, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0214] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
140,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 248.
[0215] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 98, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 119, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 246, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 161, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 182, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0216] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 246, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence
of SEQ
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ID NO: 182, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0217] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
246,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 182.
[0218] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 98, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 119, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 140, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 161, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 249, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0219] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 249, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
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[0220] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
140,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 249.
[0221] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 98, or a variant thereof comprising up to about 5 (for example about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and an HC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 119, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions; and a light chain variable domain comprising an
LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 140, or a variant thereof
comprising up
to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; an LC-CDR2
comprising the amino acid sequence of SEQ ID NO: 161, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
an LC-CDR3
comprising the amino acid sequence of SEQ ID NO: 250, or a variant thereof
comprising up
to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0222] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 140, an LC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 161, and an LC-CDR3 comprising the amino acid sequence
of SEQ
ID NO: 250, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions in the LC-CDR sequences.
[0223] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
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NO: 77, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an
HC-
CDR3 comprising the amino acid sequence of SEQ ID NO: 119; and a light chain
variable
domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:
140,
an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC-
CDR3
comprising the amino acid sequence of SEQ ID NO: 250.
[0224] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 15,
or a variant
thereof having at least about 95% (for example at least about any of 96%, 97%,
98%, or 99%)
sequence identity, and a light chain variable domain comprising the amino acid
sequence set
forth in SEQ ID NO: 36, or a variant thereof having at least about 95%
(including for
example at least about any of 96%, 97%, 98%, or 99%) sequence identity. In
some
embodiments, the anti-E7MC antibody moiety comprises a heavy chain variable
domain
comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light
chain variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 36.
[0225] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 16,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 37, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 16 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 37.
[0226] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 17,
or a variant
thereof having at least about 95% (for example at least about any of 96%, 97%,
98%, or 99%)
sequence identity, and a light chain variable domain comprising the amino acid
sequence set
forth in SEQ ID NO: 38, or a variant thereof having at least about 95%
(including for
example at least about any of 96%, 97%, 98%, or 99%) sequence identity. In
some
embodiments, the anti-E7MC antibody moiety comprises a heavy chain variable
domain
comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light
chain variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 38.
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[0227] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 18,
or a variant
thereof having at least about 95% (for example at least about any of 96%, 97%,
98%, or 99%)
sequence identity, and a light chain variable domain comprising the amino acid
sequence set
forth in SEQ ID NO: 39, or a variant thereof having at least about 95%
(including for
example at least about any of 96%, 97%, 98%, or 99%) sequence identity. In
some
embodiments, the anti-E7MC antibody moiety comprises a heavy chain variable
domain
comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light
chain variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 39.
[0228] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 19,
or a variant
thereof having at least about 95% (for example at least about any of 96%, 97%,
98%, or 99%)
sequence identity, and a light chain variable domain comprising the amino acid
sequence set
forth in SEQ ID NO: 40, or a variant thereof having at least about 95%
(including for
example at least about any of 96%, 97%, 98%, or 99%) sequence identity. In
some
embodiments, the anti-E7MC antibody moiety comprises a heavy chain variable
domain
comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light
chain variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 40.
[0229] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 20,
or a variant
thereof having at least about 95% (for example at least about any of 96%, 97%,
98%, or 99%)
sequence identity, and a light chain variable domain comprising the amino acid
sequence set
forth in SEQ ID NO: 41, or a variant thereof having at least about 95%
(including for
example at least about any of 96%, 97%, 98%, or 99%) sequence identity. In
some
embodiments, the anti-E7MC antibody moiety comprises a heavy chain variable
domain
comprising the amino acid sequence set forth in SEQ ID NO: 20 and a light
chain variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 41.
[0230] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 21,
or a variant
thereof having at least about 95% (for example at least about any of 96%, 97%,
98%, or 99%)
sequence identity, and a light chain variable domain comprising the amino acid
sequence set
forth in SEQ ID NO: 42, or a variant thereof having at least about 95%
(including for
example at least about any of 96%, 97%, 98%, or 99%) sequence identity. In
some
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embodiments, the anti-E7MC antibody moiety comprises a heavy chain variable
domain
comprising the amino acid sequence set forth in SEQ ID NO: 21 and a light
chain variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 42.
[0231] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 22,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 43, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 22 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 43.
[0232] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 23,
or a variant
thereof having at least about 95% (for example at least about any of 96%, 97%,
98%, or 99%)
sequence identity, and a light chain variable domain comprising the amino acid
sequence set
forth in SEQ ID NO: 44, or a variant thereof having at least about 95%
(including for
example at least about any of 96%, 97%, 98%, or 99%) sequence identity. In
some
embodiments, the anti-E7MC antibody moiety comprises a heavy chain variable
domain
comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light
chain variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 44.
[0233] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 24,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 45, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 24 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 45.
[0234] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 25,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
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98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 46, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 25 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 46.
[0235] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 26,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 47, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 26 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 47.
[0236] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 27,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 48, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 27 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 48.
[0237] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 28,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 49, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 28 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 49.
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[0238] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 50, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 29 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 50.
[0239] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 30,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 51, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 30 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 51.
[0240] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 31,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 52, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 31 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 52.
[0241] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 32,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 53, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
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some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 32 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 53.
[0242] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 33,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 54, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 33 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 54.
[0243] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 34,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 55, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 34 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 55.
[0244] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 56, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56.
[0245] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
233, or a
variant thereof having at least about 95% (including for example at least
about any of 96%,
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97%, 98%, or 99%) sequence identity, and a light chain variable domain
comprising the
amino acid sequence set forth in SEQ ID NO: 56, or a variant thereof having at
least about
95% (including for example at least about any of 96%, 97%, 98%, or 99%)
sequence identity.
In some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 233 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56.
[0246] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
234, or a
variant thereof having at least about 95% (including for example at least
about any of 96%,
97%, 98%, or 99%) sequence identity, and a light chain variable domain
comprising the
amino acid sequence set forth in SEQ ID NO: 56, or a variant thereof having at
least about
95% (including for example at least about any of 96%, 97%, 98%, or 99%)
sequence identity.
In some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 234 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56.
[0247] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
235, or a
variant thereof having at least about 95% (including for example at least
about any of 96%,
97%, 98%, or 99%) sequence identity, and a light chain variable domain
comprising the
amino acid sequence set forth in SEQ ID NO: 56, or a variant thereof having at
least about
95% (including for example at least about any of 96%, 97%, 98%, or 99%)
sequence identity.
In some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 235 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56.
[0248] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 238, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
238.
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[0249] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 239, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
239.
[0250] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 240, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
240.
[0251] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 241, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
241.
[0252] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 35,
or a variant
thereof having at least about 95% (including for example at least about any of
96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence set forth in SEQ ID NO: 242, or a variant thereof having at least
about 95%
(including for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
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some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 35 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
242.
[0253] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
236, or a
variant thereof having at least about 95% (including for example at least
about any of 96%,
97%, 98%, or 99%) sequence identity, and a light chain variable domain
comprising the
amino acid sequence set forth in SEQ ID NO: 243, or a variant thereof having
at least about
95% (including for example at least about any of 96%, 97%, 98%, or 99%)
sequence identity.
In some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 236 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
243.
[0254] In some embodiments, the anti-E7MC antibody moiety comprises a heavy
chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO:
237, or a
variant thereof having at least about 95% (including for example at least
about any of 96%,
97%, 98%, or 99%) sequence identity, and a light chain variable domain
comprising the
amino acid sequence set forth in SEQ ID NO: 56, or a variant thereof having at
least about
95% (including for example at least about any of 96%, 97%, 98%, or 99%)
sequence identity.
In some embodiments, the anti-E7MC antibody moiety comprises a heavy chain
variable
domain comprising the amino acid sequence set forth in SEQ ID NO: 237 and a
light chain
variable domain comprising the amino acid sequence set forth in SEQ ID NO: 56.
[0255] In some embodiments, the anti-E7MC antibody moiety competes for binding
to a
target HPV16-E7/MHC class I complex with a second anti-E7MC antibody moiety
according
to any of the anti-E7MC antibody moieties described herein. In some
embodiments, the anti-
E7MC antibody moiety binds to the same, or substantially the same, epitope as
the second
anti-E7MC antibody moiety. In some embodiments, binding of the anti-E7MC
antibody
moiety to the target HPV16-E7/MHC class I complex inhibits binding of the
second anti-
E7MC antibody moiety to the target HPV16-E7/MHC class I complex by at least
about 70%
(such as by at least about any of 75%, 80%, 85%, 90%, 95%, 98% or 99%), or
vice versa. In
some embodiments, the anti-E7MC antibody moiety and the second anti-E7MC
antibody
moiety cross-compete for binding to the target HPV16-E7/MHC class I complex,
i.e., each of
the antibody moieties competes with the other for binding to the target HPV16-
E7/MHC
class I complex.
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[0256] For example, in some embodiments, the anti-E7MC antibody moiety
competes for
binding to a target HPV16-E7/MHC class I complex with an antibody moiety
comprising i) a
heavy chain variable domain sequence comprising an HC-CDR1 comprising the
amino acid
sequence of SEQ ID NO: 183, or a variant thereof comprising up to about 3 (for
example
about any of 1, 2, or 3) amino acid substitutions, an HC-CDR2 comprising the
amino acid
sequence of SEQ ID NO: 184 or 185, or a variant thereof comprising up to about
3 (for
example about any of 1, 2, or 3) amino acid substitutions, and an HC-CDR3
comprising the
amino acid sequence of any one of SEQ ID NOs: 186-188; or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
ii) a light chain
variable domain comprising an LC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 189 or 190, or a variant thereof comprising up to about 3 (for example
about any of 1, 2,
or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid
sequence of SEQ
ID NO: 191, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or
3) amino acid substitutions.
[0257] In some embodiments, the anti-E7MC antibody moiety competes for binding
to a
target HPV16-E7/MHC class I complex with an antibody moiety comprising i) a
heavy chain
variable domain sequence comprising an HC-CDR1 comprising (and in some
embodiments
consisting of) the amino acid sequence of any one of SEQ ID NOs: 57-77; or a
variant
thereof comprising up to about 5 (for example about any of 1, 2, 3, 4, or 5)
amino acid
substitutions; an HC-CDR2 comprising (and in some embodiments consisting of)
the amino
acid sequence of any one of SEQ ID NOs: 78-98; or a variant thereof comprising
up to about
(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; and an
HC-CDR3
comprising (and in some embodiments consisting of) the amino acid sequence of
any one of
SEQ ID NOs: 99-119, 244, and 245; or a variant thereof comprising up to about
5 (for
example about any of 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a
light chain variable
domain sequence comprising an LC-CDR1 comprising (and in some embodiments
consisting
of) the amino acid sequence of any one of SEQ ID NOs: 120-140 and 246; or a
variant
thereof comprising up to about 5 (for example about any of 1, 2, 3, 4, or 5)
amino acid
substitutions; an LC-CDR2 comprising (and in some embodiments consisting of)
the amino
acid sequence of any one of SEQ ID NOs: 141-161; or a variant thereof
comprising up to
about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and an
LC-CDR3
comprising (and in some embodiments consisting of) the amino acid sequence of
any one of
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SEQ ID NOs: 162-182 and 247-250; or a variant thereof comprising up to about 5
(for
example about any of 1, 2, 3, 4, or 5) amino acid substitutions.
[0258] In some embodiments, the anti-E7MC antibody moiety competes for binding
to a
target HPV16-E7/MHC class I complex with an antibody moiety comprising i) a
heavy chain
variable domain sequence comprising an HC-CDR1 comprising (and in some
embodiments
consisting of) the amino acid sequence of any one of SEQ ID NOs: 57-77; an HC-
CDR2
comprising (and in some embodiments consisting of) the amino acid sequence of
any one of
SEQ ID NOs: 78-98; and an HC-CDR3 comprising (and in some embodiments
consisting of)
the amino acid sequence of any one of SEQ ID NOs: 99-119, 244, and 245; or a
variant
thereof comprising up to about 5 (for example about any of 1, 2, 3, 4, or 5)
amino acid
substitutions in the HC-CDR sequences; and ii) a light chain variable domain
sequence
comprising an LC-CDR1 comprising (and in some embodiments consisting of) the
amino
acid sequence of any one of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising
(and
in some embodiments consisting of) the amino acid sequence of any one of SEQ
ID NOs:
141-161; and an LC-CDR3 comprising (and in some embodiments consisting of) the
amino
acid sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a variant
thereof
comprising up to about 5 (for example about any of 1, 2, 3, 4, or 5) amino
acid substitutions
in the LC-CDR sequences.
[0259] In some embodiments, the anti-E7MC antibody moiety competes for binding
to a
target HPV16-E7/MHC class I complex with an antibody moiety comprising a heavy
chain
variable domain comprising (and in some embodiments consisting of) the amino
acid
sequence of any one of SEQ ID NOs: 15-35 and 233-237, or a variant thereof
having at least
about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity,
and a light chain variable domain comprising (and in some embodiments
consisting of) the
amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, or a variant
thereof
having at least about 95% (for example at least about any of 96%, 97%, 98%, or
99%)
sequence identity.
Full-length anti-E7MC antibodies
[0260] The anti-E7MC constructs in some embodiments are full-length antibodies
comprising an anti-E7MC antibody moiety (also referred to herein as a "full-
length anti-
E7MC antibody"). In some embodiments, the full-length antibody is a monoclonal
antibody.
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[0261] In some embodiments, the full-length anti-E7MC antibody comprises an Fc
sequence from an immunoglobulin, such as IgA, IgD, IgE, IgG, and IgM. In some
embodiments, the full-length anti-E7MC antibody comprises an Fc sequence of
IgG, such as
any of IgGl, IgG2, IgG3, or IgG4. In some embodiments, the full-length anti-
E7MC antibody
comprises an Fc sequence of a human immunoglobulin. In some embodiments, the
full-length
anti-E7MC antibody comprises an Fc sequence of a mouse immunoglobulin. In some
embodiments, the full-length anti-E7MC antibody comprises an Fc sequence that
has been
altered or otherwise changed so that it has enhanced antibody dependent
cellular cytotoxicity
(ADCC) or complement dependent cytotoxicity (CDC) effector function.
[0262] Thus, for example, in some embodiments, there is provided a full-length
anti-E7MC
antibody comprising a) an anti-E7MC antibody moiety that specifically binds to
a complex
comprising an HPV16-E7 peptide and an MHC class I protein, and b) an Fc
region. In some
embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some
embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC
class I
protein is HLA-A*02:01. In some embodiments, there is provided a full-length
anti-E7MC
antibody comprising a) an anti-E7MC antibody moiety that specifically binds to
a complex
comprising an HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A*02:01, and b) an
Fc
region. In some embodiments, the Fc region comprises an IgG1 Fc sequence. In
some
embodiments, the Fc region comprises a human IgG1 Fc sequence. In some
embodiments,
the Fc region comprises a mouse IgG1 Fc sequence. In some embodiments, the
anti-E7MC
antibody moiety cross-reacts with at least one (such as at least any of 2, 3,
4, 5, or 6) complex
comprising the MHC class I protein and a variant of the HPV16-E7 peptide
having one
amino acid substitution (such as a conservative amino acid substitution). In
some
embodiments, the anti-E7MC antibody moiety cross-reacts with at least one
(such as at least
any of 2, 3, 4, or 5) complex comprising the HPV16-E7 peptide and a different
subtype of the
MHC class I protein.
[0263] In some embodiments, there is provided a full-length anti-E7MC antibody
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 183, or a variant thereof comprising up to about 3 (for example about any
of 1, 2, or 3)
amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ
ID NO:
184 or 185, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or 3)
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amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of
any one
of SEQ ID NOs: 186-188; or a variant thereof comprising up to about 3 (for
example about
any of 1, 2, or 3) amino acid substitutions; and ii) a light chain variable
domain comprising
an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, or a
variant
thereof comprising up to about 3 (for example about any of 1, 2, or 3) amino
acid
substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:
191, or
a variant thereof comprising up to about 3 (for example about any of 1, 2, or
3) amino acid
substitutions, and b) an Fc region. In some embodiments, the Fc region
comprises an IgG1 Fc
sequence. In some embodiments, the Fc region comprises a human IgG1 Fc
sequence. In
some embodiments, the Fc region comprises a mouse IgG1 Fc sequence.
[0264] In some embodiments, there is provided a full-length anti-E7MC antibody
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 183, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or
185, and
an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 186-
188; and
ii) a light chain variable domain comprising an LC-CDR1 comprising the amino
acid
sequence of SEQ ID NO: 189 or 190, and an LC-CDR3 comprising the amino acid
sequence
of SEQ ID NO: 191, and b) an Fc region. In some embodiments, the Fc region
comprises an
IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc
sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc
sequence.
[0265] In some embodiments, there is provided a full-length anti-E7MC antibody
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain comprising an HC-CDR1 comprising the amino acid sequence of any one of
SEQ ID
NOs: 57-77, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of
any one of
SEQ ID NOs: 78-98, or a variant thereof comprising up to about 5 (such as
about any of 1,2,
3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid
sequence of
any one of SEQ ID NOs: 99-119, 244, and 245, or a variant thereof comprising
up to about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a
light chain variable
domain comprising an LC-CDR1 comprising the amino acid sequence of any one of
SEQ ID
NOs: 120-140 and 246, or a variant thereof comprising up to about 5 (such as
about any of 1,
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2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid
sequence of
any one of SEQ ID NOs: 141-161, or a variant thereof comprising up to about 3
(such as
about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising
the amino acid
sequence of any one of SEQ ID NOs: 162-182 and 247-250, or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions. In some
embodiments, the Fc region comprises an IgG1 Fc sequence. In some embodiments,
the Fc
region comprises a human IgG1 Fc sequence. In some embodiments, the Fc region
comprises
a mouse IgG1 Fc sequence.
[0266] In some embodiments, there is provided a full-length anti-E7MC antibody
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
any one
of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one
of
SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any
one of
SEQ ID NOs: 99-119, 244, and 245; or a variant thereof comprising up to about
5 (such as
about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR
sequences; and ii) a
light chain variable domain sequence comprising an LC-CDR1 comprising the
amino acid
sequence of any one of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising the
amino
acid sequence of any one of SEQ ID NOs: 141-161; and an LC-CDR3 comprising the
amino
acid sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
LC-CDR sequences; and b) an Fc region. In some embodiments, the Fc region
comprises an
IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc
sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc
sequence.
[0267] In some embodiments, there is provided a full-length anti-E7MC antibody
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
any one
of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one
of
SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any
one of
SEQ ID NOs: 99-119, 244, and 245; and ii) a light chain variable domain
sequence
comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID
NOs:
120-140 and 246; an LC-CDR2 comprising the amino acid sequence of any one of
SEQ ID
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NOs: 141-161; and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 162-182 and 247-250; and b) an Fc region. In some embodiments, the Fc
region
comprises an IgG1 Fc sequence. In some embodiments, the Fc region comprises a
human
IgG1 Fc sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc
sequence.
[0268] In some embodiments, there is provided a full-length anti-E7MC antibody
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising a heavy chain
variable domain
comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-
237, or a
variant thereof having at least about 95% (for example at least about any of
96%, 97%, 98%,
or 99%) sequence identity, and a light chain variable domain comprising the
amino acid
sequence of any one of SEQ ID NOs: 36-56 and 238-243, or a variant thereof
having at least
about 95% (including for example at least about any of 96%, 97%, 98%, or 99%)
sequence
identity; and b) an Fc region. In some embodiments, the Fc region comprises an
IgG1 Fc
sequence. In some embodiments, the Fc region comprises a human IgG1 Fc
sequence. In
some embodiments, the Fc region comprises a mouse IgG1 Fc sequence.
[0269] In some embodiments, there is provided a full-length anti-E7MC antibody
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising a heavy chain
variable domain
comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237
and a
light chain variable domain comprising the amino acid sequence of any one of
SEQ ID NOs:
36-56 and 238-243; and b) an Fc region. In some embodiments, the Fc region
comprises an
IgG1 Fc sequence. In some embodiments, the Fc region comprises a human IgG1 Fc
sequence. In some embodiments, the Fc region comprises a mouse IgG1 Fc
sequence.
[0270] In some embodiments, the full-length anti-E7MC antibody binds to a
complex
comprising an HPV16-E7 peptide and an MHC class I protein with a Kd between
about 0.1
pM to about 500 nM (such as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM,
500 pM,
1 nM, 10 nM, 50 nM, 100 nM, or 500 nM, including any ranges between these
values). In
some embodiments, the full-length anti-E7MC antibody binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein with a Kd between about 1 pM to
about 250
pM (such as about any of 1, 10, 25, 50, 75, 100, 150, 200, or 250 pM,
including any ranges
between these values).
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Multi-Specific anti-E7MC molecules
[0271] The anti-E7MC constructs in some embodiments comprise a multi-specific
anti-
E7MC molecule comprising an anti-E7MC antibody moiety and a second binding
moiety
(such as a second antigen-binding moiety). In some embodiments, the multi-
specific anti-
E7MC molecule comprises an anti-E7MC antibody moiety and a second antigen-
binding
moiety.
[0272] Multi-specific molecules are molecules that have binding specificities
for at least
two different antigens or epitopes (e.g., bispecific antibodies have binding
specificities for
two antigens or epitopes). Multi-specific molecules with more than two
valencies and/or
specificities are also contemplated. For example, trispecific antibodies can
be prepared. Tutt
et al. J. Immunol. 147: 60 (1991). It is to be appreciated that one of skill
in the art could select
appropriate features of individual multi-specific molecules described herein
to combine with
one another to form a multi-specific anti-E7MC molecule of the invention.
[0273] Thus, for example, in some embodiments, there is provided a multi-
specific (e.g.,
bispecific) anti-E7MC molecule comprising a) an anti-E7MC antibody moiety that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, and b) a second binding moiety (such as an antigen-binding moiety).
In some
embodiments, the second binding moiety specifically binds to a complex
comprising a
different HPV16-E7 peptide bound to the MHC class I protein. In some
embodiments, the
second scFv specifically binds to a complex comprising the HPV16-E7 peptide
bound to a
different MHC class I protein. In some embodiments, the second binding moiety
specifically
binds to a different epitope on the complex comprising the HPV16-E7 peptide
and the MHC
class I protein. In some embodiments, the second binding moiety specifically
binds to a
different antigen. In some embodiments, the second binding moiety specifically
binds to an
antigen on the surface of a cell, such as a cytotoxic cell. In some
embodiments, the second
binding moiety specifically binds to an antigen on the surface of a
lymphocyte, such as a T
cell, an NK cell, a neutrophil, a monocyte, a macrophage, or a dendritic cell.
In some
embodiments, the second binding moiety specifically binds to an effector T
cell, such as a
cytotoxic T cell (also known as cytotoxic T lymphocyte (CTL) or T killer
cell).
[0274] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein, and b) a second antigen-
binding moiety
that binds specifically to CD3. In some embodiments, the second antigen-
binding moiety
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specifically binds to CD3E. In some embodiments, the second antigen-binding
moiety
specifically binds to an agonistic epitope of CD3E. The term "agonistic
epitope", as used
herein, means (a) an epitope that, upon binding of the multi-specific
molecule, optionally
upon binding of several multi-specific molecules on the same cell, allows said
multi-specific
molecules to activate TCR signaling and induce T cell activation, and/or (b)
an epitope that is
solely composed of amino acid residues of the epsilon chain of CD3 and is
accessible for
binding by the multi-specific molecule, when presented in its natural context
on T cells (i.e.
surrounded by the TCR, the CD3y chain, etc.), and/or (c) an epitope that, upon
binding of the
multi-specific molecule, does not lead to stabilization of the spatial
position of CD3E relative
to CD3y.
[0275] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein, and b) a second antigen-
binding moiety
that binds specifically to an antigen on the surface of an effector cell,
including for example
CD3y, CD36, CD3E, CD3c CD28, CD16a, CD56, CD68, and GDS2D.
[0276] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein, and b) a second antigen-
binding moiety
that binds specifically to a component of the complement system, such as Clq.
C lq is a
subunit of the Cl enzyme complex that activates the serum complement system.
[0277] In some embodiments, the second antigen-binding moiety specifically
binds to an
Fc receptor. In some embodiments, the second antigen-binding moiety
specifically binds to
an Fcy receptor (FcyR). The FcyR may be an FcyRIII present on the surface of
natural killer
(NK) cells or one of FcyRI, FcyRIIA, FcyRIIBI, FcyRIIB2, and FcyRIIIB present
on the
surface of macrophages, monocytes, neutrophils and/or dendritic cells. In some
embodiments,
the second antigen-binding moiety is an Fc region or functional fragment
thereof. A
"functional fragment" as used in this context refers to a fragment of an
antibody Fc region
that is still capable of binding to an FcR, in particular to an FcyR, with
sufficient specificity
and affinity to allow an FcyR bearing effector cell, in particular a
macrophage, a monocyte, a
neutrophil and/or a dendritic cell, to kill the target cell by cytotoxic lysis
or phagocytosis. A
functional Fc fragment is capable of competitively inhibiting the binding of
the original, full-
length Fc portion to an FcR such as the activating FcyRI. In some embodiments,
a functional
Fc fragment retains at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of its
affinity to
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an activating FcyR. In some embodiments, the Fc region or functional fragment
thereof is an
enhanced Fc region or functional fragment thereof. The term "enhanced Fc
region", as used
herein, refers to an Fc region that is modified to enhance Fc receptor-
mediated effector-
functions, in particular antibody-dependent cell-mediated cytotoxicity (ADCC),
complement-
dependent cytotoxicity (CDC), and antibody-mediated phagocytosis. This can be
achieved as
known in the art, for example by altering the Fc region in a way that leads to
an increased
affinity for an activating receptor (e.g. FcyRIIIA (CD16A) expressed on
natural killer (NK)
cells) and/or a decreased binding to an inhibitory receptor (e.g. FcyRIIB1/B2
(CD32B)). In
yet other embodiments, the second antigen-binding moiety is an antibody or
antigen-binding
fragment thereof that specifically binds to an FcR, in particular to an FcyR,
with sufficient
specificity and affinity to allow an FcyR bearing effector cell, in particular
a macrophage, a
monocyte, a neutrophil and/or a dendritic cell, to kill the target cell by
cytotoxic lysis or
phagocytosis.
[0278] In some embodiments, the multi-specific anti-E7MC molecule allows
killing of
E7MC-presenting target cells and/or can effectively redirect CTLs to lyse E7MC-
presenting
target cells. In some embodiments, the multi-specific (e.g., bispecific) anti-
E7MC molecule
of the present invention shows an in vitro EC50 ranging from 10 to 500 ng/ml,
and is able to
induce redirected lysis of about 50% of the target cells through CTLs at a
ratio of CTLs to
target cells of from about 1:1 to about 50:1 (such as from about 1:1 to about
15:1, or from
about 2:1 to about 10:1).
[0279] In some embodiments, the multi-specific (e.g., bispecific) anti-E7MC
molecule is
capable of cross-linking a stimulated or unstimulated CTL and the target cell
in such a way
that the target cell is lysed. This offers the advantage that no generation of
target-specific T
cell clones or common antigen presentation by dendritic cells is required for
the multi-
specific anti-E7MC molecule to exert its desired activity. In some
embodiments, the multi-
specific anti-E7MC molecule of the present invention is capable of redirecting
CTLs to lyse
the target cells in the absence of other activating signals. In some
embodiments, the second
antigen-binding moiety of the multi-specific anti-E7MC molecule specifically
binds to CD3
(e.g., specifically binds to CDR), and signaling through CD28 and/or IL-2 is
not required for
redirecting CTLs to lyse the target cells.
[0280] Methods for measuring the preference of the multi-specific anti-E7MC
molecule to
simultaneously bind to two antigens (e.g., antigens on two different cells)
are within the
normal capabilities of a person skilled in the art. For example, when the
second binding
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moiety specifically binds to CD3, the multi-specific anti-E7MC molecule may be
contacted
with a mixture of CD3 /HPV16-ET cells and CD37HPV16-E7+ cells. The number of
multi-
specific anti-E7MC molecule-positive single cells and the number of cells
cross-linked by
multi-specific anti-E7MC molecules may then be assessed by microscopy or
fluorescence-
activated cell sorting (FACS) as known in the art.
[0281] For example, in some embodiments, there is provided a multi-specific
anti-E7MC
molecule comprising a) an anti-E7MC antibody moiety that specifically binds to
a complex
comprising an HPV16-E7 peptide and an MHC class I protein, and b) a second
antigen-
binding moiety. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19
(SEQ ID
NO: 4). In some embodiments, the MHC class I protein is HLA-A02. In some
embodiments,
the MHC class I protein is HLA-A*02:01. In some embodiments, the second
antigen-binding
moiety specifically binds to a complex comprising a different HPV16-E7 peptide
bound to
the MHC class I protein. In some embodiments, the second antigen-binding
moiety
specifically binds to a complex comprising the HPV16-E7 peptide bound to a
different MHC
class I protein. In some embodiments, the second antigen-binding moiety
specifically binds to
a different epitope on the complex comprising the HPV16-E7 peptide and the MHC
class I
protein. In some embodiments, the second antigen-binding moiety specifically
binds to
another antigen. In some embodiments, the second antigen-binding moiety
specifically binds
to an antigen on the surface of a cell, such as an E7MC-presenting cell. In
some
embodiments, the second antigen-binding moiety specifically binds to an
antigen on the
surface of a cell that does not express HPV16-E7. In some embodiments, the
second antigen-
binding moiety specifically binds to an antigen on the surface of a cytotoxic
cell. In some
embodiments, the second antigen-binding moiety specifically binds to an
antigen on the
surface of a lymphocyte, such as a T cell, an NK cell, a neutrophil, a
monocyte, a
macrophage, or a dendritic cell. In some embodiments, the second antigen-
binding moiety
specifically binds to an antigen on the surface of an effector T cell, such as
a cytotoxic T cell.
In some embodiments, the second antigen-binding moiety specifically binds to
an antigen on
the surface of an effector cell, including for example CD3y, CD36, CD3c, CD3c
CD28,
CD16a, CD56, CD68, and GDS2D. In some embodiments, the anti-E7MC antibody
moiety is
human, humanized, or semi-synthetic. In some embodiments, the second antigen-
binding
moiety is an antibody moiety. In some embodiments, the second antigen-binding
moiety is a
human, humanized, or semi-synthetic antibody moiety. In some embodiments, the
multi-
specific anti-E7MC molecule further comprises at least one (such as at least
about any of 2, 3,
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4, 5, or more) additional antigen-binding moieties. In some embodiments, the
anti-E7MC
antibody moiety cross-reacts with at least one (such as at least any of 2, 3,
4, 5, or 6) complex
comprising the MHC class I protein and a variant of the HPV16-E7 peptide
having one
amino acid substitution (such as a conservative amino acid substitution). In
some
embodiments, the anti-E7MC antibody moiety cross-reacts with at least one
(such as at least
any of 2, 3, 4, or 5) complex comprising the HPV16-E7 peptide and a different
subtype of the
MHC class I protein.
[0282] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A*02:01, and b) a second
antigen-
binding moiety.
[0283] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 183, or a variant thereof comprising up to about 3 (for example about any
of 1, 2, or 3)
amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ
ID NO:
184 or 185, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or 3)
amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of
any one
of SEQ ID NOs: 186-188, or a variant thereof comprising up to about 3 (for
example about
any of 1, 2, or 3) amino acid substitutions; and ii) a light chain variable
domain comprising
an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, or a
variant
thereof comprising up to about 3 (for example about any of 1, 2, or 3) amino
acid
substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:
191, or
a variant thereof comprising up to about 3 (for example about any of 1, 2, or
3) amino acid
substitutions, and b) a second antigen-binding moiety.
[0284] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 183, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or
185, and
an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 186-
188; and
ii) a light chain variable domain comprising an LC-CDR1 comprising the amino
acid
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sequence of SEQ ID NO: 189 or 190, and an LC-CDR3 comprising the amino acid
sequence
of SEQ ID NO: 191, and b) a second antigen-binding moiety.
[0285] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain comprising an HC-CDR1 comprising the amino acid sequence of any one of
SEQ ID
NOs: 57-77, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of
any one of
SEQ ID NOs: 78-98, or a variant thereof comprising up to about 5 (such as
about any of 1,2,
3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid
sequence of
any one of SEQ ID NOs: 99-119, 244, and 245, or a variant thereof comprising
up to about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a
light chain variable
domain comprising an LC-CDR1 comprising the amino acid sequence of any one of
SEQ ID
NOs: 120-140 and 246, or a variant thereof comprising up to about 5 (such as
about any of 1,
2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid
sequence of
any one of SEQ ID NOs: 141-161, or a variant thereof comprising up to about 3
(such as
about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising
the amino acid
sequence of any one of SEQ ID NOs: 162-182 and 247-250, or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions; and b) a second
antigen-binding moiety.
[0286] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
any one
of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one
of
SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any
one of
SEQ ID NOs: 99-119, 244, and 245; or a variant thereof comprising up to about
5 (such as
about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR
sequences; and ii) a
light chain variable domain sequence comprising an LC-CDR1 comprising the
amino acid
sequence of any one of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising the
amino
acid sequence of any one of SEQ ID NOs: 141-161; and an LC-CDR3 comprising the
amino
acid sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a variant
thereof
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comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
LC-CDR sequences; and b) a second antigen-binding moiety.
[0287] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
any one
of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one
of
SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any
one of
SEQ ID NOs: 99-119, 244, and 245; and ii) a light chain variable domain
sequence
comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID
NOs:
120-140 and 246; an LC-CDR2 comprising the amino acid sequence of any one of
SEQ ID
NOs: 141-161; and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 162-182 and 247-250; and b) a second antigen-binding moiety.
[0288] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety comprising a heavy chain variable
domain
comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-
237, or a
variant thereof having at least about 95% (for example at least about any of
96%, 97%, 98%,
or 99%) sequence identity, and a light chain variable domain comprising the
amino acid
sequence of any one of SEQ ID NOs: 36-56 and 238-243, or a variant thereof
having at least
about 95% (including for example at least about any of 96%, 97%, 98%, or 99%)
sequence
identity; and b) a second scFv.
[0289] In some embodiments, there is provided a multi-specific anti-E7MC
molecule
comprising a) an anti-E7MC antibody moiety comprising a heavy chain variable
domain
comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237
and a
light chain variable domain comprising the amino acid sequence of any one of
SEQ ID NOs:
36-56 and 238-243; and b) a second antigen-binding moiety.
[0290] In some embodiments, the multi-specific anti-E7MC molecule is, for
example, a
diabody (Db), a single-chain diabody (scDb), a tandem scDb (Tandab), a linear
dimeric scDb
(LD-scDb), a circular dimeric scDb (CD-scDb), a di-diabody, a tandem scFv, a
tandem di-
scFv (e.g., a bispecific T cell engager), a tandem tri-scFv, a tri(a)body, a
bispecific Fab2, a
di-miniantibody, a tetrabody, an scFv-Fc-scFv fusion, a dual-affinity
retargeting (DART)
antibody, a dual variable domain (DVD) antibody, an IgG-scFab, an scFab-ds-
scFv, an Fv2-
Fc, an IgG-scFv fusion, a dock and lock (DNL) antibody, a knob-into-hole (KiH)
antibody
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(bispecific IgG prepared by the KiH technology), a DuoBody (bispecific IgG
prepared by the
Duobody technology), a heteromultimeric antibody, or a heteroconjugate
antibody. In some
embodiments, the multi-specific anti-E7MC molecule is a tandem scFv (e.g., a
tandem di-
scFv, such as a bispecific T cell engager).
Tandem scFv
[0291] The multi-specific anti-E7MC molecule in some embodiments is a tandem
scFv
comprising a first scFv comprising an anti-E7MC antibody moiety and a second
scFv (also
referred to herein as a "tandem scFv multi-specific anti-E7MC antibody"). In
some
embodiments, the tandem scFv multi-specific anti-E7MC antibody further
comprises at least
one (such as at least about any of 2, 3, 4, 5, or more) additional scFv.
[0292] In some embodiments, there is provided a tandem scFv multi-specific
(e.g.,
bispecific) anti-E7MC antibody comprising a) a first scFv that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) a
second
scFv. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO:
4). In
some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the
MHC
class I protein is HLA-A*02:01. In some embodiments, the second scFv
specifically binds to
a complex comprising a different HPV16-E7 peptide bound to the MHC class I
protein. In
some embodiments, the second scFv specifically binds to a complex comprising
the HPV16-
E7 peptide bound to a different MHC class I protein. In some embodiments, the
second scFv
specifically binds to a different epitope on the complex comprising the HPV16-
E7 peptide
and the MHC class I protein. In some embodiments, the second scFv specifically
binds to
another antigen. In some embodiments, the second scFv specifically binds to an
antigen on
the surface of a cell, such as an E7MC-presenting cell. In some embodiments,
the second
scFv specifically binds to an antigen on the surface of a cell that does not
express HPV16-E7.
In some embodiments, the second scFv specifically binds to an antigen on the
surface of a
cytotoxic cell. In some embodiments, the second scFv specifically binds to an
antigen on the
surface of a lymphocyte, such as a T cell, an NK cell, a neutrophil, a
monocyte, a
macrophage, or a dendritic cell. In some embodiments, the second scFv
specifically binds to
an antigen on the surface of an effector T cell, such as a cytotoxic T cell.
In some
embodiments, the second scFv specifically binds to an antigen on the surface
of an effector
cell, including for example CD3y, CD36, CD3c, CD3c CD28, CD16a, CD56, CD68,
and
GDS2D. In some embodiments, the first scFv is human, humanized, or semi-
synthetic. In
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some embodiments, the second scFv is human, humanized, or semi-synthetic. In
some
embodiments, both the first scFv and the second scFv are human, humanized, or
semi-
synthetic. In some embodiments, the tandem scFv multi-specific anti-E7MC
antibody further
comprises at least one (such as at least about any of 2, 3, 4, 5, or more)
additional scFv. In
some embodiments, the anti-E7MC antibody moiety cross-reacts with at least one
(such as at
least any of 2, 3, 4, 5, or 6) complex comprising the MHC class I protein and
a variant of the
HPV16-E7 peptide having one amino acid substitution (such as a conservative
amino acid
substitution). In some embodiments, the anti-E7MC antibody moiety cross-reacts
with at
least one (such as at least any of 2, 3, 4, or 5) complex comprising the HPV16-
E7 peptide and
a different subtype of the MHC class I protein.
[0293] In some embodiments, there is provided a tandem scFv multi-specific
(e.g.,
bispecific) anti-E7MC antibody comprising a) a first scFv that specifically
binds to a
complex comprising an HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A*02:01,
and b)
a second scFv.
[0294] In some embodiments, there is provided a tandem scFv multi-specific
(e.g.,
bispecific) anti-E7MC antibody comprising a) a first scFv that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of SEQ ID NO: 183, or a variant thereof comprising up to about 3 (for
example
about any of 1, 2, or 3) amino acid substitutions, an HC-CDR2 comprising the
amino acid
sequence of SEQ ID NO: 184 or 185, or a variant thereof comprising up to about
3 (for
example about any of 1, 2, or 3) amino acid substitutions, and an HC-CDR3
comprising the
amino acid sequence of any one of SEQ ID NOs: 186-188, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
ii) a light chain
variable domain comprising an LC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 189 or 190, or a variant thereof comprising up to about 3 (for example
about any of 1, 2,
or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid
sequence of SEQ
ID NO: 191, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or
3) amino acid substitutions, and b) a second scFv.
[0295] In some embodiments, there is provided a tandem scFv multi-specific
(e.g.,
bispecific) anti-E7MC antibody comprising a) a first scFv that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
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sequence of SEQ ID NO: 183, an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 184 or 185, and an HC-CDR3 comprising the amino acid sequence of any one
of SEQ
ID NOs: 186-188; and ii) a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 189 or 190, and an LC-CDR3 comprising
the amino
acid sequence of SEQ ID NO: 191, and b) a second scFv.
[0296] In some embodiments, there is provided a tandem scFv multi-specific
(e.g.,
bispecific) anti-E7MC antibody comprising a) a first scFv that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain comprising an HC-CDR1 comprising the amino acid sequence
of any
one of SEQ ID NOs: 57-77, or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino
acid
sequence of any one of SEQ ID NOs: 78-98, or a variant thereof comprising up
to about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-
CDR3 comprising
the amino acid sequence of any one of SEQ ID NOs: 99-119, 244, and 245, or a
variant
thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino
acid
substitutions; and ii) a light chain variable domain comprising an LC-CDR1
comprising the
amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions, an
LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 141-161,
or a
variant thereof comprising up to about 3 (such as about any of 1, 2, or 3)
amino acid
substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 162-182 and 247-250, or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions; and b) a second scFv.
[0297] In some embodiments, there is provided a tandem scFv multi-specific
(e.g.,
bispecific) anti-E7MC antibody comprising a) a first scFv that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid
sequence of any one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino
acid
sequence of any one of SEQ ID NOs: 99-119, 244, and 245; or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions
in the HC-CDR
sequences; and ii) a light chain variable domain sequence comprising an LC-
CDR1
comprising the amino acid sequence of any one of SEQ ID NOs: 120-140 and 246;
an LC-
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CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 141-161; and
an LC-
CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 162-182 and
247-
250; or a variant thereof comprising up to about 5 (such as about any of 1,2,
3,4, or 5) amino
acid substitutions in the LC-CDR sequences; and b) a second scFv.
[0298] In some embodiments, there is provided a tandem scFv multi-specific
(e.g.,
bispecific) anti-E7MC antibody comprising a) a first scFv that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid
sequence of any one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino
acid
sequence of any one of SEQ ID NOs: 99-119, 244, and 245; and ii) a light chain
variable
domain sequence comprising an LC-CDR1 comprising the amino acid sequence of
any one
of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising the amino acid sequence
of any
one of SEQ ID NOs: 141-161; and an LC-CDR3 comprising the amino acid sequence
of any
one of SEQ ID NOs: 162-182 and 247-250; and b) a second scFv.
[0299] In some embodiments, there is provided a tandem scFv multi-specific
(e.g.,
bispecific) anti-E7MC antibody comprising a) a first scFv comprising a heavy
chain variable
domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and
233-237,
or a variant thereof having at least about 95% (for example at least about any
of 96%, 97%,
98%, or 99%) sequence identity, and a light chain variable domain comprising
the amino acid
sequence of any one of SEQ ID NOs: 36-56 and 238-243, or a variant thereof
having at least
about 95% (including for example at least about any of 96%, 97%, 98%, or 99%)
sequence
identity; and b) a second scFv.
[0300] In some embodiments, there is provided a tandem scFv multi-specific
(e.g.,
bispecific) anti-E7MC antibody comprising a) a first scFv comprising a heavy
chain variable
domain comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and
233-237
and a light chain variable domain comprising the amino acid sequence of any
one of SEQ ID
NOs: 36-56 and 238-243; and b) a second scFv.
[0301] In some embodiments, there is provided a tandem scFv multi-specific
(e.g.,
bispecific) anti-E7MC antibody comprising a) a first scFv that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) a
second
scFv, wherein the tandem scFv multi-specific anti-E7MC antibody is a tandem di-
scFv or a
tandem tri-scFv. In some embodiments, the tandem scFv multi-specific anti-E7MC
antibody
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is a tandem di-scFv. In some embodiments, the tandem scFv multi-specific anti-
E7MC
antibody is a bispecific T-cell engager.
[0302] For example, in some embodiments, there is provided a tandem di-scFv
bispecific
anti-E7MC antibody comprising a) a first scFv that specifically binds to a
complex
comprising an HPV16-E7 peptide and an MHC class I protein, and b) a second
scFv that
specifically binds to an antigen on the surface of a T cell. In some
embodiments, the HPV16-
E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC
class I
protein is HLA-A02. In some embodiments, the MHC class I protein is HLA-
A*02:01. In
some embodiments, the second scFv specifically binds to an antigen on the
surface of an
effector T cell, such as a cytotoxic T cell. In some embodiments, the second
scFv specifically
binds to an antigen selected, for example, from the group consisting of CD3y,
CD36, CD3c,
CD3c CD28, 0X40, GITR, CD137, CD27, CD4OL, and HVEM. In some embodiments, the
second scFv specifically binds to an agonistic epitope on an antigen on the
surface of a T cell,
wherein the binding of the second scFv to the antigen enhances T cell
activation. In some
embodiments, the first scFv is human, humanized, or semi-synthetic. In some
embodiments,
the second scFv is human, humanized, or semi-synthetic. In some embodiments,
both the first
scFv and the second scFv are human, humanized, or semi-synthetic.
[0303] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A*02:01, and b) a second scFv
that
specifically binds to an antigen on the surface of a T cell.
[0304] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID
NO: 183,
or a variant thereof comprising up to about 3 (for example about any of 1, 2,
or 3) amino acid
substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184
or 185,
or a variant thereof comprising up to about 3 (for example about any of 1, 2,
or 3) amino acid
substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 186-188, or a variant thereof comprising up to about 3 (for example about
any of 1, 2,
or 3) amino acid substitutions; and ii) a light chain variable domain
comprising an LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 189 or 190, or a variant
thereof
comprising up to about 3 (for example about any of 1, 2, or 3) amino acid
substitutions, and
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an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or a variant
thereof
comprising up to about 3 (for example about any of 1, 2, or 3) amino acid
substitutions, and
b) a second scFv that specifically binds to an antigen on the surface of a T
cell.
[0305] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID
NO: 183,
an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, and an
HC-
CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 186-188; and
ii) a
light chain variable domain comprising an LC-CDR1 comprising the amino acid
sequence of
SEQ ID NO: 189 or 190, and an LC-CDR3 comprising the amino acid sequence of
SEQ ID
NO: 191, and b) a second scFv that specifically binds to an antigen on the
surface of a T cell.
[0306] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable domain
comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID
NOs:
57-77, or a variant thereof comprising up to about 5 (such as about any of
1,2, 3,4, or 5)
amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any
one of
SEQ ID NOs: 78-98, or a variant thereof comprising up to about 5 (such as
about any of 1,2,
3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid
sequence of
any one of SEQ ID NOs: 99-119, 244, and 245, or a variant thereof comprising
up to about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a
light chain variable
domain comprising an LC-CDR1 comprising the amino acid sequence of any one of
SEQ ID
NOs: 120-140 and 246, or a variant thereof comprising up to about 5 (such as
about any of 1,
2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid
sequence of
any one of SEQ ID NOs: 141-161, or a variant thereof comprising up to about 3
(such as
about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising
the amino acid
sequence of any one of SEQ ID NOs: 162-182 and 247-250, or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions; and b) a second
scFv that specifically binds to an antigen on the surface of a T cell.
[0307] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable domain
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sequence comprising an HC-CDR1 comprising the amino acid sequence of any one
of SEQ
ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one of SEQ
ID
NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 99-119, 244, and 245; or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequences; and ii)
a light chain
variable domain sequence comprising an LC-CDR1 comprising the amino acid
sequence of
any one of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising the amino acid
sequence of any one of SEQ ID NOs: 141-161; and an LC-CDR3 comprising the
amino acid
sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions
in the LC-CDR
sequences, and b) a second scFv that specifically binds to an antigen on the
surface of a T
cell.
[0308] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of any one
of SEQ
ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one of SEQ
ID
NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 99-119, 244, and 245; and ii) a light chain variable domain sequence
comprising an
LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 120-140
and
246; an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs:
141-161;
and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:
162-182
and 247-250; and b) a second scFv that specifically binds to an antigen on the
surface of a T
cell.
[0309] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv comprising a heavy chain variable domain
comprising the
amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or a variant
thereof
having at least about 95% (for example at least about any of 96%, 97%, 98%, or
99%)
sequence identity, and a light chain variable domain comprising the amino acid
sequence of
any one of SEQ ID NOs: 36-56 and 238-243, or a variant thereof having at least
about 95%
(for example at least about any of 96%, 97%, 98%, or 99%) sequence identity,
and b) a
second scFv that specifically binds to an antigen on the surface of a T cell.
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[0310] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv comprising a heavy chain variable domain
comprising the
amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237 and a light
chain
variable domain comprising the amino acid sequence of any one of SEQ ID NOs:
36-56 and
238-243, and b) a second scFv that specifically binds to an antigen on the
surface of a T cell.
[0311] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein, and b) a second scFv that
specifically binds
to CD3E. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID
NO: 4).
In some embodiments, the MHC class I protein is HLA-A02. In some embodiments,
the
MHC class I protein is HLA-A*02:01. In some embodiments, the first scFv is
fused to the
second scFv through linkage with a peptide linker. In some embodiments, the
peptide linker
is between about 5 to about 20 (such as about any of 5, 10, 15, or 20,
including any ranges
between these values) amino acids in length. In some embodiments, the peptide
linker
comprises (and in some embodiments consists of) the amino acid sequence GGGGS.
In some
embodiments, the first scFv is human, humanized, or semi-synthetic. In some
embodiments,
the second scFv is human, humanized, or semi-synthetic. In some embodiments,
both the first
scFv and the second scFv are human, humanized, or semi-synthetic.
[0312] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A*02:01, and b) a second scFv
that
specifically binds to CD3E. In some embodiments, the first scFv is fused to
the second scFv
through linkage with a peptide linker. In some embodiments, the peptide linker
is between
about 5 to about 20 (such as about any of 5, 10, 15, or 20, including any
ranges between these
values) amino acids in length. In some embodiments, the peptide linker
comprises (and in
some embodiments consists of) the amino acid sequence GGGGS. In some
embodiments, the
first scFv is human, humanized, or semi-synthetic. In some embodiments, the
second scFv is
human, humanized, or semi-synthetic. In some embodiments, both the first scFv
and the
second scFv are human, humanized, or semi-synthetic.
[0313] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID
NO: 183,
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or a variant thereof comprising up to about 3 (for example about any of 1, 2,
or 3) amino acid
substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184
or 185,
or a variant thereof comprising up to about 3 (for example about any of 1, 2,
or 3) amino acid
substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 186-188, or a variant thereof comprising up to about 3 (for example about
any of 1, 2,
or 3) amino acid substitutions; and ii) a light chain variable domain
comprising an LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 189 or 190, or a variant
thereof
comprising up to about 3 (for example about any of 1, 2, or 3) amino acid
substitutions, and
an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or a variant
thereof
comprising up to about 3 (for example about any of 1, 2, or 3) amino acid
substitutions, and
b) a second scFv that specifically binds to CD3E. In some embodiments, the
first scFv is
fused to the second scFv through linkage with a peptide linker. In some
embodiments, the
peptide linker is between about 5 to about 20 (such as about any of 5, 10, 15,
or 20, including
any ranges between these values) amino acids in length. In some embodiments,
the peptide
linker comprises (and in some embodiments consists of) the amino acid sequence
GGGGS. In
some embodiments, the first scFv is human, humanized, or semi-synthetic. In
some
embodiments, the second scFv is human, humanized, or semi-synthetic. In some
embodiments, both the first scFv and the second scFv are human, humanized, or
semi-
synthetic.
[0314] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID
NO: 183,
an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, and an
HC-
CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 186-188; and
ii) a
light chain variable domain comprising an LC-CDR1 comprising the amino acid
sequence of
SEQ ID NO: 189 or 190, and an LC-CDR3 comprising the amino acid sequence of
SEQ ID
NO: 191, and b) a second scFv that specifically binds to CD3E. In some
embodiments, the
first scFv is fused to the second scFv through linkage with a peptide linker.
In some
embodiments, the peptide linker is between about 5 to about 20 (such as about
any of 5, 10,
15, or 20, including any ranges between these values) amino acids in length.
In some
embodiments, the peptide linker comprises (and in some embodiments consists
of) the amino
acid sequence GGGGS. In some embodiments, the first scFv is human, humanized,
or semi-
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synthetic. In some embodiments, the second scFv is human, humanized, or semi-
synthetic. In
some embodiments, both the first scFv and the second scFv are human,
humanized, or semi-
synthetic.
[0315] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable domain
comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID
NOs:
57-77, or a variant thereof comprising up to about 5 (such as about any of
1,2, 3,4, or 5)
amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any
one of
SEQ ID NOs: 78-98, or a variant thereof comprising up to about 5 (such as
about any of 1,2,
3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid
sequence of
any one of SEQ ID NOs: 99-119, 244, and 245, or a variant thereof comprising
up to about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a
light chain variable
domain comprising an LC-CDR1 comprising the amino acid sequence of any one of
SEQ ID
NOs: 120-140 and 246, or a variant thereof comprising up to about 5 (such as
about any of 1,
2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid
sequence of
any one of SEQ ID NOs: 141-161, or a variant thereof comprising up to about 3
(such as
about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising
the amino acid
sequence of any one of SEQ ID NOs: 162-182 and 247-250, or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions, and b) a second
scFv that specifically binds to CD3E. In some embodiments, the first scFv is
fused to the
second scFv through linkage with a peptide linker. In some embodiments, the
peptide linker
is between about 5 to about 20 (such as about any of 5, 10, 15, or 20,
including any ranges
between these values) amino acids in length. In some embodiments, the peptide
linker
comprises (and in some embodiments consists of) the amino acid sequence GGGGS.
In some
embodiments, the first scFv is human, humanized, or semi-synthetic. In some
embodiments,
the second scFv is human, humanized, or semi-synthetic. In some embodiments,
both the first
scFv and the second scFv are human, humanized, or semi-synthetic.
[0316] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of any one
of SEQ
ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one of SEQ
ID
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NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 99-119, 244, and 245; or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequences; and ii)
a light chain
variable domain sequence comprising an LC-CDR1 comprising the amino acid
sequence of
any one of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising the amino acid
sequence of any one of SEQ ID NOs: 141-161; and an LC-CDR3 comprising the
amino acid
sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions
in the LC-CDR
sequences, and b) a second scFv that specifically binds to CD3E. In some
embodiments, the
first scFv is fused to the second scFv through linkage with a peptide linker.
In some
embodiments, the peptide linker is between about 5 to about 20 (such as about
any of 5, 10,
15, or 20, including any ranges between these values) amino acids in length.
In some
embodiments, the peptide linker comprises (and in some embodiments consists
of) the amino
acid sequence GGGGS. In some embodiments, the first scFv is human, humanized,
or semi-
synthetic. In some embodiments, the second scFv is human, humanized, or semi-
synthetic. In
some embodiments, both the first scFv and the second scFv are human,
humanized, or semi-
synthetic.
[0317] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv that specifically binds to a complex
comprising an
HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of any one
of SEQ
ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one of SEQ
ID
NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 99-119, 244, and 245; and ii) a light chain variable domain sequence
comprising an
LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 120-140
and
246; an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs:
141-161;
and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:
162-182
and 247-250; and b) a second scFv that specifically binds to CD3E. In some
embodiments, the
first scFv is fused to the second scFv through linkage with a peptide linker.
In some
embodiments, the peptide linker is between about 5 to about 20 (such as about
any of 5, 10,
15, or 20, including any ranges between these values) amino acids in length.
In some
embodiments, the peptide linker comprises (and in some embodiments consists
of) the amino
acid sequence GGGGS. In some embodiments, the first scFv is human, humanized,
or semi-
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synthetic. In some embodiments, the second scFv is human, humanized, or semi-
synthetic. In
some embodiments, both the first scFv and the second scFv are human,
humanized, or semi-
synthetic.
[0318] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv comprising a heavy chain variable domain
comprising the
amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237, or a variant
thereof
having at least about 95% (for example at least about any of 96%, 97%, 98%, or
99%)
sequence identity, and a light chain variable domain comprising the amino acid
sequence of
any one of SEQ ID NOs: 36-56 and 238-243, or a variant thereof having at least
about 95%
(for example at least about any of 96%, 97%, 98%, or 99%) sequence identity,
and b) a
second scFv that specifically binds to CD3E. In some embodiments, the first
scFv is fused to
the second scFv through linkage with a peptide linker. In some embodiments,
the peptide
linker is between about 5 to about 20 (such as about any of 5, 10, 15, or 20,
including any
ranges between these values) amino acids in length. In some embodiments, the
peptide linker
comprises (and in some embodiments consists of) the amino acid sequence GGGGS.
In some
embodiments, the first scFv is human, humanized, or semi-synthetic. In some
embodiments,
the second scFv is human, humanized, or semi-synthetic. In some embodiments,
both the first
scFv and the second scFv are human, humanized, or semi-synthetic.
[0319] In some embodiments, there is provided a tandem di-scFv bispecific anti-
E7MC
antibody comprising a) a first scFv comprising a heavy chain variable domain
comprising the
amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237 and a light
chain
variable domain comprising the amino acid sequence of any one of SEQ ID NOs:
36-56 and
238-243, and b) a second scFv that specifically binds to CD3E. In some
embodiments, the
first scFv is fused to the second scFv through linkage with a peptide linker.
In some
embodiments, the peptide linker is between about 5 to about 20 (such as about
any of 5, 10,
15, or 20, including any ranges between these values) amino acids in length.
In some
embodiments, the peptide linker comprises (and in some embodiments consists
of) the amino
acid sequence GGGGS. In some embodiments, the first scFv is human, humanized,
or semi-
synthetic. In some embodiments, the second scFv is human, humanized, or semi-
synthetic. In
some embodiments, both the first scFv and the second scFv are human,
humanized, or semi-
synthetic.
[0320] In some embodiments, the tandem di-scFv bispecific anti-E7MC antibody
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein with a Kd
between
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about 0.1 pM to about 500 nM (such as about any of 0.1 pM, 1.0 pM, 10 pM, 50
pM, 100
pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM, including any ranges
between these
values). In some embodiments, the tandem di-scFv bispecific anti-E7MC antibody
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein with a Kd
between
about 1 nM to about 500 nM (such as about any of 1, 10, 25, 50, 75, 100, 150,
200, 250, 300,
350, 400, 450, or 500 nM, including any ranges between these values).
Chimeric Antigen Receptor (CAR) and CAR effector cells
[0321] The anti-E7MC construct in some embodiments is a chimeric antigen
receptor
(CAR) comprising an anti-E7MC antibody moiety (also referred to herein as an
"anti-E7MC
CAR"). Also provided is a CAR effector cell (e.g., T cell) comprising a CAR
comprising an
anti-E7MC antibody moiety (also referred to herein as an "anti-E7MC CAR
effector cell",
e.g., "anti-E7MC CAR T cell").
[0322] The anti-E7MC CAR comprises a) an extracellular domain comprising an
anti-
E7MC antibody moiety that specifically binds to a complex comprising an HPV16-
E7
peptide and an MHC class I protein and b) an intracellular signaling domain. A
transmembrane domain may be present between the extracellular domain and the
intracellular
domain.
[0323] Between the extracellular domain and the transmembrane domain of the
anti-E7MC
CAR, or between the intracellular domain and the transmembrane domain of the
anti-E7MC
CAR, there may be a spacer domain. The spacer domain can be any oligo- or
polypeptide that
functions to link the transmembrane domain to the extracellular domain or the
intracellular
domain in the polypeptide chain. A spacer domain may comprise up to about 300
amino
acids, including for example about 10 to about 100, or about 25 to about 50
amino acids.
[0324] The transmembrane domain may be derived either from a natural or from a
synthetic source. Where the source is natural, the domain may be derived from
any
membrane-bound or transmembrane protein. Transmembrane regions of particular
use in this
invention may be derived from (i.e. comprise at least the transmembrane
region(s) of) the a,
(3, 6, y, or chain of the T-cell receptor, CD28, CD3c, CD3; CD45, CD4, CD5,
CD8, CD9,
CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some
embodiments, the transmembrane domain may be synthetic, in which case it may
comprise
predominantly hydrophobic residues such as leucine and valine. In some
embodiments, a
triplet of phenylalanine, tryptophan and valine may be found at each end of a
synthetic
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transmembrane domain. In some embodiments, a short oligo- or polypeptide
linker, having a
length of, for example, between about 2 and about 10 (such as about any of 2,
3, 4, 5, 6, 7, 8,
9, or 10) amino acids in length may form the linkage between the transmembrane
domain and
the intracellular signaling domain of the anti-E7MC CAR. In some embodiments,
the linker
is a glycine-serine doublet.
[0325] In some embodiments, the transmembrane domain that naturally is
associated with
one of the sequences in the intracellular domain of the anti-E7MC CAR is used
(e.g., if an
anti-E7MC CAR intracellular domain comprises a CD28 co-stimulatory sequence,
the
transmembrane domain of the anti-E7MC CAR is derived from the CD28
transmembrane
domain). In some embodiments, the transmembrane domain can be selected or
modified by
amino acid substitution to avoid binding of such domains to the transmembrane
domains of
the same or different surface membrane proteins to minimize interactions with
other members
of the receptor complex.
[0326] The intracellular signaling domain of the anti-E7MC CAR is responsible
for
activation of at least one of the normal effector functions of the immune cell
in which the
anti-E7MC CAR has been placed in. Effector function of a T cell, for example,
may be
cytolytic activity or helper activity including the secretion of cytokines.
Thus the term
"intracellular signaling domain" refers to the portion of a protein which
transduces the
effector function signal and directs the cell to perform a specialized
function. While usually
the entire intracellular signaling domain can be employed, in many cases it is
not necessary to
use the entire chain. To the extent that a truncated portion of the
intracellular signaling
domain is used, such truncated portion may be used in place of the intact
chain as long as it
transduces the effector function signal. The term "intracellular signaling
sequence" is thus
meant to include any truncated portion of the intracellular signaling domain
sufficient to
transduce the effector function signal.
[0327] Examples of intracellular signaling domains for use in the anti-E7MC
CAR of the
invention include the cytoplasmic sequences of the T cell receptor (TCR) and
co-receptors
that act in concert to initiate signal transduction following antigen receptor
engagement, as
well as any derivative or variant of these sequences and any synthetic
sequence that has the
same functional capability.
[0328] It is known that signals generated through the TCR alone are
insufficient for full
activation of the T cell and that a secondary or co-stimulatory signal is also
required. Thus, T
cell activation can be said to be mediated by two distinct classes of
intracellular signaling
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sequence: those that initiate antigen-dependent primary activation through the
TCR (primary
signaling sequences) and those that act in an antigen-independent manner to
provide a
secondary or co-stimulatory signal (co-stimulatory signaling sequences).
[0329] Primary signaling sequences regulate primary activation of the TCR
complex either
in a stimulatory way, or in an inhibitory way. Primary signaling sequences
that act in a
stimulatory manner may contain signaling motifs which are known as
immunoreceptor
tyrosine-based activation motifs or ITAMs. The anti-E7MC CAR constructs in
some
embodiments comprise one or more ITAMs.
[0330] Examples of ITAM containing primary signaling sequences that are of
particular
use in the invention include those derived from TCR, FcRy, FcR(3, CD3y, CD3,
CD3c,
CD5, CD22, CD79a, CD79b, and CD66d.
[0331] In some embodiments, the anti-E7MC CAR comprises a primary signaling
sequence derived from CD3c For example, the intracellular signaling domain of
the CAR
can comprise the CD3 intracellular signaling sequence by itself or combined
with any other
desired intracellular signaling sequence(s) useful in the context of the anti-
E7MC CAR of the
invention. For example, the intracellular domain of the anti-E7MC CAR can
comprise a
CD3 intracellular signaling sequence and a costimulatory signaling sequence.
The
costimulatory signaling sequence can be a portion of the intracellular domain
of a
costimulatory molecule including, for example, CD27, CD28, 4-1BB (CD137),
0X40, CD30,
CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,
LIGHT,
NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.
[0332] In some embodiments, the intracellular signaling domain of the anti-
E7MC CAR
comprises the intracellular signaling sequence of CD3 and the intracellular
signaling
sequence of CD28. In some embodiments, the intracellular signaling domain of
the anti-
E7MC CAR comprises the intracellular signaling sequence of CD3 and the
intracellular
signaling sequence of 4-1BB. In some embodiments, the intracellular signaling
domain of the
anti-E7MC CAR comprises the intracellular signaling sequence of CD3 and the
intracellular
signaling sequences of CD28 and 4-1BB.
[0333] Thus, for example, in some embodiments, there is provided an anti-E7MC
CAR
comprising a) an extracellular domain comprising an anti-E7MC antibody moiety
that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, b) a transmembrane domain, and c) an intracellular signaling domain.
In some
embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some
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embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC
class I
protein is HLA-A*02:01. In some embodiments, the intracellular signaling
domain is capable
of activating an immune cell. In some embodiments, the intracellular signaling
domain
comprises a primary signaling sequence and a co-stimulatory signaling
sequence. In some
embodiments, the primary signaling sequence comprises a CD3t intracellular
signaling
sequence. In some embodiments, the co-stimulatory signaling sequence comprises
a CD28
intracellular signaling sequence. In some embodiments, the intracellular
domain comprises a
CD3 intracellular signaling sequence and a CD28 intracellular signaling
sequence. In some
embodiments, the anti-E7MC antibody moiety cross-reacts with at least one
(such as at least
any of 2, 3, 4, 5, or 6) complex comprising the MHC class I protein and a
variant of the
HPV16-E7 peptide having one amino acid substitution (such as a conservative
amino acid
substitution). In some embodiments, the anti-E7MC antibody moiety cross-reacts
with at
least one (such as at least any of 2, 3, 4, or 5) complex comprising the HPV16-
E7 peptide and
a different subtype of the MHC class I protein.
[0334] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A*02:01,
b) a
transmembrane domain, and c) an intracellular signaling domain. In some
embodiments, the
intracellular signaling domain is capable of activating an immune cell. In
some embodiments,
the intracellular signaling domain comprises a primary signaling sequence and
a co-
stimulatory signaling sequence. In some embodiments, the primary signaling
sequence
comprises a CD3 intracellular signaling sequence. In some embodiments, the co-
stimulatory
signaling sequence comprises a CD28 intracellular signaling sequence. In some
embodiments, the intracellular domain comprises a CD3 intracellular signaling
sequence and
a CD28 intracellular signaling sequence.
[0335] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of SEQ ID NO: 183, or a variant thereof comprising up to about 3 (for
example
about any of 1, 2, or 3) amino acid substitutions, an HC-CDR2 comprising the
amino acid
sequence of SEQ ID NO: 184 or 185, or a variant thereof comprising up to about
3 (for
example about any of 1, 2, or 3) amino acid substitutions, and an HC-CDR3
comprising the
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amino acid sequence of any one of SEQ ID NOs: 186-188, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
ii) a light chain
variable domain comprising an LC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 189 or 190, or a variant thereof comprising up to about 3 (for example
about any of 1, 2,
or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid
sequence of SEQ
ID NO: 191, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or
3) amino acid substitutions, b) a transmembrane domain, and c) an
intracellular signaling
domain. In some embodiments, the intracellular signaling domain is capable of
activating an
immune cell. In some embodiments, the intracellular signaling domain comprises
a primary
signaling sequence and a co-stimulatory signaling sequence. In some
embodiments, the
primary signaling sequence comprises a CD3t intracellular signaling sequence.
In some
embodiments, the co-stimulatory signaling sequence comprises a CD28
intracellular
signaling sequence. In some embodiments, the intracellular domain comprises a
CD3
intracellular signaling sequence and a CD28 intracellular signaling sequence.
[0336] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of SEQ ID NO: 183, an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 184 or 185, and an HC-CDR3 comprising the amino acid sequence of any one
of SEQ
ID NOs: 186-188; and ii) a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 189 or 190, and an LC-CDR3 comprising
the amino
acid sequence of SEQ ID NO: 191; b) an intracellular signaling domain. In some
embodiments, the intracellular signaling domain is capable of activating an
immune cell. In
some embodiments, the intracellular signaling domain comprises a primary
signaling
sequence and a co-stimulatory signaling sequence. In some embodiments, the
primary
signaling sequence comprises a CD3 intracellular signaling sequence. In some
embodiments, the co-stimulatory signaling sequence comprises a CD28
intracellular
signaling sequence. In some embodiments, the intracellular domain comprises a
CD3
intracellular signaling sequence and a CD28 intracellular signaling sequence.
[0337] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
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chain variable domain comprising an HC-CDR1 comprising the amino acid sequence
of any
one of SEQ ID NOs: 57-77, or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino
acid
sequence of any one of SEQ ID NOs: 78-98, or a variant thereof comprising up
to about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-
CDR3 comprising
the amino acid sequence of any one of SEQ ID NOs: 99-119, 244, and 245, or a
variant
thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino
acid
substitutions; and ii) a light chain variable domain comprising an LC-CDR1
comprising the
amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions, an
LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 141-161,
or a
variant thereof comprising up to about 3 (such as about any of 1, 2, or 3)
amino acid
substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 162-182 and 247-250, or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions; b) a transmembrane domain, and
c) an
intracellular signaling domain. In some embodiments, the intracellular
signaling domain is
capable of activating an immune cell. In some embodiments, the intracellular
signaling
domain comprises a primary signaling sequence and a co-stimulatory signaling
sequence. In
some embodiments, the primary signaling sequence comprises a CD3t
intracellular signaling
sequence. In some embodiments, the co-stimulatory signaling sequence comprises
a CD28
intracellular signaling sequence. In some embodiments, the intracellular
domain comprises a
CD3 intracellular signaling sequence and a CD28 intracellular signaling
sequence.
[0338] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid
sequence of any one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino
acid
sequence of any one of SEQ ID NOs: 99-119, 244, and 245; or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions
in the HC-CDR
sequences; and ii) a light chain variable domain sequence comprising an LC-
CDR1
comprising the amino acid sequence of any one of SEQ ID NOs: 120-140 and 246;
an LC-
CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 141-161; and
an LC-
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CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 162-182 and
247-
250; or a variant thereof comprising up to about 5 (such as about any of 1,2,
3,4, or 5) amino
acid substitutions in the LC-CDR sequences; b) a transmembrane domain, and c)
an
intracellular signaling domain. In some embodiments, the intracellular
signaling domain is
capable of activating an immune cell. In some embodiments, the intracellular
signaling
domain comprises a primary signaling sequence and a co-stimulatory signaling
sequence. In
some embodiments, the primary signaling sequence comprises a CD3t
intracellular signaling
sequence. In some embodiments, the co-stimulatory signaling sequence comprises
a CD28
intracellular signaling sequence. In some embodiments, the intracellular
domain comprises a
CD3 intracellular signaling sequence and a CD28 intracellular signaling
sequence.
[0339] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid
sequence of any one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino
acid
sequence of any one of SEQ ID NOs: 99-119, 244, and 245; and ii) a light chain
variable
domain sequence comprising an LC-CDR1 comprising the amino acid sequence of
any one
of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising the amino acid sequence
of any
one of SEQ ID NOs: 141-161; and an LC-CDR3 comprising the amino acid sequence
of any
one of SEQ ID NOs: 162-182 and 247-250; b) an intracellular signaling domain.
In some
embodiments, the intracellular signaling domain is capable of activating an
immune cell. In
some embodiments, the intracellular signaling domain comprises a primary
signaling
sequence and a co-stimulatory signaling sequence. In some embodiments, the
primary
signaling sequence comprises a CD3 intracellular signaling sequence. In some
embodiments, the co-stimulatory signaling sequence comprises a CD28
intracellular
signaling sequence. In some embodiments, the intracellular domain comprises a
CD3
intracellular signaling sequence and a CD28 intracellular signaling sequence.
[0340] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising a
heavy
chain variable domain comprising the amino acid sequence of any one of SEQ ID
NOs: 15-
35 and 233-237, or a variant thereof having at least about 95% (for example at
least about any
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of 96%, 97%, 98%, or 99%) sequence identity, and a light chain variable domain
comprising
the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, or a
variant thereof
having at least about 95% (including for example at least about any of 96%,
97%, 98%, or
99%) sequence identity; b) a transmembrane domain, and c) an intracellular
signaling
domain. In some embodiments, the intracellular signaling domain is capable of
activating an
immune cell. In some embodiments, the intracellular signaling domain comprises
a primary
signaling sequence and a co-stimulatory signaling sequence. In some
embodiments, the
primary signaling sequence comprises a CD3t intracellular signaling sequence.
In some
embodiments, the co-stimulatory signaling sequence comprises a CD28
intracellular
signaling sequence. In some embodiments, the intracellular domain comprises a
CD3
intracellular signaling sequence and a CD28 intracellular signaling sequence.
[0341] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising a
heavy
chain variable domain comprising the amino acid sequence of any one of SEQ ID
NOs: 15-
35 and 233-237 and a light chain variable domain comprising the amino acid
sequence of any
one of SEQ ID NOs: 36-56 and 238-243; b) an intracellular signaling domain. In
some
embodiments, the intracellular signaling domain is capable of activating an
immune cell. In
some embodiments, the intracellular signaling domain comprises a primary
signaling
sequence and a co-stimulatory signaling sequence. In some embodiments, the
primary
signaling sequence comprises a CD3 intracellular signaling sequence. In some
embodiments, the co-stimulatory signaling sequence comprises a CD28
intracellular
signaling sequence. In some embodiments, the intracellular domain comprises a
CD3
intracellular signaling sequence and a CD28 intracellular signaling sequence.
[0342] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein, b) a
transmembrane
domain, and c) an intracellular signaling domain comprising a CD3
intracellular signaling
sequence and a CD28 intracellular signaling sequence. In some embodiments, the
HPV16-E7
peptide is HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I
protein
is HLA-A02. In some embodiments, the MHC class I protein is HLA-A*02:01.
[0343] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
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complex comprising an HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A*02:01,
b) a
transmembrane domain, and c) an intracellular signaling domain comprising a
CD3
intracellular signaling sequence and a CD28 intracellular signaling sequence.
[0344] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of SEQ ID NO: 183, or a variant thereof comprising up to about 3 (for
example
about any of 1, 2, or 3) amino acid substitutions, an HC-CDR2 comprising the
amino acid
sequence of SEQ ID NO: 184 or 185, or a variant thereof comprising up to about
3 (for
example about any of 1, 2, or 3) amino acid substitutions, and an HC-CDR3
comprising the
amino acid sequence of any one of SEQ ID NOs: 186-188, or a variant thereof
comprising up
to about 3 (for example about any of 1, 2, or 3) amino acid substitutions; and
ii) a light chain
variable domain comprising an LC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 189 or 190, or a variant thereof comprising up to about 3 (for example
about any of 1, 2,
or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid
sequence of SEQ
ID NO: 191, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or
3) amino acid substitutions, b) a transmembrane domain, and c) an
intracellular signaling
domain comprising a CD3 intracellular signaling sequence and a CD28
intracellular
signaling sequence.
[0345] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of SEQ ID NO: 183, an HC-CDR2 comprising the amino acid sequence of
SEQ ID
NO: 184 or 185, and an HC-CDR3 comprising the amino acid sequence of any one
of SEQ
ID NOs: 186-188; and ii) a light chain variable domain comprising an LC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 189 or 190, and an LC-CDR3 comprising
the amino
acid sequence of SEQ ID NO: 191, b) a transmembrane domain, and c) an
intracellular
signaling domain comprising a CD3t intracellular signaling sequence and a CD28
intracellular signaling sequence.
[0346] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
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complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain comprising an HC-CDR1 comprising the amino acid sequence
of any
one of SEQ ID NOs: 57-77, or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino
acid
sequence of any one of SEQ ID NOs: 78-98, or a variant thereof comprising up
to about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-
CDR3 comprising
the amino acid sequence of any one of SEQ ID NOs: 99-119, 244, and 245, or a
variant
thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino
acid
substitutions; and ii) a light chain variable domain comprising an LC-CDR1
comprising the
amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions, an
LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 141-161,
or a
variant thereof comprising up to about 3 (such as about any of 1, 2, or 3)
amino acid
substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 162-182 and 247-250, or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions; b) a transmembrane domain, and
c) an
intracellular signaling domain comprising a CD3 intracellular signaling
sequence and a
CD28 intracellular signaling sequence.
[0347] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid
sequence of any one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino
acid
sequence of any one of SEQ ID NOs: 99-119, 244, and 245; or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions
in the HC-CDR
sequences; and ii) a light chain variable domain sequence comprising an LC-
CDR1
comprising the amino acid sequence of any one of SEQ ID NOs: 120-140 and 246;
an LC-
CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 141-161; and
an LC-
CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 162-182 and
247-
250; or a variant thereof comprising up to about 5 (such as about any of 1,2,
3,4, or 5) amino
acid substitutions in the LC-CDR sequences; b) a transmembrane domain, and c)
an
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intracellular signaling domain comprising a CD3 intracellular signaling
sequence and a
CD28 intracellular signaling sequence.
[0348] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain sequence comprising an HC-CDR1 comprising the amino acid
sequence of any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid
sequence of any one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino
acid
sequence of any one of SEQ ID NOs: 99-119, 244, and 245; and ii) a light chain
variable
domain sequence comprising an LC-CDR1 comprising the amino acid sequence of
any one
of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising the amino acid sequence
of any
one of SEQ ID NOs: 141-161; and an LC-CDR3 comprising the amino acid sequence
of any
one of SEQ ID NOs: 162-182 and 247-250; b) a transmembrane domain, and c) an
intracellular signaling domain comprising a CD3 intracellular signaling
sequence and a
CD28 intracellular signaling sequence.
[0349] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising
i) a heavy
chain variable domain comprising the amino acid sequence of any one of SEQ ID
NOs: 15-
35 and 233-237, or a variant thereof having at least about 95% (for example at
least about any
of 96%, 97%, 98%, or 99%) sequence identity, and a light chain variable domain
comprising
the amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243, or a
variant thereof
having at least about 95% (including for example at least about any of 96%,
97%, 98%, or
99%) sequence identity; b) a transmembrane domain, and c) an intracellular
signaling domain
comprising a CD3 intracellular signaling sequence and a CD28 intracellular
signaling
sequence.
[0350] In some embodiments, there is provided an anti-E7MC CAR comprising a)
an
extracellular domain comprising an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein comprising a
heavy
chain variable domain comprising the amino acid sequence of any one of SEQ ID
NOs: 15-
35 and 233-237 and a light chain variable domain comprising the amino acid
sequence of any
one of SEQ ID NOs: 36-56 and 238-243; b) a transmembrane domain, and c) an
intracellular
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signaling domain comprising a CD3t intracellular signaling sequence and a CD28
intracellular signaling sequence.
[0351] Also provided herein are effector cells (such as lymphocytes, e.g., T
cells)
expressing an anti-E7MC CAR.
[0352] Also provided is a method of producing an effector cell expressing an
anti-E7MC
CAR, the method comprising introducing a vector comprising a nucleic acid
encoding the
anti-E7MC CAR into the effector cell. In some embodiments, introducing the
vector into the
effector cell comprises transducing the effector cell with the vector. In some
embodiments,
introducing the vector into the effector cell comprises transfecting the
effector cell with the
vector. Transduction or transfection of the vector into the effector cell can
be carried about
using any method known in the art.
Immunoconjugates
[0353] The anti-E7MC constructs in some embodiments comprise an
immunoconjugate
comprising an anti-E7MC antibody moiety attached to an effector molecule (also
referred to
herein as an "anti-E7MC immunoconjugate"). In some embodiments the effector
molecule is
a therapeutic agent, such as a cancer therapeutic agent, which is either
cytotoxic, cytostatic or
otherwise provides some therapeutic benefit. In some embodiments, the effector
molecule is a
label, which can generate a detectable signal, either directly or indirectly.
[0354] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising an anti-E7MC antibody moiety and a therapeutic agent (also referred
to herein as
an "antibody-drug conjugate", or "ADC"). In some embodiments, the therapeutic
agent is a
toxin that is either cytotoxic, cytostatic or otherwise prevents or reduces
the ability of the
target cells to divide. The use of ADCs for the local delivery of cytotoxic or
cytostatic agents,
i.e., drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos
and Epenetos,
Anticancer Research 19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg.
Del. Rev.
26:151 -172 (1997); U.S. Patent No. 4,975,278) allows targeted delivery of the
drug moiety
to target cells, and intracellular accumulation therein, where systemic
administration of these
unconjugated therapeutic agents may result in unacceptable levels of toxicity
to normal cells
as well as the target cells sought to be eliminated (Baldwin et al., Lancet
(Mar. 15,
1986):603-605 (1986); Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In
Cancer
Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical
Applications,
A. Pinchera et al. (eds.), pp. 475- 506). Maximal efficacy with minimal
toxicity is sought
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thereby. Importantly, since most normal cells do not present the E7MC on their
surface, they
cannot bind the anti-E7MC immunoconjugate, and are protected from the killing
effect of the
toxin or other therapeutic agents.
[0355] Therapeutic agents used in anti-E7MC immunoconjugates include, for
example,
daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., Cancer
Immunol.
Immunother. 21:183-187 (1986)). Toxins used in anti-E7MC immunoconjugates
include
bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small
molecule toxins
such as geldanamycin (Mandler et al., J.Nat. Cancer Inst. 92(19):1573-1581
(2000); Mandler
et al., Bioorganic & Med. Chem. Letters 10:1025- 1028 (2000); Mandler et al.,
Bioconjugate
Chem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl.
Acad. Sci.
USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928
(1998);
Hinman et al., Cancer Res. 53:3336-3342 (1993)). The toxins may exert their
cytotoxic and
cytostatic effects by mechanisms including tubulin binding, DNA binding, or
topoisomerase
inhibition. Some cytotoxic drugs tend to be inactive or less active when
conjugated to large
antibodies or protein receptor ligands.
[0356] Enzymatically active toxins and fragments thereof that can be used
include, for
example, diphtheria A chain, nonbinding active fragments of diphtheria toxin,
exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A
chain,a-
sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI,
PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis
inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. See,
e.g., WO 93/21232 published October 28, 1993.
[0357] Anti-E7MC immunoconjugates of an anti-E7MC antibody moiety and one or
more
small molecule toxins, such as a calicheamicin, maytansinoids, dolastatins,
aurostatins, a
trichothecene, and CC1065, and the derivatives of these toxins that have toxin
activity, are
also contemplated herein.
[0358] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising a therapeutic agent that has an intracellular activity. In some
embodiments, the
anti-E7MC immunoconjugate is internalized and the therapeutic agent is a
cytotoxin that
blocks the protein synthesis of the cell, therein leading to cell death. In
some embodiments,
the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome-
inactivating
activity including, for example, gelonin, bouganin, saporin, ricin, ricin A
chain, bryodin,
diphtheria toxin, restrictocin, Pseudomonas exotoxin A and variants thereof.
In some
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embodiments, where the therapeutic agent is a cytotoxin comprising a
polypeptide having a
ribosome-inactivating activity, the anti-E7MC immunoconjugate must be
internalized upon
binding to the target cell in order for the protein to be cytotoxic to the
cells.
[0359] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising a therapeutic agent that acts to disrupt DNA. In some embodiments,
the
therapeutic agent that acts to disrupt DNA is, for example, selected from the
group consisting
of enediyne (e.g., calicheamicin and esperamicin) and non-enediyne small
molecule agents
(e.g., bleomycin, methidiumpropyl-EDTA-Fe(II)). Other cancer therapeutic
agents useful in
accordance with the present application include, without limitation,
daunorubicin,
doxorubicin, distamycin A, cisplatin, mitomycin C, ecteinascidins,
duocarmycin/CC-1065,
and bleomycin/pepleomycin.
[0360] The present invention further contemplates an anti-E7MC immunoconjugate
formed
between the anti-E7MC antibody moiety and a compound with nucleolytic activity
(e.g., a
ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
[0361] In some embodiments, the anti-E7MC immunoconjugate comprises an agent
that
acts to disrupt tubulin. Such agents may include, for example,
rhizoxin/maytansine,
paclitaxel, vincristine and vinblastine, colchicine, auristatin dolastatin 10
MMAE, and
peloruside A.
[0362] In some embodiments, the anti-E7MC immunoconjugate comprises an
alkylating
agent including, for example, Asaley NSC 167780, AZQ NSC 182986, BCNU NSC
409962,
Busulfan NSC 750, carboxyphthalatoplatinum NSC 271674, CBDCA NSC 241240, CCNU
NSC 79037, CHIP NSC 256927, chlorambucil NSC 3088, chlorozotocin NSC 178248,
cis-
platinum NSC 119875, clomesone NSC 338947, cyanomorpholinodoxorubicin NSC
357704,
cyclodisone NSC 348948, dianhydrogalactitol NSC 132313, fluorodopan NSC 73754,
hepsulfam NSC 329680, hycanthone NSC 142982, melphalan NSC 8806, methyl CCNU
NSC 95441 , mitomycin C NSC 26980, mitozolamide NSC 353451 , nitrogen mustard
NSC
762, PCNU NSC 95466, piperazine NSC 344007, piperazinedione NSC 135758,
pipobroman
NSC 25154, porfiromycin NSC 56410, spirohydantoin mustard NSC 172112,
teroxirone
NSC 296934, tetraplatin NSC 363812, thio-tepa NSC 6396, triethylenemelamine
NSC 9706,
uracil nitrogen mustard NSC 34462, and Yoshi-864 NSC 102627.
[0363] In some embodiments, the cancer therapeutic agent portion of the anti-
E7MC
immunoconjugate of the present application may comprise an antimitotic agent
including,
without limitation, allocolchicine NSC 406042, Halichondrin B NSC 609395,
colchicine
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NSC 757, colchicine derivative NSC 33410, dolastatin 10 NSC 376128 (NG -
auristatin
derived), maytansine NSC 153858, rhizoxin NSC 332598, taxol NSC 125973, taxol
derivative NSC 608832, thiocolchicine NSC 361792, trityl cysteine NSC 83265,
vinblastine
sulfate NSC 49842, and vincristine sulfate NSC 67574.
[0364] In some embodiments, the anti-E7MC immunoconjugate comprises a
topoisomerase
I inhibitor including, without limitation, camptothecin NSC 94600,
camptothecin, Na salt
NSC 100880, aminocamptothecin NSC 603071 , camptothecin derivative NSC 95382,
camptothecin derivative NSC 107124, camptothecin derivative NSC 643833,
camptothecin
derivative NSC 629971 , camptothecin derivative NSC 295500, camptothecin
derivative NSC
249910, camptothecin derivative NSC 606985, camptothecin derivative NSC
374028,
camptothecin derivative NSC 176323, camptothecin derivative NSC 295501 ,
camptothecin
derivative NSC 606172, camptothecin derivative NSC 606173, camptothecin
derivative NSC
610458, camptothecin derivative NSC 618939, camptothecin derivative NSC
610457,
camptothecin derivative NSC 610459, camptothecin derivative NSC 606499,
camptothecin
derivative NSC 610456, camptothecin derivative NSC 364830, camptothecin
derivative NSC
606497, and morpholinodoxorubicin NSC 354646.
[0365] In some embodiments, the anti-E7MC immunoconjugate comprises a
topoisomerase
II inhibitor including, without limitation, doxorubicin NSC 123127, amonafide
NSC 308847,
m-AMSA NSC 249992, anthrapyrazole derivative NSC 355644, pyrazoloacridine NSC
366140, bisantrene HCL NSC 337766, daunorubicin NSC 82151 , deoxydoxorubicin
NSC
267469, mitoxantrone NSC 301739, menogaril NSC 269148, N,N-dibenzyl daunomycin
NSC 268242, oxanthrazole NSC 349174, rubidazone NSC 164011 , VM-26 NSC 122819,
and VP-16 NSC 141540.
[0366] In some embodiments, the anti-E7MC immunoconjugate comprises an RNA or
DNA antimetabolite including, without limitation, L-alanosine NSC 153353, 5-
azacytidine
NSC 102816, 5-fluorouracil NSC 19893, acivicin NSC 163501 , aminopterin
derivative NSC
132483, aminopterin derivative NSC 184692, aminopterin derivative NSC 134033,
an antifol
NSC 633713, an antifol NSC 623017, Baker's soluble antifol NSC 139105,
dichlorallyl
lawsone NSC 126771 , brequinar NSC 368390, ftorafur (pro-drug) NSC 148958, 5,6-
dihydro-5-azacytidine NSC 264880, methotrexate NSC 740, methotrexate
derivative NSC
174121 , N-(phosphonoacety1)-L-aspartate (PALA) NSC 224131 , pyrazofurin NSC
143095,
trimetrexate NSC 352122, 3-HP NSC 95678, 2'-deoxy-5-fluorouridine NSC 27640, 5-
HP
NSC 107392,a-TGDR NSC 71851 , aphidicolin glycinate NSC 303812, ara-C NSC
63878, 5-
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aza-2'- deoxycytidine NSC 127716,f3-TGDR NSC 71261 , cyclocytidine NSC 145668,
guanazole NSC 1895, hydroxyurea NSC 32065, inosine glycodialdehyde NSC 118994,
macbecin Ii NSC 330500, pyrazoloimidazole NSC 51143, thioguanine NSC 752, and
thiopurine NSC 755.
[0367] In some embodiments, the anti-E7MC immunoconjugate comprises a highly
radioactive atom. A variety of radioactive isotopes are available for the
production of
radioconjugated antibodies. Examples include 211At, 1311, 125L 90y, 186Re,
188Re, 153sm, 212Bi,
32P, 212Pb and radioactive isotopes of Lu.
[0368] In some embodiments, the anti-E7MC antibody moiety can be conjugated to
a
"receptor" (such as streptavidin) for utilization in tumor pre-targeting
wherein the antibody-
receptor conjugate is administered to the patient, followed by removal of
unbound conjugate
from the circulation using a clearing agent and then administration of a
"ligand" (e.g., avidin)
that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
[0369] In some embodiments, an anti-E7MC immunoconjugate may comprise an anti-
E7MC antibody moiety conjugated to a prodrug-activating enzyme. In some such
embodiments, a prodrug-activating enzyme converts a prodrug (e.g., a peptidyl
chemotherapeutic agent, see WO 81/01145) to an active drug, such as an anti-
cancer drug.
Such anti-E7MC immunoconjugates are useful, in some embodiments, in antibody-
dependent
enzyme-mediated prodrug therapy ("ADEPT"). Enzymes that may be conjugated to
an
antibody include, but are not limited to, alkaline phosphatases, which are
useful for
converting phosphate-containing prodrugs into free drugs; arylsulfatases,
which are useful for
converting sulfate-containing prodrugs into free drugs; cytosine deaminase,
which is useful
for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-
fluorouracil; proteases,
such as serratia protease, thermolysin, subtilisin, carboxypeptidases and
cathepsins (such as
cathepsins B and L), which are useful for converting peptide-containing
prodrugs into free
drugs; D-alanylcarboxypeptidases, which are useful for converting prodrugs
that contain D-
amino acid substituents; carbohydrate-cleaving enzymes such as P-galactosidase
and
neuraminidase, which are useful for converting glycosylated prodrugs into free
drugs; f3-
lactamase, which is useful for converting drugs derivatized with f3 -lactams
into free drugs;
and penicillin amidases, such as penicillin V amidase and penicillin G
amidase, which are
useful for converting drugs derivatized at their amine nitrogens with
phenoxyacetyl or
phenylacetyl groups, respectively, into free drugs. In some embodiments,
enzymes may be
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covalently bound to antibody moieties by recombinant DNA techniques well known
in the
art. See, e.g., Neuberger et al., Nature 312:604-608 (1984).
[0370] In some embodiments, the therapeutic portion of the anti-E7MC
immunoconjugates
may be a nucleic acid. Nucleic acids that may be used include, but are not
limited to, anti-
sense RNA, genes or other polynucleotides, including nucleic acid analogs such
as
thioguanine and thiopurine.
[0371] The present application further provides anti-E7MC immunoconjugates
comprising
an anti-E7MC antibody moiety attached to an effector molecule, wherein the
effector
molecule is a label, which can generate a detectable signal, indirectly or
directly. These anti-
E7MC immunoconjugates can be used for research or diagnostic applications,
such as for the
in vivo detection of cancer. The label is preferably capable of producing,
either directly or
indirectly, a detectable signal. For example, the label may be radio-opaque or
a radioisotope,
14C, 32p, 35s, 1231, 1251 1, 131-r;
such as 3H, a fluorescent (fluorophore) or chemiluminescent
(chromophore) compound, such as fluorescein isothiocyanate, rhodamine or
luciferin; an
enzyme, such as alkaline phosphatase,f3-galactosidase or horseradish
peroxidase; an imaging
agent; or a metal ion. In some embodiments, the label is a radioactive atom
for scintigraphic
studies, for example 99Tc or 123I, or a spin label for nuclear magnetic
resonance (NMR)
imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89,
iodine-
123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,
gadolinium,
manganese or iron. Zirconium-89 may be complexed to various metal chelating
agents and
conjugated to antibodies, e.g., for PET imaging (WO 2011/056983).
[0372] In some embodiments, the anti-E7MC immunoconjugate is detectable
indirectly.
For example, a secondary antibody that is specific for the anti-E7MC
immunoconjugate and
contains a detectable label can be used to detect the anti-E7MC
immunoconjugate.
[0373] Thus, for example, in some embodiments, there is provided an anti-E7MC
immunoconjugate comprising a) an anti-E7MC antibody moiety that specifically
binds to a
complex comprising an HPV16-E7 peptide and an MHC class I protein, and b) an
effector
molecule. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID
NO:
4). In some embodiments, the MHC class I protein is HLA-A02. In some
embodiments, the
MHC class I protein is HLA-A*02:01. In some embodiments, the effector molecule
is
covalently attached to the anti-E7MC antibody moiety. In some embodiments, the
effector
molecule is a therapeutic agent selected, for example, from the group
consisting of a drug, a
toxin, a radioisotope, a protein, a peptide, and a nucleic acid. In some
embodiments, the
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effector molecular is a cancer therapeutic agent. In some embodiments, the
cancer therapeutic
agent is a chemotherapeutic. In some embodiments, the cancer therapeutic agent
is a highly
11 131
radioactive atom selected, for example, from the group consisting of - 2 At, L
125j 90y
32
186Re, 188Re, 153SM, 212 Bi, P, and 212Pb. In some embodiments, the effector
molecule is a
label that can generate a detectable signal, either directly or indirectly. In
some embodiments,
the label is a radioisotope selected, for example, from the group consisting
of 3H, 14C, 32P,
35, 1231, 1251, and 1311. In some embodiments, the anti-E7MC antibody moiety
is an scFv. In
some embodiments, the anti-E7MC antibody moiety is human, humanized, or semi-
synthetic.
In some embodiments, the anti-E7MC antibody moiety cross-reacts with at least
one (such as
at least any of 2, 3, 4, 5, or 6) complex comprising the MHC class I protein
and a variant of
the HPV16-E7 peptide having one amino acid substitution (such as a
conservative amino acid
substitution). In some embodiments, the anti-E7MC antibody moiety cross-reacts
with at
least one (such as at least any of 2, 3, 4, or 5) complex comprising the HPV16-
E7 peptide and
a different subtype of the MHC class I protein.
[0374] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 11-19 peptide (SEQ ID NO: 4) and HLA-A*02:01, and b) an effector
molecule. In some embodiments, the effector molecule is covalently attached to
the anti-
E7MC antibody moiety. In some embodiments, the effector molecule is a
therapeutic agent
selected, for example, from the group consisting of a drug, a toxin, a
radioisotope, a protein, a
peptide, and a nucleic acid. In some embodiments, the effector molecular is a
cancer
therapeutic agent. In some embodiments, the cancer therapeutic agent is a
chemotherapeutic.
In some embodiments, the cancer therapeutic agent is a highly radioactive atom
selected, for
211At, 1311, 1251, 90y, 186Re, 188Re, 1535m, 212, 32r
Bi,-.,
example, from the group consisting of and
212Pb. In some embodiments, the effector molecule is a label that can generate
a detectable
signal, either directly or indirectly. In some embodiments, the label is a
radioisotope selected,
, 32p , 35 s, 1231 , 125-%
for example, from the group consisting of 3H, 14C 1 and 131i. In some
embodiments, the anti-E7MC antibody moiety is an scFv. In some embodiments,
the anti-
E7MC antibody moiety is human, humanized, or semi-synthetic.
[0375] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
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NO: 183, or a variant thereof comprising up to about 3 (for example about any
of 1, 2, or 3)
amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ
ID NO:
184 or 185, or a variant thereof comprising up to about 3 (for example about
any of 1, 2, or 3)
amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of
any one
of SEQ ID NOs: 186-188, or a variant thereof comprising up to about 3 (for
example about
any of 1, 2, or 3) amino acid substitutions; and ii) a light chain variable
domain comprising
an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, or a
variant
thereof comprising up to about 3 (for example about any of 1, 2, or 3) amino
acid
substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:
191, or
a variant thereof comprising up to about 3 (for example about any of 1, 2, or
3) amino acid
substitutions, and b) an effector molecule.
[0376] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
SEQ ID
NO: 183, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or
185, and
an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 186-
188; and
ii) a light chain variable domain comprising an LC-CDR1 comprising the amino
acid
sequence of SEQ ID NO: 189 or 190, and an LC-CDR3 comprising the amino acid
sequence
of SEQ ID NO: 191, and b) an effector molecule.
[0377] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain comprising an HC-CDR1 comprising the amino acid sequence of any one of
SEQ ID
NOs: 57-77, or a variant thereof comprising up to about 5 (such as about any
of 1, 2, 3, 4, or
5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of
any one of
SEQ ID NOs: 78-98, or a variant thereof comprising up to about 5 (such as
about any of 1,2,
3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid
sequence of
any one of SEQ ID NOs: 99-119, 244, and 245, or a variant thereof comprising
up to about 5
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a
light chain variable
domain comprising an LC-CDR1 comprising the amino acid sequence of any one of
SEQ ID
NOs: 120-140 and 246, or a variant thereof comprising up to about 5 (such as
about any of 1,
2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid
sequence of
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any one of SEQ ID NOs: 141-161, or a variant thereof comprising up to about 3
(such as
about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising
the amino acid
sequence of any one of SEQ ID NOs: 162-182 and 247-250, or a variant thereof
comprising
up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions, and b) an
effector molecule.
[0378] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
any one
of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one
of
SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any
one of
SEQ ID NOs: 99-119, 244, and 245; or a variant thereof comprising up to about
5 (such as
about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR
sequences; and ii) a
light chain variable domain sequence comprising an LC-CDR1 comprising the
amino acid
sequence of any one of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising the
amino
acid sequence of any one of SEQ ID NOs: 141-161; and an LC-CDR3 comprising the
amino
acid sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
LC-CDR sequences, and b) an effector molecule.
[0379] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising i) a heavy chain
variable
domain sequence comprising an HC-CDR1 comprising the amino acid sequence of
any one
of SEQ ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one
of
SEQ ID NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any
one of
SEQ ID NOs: 99-119, 244, and 245; and ii) a light chain variable domain
sequence
comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID
NOs:
120-140 and 246; an LC-CDR2 comprising the amino acid sequence of any one of
SEQ ID
NOs: 141-161; and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 162-182 and 247-250, and b) an effector molecule.
[0380] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising a heavy chain
variable domain
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comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-
237, or a
variant thereof having at least about 95% (for example at least about any of
96%, 97%, 98%,
or 99%) sequence identity, and a light chain variable domain comprising the
amino acid
sequence of any one of SEQ ID NOs: 36-56 and 238-243, or a variant thereof
having at least
about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity,
and b) an effector molecule.
[0381] In some embodiments, there is provided an anti-E7MC immunoconjugate
comprising a) an anti-E7MC antibody moiety that specifically binds to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein comprising a heavy chain
variable domain
comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237
and a
light chain variable domain comprising the amino acid sequence of any one of
SEQ ID NOs:
36-56 and 238-243, b) an effector molecule.
Nucleic Acids
[0382] Nucleic acid molecules encoding the anti-E7MC constructs or anti-E7MC
antibody
moieties are also contemplated. In some embodiments, there is provided a
nucleic acid (or a
set of nucleic acids) encoding a full-length anti-E7MC antibody. In some
embodiments, there
is provided a nucleic acid (or a set of nucleic acids) encoding a multi-
specific anti-E7MC
molecule (e.g., a multi-specific anti-E7MC antibody, a bispecific anti-E7MC
antibody, or a
bispecific T-cell engager anti-E7MC antibody), or polypeptide portion thereof.
In some
embodiments, there is provided a nucleic acid (or a set of nucleic acids)
encoding an anti-
E7MC CAR. In some embodiments, there is provided a nucleic acid (or a set of
nucleic acids)
encoding an anti-E7MC immunoconjugate, or polypeptide portion thereof.
[0383] The present application also includes variants to these nucleic acid
sequences. For
example, the variants include nucleotide sequences that hybridize to the
nucleic acid
sequences encoding the anti-E7MC constructs or anti-E7MC antibody moieties of
the present
application under at least moderately stringent hybridization conditions.
[0384] The present invention also provides vectors in which a nucleic acid of
the present
invention is inserted.
[0385] In brief summary, the expression of an anti-E7MC construct (e.g., anti-
E7MC CAR)
or polypeptide portion thereof by a natural or synthetic nucleic acid encoding
the anti-E7MC
construct or polypeptide portion thereof can be achieved by inserting the
nucleic acid into an
appropriate expression vector, such that the nucleic acid is operably linked
to 5' and 3'
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regulatory elements, including for example a promoter (e.g., a lymphocyte-
specific promoter)
and a 3' untranslated region (UTR). The vectors can be suitable for
replication and
integration in eukaryotic host cells. Typical cloning and expression vectors
contain
transcription and translation terminators, initiation sequences, and promoters
useful for
regulation of the expression of the desired nucleic acid sequence.
[0386] The nucleic acids of the present invention may also be used for nucleic
acid
immunization and gene therapy, using standard gene delivery protocols. Methods
for gene
delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859,
5,589,466,
incorporated by reference herein in their entireties. In some embodiments, the
invention
provides a gene therapy vector.
[0387] The nucleic acid can be cloned into a number of types of vectors. For
example, the
nucleic acid can be cloned into a vector including, but not limited to a
plasmid, a phagemid, a
phage derivative, an animal virus, and a cosmid. Vectors of particular
interest include
expression vectors, replication vectors, probe generation vectors, and
sequencing vectors.
[0388] Further, the expression vector may be provided to a cell in the form of
a viral
vector. Viral vector technology is well known in the art and is described, for
example, in
Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor
Laboratory, New York), and in other virology and molecular biology manuals.
Viruses which
are useful as vectors include, but are not limited to, retroviruses,
adenoviruses, adeno-
associated viruses, herpes viruses, and lentiviruses. In general, a suitable
vector contains an
origin of replication functional in at least one organism, a promoter
sequence, convenient
restriction endonuclease sites, and one or more selectable markers (see, e.g.,
WO 01/96584;
WO 01/29058; and U.S. Pat. No. 6,326,193).
[0389] A number of viral based systems have been developed for gene transfer
into
mammalian cells. For example, retroviruses provide a convenient platform for
gene delivery
systems. A selected gene can be inserted into a vector and packaged in
retroviral particles
using techniques known in the art. The recombinant virus can then be isolated
and delivered
to cells of the subject either in vivo or ex vivo. A number of retroviral
systems are known in
the art. In some embodiments, adenovirus vectors are used. A number of
adenovirus vectors
are known in the art. In some embodiments, lentivirus vectors are used.
Vectors derived from
retroviruses such as the lentivirus are suitable tools to achieve long-term
gene transfer since
they allow long-term, stable integration of a transgene and its propagation in
daughter cells.
Lentiviral vectors have the added advantage over vectors derived from onco-
retroviruses such
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as murine leukemia viruses in that they can transduce non-proliferating cells,
such as
hepatocytes. They also have the added advantage of low immunogenicity.
[0390] Additional promoter elements, e.g., enhancers, regulate the frequency
of
transcriptional initiation. Typically, these are located in the region 30-110
bp upstream of the
start site, although a number of promoters have recently been shown to contain
functional
elements downstream of the start site as well. The spacing between promoter
elements
frequently is flexible, so that promoter function is preserved when elements
are inverted or
moved relative to one another. In the thymidine kinase (tk) promoter, the
spacing between
promoter elements can be increased to 50 bp apart before activity begins to
decline.
[0391] One example of a suitable promoter is the immediate early
cytomegalovirus (CMV)
promoter sequence. This promoter sequence is a strong constitutive promoter
sequence
capable of driving high levels of expression of any polynucleotide sequence
operatively
linked thereto. Another example of a suitable promoter is Elongation Growth
Factor-1a (EF-
1 a). However, other constitutive promoter sequences may also be used,
including, but not
limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor
virus
(MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR)
promoter,
MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus
immediate
early promoter, a Rous sarcoma virus promoter, as well as human gene promoters
such as,
but not limited to, the actin promoter, the myosin promoter, the hemoglobin
promoter, and
the creatine kinase promoter. Further, the invention should not be limited to
the use of
constitutive promoters. Inducible promoters are also contemplated as part of
the invention.
The use of an inducible promoter provides a molecular switch capable of
turning on
expression of the polynucleotide sequence which it is operatively linked when
such
expression is desired, or turning off the expression when expression is not
desired. Examples
of inducible promoters include, but are not limited to a metallothionine
promoter, a
glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
[0392] In order to assess the expression of a polypeptide or portions thereof,
the expression
vector to be introduced into a cell can also contain either a selectable
marker gene or a
reporter gene or both to facilitate identification and selection of expressing
cells from the
population of cells sought to be transfected or infected through viral
vectors. In other aspects,
the selectable marker may be carried on a separate piece of DNA and used in a
co-
transfection procedure. Both selectable markers and reporter genes may be
flanked with
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appropriate regulatory sequences to enable expression in the host cells.
Useful selectable
markers include, for example, antibiotic-resistance genes, such as neo and the
like.
[0393] Reporter genes are used for identifying potentially transfected cells
and for
evaluating the functionality of regulatory sequences. In general, a reporter
gene is a gene that
is not present in or expressed by the recipient organism or tissue and that
encodes a
polypeptide whose expression is manifested by some easily detectable property,
e.g.,
enzymatic activity. Expression of the reporter gene is assayed at a suitable
time after the
DNA has been introduced into the recipient cells. Suitable reporter genes may
include genes
encoding luciferase, P-galactosidase, chloramphenicol acetyl transferase,
secreted alkaline
phosphatase, or the green fluorescent protein gene (e.g., Ui-Tel et al., 2000
FEBS Letters
479: 79-82). Suitable expression systems are well known and may be prepared
using known
techniques or obtained commercially. In general, the construct with the
minimal 5' flanking
region showing the highest level of expression of reporter gene is identified
as the promoter.
Such promoter regions may be linked to a reporter gene and used to evaluate
agents for the
ability to modulate promoter-driven transcription.
[0394] Methods of introducing and expressing genes into a cell are known in
the art. In the
context of an expression vector, the vector can be readily introduced into a
host cell, e.g.,
mammalian, bacterial, yeast, or insect cell by any method in the art. For
example, the
expression vector can be transferred into a host cell by physical, chemical,
or biological
means.
[0395] Physical methods for introducing a polynucleotide into a host cell
include calcium
phosphate precipitation, lipofection, particle bombardment, microinjection,
electroporation,
and the like. Methods for producing cells comprising vectors and/or exogenous
nucleic acids
are well-known in the art. See, for example, Sambrook et al. (2001, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some
embodiments, the
introduction of a polynucleotide into a host cell is carried out by calcium
phosphate
transfection.
[0396] Biological methods for introducing a polynucleotide of interest into a
host cell
include the use of DNA and RNA vectors. Viral vectors, and especially
retroviral vectors,
have become the most widely used method for inserting genes into mammalian,
e.g., human
cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes
simplex virus 1,
adenoviruses and adeno-associated viruses, and the like. See, for example,
U.S. Pat. Nos.
5,350,674 and 5,585,362.
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[0397] Chemical means for introducing a polynucleotide into a host cell
include colloidal
dispersion systems, such as macromolecule complexes, nanocapsules,
microspheres, beads,
and lipid-based systems including oil-in-water emulsions, micelles, mixed
micelles, and
liposomes. An exemplary colloidal system for use as a delivery vehicle in
vitro and in vivo is
a liposome (e.g., an artificial membrane vesicle).
[0398] In the case where a non-viral delivery system is utilized, an exemplary
delivery
vehicle is a liposome. The use of lipid formulations is contemplated for the
introduction of
the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another
aspect, the nucleic
acid may be associated with a lipid. The nucleic acid associated with a lipid
may be
encapsulated in the aqueous interior of a liposome, interspersed within the
lipid bilayer of a
liposome, attached to a liposome via a linking molecule that is associated
with both the
liposome and the oligonucleotide, entrapped in a liposome, complexed with a
liposome,
dispersed in a solution containing a lipid, mixed with a lipid, combined with
a lipid,
contained as a suspension in a lipid, contained or complexed with a micelle,
or otherwise
associated with a lipid. Lipid, lipid/DNA or lipid/expression vector
associated compositions
are not limited to any particular structure in solution. For example, they may
be present in a
bilayer structure, as micelles, or with a "collapsed" structure. They may also
simply be
interspersed in a solution, possibly forming aggregates that are not uniform
in size or shape.
Lipids are fatty substances which may be naturally occurring or synthetic
lipids. For example,
lipids include the fatty droplets that naturally occur in the cytoplasm as
well as the class of
compounds which contain long-chain aliphatic hydrocarbons and their
derivatives, such as
fatty acids, alcohols, amines, amino alcohols, and aldehydes.
[0399] Regardless of the method used to introduce exogenous nucleic acids into
a host cell
or otherwise expose a cell to the inhibitor of the present invention, in order
to confirm the
presence of the recombinant DNA sequence in the host cell, a variety of assays
may be
performed. Such assays include, for example, "molecular biological" assays
well known to
those of skill in the art, such as Southern and Northern blotting, RT-PCR and
PCR;
"biochemical" assays, such as detecting the presence or absence of a
particular peptide, e.g.,
by immunological means (ELISAs and Western blots) or by assays described
herein to
identify agents falling within the scope of the invention.
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MHC class I proteins
[0400] MHC class I proteins are one of two primary classes of major
histocompatibility
complex (MHC) molecules (the other being MHC class II) and are found on nearly
every
nucleated cell of the body. Their function is to display fragments of proteins
from within the
cell to T cells; healthy cells will be ignored, while cells containing foreign
proteins will be
attacked by the immune system. Because MHC class I proteins present peptides
derived from
cytosolic proteins, the pathway of MHC class I presentation is often called
the cytosolic or
endogenous pathway. Class I MHC molecules bind peptides generated mainly from
degradation of cytosolic proteins by the proteasome. The MHC I:peptide complex
is then
inserted into the plasma membrane of the cell. The peptide is bound to the
extracellular part
of the class I MHC molecule. Thus, the function of the class I MHC is to
display intracellular
proteins to cytotoxic T cells (CTLs). However, class I MHC can also present
peptides
generated from exogenous proteins, in a process known as cross-presentation.
[0401] MHC class I proteins consist of two polypeptide chains, a and (32-
microglobulin
(132M). The two chains are linked noncovalently via interaction of b2m and the
a3 domain.
Only the a chain is polymorphic and encoded by a HLA gene, while the b2m
subunit is not
polymorphic and encoded by the (3-2 microglobulin gene. The a3 domain is
plasma
membrane-spanning and interacts with the CD8 co-receptor of T-cells. The a3-
CD8
interaction holds the MHC I molecule in place while the T cell receptor (TCR)
on the surface
of the cytotoxic T cell binds its al-a2 heterodimer ligand, and checks the
coupled peptide for
antigenicity. The al and a2 domains fold to make up a groove for peptides to
bind. MHC
class I proteins bind peptides that are 8-10 amino acid in length.
[0402] The human leukocyte antigen (HLA) genes are the human versions of the
MHC
genes. The three major MHC class I proteins in humans are HLA-A, HLA-B, and
HLA-C,
while the 3 minor ones are HLA-E, HLA-F, and HLA-G. HLA-A is ranked among the
genes
in humans with the fastest-evolving coding sequence. As of December 2013,
there were 2432
known HLA-A alleles coding for 1740 active proteins and 117 null proteins. The
HLA-A
gene is located on the short arm of chromosome 6 and encodes the larger, a-
chain, constituent
of HLA-A. Variation of HLA-A a-chain is key to HLA function. This variation
promotes
genetic diversity in the population. Since each HLA has a different affinity
for peptides of
certain structures, greater variety of HLAs means greater variety of antigens
to be 'presented'
on the cell surface, enhancing the likelihood that a subset of the population
will be resistant to
any given foreign invader. This decreases the likelihood that a single
pathogen has the
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capability to wipe out the entire human population. Each individual can
express up to two
types of HLA-A, one from each of their parents. Some individuals will inherit
the same
HLA-A from both parents, decreasing their individual HLA diversity; however,
the majority
of individuals will receive two different copies of HLA-A. This same pattern
follows for all
HLA groups. In other words, a person can only express either one or two of the
2432 known
HLA-A alleles.
[0403] All alleles receive at least a four digit classification, e.g., HLA-
A*02:12. The A
signifies which HLA gene the allele belongs to. There are many HLA-A alleles,
so that
classification by serotype simplifies categorization. The next pair of digits
indicates this
assignment. For example, HLA-A*02:02, HLA-A*02:04, and HLA-A*02:324 are all
members of the A2 serotype (designated by the *02 prefix). This group is the
primary factor
responsible for HLA compatibility. All numbers after this cannot be determined
by
serotyping and are designated through gene sequencing. The second set of
digits indicates
what HLA protein is produced. These are assigned in order of discovery and as
of December
2013 there are 456 different HLA-A02 proteins known (assigned names HLA-
A*02:01 to
HLA-A*02:456). The shortest possible HLA name includes both of these details.
Each
extension beyond that signifies a nucleotide change that may or may not change
the protein.
[0404] In some embodiments, the anti-E7MC antibody moiety specifically binds
to a
complex comprising an HPV16-E7 peptide and an MHC class I protein, wherein the
MHC
class I protein is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In some
embodiments, the MHC class I protein is HLA-A, HLA-B, or HLA-C. In some
embodiments,
the MHC class I protein is HLA-A. In some embodiments, the MHC class I protein
is HLA-
B. In some embodiments, the MHC class I protein is HLA-C. In some embodiments,
the
MHC class I protein is HLA-A01, HLA-A02, HLA-A03, HLA-A09, HLA-A10, HLA-All,
HLA-A19, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30,
HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68,
HLA-A69, HLA-A74, or HLA-A80. In some embodiments, the MHC class I protein is
HLA-
A02. In some embodiments, the MHC class I protein is any one of HLA-A*02:01-
555, such
as HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-
A*02:06, HLA-A*02:07, HLA-A*02:08, HLA-A*02:09, HLA-A*02:10, HLA-A*02:11,
HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:15, HLA-A*02:16, HLA-
A*02:17, HLA-A*02:18, HLA-A*02:19, HLA-A*02:20, HLA-A*02:21, HLA-A*02:22, or
HLA-A*02:24. In some embodiments, the MHC class I protein is HLA-A*02:01. HLA-
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A*02:01 is expressed in 39-46% of all Caucasians, and therefore represents a
suitable choice
of MHC class I protein for use in the present invention.
[0405] HPV16-E7 peptides suitable for use in generating anti-E7MC antibody
moieties can
be determined, for example, based on the presence of HLA-A*02:01-binding
motifs and
cleavage sites for proteasomes and immune-proteasomes using computer
prediction models
known to those of skill in the art. For predicting MHC binding sites, such
models include, but
are not limited to, IEDB (Vita et al., The immune epitope database (IEDB) 3Ø
Nucleic Acids
Res. 2014 Oct 9. pii: gku938), ProPredl (described in more detail in Singh and
Raghava,
ProPred: prediction of HIA-DR binding sites. BIOINFORMA TICS 17(12):1236-1237,
2001),
and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell
Epitope
Prediction. in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-
93, 2007).
[0406] Once appropriate peptides have been identified, peptide synthesis may
be done in
accordance with protocols well known to those of skill in the art. Because of
their relatively
small size, the peptides of the invention may be directly synthesized in
solution or on a solid
support in accordance with conventional peptide synthesis techniques. Various
automatic
synthesizers are commercially available and can be used in accordance with
known protocols.
The synthesis of peptides in solution phase has become a well-established
procedure for
large-scale production of synthetic peptides and as such is a suitable
alternative method for
preparing the peptides of the invention (See for example, Solid Phase Peptide
Synthesis by
John Morrow Stewart and Martin et al. Application of Almez-mediated Amidation
Reactions
to Solution Phase Peptide Synthesis, Tetrahedron Letters Vol. 39, pages 1517-
1520, 1998).
[0407] The binding activity of candidate HPV16-E7 peptides can be tested using
the
antigen-processing-deficient T2 cell line, which increases expression of HLA-A
when
stabilized by a peptide in the antigen-presenting groove. T2 cells are pulsed
with the
candidate peptide for a time sufficient to stabilize HLA-A expression on the
cell surface,
which can be measured using any methods known in the art, such as by
immunostaining with
a fluorescently labeled monoclonal antibody specific for HLA-A (for example,
BB7.2)
followed by fluorescence-activated cell-sorting (FACS) analysis.
Preparation of anti-E7MC antibodies and anti-E7MC antibody moieties
[0408] In some embodiments, the anti-E7MC antibody or anti-E7MC antibody
moiety is a
monoclonal antibody. Monoclonal antibodies can be prepared, e.g., using
hybridoma
methods, such as those described by Kohler and Milstein, Nature, 256:495
(1975) and
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Sergeeva et al., Blood, 117(16):4262-4272, using the phage display methods
described herein
and in the Examples below, or using recombinant DNA methods (see, e.g., US
Patent No.
4,816,567).
[0409] In a hybridoma method, a hamster, mouse, or other appropriate host
animal is
typically immunized with an immunizing agent to elicit lymphocytes that
produce or are
capable of producing antibodies that will specifically bind to the immunizing
agent.
Alternatively, the lymphocytes can be immunized in vitro. The immunizing agent
can include
a polypeptide or a fusion protein of the protein of interest, or a complex
comprising at least
two molecules, such as a complex comprising an HPV16-E7 peptide and an MHC
class I
protein. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of
human origin
are desired, or spleen cells or lymph node cells are used if non-human
mammalian sources
are desired. The lymphocytes are then fused with an immortalized cell line
using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell. See,
e.g., Goding,
Monoclonal Antibodies: Principles and Practice (New York: Academic Press,
1986), pp. 59-
103. Immortalized cell lines are usually transformed mammalian cells,
particularly myeloma
cells of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell
lines are
employed. The hybridoma cells can be cultured in a suitable culture medium
that preferably
contains one or more substances that inhibit the growth or survival of the
unfused,
immortalized cells. For example, if the parental cells lack the enzyme
hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas
typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which
prevents the growth of HGPRT-deficient cells.
[0410] In some embodiments, the immortalized cell lines fuse efficiently,
support stable
high-level expression of antibody by the selected antibody-producing cells,
and are sensitive
to a medium such as HAT medium. In some embodiments, the immortalized cell
lines are
murine myeloma lines, which can be obtained, for instance, from the Salk
Institute Cell
Distribution Center, San Diego, California and the American Type Culture
Collection,
Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines
also have
been described for the production of human monoclonal antibodies. Kozbor, J.
Immunol.,
133:3001 (1984); Brodeur et al. Monoclonal Antibody Production Techniques and
Applications (Marcel Dekker, Inc.: New York, 1987) pp. 51-63.
[0411] The culture medium in which the hybridoma cells are cultured can then
be assayed
for the presence of monoclonal antibodies directed against the polypeptide.
The binding
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specificity of monoclonal antibodies produced by the hybridoma cells can be
determined by
immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay
(RIA) or
enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are
known in
the art. The binding affinity of the monoclonal antibody can, for example, be
determined by
the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
[0412] After the desired hybridoma cells are identified, the clones can be sub
cloned by
limiting dilution procedures and grown by standard methods. Goding, supra.
Suitable culture
media for this purpose include, for example, Dulbecco's Modified Eagle's
Medium and
RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as
ascites in a
mammal.
[0413] The monoclonal antibodies secreted by the sub clones can be isolated or
purified
from the culture medium or ascites fluid by conventional immunoglobulin
purification
procedures such as, for example, protein A-Sepharose, hydroxylapatite
chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0414] The anti-E7MC antibodies or antibody moieties may also be identified by
screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a
variety of methods are known in the art for generating phage display libraries
and screening
such libraries for antibodies possessing the desired binding characteristics.
Such methods are
reviewed, e.g., in Hoogenboom et al., Methods in Molecular Biology 178:1-37
(O'Brien et
al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in
McCafferty et al.,
Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al.,
J. Mol. Biol.
222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-
175 (Lo,
ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-
310 (2004);
Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.
Acad. Sci. USA
101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-
132(2004).
[0415] In certain phage display methods, repertoires of VH and VL genes are
separately
cloned by polymerase chain reaction (PCR) and recombined randomly in phage
libraries,
which can then be screened for antigen-binding phage as described in Winter et
al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody fragments,
either as single-
chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized
sources provide
high-affinity antibodies to the immunogen without the requirement of
constructing
hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from
human) to provide a
single source of antibodies to a wide range of non-self and also self antigens
without any
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immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993).
Finally, naive
libraries can also be made synthetically by cloning unrearranged V-gene
segments from stem
cells, and using PCR primers containing random sequence to encode the highly
variable
CDR3 regions and to accomplish rearrangement in vitro, as described by
Hoogenboom and
Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing
human antibody
phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos.
2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,
2007/0237764,
2007/0292936, and 2009/0002360.
[0416] The antibodies or antigen-binding fragments thereof can be prepared
using phage
display to screen libraries for antibodies specific to a complex comprising an
HPV16-E7
peptide and an MHC class I protein. The library can be a human scFv phage
display library
having a diversity of at least one x 109 (such as at least about any of 1 x
109, 2.5 x 109, 5 x
109, 7.5 x 109, 1 x 1010, 2.5 x 1010, 5 x 1010, 7.5 x 1010, or 1 x 1011)
unique human antibody
fragments. In some embodiments, the library is a naïve human library
constructed from DNA
extracted from human PMBCs and spleens from healthy donors, encompassing all
human
heavy and light chain subfamilies. In some embodiments, the library is a naïve
human library
constructed from DNA extracted from PBMCs isolated from patients with various
diseases,
such as patients with autoimmune diseases, cancer patients, and patients with
infectious
diseases. In some embodiments, the library is a semi-synthetic human library,
wherein heavy
chain CDR3 is completely randomized, with all amino acids (with the exception
of cysteine)
equally likely to be present at any given position (see, e.g., Hoet, R.M. et
al., Nat. Biotechnol.
23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 of the semi-
synthetic
human library has a length from about 5 to about 24 (such as about any of 5,
6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) amino acids. In some
embodiments, the
library is a non-human phage display library.
[0417] Phage clones that bind to the E7MC with high affinity can be selected
by iterative
binding of phage to the E7MC, which is bound to a solid support (such as, for
example, beads
for solution panning or mammalian cells for cell panning), followed by removal
of non-
bound phage and by elution of specifically bound phage. In an example of
solution panning,
the E7MC can be biotinylated for immobilization to a solid support. The
biotinylated E7MC
is mixed with the phage library and a solid support, such as streptavidin-
conjugated
Dynabeads M-280, and then E7MC-phage-bead complexes are isolated. The bound
phage
clones are then eluted and used to infect an appropriate host cell, such as E.
coli XL1-Blue,
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for expression and purification. In an example of cell panning, T2 cells (a
TAP-deficient,
HLA-A*02:01+ lymphoblast cell line) loaded with the HPV16-E7 peptide of the
E7MC are
mixed with the phage library, after which the cells are collected and the
bound clones are
eluted and used to infect an appropriate host cell for expression and
purification. The panning
can be performed for multiple (such as about any of 2, 3, 4, 5, 6 or more)
rounds with either
solution panning, cell panning, or a combination of both, to enrich for phage
clones binding
specifically to the E7MC. Enriched phage clones can be tested for specific
binding to the
E7MC by any methods known in the art, including for example ELISA and FACS.
[0418] Monoclonal antibodies can also be made by recombinant DNA methods, such
as
those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies of
the invention can be readily isolated and sequenced using conventional
procedures (e.g., by
using oligonucleotide probes that are capable of binding specifically to genes
encoding the
heavy and light chains of murine antibodies). Hybridoma cells as described
above or E7MC-
specific phage clones of the invention can serve as a source of such DNA. Once
isolated, the
DNA can be placed into expression vectors, which are then transfected into
host cells such as
simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do
not
otherwise produce immunoglobulin protein, to obtain the synthesis of
monoclonal antibodies
in the recombinant host cells. The DNA also can be modified, for example, by
substituting
the coding sequence for human heavy- and light-chain constant domains and/or
framework
regions in place of the homologous non-human sequences (U.S. Patent No.
4,816,567;
Morrison et al., supra) or by covalently joining to the immunoglobulin coding
sequence all or
part of the coding sequence for a nonimmunoglobulin polypeptide. Such a non-
immunoglobulin polypeptide can be substituted for the constant domains of an
antibody of
the invention, or can be substituted for the variable domains of one antigen-
combining site of
an antibody of the invention to create a chimeric bivalent antibody.
[0419] The antibodies can be monovalent antibodies. Methods for preparing
monovalent
antibodies are known in the art. For example, one method involves recombinant
expression of
immunoglobulin light chain and modified heavy chain. The heavy chain is
truncated
generally at any point in the Fc region so as to prevent heavy-chain
crosslinking.
Alternatively, the relevant cysteine residues are substituted with another
amino acid residue
or are deleted so as to prevent crosslinking.
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[0420] In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of
antibodies to produce fragments thereof, particularly Fab fragments, can be
accomplished
using any method known in the art.
[0421] Antibody variable domains with the desired binding specificities
(antibody-antigen
combining sites) can be fused to immunoglobulin constant-domain sequences. The
fusion
preferably is with an immunoglobulin heavy-chain constant domain, comprising
at least part
of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy-chain
constant
region (CH1) containing the site necessary for light-chain binding is present
in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if
desired, the
immunoglobulin light chain, are inserted into separate expression vectors, and
are co-
transfected into a suitable host organism. For further details of generating
bispecific
antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210
(1986).
Human and Humanized Antibodies
[0422] The anti-E7MC antibodies or antibody moieties can be humanized
antibodies or
human antibodies. Humanized forms of non-human (e.g., murine) antibodies are
chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab,
Fab',
F(ab')2, scFv, or other antigen-binding subsequences of antibodies) that
typically contain
minimal sequence derived from non-human immunoglobulin. Humanized antibodies
include
human immunoglobulins (recipient antibody) in which residues from a CDR of the
recipient
are replaced by residues from a CDR of a non-human species (donor antibody)
such as
mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
In some instances,
Fv framework residues of the human immunoglobulin are replaced by
corresponding non-
human residues. Humanized antibodies can also comprise residues that are found
neither in
the recipient antibody nor in the imported CDR or framework sequences. In
general, the
humanized antibody can comprise substantially all of at least one, and
typically two, variable
domains, in which all or substantially all of the CDR regions correspond to
those of a non-
human immunoglobulin, and all or substantially all of the FR regions are those
of a human
immunoglobulin consensus sequence. In some embodiments, the humanized antibody
will
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a
human immunoglobulin. See, e.g., Jones et al., Nature, 321: 522-525 (1986);
Riechmann et
al., Nature, 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596
(1992).
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[0423] Generally, a humanized antibody has one or more amino acid residues
introduced
into it from a source that is non-human. These non-human amino acid residues
are often
referred to as "import" residues, which are typically taken from an "import"
variable domain.
According to some embodiments, humanization can be essentially performed
following the
method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986);
Riechmann et
al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536
(1988)), by
substituting rodent CDRs or CDR sequences for the corresponding sequences of a
human
antibody. Accordingly, such "humanized" antibodies are antibodies (U.S. Patent
No.
4,816,567), wherein substantially less than an intact human variable domain
has been
substituted by the corresponding sequence from a non-human species. In
practice, humanized
antibodies are typically human antibodies in which some CDR residues and
possibly some
FR residues are substituted by residues from analogous sites in rodent
antibodies.
[0424] As an alternative to humanization, human antibodies can be generated.
For example,
it is now possible to produce transgenic animals (e.g., mice) that are
capable, upon
immunization, of producing a full repertoire of human antibodies in the
absence of
endogenous immunoglobulin production. For example, it has been described that
the
homozygous deletion of the antibody heavy-chain joining region (JH) gene in
chimeric and
germ-line mutant mice results in complete inhibition of endogenous antibody
production.
Transfer of the human germ-line immunoglobulin gene array into such germ-line
mutant
mice will result in the production of human antibodies upon antigen challenge.
See, e.g.,
Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature,
362:255-258 (1993);
Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,806,
5,569,825,
5,591,669; 5,545,807; and WO 97/17852. Alternatively, human antibodies can be
made by
introducing human immunoglobulin loci into transgenic animals, e.g., mice in
which the
endogenous immunoglobulin genes have been partially or completely inactivated.
Upon
challenge, human antibody production is observed that closely resembles that
seen in humans
in all respects, including gene rearrangement, assembly, and antibody
repertoire. This
approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825;
5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio/Technology, 10: 779-
783 (1992);
Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813
(1994);
Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature
Biotechnology, 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13:
65-93
(1995).
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[0425] Human antibodies may also be generated by in vitro activated B cells
(see U.S.
Patents 5,567,610 and 5,229,275) or by using various techniques known in the
art, including
phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
Marks et al.,
J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et
al. are also
available for the preparation of human monoclonal antibodies. Cole et al.,
Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al.,
J. Immunol.,
147(1): 86-95 (1991).
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Multi-specific Antibodies
[0426] In some embodiments, the anti-E7MC construct is a multi-specific
antibody.
Suitable methods for making multi-specific (e.g., bispecific) antibodies are
well known in the
art. For example, the production of bispecific antibodies can based on the co-
expression of
two immunoglobulin heavy-chain/light-chain pairs, where the two pairs each
have different
specificities, and upon association result in a heterodimeric antibody (see,
e.g., Milstein and
Cuello, Nature, 305: 537-539 (1983); WO 93/08829, and Traunecker et al., EMBO
J. 10:
3655 (1991)). Because of the random assortment of immunoglobulin heavy and
light chains,
these hybridomas (quadromas) produce a potential mixture of ten different
antibody
molecules, of which only one has the correct bispecific structure. The
purification of the
correct molecule is usually accomplished by affinity chromatography steps.
Similar
procedures are disclosed in WO 93/08829 and in Traunecker et al., EMBO, 10:
3655-3659
(1991). Alternatively, the combining of heavy and light chains can be directed
by taking
advantage of species-restricted pairing (see, e.g., Lindhofer et al., J.
Immunol., 155:219-225
(1995)) and the pairing of heavy chains can be directed by use of "knob-into
hole"
engineering of CH3 domains (see, e.g., U.S. Pat. No. 5,731,168; Ridgway et
al., Protein
Eng., 9(7):617-621 (1996)). Multi-specific antibodies may also be made by
engineering
electrostatic steering effects for making antibody Fc-heterodimeric molecules
(see, e.g., WO
2009/089004A1). In yet another method, stable bispecific antibodies can be
generated by
controlled Fab-arm exchange, where two parental antibodies having distinct
antigen
specificity and matched point mutations in the CH3 domains are mixed in
reducing condition
to allow for separation, reassembly, and reoxidation to form highly pure
bispecific antibodies.
Labrigin et al., Proc. Natl. Acad. Sci., 110(13):5145-5150 (2013). Such
antibodies,
comprising a mixture of heavy-chain/light-chain pairs, are also referred to
herein as
"heteromultimeric antibodies".
[0427] Antibodies or antigen-binding fragments thereof having different
specificities can
also be chemically cross-linked to generate multi-specific heteroconjugate
antibodies. For
example, two F(ab')2 molecules, each having specificity for a different
antigen, can be
chemically linked. Pullarkat et al., Trends Biotechnol., 48:9-21 (1999). Such
antibodies have,
for example, been proposed to target immune-system cells to unwanted cells
(U.S. Patent No.
4,676,980), and for treatment of HIV infection. WO 91/00360; WO 92/200373; EP
03089. It
is contemplated that the antibodies can be prepared in vitro using known
methods in synthetic
protein chemistry, including those involving crosslinking agents. For example,
immunotoxins
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can be constructed using a disulfide-exchange reaction or by forming a
thioether bond.
Examples of suitable reagents for this purpose include iminothiolate and
methy1-4-
mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.
4,676,980.
[0428] In some embodiments, multi-specific antibodies can be prepared using
recombinant
DNA techniques. For example, a bispecific antibody can be engineered by fusing
two scFvs,
such as by fusing them through a peptide linker, resulting in a tandem scFv.
One example of
a tandem scFv is a bispecific T cell engager. Bispecific T cell engagers are
made by linking
an anti-CD3 scFv to an scFv specific for a surface antigen of a target cell,
such as a tumor-
associated antigen (TAA), resulting in the redirection of T cells to the
target cells. Mack et
al., Proc. Natl. Acad. Sci., 92:7021-7025 (1995); Brischwein et al., Mol.
Immunol.,
43(8):1129-1143 (2006). By shortening the length of a peptide linker between
two variable
domains, they can be prevented from self-assembling and forced to pair with
domains on a
second polypeptide, resulting in a compact bispecific antibody called a
diabody (Db).
Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448 (1993). The two
polypeptides of a Db
each comprise a VH connected to a VL by a linker which is too short to allow
pairing
between the two domains on the same chain. Accordingly, the VH and VL domains
of one
polypeptide are forced to pair with the complementary VL and VH domains of
another
polypeptide, thereby forming two antigen-binding sites. In a modification of
this format, the
two polypeptides are linked by another peptide linker, resulting in a single
chain diabody
(scDb). In yet another modification of the Db format, dual-affinity
retargeting (DART)
bispecific antibodies can be generated by introducing a disulfide linkage
between cysteine
residues at the C-terminus of each polypeptide, optionally including domains
prior to the C-
terminal cysteine residues that drive assembly of the desired heterodimeric
structure. Veri et
al., Arthritis Rheum., 62(7):1933-1943 (2010). Dual-variable-domain
immunoglobulins
(DVD-IgTm), in which the target-binding variable domains of two monoclonal
antibodies are
combined via naturally occurring linkers to yield a tetravalent, bispecific
antibody, are also
known in the art. Gu and Ghayur, Methods Enzymol., 502:25-41 (2012). In yet
another
format, Dock and Lock (DNL), bispecific antibodies are prepared by taking
advantage of the
dimerization of a peptide (DDD2) derived from the regulatory subunit of human
cAMP-
dependent protein kinase (PKA) with a peptide (AD2) derived from the anchoring
domains of
human A kinase anchor proteins (AKAPs). Rossi et al., Proc. Natl. Acad. Sci.,
103:6841-
6846 (2006).
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[0429] Various techniques for making and isolating bispecific antibody
fragments directly
from recombinant cell culture have also been described. For example,
bispecific antibodies
have been produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553
(1992). This method can also be utilized for the production of antibody
homodimers.
Anti-E7MC variants
[0430] In some embodiments, amino acid sequence variants of the antibody
moieties
provided herein are contemplated. For example, it may be desirable to improve
the binding
affinity and/or other biological properties of the antibody moiety. Amino acid
sequence
variants of an antibody moiety may be prepared by introducing appropriate
modifications into
the nucleotide sequence encoding the antibody moiety, or by peptide synthesis.
Such
modifications include, for example, deletions from, and/or insertions into
and/or substitutions
of residues within the amino acid sequences of the antibody moiety. Any
combination of
deletion, insertion, and substitution can be made to arrive at the final
construct, provided that
the final construct possesses the desired characteristics, e.g., antigen-
binding.
[0431] In some embodiments, antibody moiety variants having one or more amino
acid
substitutions are provided. Sites of interest for substitutional mutagenesis
include the HVRs
and FRs. Amino acid substitutions may be introduced into an antibody moiety of
interest and
the products screened for a desired activity, e.g., retained/improved antigen
binding,
decreased immunogenicity, or improved ADCC or CDC.
[0432] Conservative substitutions are shown in Table 5 below.
TABLE 5: CONSERVATIVE SUBS TITITIONS
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) Val; Leu; Ile Val
Arg (R) Lys; Gln; Asn Lys
Asn (N) Gln; His; Asp, Lys; Arg Gln
Asp (D) Glu; Asn Glu
Cys (C) Ser; Ala Ser
Gln (Q) Asn; Glu Asn
Glu (E) Asp; Gln Asp
Gly (G) Ala Ala
His (H) Asn; Gln; Lys; Arg Arg
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Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu
Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile
Lys (K) Arg; Gln; Asn Arg
Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr
Pro (P) Ala Ala
Ser (S) Thr Thr
Thr (T) Val; Ser Ser
Trp (W) Tyr; Phe Tyr
Tyr (Y) Trp; Phe; Thr; Ser Phe
Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0433] Amino acids may be grouped into different classes according to common
side-chain
properties:
a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
c. acidic: Asp, Glu;
d. basic: His, Lys, Arg;
e. residues that influence chain orientation: Gly, Pro;
f. aromatic: Trp, Tyr, Phe.
[0434] Non-conservative substitutions will entail exchanging a member of one
of these
classes for another class.
[0435] An exemplary substitutional variant is an affinity matured antibody
moiety, which
may be conveniently generated, e.g., using phage display-based affinity
maturation
techniques. Briefly, one or more CDR residues are mutated and the variant
antibody moieties
displayed on phage and screened for a particular biological activity (e.g.
binding affinity).
Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve
antibody moiety
affinity. Such alterations may be made in HVR "hotspots," i.e., residues
encoded by codons
that undergo mutation at high frequency during the somatic maturation process
(see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or specificity
determining
residues (SDRs), with the resulting variant VH or VL being tested for binding
affinity.
Affinity maturation by constructing and reselecting from secondary libraries
has been
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described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37
(O'Brien et
al., ed., Human Press, Totowa, NJ, (2001).)
[0436] In some embodiments of affinity maturation, diversity is introduced
into the
variable genes chosen for maturation by any of a variety of methods (e.g.,
error-prone PCR,
chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library
is then
created. The library is then screened to identify any antibody moiety variants
with the desired
affinity. Another method to introduce diversity involves HVR-directed
approaches, in which
several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in
antigen binding may be specifically identified, e.g., using alanine scanning
mutagenesis or
modeling. CDR-H3 and CDR-L3 in particular are often targeted.
[0437] In some embodiments, substitutions, insertions, or deletions may occur
within one
or more HVRs so long as such alterations do not substantially reduce the
ability of the
antibody moiety to bind antigen. For example, conservative alterations (e.g.,
conservative
substitutions as provided herein) that do not substantially reduce binding
affinity may be
made in HVRs. Such alterations may be outside of HVR "hotspots" or SDRs. In
some
embodiments of the variant VH and VL sequences provided above, each HVR either
is
unaltered, or contains no more than one, two or three amino acid
substitutions.
[0438] A useful method for identification of residues or regions of an
antibody moiety that
may be targeted for mutagenesis is called "alanine scanning mutagenesis" as
described by
Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue
or group of
target residues (e.g., charged residues such as arg, asp, his, lys, and glu)
are identified and
replaced by a neutral or negatively charged amino acid (e.g., alanine or
polyalanine) to
determine whether the interaction of the antibody moiety with antigen is
affected. Further
substitutions may be introduced at the amino acid locations demonstrating
functional
sensitivity to the initial substitutions. Alternatively, or additionally, a
crystal structure of an
antigen-antibody moiety complex can be determined to identify contact points
between the
antibody moiety and antigen. Such contact residues and neighboring residues
may be targeted
or eliminated as candidates for substitution. Variants may be screened to
determine whether
they contain the desired properties.
[0439] Amino acid sequence insertions include amino- and/or carboxyl-terminal
fusions
ranging in length from one residue to polypeptides containing a hundred or
more residues, as
well as intrasequence insertions of single or multiple amino acid residues.
Examples of
terminal insertions include an antibody moiety with an N-terminal methionyl
residue. Other
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insertional variants of the antibody moiety include the fusion to the N- or C-
terminus of the
antibody moiety to an enzyme (e.g. for ADEPT) or a polypeptide which increases
the serum
half-life of the antibody moiety.
Fc Region Variants
[0440] In some embodiments, one or more amino acid modifications may be
introduced
into the Fc region of a full-length anti-E7MC antibody provided herein,
thereby generating an
Fc region variant. In some embodiments, the Fc region variant has enhanced
antibody
dependent cellular cytotoxicity (ADCC) effector function, often related to
binding to Fc
receptors (FcRs). In some embodiments, the Fc region variant has decreased
ADCC effector
function. There are many examples of changes or mutations to Fc sequences that
can alter
effector function. For example, WO 00/42072 and Shields et al. J Biol. Chem.
9(2): 6591-
6604 (2001) describe antibody variants with improved or diminished binding to
FcRs. The
contents of those publications are specifically incorporated herein by
reference.
[0441] Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) is a mechanism of
action
of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune
defense
whereby an effector cell of the immune system actively lyses a target cell
(e.g., a cancer cell),
whose membrane-surface antigens have been bound by specific antibodies (e.g.,
an anti-
E7MC antibody). The typical ADCC involves activation of NK cells by
antibodies. An NK
cell expresses CD16 which is an Fc receptor. This receptor recognizes, and
binds to, the Fc
portion of an antibody bound to the surface of a target cell. The most common
Fc receptor on
the surface of an NK cell is called CD16 or FcyRIII. Binding of the Fc
receptor to the Fc
region of an antibody results in NK cell activation, release of cytolytic
granules and
consequent target cell apoptosis. The contribution of ADCC to tumor cell
killing can be
measured with a specific test that uses NK-92 cells that have been transfected
with a high-
affinity FcR. Results are compared to wild-type NK-92 cells that do not
express the FcR.
[0442] In some embodiments, the invention contemplates an anti-E7MC construct
variant
comprising an FC region that possesses some but not all effector functions,
which makes it a
desirable candidate for applications in which the half-life of the anti-E7MC
construct in vivo
is important yet certain effector functions (such as CDC and ADCC) are
unnecessary or
deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to
confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor
(FcR) binding
assays can be conducted to ensure that the antibody lacks FcyR binding (hence
likely lacking
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ADCC activity), but retains FcRn binding ability. The primary cells for
mediating ADCC,
NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and
FcyRIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page 464 of
Ravetch and
Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro
assays to
assess ADCC activity of a molecule of interest is described in U.S. Pat. No.
5,500,362 (see,
e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and
Hellstrom, Jet
al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337
(see
Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-
radioactive
assay methods may be employed (see, for example, ACTITm non-radioactive
cytotoxicity
assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and
CytoTox 96TM
non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector
cells for such
assays include peripheral blood mononuclear cells (PBMC) and Natural Killer
(NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of interest may
be assessed in
vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc.
Nat'l Acad. Sci.
USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm
that the
antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq
and C3c binding
ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a
CDC
assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol.
Methods
202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M.
S. and M. J.
Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half
life
determinations can also be performed using methods known in the art (see,
e.g., Petkova, S.
B. et al., Intl. Immunol. 18(12):1759-1769 (2006)).
[0443] Antibodies with reduced effector function include those with
substitution of one or
more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.
6,737,056).
Such Fc mutants include Fc mutants with substitutions at two or more of amino
acid positions
265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with
substitution of
residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
[0444] Certain antibody variants with improved or diminished binding to FcRs
are
described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et
al., J. Biol.
Chem. 9(2): 6591-6604 (2001).)
[0445] In some embodiments, there is provided an anti-E7MC construct (e.g., a
full-length
anti-E7MC antibody) variant comprising a variant Fc region comprising one or
more amino
acid substitutions which improve ADCC. In some embodiments, the variant Fc
region
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comprises one or more amino acid substitutions which improve ADCC, wherein the
substitutions are at positions 298, 333, and/or 334 of the variant Fc region
(EU numbering of
residues). In some embodiments, the anti-E7MC construct (e.g., full-length
anti-E7MC
antibody) variant comprises the following amino acid substitution in its
variant Fc region:
S298A, E333A, and K334A.
[0446] In some embodiments, alterations are made in the Fc region that result
in altered
(i.e., either improved or diminished) Clq binding and/or Complement Dependent
Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO
99/51642, and
Idusogie et al., J. Immunol. 164: 4178-4184 (2000).
[0447] In some embodiments, there is provided an anti-E7MC construct (e.g., a
full-length
anti-E7MC antibody) variant comprising a variant Fc region comprising one or
more amino
acid substitutions which increase half-life and/or improve binding to the
neonatal Fc receptor
(FcRn). Antibodies with increased half-lives and improved binding to FcRn are
described in
U52005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with
one or
more substitutions therein which improve binding of the Fc region to FcRn.
Such Fc variants
include those with substitutions at one or more of Fc region residues: 238,
256, 265, 272,
286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382,
413, 424 or 434,
e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).
[0448] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.
5,648,260;
U.S. Pat. No. 5,624,821; and WO 94/29351 concerning other examples of Fc
region variants.
[0449] Anti-E7MC constructs (such as full-length anti-E7MC antibodies)
comprising any
of the Fc variants described herein, or combinations thereof, are
contemplated.
Glycosylation Variants
[0450] In some embodiments, an anti-E7MC construct provided herein is altered
to
increase or decrease the extent to which the anti-E7MC construct is
glycosylated. Addition or
deletion of glycosylation sites to an anti-E7MC construct may be conveniently
accomplished
by altering the amino acid sequence of the anti-E7MC construct or polypeptide
portion
thereof such that one or more glycosylation sites is created or removed.
[0451] Where the anti-E7MC construct comprises an Fc region, the carbohydrate
attached
thereto may be altered. Native antibodies produced by mammalian cells
typically comprise a
branched, biantennary oligosaccharide that is generally attached by an N-
linkage to Asn297
of the CH2 domain of the Fc region. See, e.g., Wright et al., TIB TECH 15:26-
32 (1997). The
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oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl
glucosamine
(G1cNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc
in the "stem"
of the biantennary oligosaccharide structure. In some embodiments,
modifications of the
oligosaccharide in an anti-E7MC construct of the invention may be made in
order to create
anti-E7MC construct variants with certain improved properties.
[0452] In some embodiments, anti-E7MC construct (such as full-length anti-E7MC
antibody) variants are provided comprising an Fc region wherein a carbohydrate
structure
attached to the Fc region has reduced fucose or lacks fucose, which may
improve ADCC
function. Specifically, anti-E7MC constructs are contemplated herein that have
reduced
fusose relative to the amount of fucose on the same anti-E7MC construct
produced in a wild-
type CHO cell. That is, they are characterized by having a lower amount of
fucose than they
would otherwise have if produced by native CHO cells (e.g., a CHO cell that
produce a
native glycosylation pattern, such as, a CHO cell containing a native FUT8
gene). In some
embodiments, the anti-E7MC construct is one wherein less than about 50%, 40%,
30%, 20%,
10%, or 5% of the N-linked glycans thereon comprise fucose. For example, the
amount of
fucose in such an anti-E7MC construct may be from 1% to 80%, from 1% to 65%,
from 5%
to 65% or from 20% to 40%. In some embodiments, the anti-E7MC construct is one
wherein
none of the N-linked glycans thereon comprise fucose, i.e., wherein the anti-
E7MC construct
is completely without fucose, or has no fucose or is afucosylated. The amount
of fucose is
determined by calculating the average amount of fucose within the sugar chain
at Asn297,
relative to the sum of all glycostructures attached to Asn 297 (e. g. complex,
hybrid and high
mannose structures) as measured by MALDI-TOF mass spectrometry, as described
in WO
2008/077546, for example. Asn297 refers to the asparagine residue located at
about position
297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may
also be
located about 3 amino acids upstream or downstream of position 297, i.e.,
between positions
294 and 300, due to minor sequence variations in antibodies. Such fucosylation
variants may
have improved ADCC function. See, e.g., US Patent Publication Nos. US
2003/0157108
(Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of
publications
related to "defucosylated" or "fucose-deficient" antibody variants include: US
2003/0157108;
WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US
2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US
2004/0109865;
WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778;
W02005/053742; W02002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249
(2004);
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Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines
capable of
producing defucosylated antibodies include Lec13 CHO cells deficient in
protein
fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat
Appl No US
2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at
Example
11), and knockout cell lines, such asa-1,6-fucosyltransferase gene, FUT8,
knockout CHO
cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al.,
Biotechnol. Bioeng., 94(4):680-688 (2006); and W02003/085107).
[0453] Anti-E7MC construct (such as full-length anti-E7MC antibody) variants
are further
provided with bisected oligosaccharides, e.g., in which a biantennary
oligosaccharide
attached to the Fc region of the anti-E7MC construct is bisected by GlcNAc.
Such anti-
E7MC construct (such as full-length anti-E7MC antibody) variants may have
reduced
fucosylation and/or improved ADCC function. Examples of such antibody variants
are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No.
6,602,684 (Umana et
al.); US 2005/0123546 (Umana et al.), and Ferrara et al., Biotechnology and
Bioengineering,
93(5): 851-861 (2006). Anti-E7MC construct (such as full-length anti-E7MC
antibody)
variants with at least one galactose residue in the oligosaccharide attached
to the Fc region
are also provided. Such anti-E7MC construct variants may have improved CDC
function.
Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.);
WO 1998/58964
(Raju, S.); and WO 1999/22764 (Raju, S.).
[0454] In some embodiments, the anti-E7MC construct (such as full-length anti-
E7MC
antibody) variants comprising an Fc region are capable of binding to an
FcyRIII. In some
embodiments, the anti-E7MC construct (such as full-length anti-E7MC antibody)
variants
comprising an Fc region have ADCC activity in the presence of human effector
cells or have
increased ADCC activity in the presence of human effector cells compared to
the otherwise
same anti-E7MC construct (such as full-length anti-E7MC antibody) comprising a
human
wild-type IgGlFc region.
Cysteine Engineered Variants
[0455] In some embodiments, it may be desirable to create cysteine engineered
anti-E7MC
constructs (such as full-length anti-E7MC antibodies) in which one or more
amino acid
residues are substituted with cysteine residues. In some embodiments, the
substituted residues
occur at accessible sites of the anti-E7MC construct. By substituting those
residues with
cysteine, reactive thiol groups are thereby positioned at accessible sites of
the anti-E7MC
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construct and may be used to conjugate the anti-E7MC construct to other
moieties, such as
drug moieties or linker-drug moieties, to create an anti-E7MC immunoconjugate,
as
described further herein. Cysteine engineered anti-E7MC constructs (such as
full-length anti-
E7MC antibodies) may be generated as described, e.g., in U.S. Pat. No.
7,521,541.
Derivatives
[0456] In some embodiments, an anti-E7MC construct provided herein may be
further
modified to contain additional nonproteinaceous moieties that are known in the
art and
readily available. The moieties suitable for derivatization of the anti-E7MC
construct include
but are not limited to water soluble polymers. Non-limiting examples of water
soluble
polymers include, but are not limited to, polyethylene glycol (PEG),
copolymers of ethylene
glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol,
polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic
anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and dextran or
poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl
alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have
advantages in
manufacturing due to its stability in water. The polymer may be of any
molecular weight, and
may be branched or unbranched. The number of polymers attached to the anti-
E7MC
construct may vary, and if more than one polymer are attached, they can be the
same or
different molecules. In general, the number and/or type of polymers used for
derivatization
can be determined based on considerations including, but not limited to, the
particular
properties or functions of the anti-E7MC construct to be improved, whether the
anti-E7MC
construct derivative will be used in a therapy under defined conditions, etc.
[0457] In some embodiments, conjugates of an anti-E7MC construct and
nonproteinaceous
moiety that may be selectively heated by exposure to radiation are provided.
In some
embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al.,
Proc. Natl.
Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any
wavelength, and
includes, but is not limited to, wavelengths that do not harm ordinary cells,
but which heat the
nonproteinaceous moiety to a temperature at which cells proximal to the anti-
E7MC
construct-nonproteinaceous moiety are killed.
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CAR Effector Cell Preparation
[0458] The present invention in one aspect provides effector cells (such as
lymphocytes,
for example T cells) expressing an anti-E7MC CAR. Exemplary methods of
preparing
effector cells (such as T cells) expressing the anti-E7MC CARs (anti-E7MC CAR
effector
cells, such as anti-E7MC CAR T cells) are provided herein.
[0459] In some embodiments, an anti-E7MC CAR effector cell (such as T cell)
can be
generated by introducing a vector (including for example a lentiviral vector)
comprising an
anti-E7MC CAR (for example a CAR comprising an anti-E7MC antibody moiety and
CD28
and CD3 intracellular signaling sequences) into the effector cell (such as T
cell). In some
embodiments, the anti-E7MC CAR effector cells (such as T cells) of the
invention are able to
replicate in vivo, resulting in long-term persistence that can lead to
sustained control of an
HPV16-E7-positive disease (such as cancer, e.g., squamous cell carcinoma,
cervical cancer,
or anal cancer).
[0460] In some embodiments, the invention relates to administering a
genetically modified
T cell expressing an anti-E7MC CAR for the treatment of a patient having an
HPV16-E7-
positive disease or at risk of having an HPV16-E7-positive disease using
lymphocyte
infusion. In some embodiments, autologous lymphocyte infusion is used in the
treatment.
Autologous PBMCs are collected from a patient in need of treatment and T cells
are activated
and expanded using the methods described herein and known in the art and then
infused back
into the patient.
[0461] In some embodiments, the anti-E7MC CAR T cell expresses an anti-E7MC
CAR
comprising an anti-E7MC antibody moiety (also referred to herein as an "anti-
E7MC CAR T
cell"). In some embodiments, the anti-E7MC CAR T cell expresses an anti-E7MC
CAR
comprising an extracellular domain comprising an anti-E7MC antibody moiety and
an
intracellular domain comprising intracellular signaling sequences of CD3 and
CD28. The
anti-E7MC CAR T cells of the invention can undergo robust in vivo T cell
expansion and can
establish E7MC-specific memory cells that persist at high levels for an
extended amount of
time in blood and bone marrow. In some embodiments, the anti-E7MC CAR T cells
of the
invention infused into a patient can eliminate E7MC-presenting cells, such as
E7MC-
presenting cancer cells, in vivo in patients having an HPV16-E7-positive
disease. In some
embodiments, the anti-E7MC CAR T cells of the invention infused into a patient
can
eliminate E7MC-presenting cells, such as E7MC-presenting cancer cells, in vivo
in patients
having an HPV16-E7-positive disease that is refractory to at least one
conventional treatment.
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[0462] Prior to expansion and genetic modification of the T cells, a source of
T cells is
obtained from a subject. T cells can be obtained from a number of sources,
including
peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord
blood, thymus
tissue, tissue from a site of infection, ascites, pleural effusion, spleen
tissue, and tumors. In
some embodiments of the present invention, any number of T cell lines
available in the art
may be used. In some embodiments of the present invention, T cells can be
obtained from a
unit of blood collected from a subject using any number of techniques known to
the skilled
artisan, such as FicollTM separation. In some embodiments, cells from the
circulating blood of
an individual are obtained by apheresis. The apheresis product typically
contains
lymphocytes, including T cells, monocytes, granulocytes, B cells, other
nucleated white
blood cells, red blood cells, and platelets. In some embodiments, the cells
collected by
apheresis may be washed to remove the plasma fraction and to place the cells
in an
appropriate buffer or media for subsequent processing steps. In some
embodiments, the cells
are washed with phosphate buffered saline (PBS). In some embodiments, the wash
solution
lacks calcium and may lack magnesium or may lack many if not all divalent
cations. As those
of ordinary skill in the art would readily appreciate a washing step may be
accomplished by
methods known to those in the art, such as by using a semi-automated "flow-
through"
centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or
the
Haemonetics Cell Saver 5) according to the manufacturer's instructions. After
washing, the
cells may be resuspended in a variety of biocompatible buffers, such as Ca2 -
free, Mg2 -free
PBS, PlasmaLyte A, or other saline solutions with or without buffer.
Alternatively, the
undesirable components of the apheresis sample may be removed and the cells
directly
resuspended in culture media.
[0463] In some embodiments, T cells are isolated from peripheral blood
lymphocytes by
lysing the red blood cells and depleting the monocytes, for example, by
centrifugation
through a PERCOLLTM gradient or by counterflow centrifugal elutriation. A
specific
subpopulation of T cells, such as CD3+, CD28 , CD4+, CD8+, CD45RA , and
CD45R0+ T
cells, can be further isolated by positive or negative selection techniques.
For example, in
some embodiments, T cells are isolated by incubation with anti-CD3/anti-CD28
(i.e., 3x28)-
conjugated beads, such as DYNABEADS M-450 CD3/CD28 T, for a time period
sufficient
for positive selection of the desired T cells. In some embodiments, the time
period is about 30
minutes. In some embodiments, the time period ranges from 30 minutes to 36
hours or longer
and all integer values there between. In some embodiments, the time period is
at least one, 2,
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3, 4, 5, or 6 hours. In some embodiments, the time period is 10 to 24 hours.
In some
embodiments, the incubation time period is 24 hours. For isolation of T cells
from patients
with leukemia, use of longer incubation times, such as 24 hours, can increase
cell yield.
Longer incubation times may be used to isolate T cells in any situation where
there are few T
cells as compared to other cell types, such as in isolating tumor infiltrating
lymphocytes
(TIL) from tumor tissue or from immune-compromised individuals. Further, use
of longer
incubation times can increase the efficiency of capture of CD8+ T cells. Thus,
by simply
shortening or lengthening the time T cells are allowed to bind to the CD3/CD28
beads and/or
by increasing or decreasing the ratio of beads to T cells, subpopulations of T
cells can be
preferentially selected for or against at culture initiation or at other time
points during the
process. Additionally, by increasing or decreasing the ratio of anti-CD3
and/or anti-CD28
antibodies on the beads or other surface, subpopulations of T cells can be
preferentially
selected for or against at culture initiation or at other desired time points.
The skilled artisan
would recognize that multiple rounds of selection can also be used in the
context of this
invention. In some embodiments, it may be desirable to perform the selection
procedure and
use the "unselected" cells in the activation and expansion process.
"Unselected" cells can also
be subjected to further rounds of selection.
[0464] Enrichment of a T cell population by negative selection can be
accomplished with a
combination of antibodies directed to surface markers unique to the negatively
selected cells.
One method is cell sorting and/or selection via negative magnetic
immunoadherence or flow
cytometry that uses a cocktail of monoclonal antibodies directed to cell
surface markers
present on the cells negatively selected. For example, to enrich for CD4+
cells by negative
selection, a monoclonal antibody cocktail typically includes antibodies to CD
14, CD20,
CD11b, CD 16, HLA-DR, and CD8. In some embodiments, it may be desirable to
enrich for
or positively select for regulatory T cells which typically express CD4+, CD25
, CD62Lhi,
GITR , and FoxP3 . Alternatively, in some embodiments, T regulatory cells are
depleted by
anti-CD25 conjugated beads or other similar methods of selection.
[0465] For isolation of a desired population of cells by positive or negative
selection, the
concentration of cells and surface (e.g., particles such as beads) can be
varied. In some
embodiments, it may be desirable to significantly decrease the volume in which
beads and
cells are mixed together (i.e., increase the concentration of cells), to
ensure maximum contact
of cells and beads. For example, in some embodiments, a concentration of about
2 billion
cells/ml is used. In some embodiments, a concentration of about 1 billion
cells/ml is used. In
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some embodiments, greater than about 100 million cells/ml is used. In some
embodiments, a
concentration of cells of about any of 10, 15, 20, 25, 30, 35, 40, 45, or 50
million cells/ml is
used. In some embodiments, a concentration of cells of about any of 75, 80,
85, 90, 95, or
100 million cells/ml is used. In some embodiments, a concentration of about
125 or about
150 million cells/ml is used. Using high concentrations can result in
increased cell yield, cell
activation, and cell expansion. Further, use of high cell concentrations
allows more efficient
capture of cells that may weakly express target antigens of interest, such as
CD28-negative T
cells, or from samples where there are many tumor cells present (i.e.,
leukemic blood, tumor
tissue, etc.). Such populations of cells may have therapeutic value and would
be desirable to
obtain. For example, using high concentration of cells allows more efficient
selection of
CD8+ T cells that normally have weaker CD28 expression.
[0466] In some embodiments of the present invention, T cells are obtained from
a patient
directly following treatment. In this regard, it has been observed that
following certain cancer
treatments, in particular treatments with drugs that damage the immune system,
shortly after
treatment during the period when patients would normally be recovering from
the treatment,
the quality of T cells obtained may be optimal or improved for their ability
to expand ex vivo.
Likewise, following ex vivo manipulation using the methods described herein,
these cells may
be in a preferred state for enhanced engraftment and in vivo expansion. Thus,
it is
contemplated within the context of the present invention to collect blood
cells, including T
cells, dendritic cells, or other cells of the hematopoietic lineage, during
this recovery phase.
Further, in some embodiments, mobilization (for example, mobilization with GM-
CSF) and
conditioning regimens can be used to create a condition in a subject wherein
repopulation,
recirculation, regeneration, and/or expansion of particular cell types is
favored, especially
during a defined window of time following therapy. Illustrative cell types
include T cells, B
cells, dendritic cells, and other cells of the immune system.
[0467] Whether prior to or after genetic modification of the T cells to
express a desirable
anti-E7MC CAR, the T cells can be activated and expanded generally using
methods as
described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680;
6,692,964;
5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566;
7,175,843;
5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application
Publication No.
20060121005.
[0468] Generally, the T cells of the invention are expanded by contact with a
surface
having attached thereto an agent that stimulates a CD3/TCR complex associated
signal and a
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ligand that stimulates a co-stimulatory molecule on the surface of the T
cells. In particular, T
cell populations may be stimulated, such as by contact with an anti-CD3
antibody, or antigen-
binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or
by contact
with a protein kinase C activator (e.g., bryostatin) in conjunction with a
calcium ionophore.
For co-stimulation of an accessory molecule on the surface of the T cells, a
ligand that binds
the accessory molecule is used. For example, a population of T cells can be
contacted with an
anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for
stimulating
proliferation of the T cells. To stimulate proliferation of either CD4+ T
cells or CD8+ T cells,
an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28
antibody
include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can
other methods
commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977,
1998; Haanen et
al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol. Meth.
227(1-2):53-63,
1999).
Immunoconjugate preparation
[0469] The anti-E7MC immunoconjugates may be prepared using any methods known
in
the art. See, e.g., WO 2009/067800, WO 2011/133886, and U.S. Patent
Application
Publication No. 2014322129, incorporated by reference herein in their
entirety.
[0470] The anti-E7MC antibody moiety of an anti-E7MC immunoconjugate may be
"attached to" the effector molecule by any means by which the anti-E7MC
antibody moiety
can be associated with, or linked to, the effector molecule. For example, the
anti-E7MC
antibody moiety of an anti-E7MC immunoconjugate may be attached to the
effector molecule
by chemical or recombinant means. Chemical means for preparing fusions or
conjugates are
known in the art and can be used to prepare the anti-E7MC immunoconjugate. The
method
used to conjugate the anti-E7MC antibody moiety and effector molecule must be
capable of
joining the binding protein with the effector molecule without interfering
with the ability of
the binding protein to bind to the antigen on the target cell.
[0471] The anti-E7MC antibody moiety of an anti-E7MC immunoconjugate may be
linked
indirectly to the effector molecule. For example, the anti-E7MC antibody
moiety of an anti-
E7MC immunoconjugate may be directly linked to a liposome containing the
effector
molecule of one of several types. The effector molecule(s) and/or the anti-
E7MC antibody
moiety may also be bound to a solid surface.
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[0472] In some embodiments, the anti-E7MC antibody moiety of an anti-E7MC
immunoconjugate and the effector molecule are both proteins and can be
conjugated using
techniques well known in the art. There are several hundred crosslinkers
available that can
conjugate two proteins. (See for example "Chemistry of Protein Conjugation and
Crosslinking". 1991 , Shans Wong, CRC Press, Ann Arbor). The crosslinker is
generally
chosen based on the reactive functional groups available or inserted on the
anti-E7MC
antibody moiety and/or effector molecule. In addition, if there are no
reactive groups, a
photoactivatible crosslinker can be used. In certain instances, it may be
desirable to include a
spacer between the anti-E7MC antibody moiety and the effector molecule.
Crosslinking
agents known to the art include the homobifunctional agents: glutaraldehyde,
dimethyladipimidate and Bis(diazobenzidine) and the heterobifunctional agents:
m
Maleimidobenzoyl-N-Hydroxysuccinimide and Sulfo-m Maleimidobenzoyl-N-
Hydroxysuccinimide.
[0473] In some embodiments, the anti-E7MC antibody moiety of an anti-E7MC
immunoconjugate may be engineered with specific residues for chemical
attachment of the
effector molecule. Specific residues used for chemical attachment of molecule
known to the
art include lysine and cysteine. The crosslinker is chosen based on the
reactive functional
groups inserted on the anti-E7MC antibody moiety, and available on the
effector molecule.
[0474] An anti-E7MC immunoconjugate may also be prepared using recombinant DNA
techniques. In such a case a DNA sequence encoding the anti-E7MC antibody
moiety is
fused to a DNA sequence encoding the effector molecule, resulting in a
chimeric DNA
molecule. The chimeric DNA sequence is transfected into a host cell that
expresses the fusion
protein. The fusion protein can be recovered from the cell culture and
purified using
techniques known in the art.
[0475] Examples of attaching an effector molecule, which is a label, to the
binding protein
include the methods described in Hunter, et al., Nature 144:945 (1962); David,
et al.,
Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981);
Nygren, J.
Histochem. and Cytochem. 30:407 (1982); Wensel and Meares, Radioimmunoimaging
And
Radioimmunotherapy, Elsevier, N.Y. (1983); and Colcher et al., "Use Of
Monoclonal
Antibodies As Radiopharmaceuticals For The Localization Of Human Carcinoma
Xenografts
In Athymic Mice", Meth. Enzymol., 121:802-16 (1986).
[0476] The radio- or other labels may be incorporated in the immunoconjugate
in known
ways. For example, the peptide may be biosynthesized or may be synthesized by
chemical
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amino acid synthesis using suitable amino acid precursors involving, for
example, fluorine-19
,
in place of hydrogen. Labels such as 99To or 1231 186Re, 188 Re and 111In can
be attached via a
cysteine residue in the peptide. Yttrium-90 can be attached via a lysine
residue. The
IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun. 80:49-57 (1978))
can be
used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy"
(Chatal,
CRC Press 1989) describes other methods in detail.
[0477] Immunoconjugates of the antibody moiety and a cytotoxic agent may be
made using
a variety of bifunctional protein coupling agents such as N-succinimidy1-3-(2-
pyridyldithio)
propionate (SPDP), succinimidy1-4-(N-maleimidomethyl) cyclohexane-1 -
carboxylate
(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as
dimethyl
adipimidate HCI), active esters (such as disuccinimidyl suberate), aldehydes
(such as
glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-
diazonium derivatives (such as bis-(p-diazoniumbenzoy1)- ethylenediamine),
diisocyanates
(such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as
1 ,5-difluoro-
2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as
described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-
isothiocyanatobenzy1-3-
methyldiethylene tnaminepentaacetic acid (MX-DTPA) is an exemplary chelating
agent for
conjugation of radionucleotide to the antibody. See, e.g., W094/11026. The
linker may be a
"cleavable linker" facilitating release of the cytotoxic drug in the cell. For
example, an acid-
labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker
or disulfide-
containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Patent
No.
5,208,020) may be used.
[0478] The anti-E7MC immunoconjugates of the invention expressly contemplate,
but are
not limited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,
HBVS, LC-
SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-
GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidy1-(4-vinylsulfone)benzoate) which are commercially available
(e.g., from Pierce
Biotechnology, Inc., Rockford, IL, U.S.A). See pages 467-498, 2003-2004
Applications
Handbook and Catalog.
Pharmaceutical Compositions
[0479] Also provided herein are compositions (such as pharmaceutical
compositions, also
referred to herein as formulations) comprising an anti-E7MC construct. In some
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embodiments, the composition further comprises a cell (such as an effector
cell, e.g., a T cell)
associated with the anti-E7MC construct. In some embodiments, there is
provided a
pharmaceutical composition comprising an anti-E7MC construct and a
pharmaceutically
acceptable carrier. In some embodiments, the pharmaceutical composition
further comprises
a cell (such as an effector cell, e.g., a T cell) associated with the anti-
E7MC construct.
[0480] Suitable formulations of the anti-E7MC constructs are obtained by
mixing an anti-
E7MC construct having the desired degree of purity with optional
pharmaceutically
acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical
Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and
concentrations employed, and include buffers such as phosphate, citrate, and
other organic
acids; antioxidants including ascorbic acid and methionine; preservatives
(such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium
chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as
methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-
cresol); low
molecular weight (less than about 10 residues) polypeptides; proteins, such as
serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone;
amino acids
such as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose, or
dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming
counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes);
and/or non-ionic
surfactants such as TWEENTm, PLURONICSTM or polyethylene glycol (PEG).
Exemplary
formulations are described in W098/56418, expressly incorporated herein by
reference.
Lyophilized formulations adapted for subcutaneous administration are described
in
W097/04801. Such lyophilized formulations may be reconstituted with a suitable
diluent to a
high protein concentration and the reconstituted formulation may be
administered
subcutaneously to the individual to be treated herein. Lipofectins or
liposomes can be used to
deliver the anti-E7MC constructs of this invention into cells.
[0481] The formulation herein may also contain one or more active compounds in
addition
to the anti-E7MC construct as necessary for the particular indication being
treated, preferably
those with complementary activities that do not adversely affect each other.
For example, it
may be desirable to further provide an anti-neoplastic agent, a growth
inhibitory agent, a
cytotoxic agent, or a chemotherapeutic agent in addition to the anti-E7MC
construct. Such
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molecules are suitably present in combination in amounts that are effective
for the purpose
intended. The effective amount of such other agents depends on the amount of
anti-E7MC
construct present in the formulation, the type of disease or disorder or
treatment, and other
factors discussed above. These are generally used in the same dosages and with
administration routes as described herein or about from 1 to 99% of the
heretofore employed
dosages.
[0482] The anti-E7MC constructs may also be entrapped in microcapsules
prepared, for
example, by coacervation techniques or by interfacial polymerization, for
example,
hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems (for example,
liposomes,
albumin microspheres, microemulsions, nano-particles and nanocapsules) or in
macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical
Sciences 16th
edition, Osol, A. Ed. (1980). Sustained-release preparations may be prepared.
[0483] Sustained-release preparations of the anti-E7MC constructs can be
prepared.
Suitable examples of sustained-release preparations include semipermeable
matrices of solid
hydrophobic polymers containing the antibody (or fragment thereof), which
matrices are in
the form of shaped articles, e.g., films, or microcapsules. Examples of
sustained-release
matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-
methacrylate ), or
poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-
glutamic acid
and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable
lactic acid-glycolic
acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed
of
lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-
hydroxybutyric
acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic
acid enable
release of molecules for over 100 days, certain hydro gels release proteins
for shorter time
periods. When encapsulated antibodies remain in the body for a long time, they
can denature
or aggregate as a result of exposure to moisture at 37 C, resulting in a loss
of biological
activity and possible changes in immunogenicity. Rational strategies can be
devised for
stabilization of anti-E7MC constructs depending on the mechanism involved. For
example, if
the aggregation mechanism is discovered to be intermolecular S-S bond
formation through
thio-disulfide interchange, stabilization can be achieved by modifying
sulfhydryl residues,
lyophilizing from acidic solutions, controlling moisture content, using
appropriate additives,
and developing specific polymer matrix compositions.
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[0484] In some embodiments, the anti-E7MC construct is formulated in a buffer
comprising a citrate, NaC1, acetate, succinate, glycine, polysorbate 80 (Tween
80), or any
combination of the foregoing. In some embodiments, the anti-E7MC construct is
formulated
in a buffer comprising about 100 mM to about 150 mM glycine. In some
embodiments, the
anti-E7MC construct is formulated in a buffer comprising about 50mM to about
100 mM
NaCl. In some embodiments, the anti-E7MC construct is formulated in a buffer
comprising
about 10mM to about 50 mM acetate. In some embodiments, the anti-E7MC
construct is
formulated in a buffer comprising about 10mM to about 50 mM succinate. In some
embodiments, the anti-E7MC construct is formulated in a buffer comprising
about 0.005% to
about 0.02% polysorbate 80. In some embodiments, the anti-E7MC construct is
formulated in
a buffer having a pH between about 5.1 and 5.6. In some embodiments, the anti-
E7MC
construct is formulated in a buffer comprising 10 mM citrate, 100 mM NaC1,
100mM
glycine, and 0.01% polysorbate 80, wherein the formulation is at pH 5.5.
[0485] The formulations to be used for in vivo administration must be sterile.
This is
readily accomplished by, e.g., filtration through sterile filtration
membranes.
Methods for treatment using anti-E7MC constructs
[0486] The anti-E7MC constructs and/or compositions of the invention can be
administered
to individuals (e.g., mammals such as humans) to treat a disease and/or
disorder associated
with HPV16-E7 expression (also referred to herein as an "HPV16-E7-positive"
disease or
disorder), including, for example, HPV16-E7-positive cancer (such as squamous
cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
and neck cancer, or oropharyngeal cancer). The present application thus in
some
embodiments provides a method of treating an HPV16-E7-positive disease (such
as cancer,
e.g., squamous cell carcinoma) in an individual comprising administering to
the individual an
effective amount of a composition (such as a pharmaceutical composition)
comprising an
anti-E7MC construct comprising an anti-E7MC antibody moiety, such as any one
of the anti-
E7MC constructs described herein. In some embodiments, the composition further
comprises
a cell (such as an effector cell) associated with the anti-E7MC construct. In
some
embodiments, the cancer is selected, for example, from the group consisting of
squamous cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
and neck cancer, or oropharyngeal cancer.
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[0487] For example, in some embodiments, there is provided a method of
treating an
HPV16-E7-positive disease in an individual comprising administering to the
individual an
effective amount of a composition comprising an anti-E7MC construct comprising
an anti-
E7MC antibody moiety that specifically binds to a complex comprising an HPV16-
E7
peptide and an MHC class I protein. In some embodiments, the HPV16-E7 peptide
is
HPV16-E7 11-19 (SEQ ID NO: 4). In some embodiments, the MHC class I protein is
HLA-
A02. In some embodiments, the MHC class I protein is HLA-A*02:01. In some
embodiments, the anti-E7MC construct is non-naturally occurring. In some
embodiments, the
anti-E7MC construct is a full-length antibody. In some embodiments, the anti-
E7MC
construct is a multi-specific (such as bispecific) molecule. In some
embodiments, the anti-
E7MC construct is a chimeric antigen receptor. In some embodiments, the anti-
E7MC
construct is an immunoconjugate. In some embodiments, the composition further
comprises a
cell (such as an effector cell) associated with the anti-E7MC construct. In
some
embodiments, the HPV16-E7-positive disease is cancer. In some embodiments, the
cancer is,
for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal
cancer, vulvar
cancer, penile cancer, head and neck cancer, or oropharyngeal cancer. In some
embodiments,
the cancer is an HPV16-E7-positive squamous cell carcinoma. In some
embodiments, the
individual is human.
[0488] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an anti-E7MC construct comprising an anti-E7MC antibody
moiety
that specifically binds to a complex comprising an HPV16-E7 11-19 peptide (SEQ
ID NO: 4)
and HLA-A*02:01. In some embodiments, the anti-E7MC construct is non-naturally
occurring. In some embodiments, the anti-E7MC construct is a full-length
antibody. In some
embodiments, the anti-E7MC construct is a multi-specific (such as bispecific)
molecule. In
some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In
some
embodiments, the anti-E7MC construct is an immunoconjugate. In some
embodiments, the
composition further comprises a cell (such as an effector cell) associated
with the anti-E7MC
construct. In some embodiments, the HPV16-E7-positive disease is cancer. In
some
embodiments, the cancer is, for example, squamous cell carcinoma, cervical
cancer, anal
cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or
oropharyngeal
cancer. In some embodiments, the cancer is an HPV16-E7-positive squamous cell
carcinoma.
In some embodiments, the individual is human.
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[0489] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an anti-E7MC construct comprising an anti-E7MC antibody
moiety
that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the anti-E7MC antibody moiety comprises: i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID
NO: 183,
or a variant thereof comprising up to about 3 (for example about any of 1, 2,
or 3) amino acid
substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184
or 185,
or a variant thereof comprising up to about 3 (for example about any of 1, 2,
or 3) amino acid
substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 186-188, or a variant thereof comprising up to about 3 (for example about
any of 1, 2,
or 3) amino acid substitutions; and ii) a light chain variable domain
comprising an LC-CDR1
comprising the amino acid sequence of SEQ ID NO: 189 or 190, or a variant
thereof
comprising up to about 3 (for example about any of 1, 2, or 3) amino acid
substitutions, and
an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or a variant
thereof
comprising up to about 3 (for example about any of 1, 2, or 3) amino acid
substitutions. In
some embodiments, the anti-E7MC construct is non-naturally occurring. In some
embodiments, the anti-E7MC construct is a full-length antibody. In some
embodiments, the
anti-E7MC construct is a multi-specific (such as bispecific) molecule. In some
embodiments,
the anti-E7MC construct is a chimeric antigen receptor. In some embodiments,
the anti-
E7MC construct is an immunoconjugate. In some embodiments, the composition
further
comprises a cell (such as an effector cell) associated with the anti-E7MC
construct. In some
embodiments, the HPV16-E7-positive disease is cancer. In some embodiments, the
cancer is,
for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal
cancer, vulvar
cancer, penile cancer, head and neck cancer, or oropharyngeal cancer. In some
embodiments,
the cancer is an HPV16-E7-positive squamous cell carcinoma. In some
embodiments, the
individual is human.
[0490] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an anti-E7MC construct comprising an anti-E7MC antibody
moiety
that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the anti-E7MC antibody moiety comprises: i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID
NO: 183,
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an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 184 or 185, and an
HC-
CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 186-188; and
ii) a
light chain variable domain comprising an LC-CDR1 comprising the amino acid
sequence of
SEQ ID NO: 189 or 190, and an LC-CDR3 comprising the amino acid sequence of
SEQ ID
NO: 191. In some embodiments, the anti-E7MC construct is non-naturally
occurring. In some
embodiments, the anti-E7MC construct is a full-length antibody. In some
embodiments, the
anti-E7MC construct is a multi-specific (such as bispecific) molecule. In some
embodiments,
the anti-E7MC construct is a chimeric antigen receptor. In some embodiments,
the anti-
E7MC construct is an immunoconjugate. In some embodiments, the composition
further
comprises a cell (such as an effector cell) associated with the anti-E7MC
construct. In some
embodiments, the HPV16-E7-positive disease is cancer. In some embodiments, the
cancer is,
for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal
cancer, vulvar
cancer, penile cancer, head and neck cancer, or oropharyngeal cancer. In some
embodiments,
the cancer is an HPV16-E7-positive squamous cell carcinoma. In some
embodiments, the
individual is human.
[0491] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an anti-E7MC construct comprising an anti-E7MC antibody
moiety
that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the anti-E7MC antibody moiety comprises: i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of any one
of SEQ
ID NOs: 57-77, or a variant thereof comprising up to about 5 (for example
about any of 1, 2,
3, 4, or 5) amino acid substitutions; an HC-CDR2 comprising the amino acid
sequence of any
one of SEQ ID NOs: 78-98, or a variant thereof comprising up to about 5 (for
example about
any of 1, 2, 3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising
the amino acid
sequence of any one of SEQ ID NOs: 99-119, 244, and 245; or a variant thereof
comprising
up to about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid
substitutions; and ii) a
light chain variable domain sequence comprising an LC-CDR1 comprising the
amino acid
sequence of any one of SEQ ID NOs: 120-140 and 246, or a variant thereof
comprising up to
about 5 (for example about any of 1, 2, 3, 4, or 5) amino acid substitutions;
an LC-CDR2
comprising the amino acid sequence of any one of SEQ ID NOs: 141-161, or a
variant
thereof comprising up to about 3 (for example about any of 1, 2, or 3) amino
acid
substitutions; and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
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NOs: 162-182 and 247-250; or a variant thereof comprising up to about 5 (for
example about
any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the
anti-E7MC
construct is non-naturally occurring. In some embodiments, the anti-E7MC
construct is a full-
length antibody. In some embodiments, the anti-E7MC construct is a multi-
specific (such as
bispecific) molecule. In some embodiments, the anti-E7MC construct is a
chimeric antigen
receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate.
In some
embodiments, the composition further comprises a cell (such as an effector
cell) associated
with the anti-E7MC construct. In some embodiments, the HPV16-E7-positive
disease is
cancer. In some embodiments, the cancer is, for example, squamous cell
carcinoma, cervical
cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and
neck cancer, or
oropharyngeal cancer. In some embodiments, the cancer is an HPV16-E7-positive
squamous
cell carcinoma. In some embodiments, the individual is human.
[0492] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an anti-E7MC construct comprising an anti-E7MC antibody
moiety
that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the anti-E7MC antibody moiety comprises: i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of any one
of SEQ
ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one of SEQ
ID
NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 99-119, 244, and 245; or a variant thereof comprising up to about 5 (for
example about
any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequences; and
ii) a light
chain variable domain sequence comprising an LC-CDR1 comprising the amino acid
sequence of any one of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising the
amino
acid sequence of any one of SEQ ID NOs: 141-161; and an LC-CDR3 comprising the
amino
acid sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a variant
thereof
comprising up to about 5 (for example about any of 1, 2, 3, 4, or 5) amino
acid substitutions
in the LC-CDR sequences. In some embodiments, the anti-E7MC construct is non-
naturally
occurring. In some embodiments, the anti-E7MC construct is a full-length
antibody. In some
embodiments, the anti-E7MC construct is a multi-specific (such as bispecific)
molecule. In
some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In
some
embodiments, the anti-E7MC construct is an immunoconjugate. In some
embodiments, the
composition further comprises a cell (such as an effector cell) associated
with the anti-E7MC
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construct. In some embodiments, the HPV16-E7-positive disease is cancer. In
some
embodiments, the cancer is, for example, squamous cell carcinoma, cervical
cancer, anal
cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or
oropharyngeal
cancer. In some embodiments, the cancer is an HPV16-E7-positive squamous cell
carcinoma.
In some embodiments, the individual is human.
[0493] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an anti-E7MC construct comprising an anti-E7MC antibody
moiety
that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the anti-E7MC antibody moiety comprises: i) a heavy chain
variable domain
sequence comprising an HC-CDR1 comprising the amino acid sequence of any one
of SEQ
ID NOs: 57-77; an HC-CDR2 comprising the amino acid sequence of any one of SEQ
ID
NOs: 78-98; and an HC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 99-119, 244, and 245; and ii) a light chain variable domain sequence
comprising an
LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 120-140
and
246; an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs:
141-161;
and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:
162-182
and 247-250. In some embodiments, the anti-E7MC construct is non-naturally
occurring. In
some embodiments, the anti-E7MC construct is a full-length antibody. In some
embodiments,
the anti-E7MC construct is a multi-specific (such as bispecific) molecule. In
some
embodiments, the anti-E7MC construct is a chimeric antigen receptor. In some
embodiments,
the anti-E7MC construct is an immunoconjugate. In some embodiments, the
composition
further comprises a cell (such as an effector cell) associated with the anti-
E7MC construct. In
some embodiments, the HPV16-E7-positive disease is cancer. In some
embodiments, the
cancer is, for example, squamous cell carcinoma, cervical cancer, anal cancer,
vaginal cancer,
vulvar cancer, penile cancer, head and neck cancer, or oropharyngeal cancer.
In some
embodiments, the cancer is an HPV16-E7-positive squamous cell carcinoma. In
some
embodiments, the individual is human.
[0494] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an anti-E7MC construct comprising an anti-E7MC antibody
moiety
that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the anti-E7MC antibody moiety comprises a heavy chain
variable domain
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comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-
237, or a
variant thereof having at least about 95% (for example at least about any of
96%, 97%, 98%,
or 99%) sequence identity, and a light chain variable domain comprising the
amino acid
sequence of any one of SEQ ID NOs: 36-56 and 238-243, or a variant thereof
having at least
about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence
identity. In
some embodiments, the anti-E7MC construct is non-naturally occurring. In some
embodiments, the anti-E7MC construct is a full-length antibody. In some
embodiments, the
anti-E7MC construct is a multi-specific (such as bispecific) molecule. In some
embodiments,
the anti-E7MC construct is a chimeric antigen receptor. In some embodiments,
the anti-
E7MC construct is an immunoconjugate. In some embodiments, the composition
further
comprises a cell (such as an effector cell) associated with the anti-E7MC
construct. In some
embodiments, the HPV16-E7-positive disease is cancer. In some embodiments, the
cancer is,
for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal
cancer, vulvar
cancer, penile cancer, head and neck cancer, or oropharyngeal cancer. In some
embodiments,
the cancer is an HPV16-E7-positive squamous cell carcinoma. In some
embodiments, the
individual is human.
[0495] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an anti-E7MC construct comprising an anti-E7MC antibody
moiety
that specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I
protein, wherein the anti-E7MC antibody moiety comprises a heavy chain
variable domain
comprising the amino acid sequence of any one of SEQ ID NOs: 15-35 and 233-237
and a
light chain variable domain comprising the amino acid sequence of any one of
SEQ ID NOs:
36-56 and 238-243. In some embodiments, the anti-E7MC construct is non-
naturally
occurring. In some embodiments, the anti-E7MC construct is a full-length
antibody. In some
embodiments, the anti-E7MC construct is a multi-specific (such as bispecific)
molecule. In
some embodiments, the anti-E7MC construct is a chimeric antigen receptor. In
some
embodiments, the anti-E7MC construct is an immunoconjugate. In some
embodiments, the
composition further comprises a cell (such as an effector cell) associated
with the anti-E7MC
construct. In some embodiments, the HPV16-E7-positive disease is cancer. In
some
embodiments, the cancer is, for example, squamous cell carcinoma, cervical
cancer, anal
cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or
oropharyngeal
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cancer. In some embodiments, the cancer is an HPV16-E7-positive squamous cell
carcinoma.
In some embodiments, the individual is human.
[0496] In some embodiments of any of the methods for treating an HPV16-E7-
positive
disease described above, the anti-E7MC construct is conjugated to a cell (such
as an immune
cell, e.g., a T cell) prior to being administered to the individual. Thus, for
example, there is
provided a method of treating an HPV16-E7-positive disease in an individual
comprising a)
conjugating any one of the anti-E7MC constructs described herein to a cell
(such as an
immune cell, e.g., a T cell) to form an anti-E7MC construct/cell conjugate,
and b)
administering to the individual an effective amount of a composition
comprising the anti-
E7MC construct/cell conjugate. In some embodiments, the cell is derived from
the individual.
In some embodiments, the cell is not derived from the individual. In some
embodiments, the
anti-E7MC construct is conjugated to the cell by covalent linkage to a
molecule on the
surface of the cell. In some embodiments, the anti-E7MC construct is
conjugated to the cell
by non-covalent linkage to a molecule on the surface of the cell. In some
embodiments, the
anti-E7MC construct is conjugated to the cell by insertion of a portion of the
anti-E7MC
construct into the outer membrane of the cell. In some embodiments, the anti-
E7MC
construct is non-naturally occurring. In some embodiments, the anti-E7MC
construct is a full-
length antibody. In some embodiments, the anti-E7MC construct is a multi-
specific (such as
bispecific) molecule. In some embodiments, the anti-E7MC construct is a
chimeric antigen
receptor. In some embodiments, the anti-E7MC construct is an immunoconjugate.
In some
embodiments, the HPV16-E7-positive disease is cancer. In some embodiments, the
cancer is,
for example, squamous cell carcinoma, cervical cancer, anal cancer, vaginal
cancer, vulvar
cancer, penile cancer, head and neck cancer, or oropharyngeal cancer. In some
embodiments,
the cancer is an HPV16-E7-positive squamous cell carcinoma. In some
embodiments, the
individual is human.
[0497] In some embodiments, the individual is a mammal (e.g., human, non-human
primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In some
embodiments, the
individual is a human. In some embodiments, the individual is a clinical
patient, a clinical
trial volunteer, an experimental animal, etc. In some embodiments, the
individual is younger
than about 60 years old (including for example younger than about any of 50,
40, 30, 25, 20,
15, or 10 years old). In some embodiments, the individual is older than about
60 years old
(including for example older than about any of 70, 80, 90, or 100 years old).
In some
embodiments, the individual is diagnosed with or genetically prone to one or
more of the
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diseases or disorders described herein (such as squamous cell carcinoma,
cervical cancer,
anal cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck
cancer, or
oropharyngeal cancer). In some embodiments, the individual has one or more
risk factors
associated with one or more diseases or disorders described herein.
[0498] The present application in some embodiments provides a method of
delivering an
anti-E7MC construct (such as any one of the anti-E7MC constructs described
herein) to a cell
presenting on its surface a complex comprising an HPV16-E7 peptide and an MHC
class I
protein in an individual, the method comprising administering to the
individual a composition
comprising the anti-E7MC construct. In some embodiments, the anti-E7MC
construct to be
delivered is associated with a cell (such as an effector cell, e.g., a T
cell).
[0499] Many diagnostic methods for HPV16-E7-positive cancer (such as squamous
cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
and neck cancer, or oropharyngeal cancer) or any other disease exhibiting
HPV16-E7
expression and the clinical delineation of those diseases are known in the
art. Such methods
include, but are not limited to, e.g., immunohistochemistry, PCR, and
fluorescent in situ
hybridization (FISH).
[0500] In some embodiments, the anti-E7MC constructs and/or compositions of
the
invention are administered in combination with a second, third, or fourth
agent (including,
e.g., an antineoplastic agent, a growth inhibitory agent, a cytotoxic agent,
or a
chemotherapeutic agent) to treat diseases or disorders involving HPV16-E7
expression. In
some embodiments, the anti-E7MC construct is administered in combination with
an agent
that increases the expression of MHC class I proteins and/or enhances the
surface
presentation of HPV16-E7 peptides by MHC class I proteins. In some
embodiments, the
agent includes, for example, IFN receptor agonists, Hsp90 inhibitors,
enhancers of p53
expression, and chemotherapeutic agents. In some embodiments, the agent is an
IFN receptor
agonist including, for example, IFNy, IFNP, and IFNa. In some embodiments, the
agent is an
Hsp90 inhibitor including, for example, tanespimycin (17-AAG), alvespimycin
(17-DMAG),
retaspimycin (IPI-504), IPI-493, CNF2024/BIIB021, MPC-3100, Debio 0932 (CUDC-
305),
PU-H71, Ganetespib (STA-9090), NVP-AUY922 (VER-52269), H5P990, KW-2478,
AT13387, SNX-5422, DS-2248, and XL888. In some embodiments, the agent is an
enhancer
of p53 expression including, for example, 5-fluorouracil and nutlin-3. In some
embodiments,
the agent is a chemotherapeutic agent including, for example, topotecan,
etoposide, cisplatin,
paclitaxel, and vinblastine.
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[0501] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual, wherein the cells expressing HPV16-E7 do not
normally present, or
present at relatively low levels, a complex comprising an HPV16-E7 protein and
an MHC
class I protein on their surface, the method comprising administering to the
individual a
composition comprising an anti-E7MC construct in combination with an agent
that increases
the expression of MHC class I proteins and/or enhances the surface
presentation of HPV16-
E7 peptides by MHC class I proteins. In some embodiments, the agent includes,
for example,
IFN receptor agonists, Hsp90 inhibitors, enhancers of p53 expression, and
chemotherapeutic
agents. In some embodiments, the agent is an IFN receptor agonist including,
for example,
IFNy, IFNP, and IFNa. In some embodiments, the agent is an Hsp90 inhibitor
including, for
example, tanespimycin (17-AAG), alvespimycin (17-DMAG), retaspimycin (IPI-
504), IPI-
493, CNF2024/BIIB021, MPC-3100, Debio 0932 (CUDC-305), PU-H71, Ganetespib (STA-
9090), NVP-AUY922 (VER-52269), HSP990, KW-2478, AT13387, SNX-5422, DS-2248,
and XL888. In some embodiments, the agent is an enhancer of p53 expression
including, for
example, 5-fluorouracil and nutlin-3. In some embodiments, the agent is a
chemotherapeutic
agent including, for example, topotecan, etoposide, cisplatin, paclitaxel, and
vinblastine.
[0502] Cancer treatments can be evaluated, for example, by tumor regression,
tumor weight
or size shrinkage, time to progression, duration of survival, progression free
survival, overall
response rate, duration of response, quality of life, protein expression
and/or activity.
Approaches to determining efficacy of the therapy can be employed, including
for example,
measurement of response through radiological imaging.
[0503] In some embodiments, the efficacy of treatment is measured as the
percentage
tumor growth inhibition (% TGI), calculated using the equation 100-(T/C x
100), where T is
the mean relative tumor volume of the treated tumor, and C is the mean
relative tumor
volume of a non-treated tumor. In some embodiments, the %TGI is about 10%,
about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,
about
91%, about 92%, about 93%, about 94% , about 95%, or more than 95%.
Dosing and Method of Administering the anti-E7MC construct Compositions
[0504] The dose of the anti-E7MC construct compositions administered to an
individual
(such as a human) may vary with the particular composition, the mode of
administration, and
the type of disease being treated. In some embodiments, the amount of the
composition is
effective to result in an objective response (such as a partial response or a
complete
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response). In some embodiments, the amount of the anti-E7MC construct
composition is
sufficient to result in a complete response in the individual. In some
embodiments, the
amount of the anti-E7MC construct composition is sufficient to result in a
partial response in
the individual. In some embodiments, the amount of the anti-E7MC construct
composition
administered (for example when administered alone) is sufficient to produce an
overall
response rate of more than about any of 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%,
64%, 65%, 70%, 75%, 80%, 85%, or 90% among a population of individuals treated
with the
anti-E7MC construct composition. Responses of an individual to the treatment
of the
methods described herein can be determined, for example, based on RECIST
levels.
[0505] In some embodiments, the amount of the composition is sufficient to
prolong
progress-free survival of the individual. In some embodiments, the amount of
the
composition is sufficient to prolong overall survival of the individual. In
some embodiments,
the amount of the composition (for example when administered along) is
sufficient to
produce clinical benefit of more than about any of 50%, 60%, 70%, or 77% among
a
population of individuals treated with the anti-E7MC construct composition.
[0506] In some embodiments, the amount of the composition, alone or in
combination with
a second, third, and/or fourth agent, is an amount sufficient to decrease the
size of a tumor,
decrease the number of cancer cells, or decrease the growth rate of a tumor by
at least about
any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to
the
corresponding tumor size, number of cancer cells, or tumor growth rate in the
same subject
prior to treatment or compared to the corresponding activity in other subjects
not receiving
the treatment. Standard methods can be used to measure the magnitude of this
effect, such as
in vitro assays with purified enzyme, cell-based assays, animal models, or
human testing.
[0507] In some embodiments, the amount of the anti-E7MC construct (e.g., full-
length
anti-E7MC antibody, multi-specific anti-E7MC molecule, anti-E7MC CAR, or anti-
E7MC
immunoconjugate) in the composition is below the level that induces a
toxicological effect
(i.e., an effect above a clinically acceptable level of toxicity) or is at a
level where a potential
side effect can be controlled or tolerated when the composition is
administered to the
individual.
[0508] In some embodiments, the amount of the composition is close to a
maximum
tolerated dose (MTD) of the composition following the same dosing regimen. In
some
embodiments, the amount of the composition is more than about any of 80%, 90%,
95%, or
98% of the MTD.
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[0509] In some embodiments, the amount of an anti-E7MC construct (e.g., full-
length anti-
E7MC antibody, multi-specific anti-E7MC molecule, anti-E7MC CAR, or anti-E7MC
immunoconjugate) in the composition is included in a range of about 0.001
i.t.g to about 1000
Idg=
[0510] In some embodiments of any of the above aspects, the effective amount
of an anti-
E7MC construct (e.g., full-length anti-E7MC antibody, multi-specific anti-E7MC
molecule,
anti-E7MC CAR, or anti-E7MC immunoconjugate) in the composition is in the
range of
about 0.1 vg/kg to about 100 mg/kg of total body weight.
[0511] The anti-E7MC construct compositions can be administered to an
individual (such
as human) via various routes, including, for example, intravenous, intra-
arterial,
intraperitoneal, intrapulmonary, oral, inhalation, intravesicular,
intramuscular, intra-tracheal,
subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some
embodiments,
sustained continuous release formulation of the composition may be used. In
some
embodiments, the composition is administered intravenously. In some
embodiments, the
composition is administered intraportally. In some embodiments, the
composition is
administered intraarterially. In some embodiments, the composition is
administered
intraperitoneally. In some embodiments, the composition is administered
intrahepatically. In
some embodiments, the composition is administered by hepatic arterial
infusion.
Anti-E7MC CAR Effector Cell Therapy
[0512] The present application also provides methods of using an anti-E7MC CAR
to
redirect the specificity of an effector cell (such as a primary T cell) to a
complex comprising
an HPV16-E7 peptide and an MHC class I protein. Thus, the present invention
also provides
a method of stimulating an effector cell-mediated response (such as a T cell-
mediated
immune response) to a target cell population or tissue comprising E7MC-
presenting cells in a
mammal, comprising the step of administering to the mammal an effector cell
(such as a T
cell) that expresses an anti-E7MC CAR.
[0513] Anti-E7MC CAR effector cells (such as T cells) expressing the anti-E7MC
CAR
can be infused to a recipient in need thereof. The infused cell is able to
kill E7MC-presenting
cells in the recipient. In some embodiments, unlike antibody therapies, anti-
E7MC CAR
effector cells (such as T cells) are able to replicate in vivo resulting in
long-term persistence
that can lead to sustained tumor control.
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[0514] In some embodiments, the anti-E7MC CAR effector cells are anti-E7MC CAR
T
cells that can undergo robust in vivo T cell expansion and can persist for an
extended amount
of time. In some embodiments, the anti-E7MC CAR T cells of the invention
develop into
specific memory T cells that can be reactivated to inhibit any additional
tumor formation or
growth.
[0515] The anti-E7MC CAR T cells of the invention may also serve as a type of
vaccine
for ex vivo immunization and/or in vivo therapy in a mammal. In some
embodiments, the
mammal is a human.
[0516] With respect to ex vivo immunization, of least one of the following
occurs in vitro
prior to administering the cell into a mammal: i) expansion of the cells, ii)
introducing a
nucleic acid encoding an anti-E7MC CAR to the cells, and/or iii)
cryopreservation of the
cells.
[0517] Ex vivo procedures are well known in the art and are discussed more
fully below.
Briefly, cells are isolated from a mammal (preferably a human) and genetically
modified (i.e.,
transduced or transfected in vitro) with a vector expressing an anti-E7MC CAR
disclosed
herein. The anti-E7MC CAR cell can be administered to a mammalian recipient to
provide a
therapeutic benefit. The mammalian recipient may be a human and the anti-E7MC
CAR cell
can be autologous with respect to the recipient. Alternatively, the cells can
be allogeneic,
syngeneic or xenogeneic with respect to the recipient.
[0518] The procedure for ex vivo expansion of hematopoietic stem and
progenitor cells is
described in U.S. Pat. No. 5,199,942, incorporated herein by reference, can be
applied to the
cells of the present invention. Other suitable methods are known in the art,
therefore the
present invention is not limited to any particular method of ex vivo expansion
of the cells.
Briefly, ex vivo culture and expansion of T cells comprises: (1) collecting
CD34+
hematopoietic stem and progenitor cells from a mammal from peripheral blood
harvest or
bone marrow explants; and (2) expanding such cells ex vivo. In addition to the
cellular growth
factors described in U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-
1, IL-3 and c-kit
ligand, can be used for culturing and expansion of the cells.
[0519] In addition to using a cell-based vaccine in terms of ex vivo
immunization, the
present invention also provides compositions and methods for in vivo
immunization to elicit
an immune response directed against an antigen in a patient.
[0520] The anti-E7MC CAR effector cells (such as T cells) of the present
invention may be
administered either alone, or as a pharmaceutical composition in combination
with diluents
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and/or with other components such as IL-2 or other cytokines or cell
populations. Briefly,
pharmaceutical compositions of the present invention may comprise anti-E7MC
CAR
effector cells (such as T cells), in combination with one or more
pharmaceutically or
physiologically acceptable carriers, diluents or excipients. Such compositions
may comprise
buffers such as neutral buffered saline, phosphate buffered saline and the
like; carbohydrates
such as glucose, mannose, sucrose or dextrans, mannitol; proteins;
polypeptides or amino
acids such as glycine; antioxidants; chelating agents such as EDTA or
glutathione; adjuvants
(e.g., aluminum hydroxide); and preservatives. In some embodiments, anti-E7MC
CAR
effector cell (such as T cell) compositions are formulated for intravenous
administration.
[0521] The precise amount of the anti-E7MC CAR effector cell (such as T cell)
compositions of the present invention to be administered can be determined by
a physician
with consideration of individual differences in age, weight, tumor size,
extent of infection or
metastasis, and condition of the patient (subject). In some embodiments, a
pharmaceutical
composition comprising the anti-E7MC CAR effector cells (such as T cells) is
administered
at a dosage of about 104 to about 109 cells/kg body weight, such any of about
104 to about
105, about 105 to about 106, about 106 to about 107, about 107 to about 108,
or about 108 to
about 109 cells/kg body weight, including all integer values within those
ranges. Anti-E7MC
CAR effect cell (such as T cell) compositions may also be administered
multiple times at
these dosages. The cells can be administered by using infusion techniques that
are commonly
known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.
319:1676, 1988).
The optimal dosage and treatment regimen for a particular patient can readily
be determined
by one skilled in the art of medicine by monitoring the patient for signs of
disease and
adjusting the treatment accordingly.
[0522] In some embodiments, it may be desired to administer activated anti-
E7MC CAR T
cells to a subject and then subsequently redraw blood (or have an apheresis
performed),
activate T cells therefrom according to the present invention, and reinfuse
the patient with
these activated and expanded T cells. This process can be carried out multiple
times every
few weeks. In some embodiments, T cells can be activated from blood draws of
from 10 cc to
400 cc. In some embodiments, T cells are activated from blood draws of 20 cc,
30 cc, 40 cc,
50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
[0523] The administration of the anti-E7MC CAR effector cells (such as T
cells) may be
carried out in any convenient manner, including by aerosol inhalation,
injection, ingestion,
transfusion, implantation or transplantation. The compositions described
herein may be
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administered to a patient subcutaneously, intradermally, intratumorally,
intranodally,
intramedullary, intramuscularly, by intravenous (i.v.) injection, or
intraperitoneally. In some
embodiments, the anti-E7MC CAR effector cell (such as T cell) compositions of
the present
invention are administered to a patient by intradermal or subcutaneous
injection. In some
embodiments, the anti-E7MC CAR effector cell (such as T cell) compositions of
the present
invention are administered by i.v. injection. The compositions of anti-E7MC
CAR effector
cells (such as T cells) may be injected directly into a tumor, lymph node, or
site of infection.
[0524] Thus, for example, in some embodiments, there is provided a method of
treating an
HPV16-E7-positive disease in an individual comprising administering to the
individual an
effective amount of a composition comprising an effector cell (such as a T
cell) expressing an
anti-E7MC CAR comprising a) an extracellular domain comprising an anti-E7MC
antibody
moiety that specifically binds to a complex comprising an HPV16-E7 peptide and
an MHC
class I protein, b) a transmembrane domain, and c) an intracellular signaling
domain
comprising a CD3 intracellular signaling sequence and a CD28 intracellular
signaling
sequence. In some embodiments, the HPV16-E7 peptide is HPV16-E7 11-19 (SEQ ID
NO:
4). In some embodiments, the MHC class I protein is HLA-A02. In some
embodiments, the
MHC class I protein is HLA-A*02:01. In some embodiments, the HPV16-E7-positive
disease
is cancer. In some embodiments, the cancer is, for example, squamous cell
carcinoma,
cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile cancer,
head and neck
cancer, or oropharyngeal cancer. In some embodiments, the cancer is an HPV16-
E7-positive
squamous cell carcinoma. In some embodiments, the individual is human.
[0525] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an effector cell (such as a T cell) expressing an anti-
E7MC CAR
comprising a) an extracellular domain comprising an anti-E7MC antibody moiety
that
specifically binds to a complex comprising an HPV16-E7 11-19 peptide (SEQ ID
NO: 4) and
HLA-A*02:01, b) a transmembrane domain, and c) an intracellular signaling
domain
comprising a CD3 intracellular signaling sequence and a CD28 intracellular
signaling
sequence. In some embodiments, the HPV16-E7-positive disease is cancer. In
some
embodiments, the cancer is, for example, squamous cell carcinoma, cervical
cancer, anal
cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or
oropharyngeal
cancer. In some embodiments, the cancer is an HPV16-E7-positive squamous cell
carcinoma.
In some embodiments, the individual is human.
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[0526] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an effector cell (such as a T cell) expressing an anti-
E7MC CAR
comprising a) an extracellular domain comprising an anti-E7MC antibody moiety
that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I protein
comprising i) a heavy chain variable domain sequence comprising an HC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 183, or a variant thereof comprising up
to about 3
(for example about any of 1, 2, or 3) amino acid substitutions, an HC-CDR2
comprising the
amino acid sequence of SEQ ID NO: 184 or 185, or a variant thereof comprising
up to about
3 (for example about any of 1, 2, or 3) amino acid substitutions, and an HC-
CDR3
comprising the amino acid sequence of any one of SEQ ID NOs: 186-188, or a
variant
thereof comprising up to about 3 (for example about any of 1, 2, or 3) amino
acid
substitutions; and ii) a light chain variable domain comprising an LC-CDR1
comprising the
amino acid sequence of SEQ ID NO: 189 or 190, or a variant thereof comprising
up to about
3 (for example about any of 1, 2, or 3) amino acid substitutions, and an LC-
CDR3 comprising
the amino acid sequence of SEQ ID NO: 191, or a variant thereof comprising up
to about 3
(for example about any of 1, 2, or 3) amino acid substitutions, b) a
transmembrane domain,
and c) an intracellular signaling domain comprising a CD3 intracellular
signaling sequence
and a CD28 intracellular signaling sequence. In some embodiments, the HPV16-E7-
positive
disease is cancer. In some embodiments, the cancer is, for example, squamous
cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
and neck cancer, or oropharyngeal cancer. In some embodiments, the cancer is
an HPV16-
E7-positive squamous cell carcinoma. In some embodiments, the individual is
human.
[0527] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an effector cell (such as a T cell) expressing an anti-
E7MC CAR
comprising a) an extracellular domain comprising an anti-E7MC antibody moiety
that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I protein
comprising i) a heavy chain variable domain sequence comprising an HC-CDR1
comprising
the amino acid sequence of SEQ ID NO: 183, an HC-CDR2 comprising the amino
acid
sequence of SEQ ID NO: 184 or 185, and an HC-CDR3 comprising the amino acid
sequence
of any one of SEQ ID NOs: 186-188; and ii) a light chain variable domain
comprising an LC-
CDR1 comprising the amino acid sequence of SEQ ID NO: 189 or 190, and an LC-
CDR3
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comprising the amino acid sequence of SEQ ID NO: 191, b) a transmembrane
domain, and c)
an intracellular signaling domain comprising a CD3 intracellular signaling
sequence and a
CD28 intracellular signaling sequence. In some embodiments, the HPV16-E7-
positive
disease is cancer. In some embodiments, the cancer is, for example, squamous
cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
and neck cancer, or oropharyngeal cancer. In some embodiments, the cancer is
an HPV16-
E7-positive squamous cell carcinoma. In some embodiments, the individual is
human.
[0528] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an effector cell (such as a T cell) expressing an anti-
E7MC CAR
comprising a) an extracellular domain comprising an anti-E7MC antibody moiety
that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I protein
comprising i) a heavy chain variable domain comprising an HC-CDR1 comprising
the amino
acid sequence of any one of SEQ ID NOs: 57-77, or a variant thereof comprising
up to about
(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2
comprising the
amino acid sequence of any one of SEQ ID NOs: 78-98, or a variant thereof
comprising up to
about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and
an HC-CDR3
comprising the amino acid sequence of any one of SEQ ID NOs: 99-119, 244, and
245, or a
variant thereof comprising up to about 5 (such as about any of 1,2, 3,4, or 5)
amino acid
substitutions; and ii) a light chain variable domain comprising an LC-CDR1
comprising the
amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, or a variant
thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions, an
LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 141-161,
or a
variant thereof comprising up to about 3 (such as about any of 1, 2, or 3)
amino acid
substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 162-182 and 247-250, or a variant thereof comprising up to about 5 (such
as about any
of 1, 2, 3, 4, or 5) amino acid substitutions; b) a transmembrane domain, and
c) an
intracellular signaling domain comprising a CD3 intracellular signaling
sequence and a
CD28 intracellular signaling sequence. In some embodiments, the HPV16-E7-
positive
disease is cancer. In some embodiments, the cancer is, for example, squamous
cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
and neck cancer, or oropharyngeal cancer. In some embodiments, the cancer is
an HPV16-
E7-positive squamous cell carcinoma. In some embodiments, the individual is
human.
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[0529] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an effector cell (such as a T cell) expressing an anti-
E7MC CAR
comprising a) an extracellular domain comprising an anti-E7MC antibody moiety
that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I protein
comprising i) a heavy chain variable domain sequence comprising an HC-CDR1
comprising
the amino acid sequence of any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising
the
amino acid sequence of any one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising
the
amino acid sequence of any one of SEQ ID NOs: 99-119, 244, and 245; or a
variant thereof
comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid
substitutions in the
HC-CDR sequences; and ii) a light chain variable domain sequence comprising an
LC-CDR1
comprising the amino acid sequence of any one of SEQ ID NOs: 120-140 and 246;
an LC-
CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 141-161; and
an LC-
CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 162-182 and
247-
250; or a variant thereof comprising up to about 5 (such as about any of 1,2,
3,4, or 5) amino
acid substitutions in the LC-CDR sequences; b) a transmembrane domain, and c)
an
intracellular signaling domain comprising a CD3 intracellular signaling
sequence and a
CD28 intracellular signaling sequence. In some embodiments, the HPV16-E7-
positive
disease is cancer. In some embodiments, the cancer is, for example, squamous
cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
and neck cancer, or oropharyngeal cancer. In some embodiments, the cancer is
an HPV16-
E7-positive squamous cell carcinoma. In some embodiments, the individual is
human.
[0530] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an effector cell (such as a T cell) expressing an anti-
E7MC CAR
comprising a) an extracellular domain comprising an anti-E7MC antibody moiety
that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I protein
comprising i) a heavy chain variable domain sequence comprising an HC-CDR1
comprising
the amino acid sequence of any one of SEQ ID NOs: 57-77; an HC-CDR2 comprising
the
amino acid sequence of any one of SEQ ID NOs: 78-98; and an HC-CDR3 comprising
the
amino acid sequence of any one of SEQ ID NOs: 99-119, 244, and 245; and ii) a
light chain
variable domain sequence comprising an LC-CDR1 comprising the amino acid
sequence of
any one of SEQ ID NOs: 120-140 and 246; an LC-CDR2 comprising the amino acid
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sequence of any one of SEQ ID NOs: 141-161; and an LC-CDR3 comprising the
amino acid
sequence of any one of SEQ ID NOs: 162-182 and 247-250; b) a transmembrane
domain, and
c) an intracellular signaling domain comprising a CD3 intracellular signaling
sequence and a
CD28 intracellular signaling sequence. In some embodiments, the HPV16-E7-
positive
disease is cancer. In some embodiments, the cancer is, for example, squamous
cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
and neck cancer, or oropharyngeal cancer. In some embodiments, the cancer is
an HPV16-
E7-positive squamous cell carcinoma. In some embodiments, the individual is
human.
[0531] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an effector cell (such as a T cell) expressing an anti-
E7MC CAR
comprising a) an extracellular domain comprising an anti-E7MC antibody moiety
that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I protein
comprising i) a heavy chain variable domain comprising the amino acid sequence
of any one
of SEQ ID NOs: 15-35 and 233-237, or a variant thereof having at least about
95% (for
example at least about any of 96%, 97%, 98%, or 99%) sequence identity, and a
light chain
variable domain comprising the amino acid sequence of any one of SEQ ID NOs:
36-56 and
238-243, or a variant thereof having at least about 95% (including for example
at least about
any of 96%, 97%, 98%, or 99%) sequence identity; b) a transmembrane domain,
and c) an
intracellular signaling domain comprising a CD3 intracellular signaling
sequence and a
CD28 intracellular signaling sequence. In some embodiments, the HPV16-E7-
positive
disease is cancer. In some embodiments, the cancer is, for example, squamous
cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
and neck cancer, or oropharyngeal cancer. In some embodiments, the cancer is
an HPV16-
E7-positive squamous cell carcinoma. In some embodiments, the individual is
human.
[0532] In some embodiments, there is provided a method of treating an HPV16-E7-
positive
disease in an individual comprising administering to the individual an
effective amount of a
composition comprising an effector cell (such as a T cell) expressing an anti-
E7MC CAR
comprising a) an extracellular domain comprising an anti-E7MC antibody moiety
that
specifically binds to a complex comprising an HPV16-E7 peptide and an MHC
class I protein
comprising a heavy chain variable domain comprising the amino acid sequence of
any one of
SEQ ID NOs: 15-35 and 233-237 and a light chain variable domain comprising the
amino
acid sequence of any one of SEQ ID NOs: 36-56 and 238-243; b) a transmembrane
domain,
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and c) an intracellular signaling domain comprising a CD3 intracellular
signaling sequence
and a CD28 intracellular signaling sequence. In some embodiments, the HPV16-E7-
positive
disease is cancer. In some embodiments, the cancer is, for example, squamous
cell
carcinoma, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, penile
cancer, head
and neck cancer, or oropharyngeal cancer. In some embodiments, the cancer is
an HPV16-
E7-positive squamous cell carcinoma. In some embodiments, the individual is
human.
Cancers
[0533] The anti-E7MC constructs and anti-E7MC CAR cells in some embodiments
can be
useful for treating HPV16-E7-positive cancer. Cancers that may be treated
using any of the
methods described herein include tumors that are not vascularized, or not yet
substantially
vascularized, as well as vascularized tumors. The cancers may comprise non-
solid tumors
(such as hematological tumors, for example, leukemias and lymphomas) or may
comprise
solid tumors. Types of cancers to be treated with the anti-E7MC constructs and
anti-E7MC
CAR cells of the invention include, but are not limited to, carcinoma,
blastoma, and sarcoma,
and certain leukemia or lymphoid malignancies, benign and malignant tumors,
and
malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers
and pediatric
tumors/cancers are also included.
[0534] Hematologic cancers are cancers of the blood or bone marrow. Examples
of
hematological (or hematogenous) cancers include leukemias, including acute
leukemias (such
as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous
leukemia and
myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia),
chronic
leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic
myelogenous
leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma,
Hodgkin's
disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple
myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic
syndrome, hairy
cell leukemia and myelodysplasia.
[0535] Solid tumors are abnormal masses of tissue that usually do not contain
cysts or
liquid areas. Solid tumors can be benign or malignant. Different types of
solid tumors are
named for the type of cells that form them (such as sarcomas, carcinomas, and
lymphomas).
Examples of solid tumors, such as sarcomas and carcinomas, include
fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas,
synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon
carcinoma,
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lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian
cancer, prostate
cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell
carcinoma,
adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary
thyroid
carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma,
papillary
adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell
carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer
(e.g.,
cervical carcinoma and pre-invasive cervical dysplasia), cancer of the anus,
anal canal, or
anorectum, vaginal cancer, cancer of the vulva (e.g., squamous cell carcinoma,
intraepithelial
carcinoma, adenocarcinoma, and fibrosarcoma), penile cancer, oropharyngeal
cancer, head
cancers (e.g., squamous cell carcinoma), neck cancers (e.g., squamous cell
carcinoma),
testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma,
teratocarcinoma,
choriocarcinoma, sarcoma, Leydig cell tumor, fibroma, fibroadenoma,
adenomatoid tumors,
and lipoma), bladder carcinoma, melanoma, cancer of the uterus (e.g.,
endometrial
carcinoma), urothelial cancers (e.g., squamous cell carcinoma, transitional
cell carcinoma,
adenocarcinoma, ureter cancer, and urinary bladder cancer), and CNS tumors
(such as a
glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known
as
glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma,
medulloblastoma,
Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma,
acoustic
neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and
brain
metastases).
[0536] Cancer treatments can be evaluated, for example, by tumor regression,
tumor weight
or size shrinkage, time to progression, duration of survival, progression free
survival, overall
response rate, duration of response, quality of life, protein expression
and/or activity.
Approaches to determining efficacy of the therapy can be employed, including
for example,
measurement of response through radiological imaging.
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Methods for Diagnosis and Imaging Using anti-E7MC constructs
[0537] Labeled anti-E7MC antibody moieties and derivatives and analogs
thereof, which
specifically bind to an E7MC on the surface of a cell, can be used for
diagnostic purposes to
detect, diagnose, or monitor diseases and/or disorders associated with the
expression of
HPV16-E7, including any of the diseases and disorders described above, such as
cancer. For
example, the anti-E7MC antibody moieties of the invention can be used in in
situ, in vivo, ex
vivo, and in vitro diagnostic assays or imaging assays.
[0538] Additional embodiments of the invention include methods of diagnosing a
disease
or disorder associated with expression of HPV16-E7 in an individual (e.g., a
mammal such as
a human). The methods comprise detecting E7MC-presenting cells in the
individual. In some
embodiments, there is provided a method of diagnosing a disease or disorder
associated with
expression of HPV16-E7 in an individual (e.g., a mammal, such as a human)
comprising (a)
administering an effective amount of a labeled anti-E7MC antibody moiety
according to any
of the embodiments described above to the individual; and (b) determining the
level of the
label in the individual, such that a level of the label above a threshold
level indicates that the
individual has the disease or disorder. The threshold level can be determined
by various
methods, including, for example, by detecting the label according to the
method of
diagnosing described above in a first set of individuals that have the disease
or disorder and a
second set of individuals that do not have the disease or disorder, and
setting the threshold to
a level that allows for discrimination between the first and second sets. In
some
embodiments, the threshold level is zero, and the method comprises determining
the presence
or absence of the label in the individual. In some embodiments, the method
further comprises
waiting for a time interval following the administering of step (a) to permit
the labeled anti-
E7MC antibody moiety to preferentially concentrate at sites in the individual
where the
E7MC is expressed (and for unbound labeled anti-E7MC antibody moiety to be
cleared). In
some embodiments, the method further comprises subtracting a background level
of the label.
Background level can be determined by various methods, including, for example,
by
detecting the label in the individual prior to administration of the labeled
anti-E7MC antibody
moiety, or by detecting the label according to the method of diagnosing
described above in an
individual that does not have the disease or disorder. In some embodiments,
the disease or
disorder is cancer. In some embodiments, the cancer is selected, for example,
from the group
consisting of squamous cell carcinoma, cervical cancer, anal cancer, vaginal
cancer, vulvar
cancer, penile cancer, head and neck cancer, or oropharyngeal cancer.
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[0539] Anti-E7MC antibody moieties of the invention can be used to assay
levels of
E7MC-presenting cell in a biological sample using methods known to those of
skill in the art.
Suitable antibody labels are known in the art and include enzyme labels, such
as, glucose
oxidase; radioisotopes, such as iodine (1311, 125i, 1231, 121I), carbon (14C),
sulfur (355), tritium
(3H), indium (1 ismin, 113min, 112k, 111-rin) .,
technetium (99Tc, 99mTc), thallium (201Ti), gallium
(68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine
(18F), samarium
(1535m), lutetium (177Lu), gadolinium (159Gd), promethium (149Pm), lanthanum
(140La),
ytterbium (175Yb) , holmium (166Ho), yttrium (90Y), scandium (475c), rhenium
(186Re, 188Re),
praseodymium (142Pr), rhodium (105Rh), and ruthenium (97Ru); luminol;
fluorescent labels,
such as fluorescein and rhodamine; and biotin.
[0540] Techniques known in the art may be applied to labeled anti-E7MC
antibody
moieties of the invention. Such techniques include, but are not limited to,
the use of
bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;
5,714,631; 5,696,239;
5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;
4,994,560;
and 5,808,003). Aside from the above assays, various in vivo and ex vivo
assays are available
to the skilled practitioner. For example, one can expose cells within the body
of the subject to
an anti-E7MC antibody moiety which is optionally labeled with a detectable
label, e.g., a
radioactive isotope, and binding of the anti-E7MC antibody moiety to the cells
can be
evaluated, e.g., by external scanning for radioactivity or by analyzing a
sample (e.g., a biopsy
or other biological sample) derived from a subject previously exposed to the
anti-E7MC
antibody moiety.
Articles of Manufacture and Kits
[0541] In some embodiments of the invention, there is provided an article of
manufacture
containing materials useful for the treatment of an HPV16-E7-positive disease
such as cancer
(for example squamous cell carcinoma, cervical cancer, anal cancer, vaginal
cancer, vulvar
cancer, penile cancer, head and neck cancer, or oropharyngeal cancer), for
delivering an anti-
E7MC construct to a cell presenting an E7MC on its surface, or for isolation
or detection of
E7MC-presenting cells in an individual. The article of manufacture can
comprise a container
and a label or package insert on or associated with the container. Suitable
containers include,
for example, bottles, vials, syringes, etc. The containers may be formed from
a variety of
materials such as glass or plastic. Generally, the container holds a
composition which is
effective for treating a disease or disorder described herein, and may have a
sterile access port
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(for example the container may be an intravenous solution bag or a vial having
a stopper
pierceable by a hypodermic injection needle). At least one active agent in the
composition is
an anti-E7MC construct of the invention. The label or package insert indicates
that the
composition is used for treating the particular condition. The label or
package insert will
further comprise instructions for administering the anti-E7MC construct
composition to the
patient. Articles of manufacture and kits comprising combinatorial therapies
described herein
are also contemplated.
[0542] Package insert refers to instructions customarily included in
commercial packages
of therapeutic products that contain information about the indications, usage,
dosage,
administration, contraindications and/or warnings concerning the use of such
therapeutic
products. In some embodiments, the package insert indicates that the
composition is used for
treating HPV16-E7-positive cancer (such as squamous cell carcinoma, cervical
cancer, anal
cancer, vaginal cancer, vulvar cancer, penile cancer, head and neck cancer, or
oropharyngeal
cancer).
[0543] Additionally, the article of manufacture may further comprise a second
container
comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection
(BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It
may further
include other materials desirable from a commercial and user standpoint,
including other
buffers, diluents, filters, needles, and syringes.
[0544] Kits are also provided that are useful for various purposes, e.g., for
treatment of an
HPV16-E7-positive disease or disorder described herein, for delivering an anti-
E7MC
construct to a cell presenting an E7MC on its surface, or for isolation or
detection of E7MC-
presenting cells in an individual, optionally in combination with the articles
of manufacture.
Kits of the invention include one or more containers comprising an anti-E7MC
construct
composition (or unit dosage form and/or article of manufacture), and in some
embodiments,
further comprise another agent (such as the agents described herein) and/or
instructions for
use in accordance with any of the methods described herein. The kit may
further comprise a
description of selection of individuals suitable for treatment. Instructions
supplied in the kits
of the invention are typically written instructions on a label or package
insert (e.g., a paper
sheet included in the kit), but machine-readable instructions (e.g.,
instructions carried on a
magnetic or optical storage disk) are also acceptable.
[0545] For example, in some embodiments, the kit comprises a composition
comprising an
anti-E7MC construct (e.g., a full-length anti-E7MC antibody, a multi-specific
anti-E7MC
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molecule (such as a bispecific anti-E7MC antibody), or an anti-E7MC
immunoconjugate). In
some embodiments, the kit comprises a) a composition comprising an anti-E7MC
construct,
and b) an effective amount of at least one other agent, wherein the other
agent increases the
expression of MHC class I proteins and/or enhances the surface presentation of
HPV16-E7
peptides by MHC class I proteins (e.g., IFNy, IFNP, IFNa, or Hsp90 inhibitor).
In some
embodiments, the kit comprises a) a composition comprising an anti-E7MC
construct, and b)
instructions for administering the anti-E7MC construct composition to an
individual for
treatment of an HPV16-E7-positive disease, including for example squamous cell
carcinoma,
cervical cancer, or anal cancer. In some embodiments, the kit comprises a) a
composition
comprising an anti-E7MC construct, b) an effective amount of at least one
other agent,
wherein the other agent increases the expression of MHC class I proteins
and/or enhances the
surface presentation of HPV16-E7 peptides by MHC class I proteins (e.g., IFNy,
IFNP, IFNa,
or Hsp90 inhibitor), and c) instructions for administering the anti-E7MC
construct
composition and the other agent(s) to an individual for treatment of an HPV16-
E7-positive
disease, including for example squamous cell carcinoma, cervical cancer, or
anal cancer. The
anti-E7MC construct and the other agent(s) can be present in separate
containers or in a
single container. For example, the kit may comprise one distinct composition
or two or more
compositions wherein one composition comprises an anti-E7MC construct and
another
composition comprises another agent.
[0546] In some embodiments, the kit comprises a) a composition comprising an
anti-E7MC
construct (e.g., a full-length anti-E7MC antibody, a multi-specific anti-E7MC
molecule (such
as a bispecific anti-E7MC antibody), or an anti-E7MC immunoconjugate), and b)
instructions
for combining the anti-E7MC construct with cells (such as cells, e.g., immune
cells, derived
from an individual) to form a composition comprising anti-E7MC construct/cell
conjugates
and administering the anti-E7MC construct/cell conjugate composition to the
individual for
treatment of an HPV16-E7-positive disease (including for example squamous cell
carcinoma,
cervical cancer, or anal cancer). In some embodiments, the kit comprises a) a
composition
comprising an anti-E7MC construct, and b) a cell (such as a cytotoxic cell).
In some
embodiments, the kit comprises a) a composition comprising an anti-E7MC
construct, b) a
cell (such as a cytotoxic cell), and c) instructions for combining the anti-
E7MC construct
with the cell to form a composition comprising anti-E7MC construct/cell
conjugates and
administering the anti-E7MC construct/cell conjugate composition to an
individual for the
treatment of an HPV16-E7-positive disease, including for example squamous cell
carcinoma,
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cervical cancer, or anal cancer. In some embodiments, the kit comprises a
composition
comprising an anti-E7MC construct in association with a cell (such as a
cytotoxic cell). In
some embodiments, the kit comprises a) a composition comprising an anti-E7MC
construct
in association with a cell (such as a cytotoxic cell), and b) instructions for
administering the
composition to an individual for the treatment of an HPV16-E7-positive
disease, including
for example squamous cell carcinoma, cervical cancer, or anal cancer. In some
embodiments,
the association is by conjugation of the anti-E7MC construct to a molecule on
the surface of
the cell. In some embodiments, the association is by insertion of a portion of
the anti-E7MC
construct into the outer membrane of the cell.
[0547] In some embodiments, the kit comprises a nucleic acid (or set of
nucleic acids)
encoding an anti-E7MC construct (e.g., a full-length anti-E7MC antibody, a
multi-specific
anti-E7MC molecule (such as a bispecific anti-E7MC antibody), an anti-E7MC
CAR, or an
anti-E7MC immunoconjugate) or polypeptide portions thereof. In some
embodiments, the kit
comprises a) a nucleic acid (or set of nucleic acids) encoding an anti-E7MC
construct or
polypeptide portions thereof, and b) a host cell (such as an effector cell)
for expressing the
nucleic acid (or set of nucleic acids). In some embodiments, the kit comprises
a) a nucleic
acid (or set of nucleic acids) encoding an anti-E7MC construct or polypeptide
portions
thereof, and b) instructions for i) expressing the anti-E7MC construct in a
host cell (such as
an effector cell, e.g., a T cell), ii) preparing a composition comprising the
anti-E7MC
construct or the host cell expressing the anti-E7MC construct, and iii)
administering the
composition comprising the anti-E7MC construct or the host cell expressing the
anti-E7MC
construct to an individual for the treatment of an HPV16-E7-positive disease,
including for
example squamous cell carcinoma, cervical cancer, or anal cancer. In some
embodiments, the
host cell is derived from the individual. In some embodiments, the kit
comprises a) a nucleic
acid (or set of nucleic acids) encoding an anti-E7MC construct or polypeptide
portions
thereof, b) a host cell (such as an effector cell) for expressing the nucleic
acid (or set of
nucleic acids), and c) instructions for i) expressing the anti-E7MC construct
in the host cell,
ii) preparing a composition comprising the anti-E7MC construct or the host
cell expressing
the anti-E7MC construct, and iii) administering the composition comprising the
anti-E7MC
construct or the host cell expressing the anti-E7MC construct to an individual
for the
treatment of an HPV16-E7-positive disease, including for example squamous cell
carcinoma,
cervical cancer, or anal cancer.
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[0548] In some embodiments, the kit comprises a nucleic acid encoding an anti-
E7MC
CAR. In some embodiments, the kit comprises a vector comprising a nucleic acid
encoding
an anti-E7MC CAR. In some embodiments, the kit comprises a) a vector
comprising a
nucleic acid encoding an anti-E7MC CAR, and b) instructions for i) introducing
the vector
into effector cells, such as T cells derived from an individual, ii) preparing
a composition
comprising the anti-E7MC CAR effector cells, and iii) administering the anti-
E7MC CAR
effector cell composition to the individual for treatment of an HPV16-E7-
positive disease,
including for example squamous cell carcinoma, cervical cancer, or anal
cancer.
[0549] The kits of the invention are in suitable packaging. Suitable packaging
includes, but
is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed
Mylar or plastic bags),
and the like. Kits may optionally provide additional components such as
buffers and
interpretative information. The present application thus also provides
articles of manufacture,
which include vials (such as sealed vials), bottles, jars, flexible packaging,
and the like.
[0550] The instructions relating to the use of the anti-E7MC construct
compositions
generally include information as to dosage, dosing schedule, and route of
administration for
the intended treatment. The containers may be unit doses, bulk packages (e.g.,
multi-dose
packages) or sub-unit doses. For example, kits may be provided that contain
sufficient
dosages of an anti-E7MC construct (e.g., a full-length anti-E7MC antibody, a
multi-specific
anti-E7MC molecule (such as a bispecific anti-E7MC antibody), an anti-E7MC
CAR, or an
anti-E7MC immunoconjugate) as disclosed herein to provide effective treatment
of an
individual for an extended period, such as any of a week, 8 days, 9 days, 10
days, 11 days, 12
days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4
months, 5 months,
7 months, 8 months, 9 months, or more. Kits may also include multiple unit
doses of the anti-
E7MC construct and pharmaceutical compositions and instructions for use and
packaged in
quantities sufficient for storage and use in pharmacies, for example, hospital
pharmacies and
compounding pharmacies.
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EXEMPLARY EMBODIMENTS
[0551] Embodiment 1. In some embodiments, there is provided an isolated anti-
E7MC
construct comprising an antibody moiety that specifically binds to a complex
comprising
human papilloma virus subtype 16 (HPV16) E7 peptide and a major
histocompatibility
(MHC) class I protein (an HPV16-E7/MHC class I complex, or E7MC).
[0552] Embodiment 2. In some further embodiments of embodiment 1, the HPV16-
E7/MHC class I complex is present on a cell surface.
[0553] Embodiment 3. In some further embodiments of embodiment 1, the HPV16-
E7/MHC class I complex is present on the surface of a cancer cell.
[0554] Embodiment 4. In some further embodiments of any one of embodiments 1-
3, the
MHC class I protein is human leukocyte antigen (HLA)-A.
[0555] Embodiment 5. In some further embodiments of embodiment 4, the MHC
class I
protein is HLA-A02.
[0556] Embodiment 6. In some further embodiments of embodiment 5, the MHC
class I
protein is selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-
A*02:06, HLA-A*02:07, and HLA-A*02:11.
[0557] Embodiment 7. In some further embodiments of embodiment 6, the MHC
class I
protein is HLA-A*02:01
[0558] Embodiment 8. In some further embodiments of any one of embodiments 1-
7, the
antibody moiety cross-reacts with a complex comprising the HPV16-E7 peptide
and a second
MHC class I protein having a different HLA allele than the MHC class I
protein.
[0559] Embodiment 9. In some further embodiments of any one of embodiments 1-
8, the
HPV16-E7 peptide is 8 to 12 amino acids in length.
[0560] Embodiment 10. In some further embodiments of any one of embodiments 1-
9, the
HPV16-E7 peptide is derived from the E7 protein of human papilloma virus
subtype 16.
[0561] Embodiment 11. In some further embodiments of any one of embodiments 1-
10, the
HPV16-E7 peptide has an amino acid sequence selected from the group consisting
of SEQ ID
NOs: 3-14.
[0562] Embodiment 12. In some further embodiments of embodiment 10, the HPV16-
E7
peptide has the amino acid sequence of YMLDLQPET (SEQ ID NO: 4).
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[0563] Embodiment 13. In some further embodiments of embodiment 12, the
isolated anti-
E7MC construct cross-reacts with a complex comprising a variant of the HPV16-
E7 peptide
having the amino acid sequence of YMLDVQPET (SEQ ID NO: 11) and the MHC class
I
protein.
[0564] Embodiment 14. In some further embodiments of any one of embodiments 1-
13, the
antibody moiety is human, humanized, or semi-synthetic.
[0565] Embodiment 15. In some further embodiments of any one of embodiments 1-
14, the
antibody moiety is a full-length antibody, a Fab, a Fab', a (Fab')2, an Fv, or
a single chain Fv
(scFv).
[0566] Embodiment 16. In some further embodiments of any one of embodiments 1-
15, the
antibody moiety binds to the HPV16-E7/MHC class I complex with an equilibrium
dissociation constant (Kd) from about 0.1 pM to about 500 nM.
[0567] Embodiment 17. In some further embodiments of any one of embodiments 1-
16, the
isolated anti-E7MC construct binds to the HPV16-E7/MHC class I complex with a
Kd from
about 0.1 pM to about 500 nM.
[0568] Embodiment 18. In some further embodiments of any one of embodiments 1-
17, the
antibody moiety comprises:
i) a heavy chain variable domain comprising a heavy chain complementarity
determining region (HC-CDR) 1 comprising the amino acid sequence of G-F/G/Y-
S/T-F-S/T-
S-Y-A/G (SEQ ID NO: 183), or a variant thereof comprising up to about 3 amino
acid
substitutions, an HC-CDR2 comprising the amino acid sequence of I-N/I P X X G
G/T/I-
T/A/P or I-S-X-S/D-G/N-G/S-N-T/I/K (SEQ ID NO: 184 or 185), or a variant
thereof
comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising
the amino
acid sequence of any one of A-R-S/R-Y/S/G-Y/V Y/W G X Y D, ARG XX X Y Y/G/S,
or ARGXXY Q/W-W-S-X-D-D (SEQ ID NOs: 186-188), or a variant thereof
comprising
up to about 3 amino acid substitutions; and
ii) a light chain variable domain comprising a light chain complementarity
determining region (LC-CDR) 1 comprising the amino acid sequence of N-I-G-S-
N/K or L-
R-S/N-X-Y (SEQ ID NO: 189 or 190), or a variant thereof comprising up to about
3 amino
acid substitutions, and an LC-CDR3 comprising the amino acid sequence of A/Q/N-
S/A/V-
W/Y/R-D-S/D-S-L/S/G-X-X-X-V (SEQ ID NO: 191), or a variant thereof comprising
up to
about 3 amino acid substitutions, wherein X can be any amino acid.
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[0569] Embodiment 19. In some further embodiments of any one of embodiments 1-
17, the
antibody moiety comprises:
i) a heavy chain variable domain comprising an HC-CDR1 comprising the amino
acid sequence of any one of SEQ ID NOs: 57-77, or a variant thereof comprising
up to about
amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any
one of
SEQ ID NOs: 78-98, or a variant thereof comprising up to about 5 amino acid
substitutions,
and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 99-
119,
244, and 245; or a variant thereof comprising up to about 5 amino acid
substitutions; and
ii) a light chain variable domain comprising an LC-CDR1 comprising the amino
acid sequence of any one of SEQ ID NOs: 120-140 and 246, or a variant thereof
comprising
up to about 5 amino acid substitutions, an LC-CDR2 comprising the amino acid
sequence of
any one of SEQ ID NOs: 141-161, or a variant thereof comprising up to about 3
amino acid
substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of
SEQ ID
NOs: 162-182 and 247-250; or a variant thereof comprising up to about 5 amino
acid
substitutions.
[0570] Embodiment 20. In some further embodiments of any one of embodiments 1-
17, the
antibody moiety comprises:
i) a heavy chain (HC) variable domain comprising an HC-CDR1 comprising the
amino acid sequence of any one of SEQ ID NOs: 57-77, an HC-CDR2 comprising the
amino
acid sequence of any one of SEQ ID NOs: 78-98, and an HC-CDR3 comprising the
amino
acid sequence of any one of SEQ ID NOs: 99-119, 244, and 245; or a variant
thereof
comprising up to about 5 amino acid substitutions in the HC-CDR regions; and
ii) a light chain (LC) variable domain comprising an LC-CDR1 comprising the
amino acid sequence of any one of SEQ ID NOs: 120-140 and 246, an LC-CDR2
comprising
the amino acid sequence of any one of SEQ ID NOs: 141-161, and an LC-CDR3
comprising
the amino acid sequence of any one of SEQ ID NOs: 162-182 and 247-250; or a
variant
thereof comprising up to about 5 amino acid substitutions in the LC-CDR
regions.
[0571] Embodiment 21. In some further embodiments of embodiment 19 or 20, the
antibody moiety comprises a) a heavy chain variable domain comprising the
amino acid
sequence of any one of SEQ ID NOs: 15-35 and 233-237, or a variant thereof
having at least
about 95% sequence identify to any one of SEQ ID NOs: 15-35 and 233-237; and
b) a light
chain variable domain comprising the amino acid sequence of any one of SEQ ID
NOs: 36-
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56 and 238-243, or a variant thereof having at least about 95% sequence
identity to any one
of SEQ ID NOs: 36-56 and 238-243.
[0572] Embodiment 22. In some further embodiments of embodiment 21, the
antibody
moiety comprises a heavy chain variable domain comprising the amino acid
sequence of any
one of SEQ ID NOs: 15-35 and 233-237 and a light chain variable domain
comprising the
amino acid sequence of any one of SEQ ID NOs: 36-56 and 238-243.
[0573] Embodiment 23. In some further embodiments of any one of embodiments 1-
22, the
isolated anti-E7MC construct is a full-length antibody.
[0574] Embodiment 24. In some further embodiments of any one of embodiments 1-
23, the
isolated anti-E7MC construct is monospecific.
[0575] Embodiment 25. In some further embodiments of any one of embodiments 1-
23, the
isolated anti-E7MC construct is multispecific.
[0576] Embodiment 26. In some further embodiments of embodiment 25, the
isolated anti-
E7MC construct is bispecific.
[0577] Embodiment 27. In some further embodiments of embodiment 25 or 26, the
isolated
anti-E7MC construct is a tandem scFv, a diabody (Db), a single chain diabody
(scDb), a dual-
affinity retargeting (DART) antibody, a dual variable domain (DVD) antibody, a
knob-into-
hole (KiH) antibody, a dock and lock (DNL) antibody, a chemically cross-linked
antibody, a
heteromultimeric antibody, or a heteroconjugate antibody.
[0578] Embodiment 28. In some further embodiments of embodiment 27, the
isolated anti-
E7MC construct is a tandem scFv comprising two scFvs linked by a peptide
linker.
[0579] Embodiment 29. In some further embodiments of embodiment 28, the
peptide linker
comprises the amino acid sequence GGGGS.
[0580] Embodiment 30. In some further embodiments of any one of embodiments 25-
29,
the isolated anti-E7MC construct further comprises a second antibody moiety
that
specifically binds to a second antigen.
[0581] Embodiment 31. In some further embodiments of embodiment 30, the second
antigen is an antigen on the surface of a T cell.
[0582] Embodiment 32. In some further embodiments of embodiment 31, the second
antigen is selected from the group consisting of CD3y, CD36, CD3c, CD3; CD28,
0X40,
GITR, CD137, CD27, CD4OL and HVEM.
[0583] Embodiment 33. In some further embodiments of embodiment 31, the second
antigen is CD3c, and wherein the isolated anti-E7MC construct is a tandem scFv
comprising
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an N-terminal scFv specific for the HPV16-E7/MHC class I complex and a C-
terminal scFv
specific for CD3E.
[0584] Embodiment 34. In some further embodiments of embodiment 31, the T cell
is
selected from the group consisting of a cytotoxic T cell, a helper T cell, and
a natural killer T
cell.
[0585] Embodiment 35. In some further embodiments of embodiment 30, the second
antigen is an antigen on the surface of a natural killer cell, a neutrophil, a
monocyte, a
macrophage or a dendritic cell.
[0586] Embodiment 36. In some further embodiments of any one of embodiments 1-
22, the
isolated anti-E7MC construct is a chimeric antigen receptor.
[0587] Embodiment 37. In some further embodiments of embodiment 36, the
chimeric
antigen receptor comprises an extracellular domain comprising the antibody
moiety, a
transmembrane domain, and an intracellular signaling domain comprising a CD3
intracellular signaling sequence and a CD28 intracellular signaling sequence.
[0588] Embodiment 38. In some further embodiments of any one of embodiments 1-
22, the
isolated anti-E7MC construct is an immunoconjugate comprising the antibody
moiety and an
effector molecule.
[0589] Embodiment 39. In some further embodiments of embodiment 37, the
effector
molecule is a therapeutic agent selected from the group consisting of a drug,
a toxin, a
radioisotope, a protein, a peptide, and a nucleic acid.
[0590] Embodiment 40. In some further embodiments of embodiment 39, the
therapeutic
agent is a drug or a toxin.
[0591] Embodiment 41. In some further embodiments of embodiment 38, the
effector
molecule is a label.
[0592] Embodiment 42. In some embodiments, there is provided a pharmaceutical
composition comprising the isolated anti-E7MC construct of any one of
embodiments 1-40.
[0593] Embodiment 43. In some embodiments, there is provided a host cell
expressing the
isolated anti-E7MC construct of any one of embodiments 1-41.
[0594] Embodiment 44. In some embodiments, there is provided a nucleic acid
encoding
the polypeptide components of the isolated anti-E7MC construct of any one of
embodiments
1-41.
[0595] Embodiment 45. In some embodiments, there is provided a vector
comprising the
nucleic acid of embodiment 44.
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[0596] Embodiment 46. In some embodiments, there is provided an effector cell
expressing
the isolated anti-E7MC construct of embodiment 36 or 37.
[0597] Embodiment 47. In some further embodiments of embodiment 46, the
effector cell
is a T cell.
[0598] Embodiment 48. In some embodiments, there is provided a method for
detecting a
cell presenting a complex comprising an HPV16-E7 peptide and an MHC class I
protein on
its surface, comprising contacting the cell with the isolated anti-E7MC
construct of
embodiment 41 and detecting the presence of the label on the cell.
[0599] Embodiment 49. In some embodiments, there is provided a method for
treating an
individual having an HPV16-E7-positive disease, comprising administering to
the individual
an effective amount of the pharmaceutical composition of embodiment 42.
[0600] Embodiment 50. In some embodiments, there is provided a method for
treating an
individual having an HPV16-E7-positive disease, comprising administering to
the individual
an effective amount of the effector cell of embodiment 46 or 47.
[0601] Embodiment 51. In some embodiments, there is provided a method of
diagnosing an
individual having an HPV16-E7-positive disease, comprising:
a) administering an effective amount of the isolated anti-E7MC construct of
embodiment 41 to the individual; and
b) determining the level of the label in the individual, wherein a level of
the label
above a threshold level indicates that the individual has the HPV16-E7-
positive disease.
[0602] Embodiment 52. In some embodiments, there is provided a method of
diagnosing an
individual having an HPV16-E7-positive disease, comprising:
a) contacting a sample derived from the individual with the isolated anti-E7MC
construct of embodiment 41; and
b) determining the number of cells bound with the isolated anti-E7MC construct
in
the sample, wherein a value for the number of cells bound with the isolated
anti-E7MC
construct above a threshold level indicates that the individual has the HPV16-
E7-positive
disease.
[0603] Embodiment 53. In some further embodiments of embodiments 49-52, the
HPV16-
E7-positive disease is HPV16-E7-positive cancer.
[0604] Embodiment 54. In some further embodiments of embodiment 53, the HPV16-
E7-
positive cancer is squamous cell carcinoma.
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[0605] Embodiment 55. In some further embodiments of embodiment 53, the HPV16-
E7-
positive cancer is cervical cancer, anogenital cancer, head and neck cancer,
or oropharyngeal
cancer.
EXAMPLES
[0606] Those skilled in the art will recognize that several embodiments are
possible within
the scope and spirit of this invention. The invention will now be described in
greater detail by
reference to the following non-limiting examples. The following examples
further illustrate
the invention but, of course, should not be construed as in any way limiting
its scope.
Materials
Cell Samples, Cell Lines, and Antibodies
[0607] The cell lines include: CaSki (ATCC CRL-1550; HLA-A2+, HPV16 ), head
and
neck squamous cell carcinoma cell line UM-SCC-104 (Millipore; HLA-A2+, HPV16
), liver
adenocarcinoma cell line SK-HEP-1 (ATCC HTB-52; HLA-A2+, HPV16-), cervical
squamous cell carcinoma cell line SiHa (ATCC HTB-35; HLA-A2-, HPV16 ),
cervical
cancer cell line C33A (ATCC HTB-31; HLA-A2+, HPV16-), cervical carcinoma cell
line
MS-751 (ATCC HTB-34; HLA-A2+, HPV16-), cervical adenocarcinoma cell line HeLa
(ATCC CCL-2; HLA-A2-, HPV16-), hepatocellular carcinoma cell line Hep3B (ATCC
HB-
8064; HLA-A2-, HPV16-), hepatocellular carcinoma cell line HepG2 (ATCC HB-
8065;
HLA-A2-, HPV16-), lymphoma cell line Raji (ATCC CCL-86; HLA-A2-, HPV16-), T
cell
leukemia cell line Jurkat (ATCC TIB-152; HLA-A2-, HPV16-), lymphoma cell line
Daudi
(ATCC CCL-213; HPA-A2-, HPV16-), leukemia cell line K562 (ATCC CCL-243; HLA-A2-
,
HPV16-), and lymphoblast cell line T2 (ATCC CRL-1992; HLA-A2+, HPV16-). T2 is
a TAP-
deficient cell line. The cell lines were cultured in RPMI 1640 supplemented
with 5% FCS,
penicillin, streptomycin, 2 mmol/L glutamine, and 2-mercaptoethanol at 37
C/5% CO2.
[0608] All peptides were purchased and synthesized by Genemed Synthesis, Inc.
(San
Antonio, Tex.). Peptides were >90% pure. The peptides were dissolved in DMSO
and diluted
in saline at 5 mg/mL and frozen at -180 C. Biotinylated single chain HPV16-E7
peptide/HLA-A*02:01 and control peptides/HLA-A*02:01 complexes were
synthesized by
refolding the peptides with recombinant HLA-A02 and beta-2 microglobulin (02M)
(-2M).
11 control peptides (SEQ ID NOs: 193-203) that bind HLA-A*02:01 were generated
from
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the following 10 genes: BCR, BTG2, CALR, CD247, CTSG, DDX5, HLA-E, IFI30,
PPP2R1B, SSR1.
Example 1. Production of biotinylated HPV16-E7/HLA-A*02:01 complex monomer
[0609] Biotinylated HPV16-E7/HLA-A*02:01 complex monomers were prepared
according to standard protocols (John D. Altman and Mark M. Davis, Current
Protocols in
Immunology 17.3.1-17.3.33, 2003). In brief, DNA encoding full-length human
beta-2
microglobulin (02m) was synthesized by Genewiz and cloned into vector pET-27b.
The BirA
substrate peptide (BSP) was added to the C-terminus of HLA-A*02:01
extracellular domain
(ECD). DNA encoding HLA-A*02:01 ECD-BSP was also synthesized by Genewiz and
cloned into vector pET-27b. The vectors expressing human f32m and HLA-A*02:01
ECD-
BSP were transformed into E.coli BL21 cells separately, and expressed proteins
were isolated
as inclusion bodies from bacterial culture. Peptide ligand HPV16-E7 peptide 11-
19 was
refolded with human f32m and HLA-A*02:01 ECD-BSP to form HPV16-E7 peptide/HLA-
A*02:01 complex monomer. Folded peptide/HLA-A*02:01 monomers were concentrated
by
ultrafiltration and further purified through size-exclusion chromatography.
HiPrep 26/60
Sephacryl S-300 HR was equilibrated with 1.5 column volumes of Hyclone
Dulbecco's
Phosphate Buffered Saline solution (Thermo Scientific, Cat No. 5H3002802). The
unpurified
sample was loaded and eluted with 1 column volume. The first peak,
corresponding to
misfolded aggregates, eluted at approximately 111 mL, the peak corresponding
to the
properly folded MHC complex was observed at 212 mL, and the peak corresponding
to free
132M was observed at 267 mL (FIG. 1). Peptide/HLA-A*02:01 monomers were
biotinylated
via BirA-mediated enzymatic reaction and subsequently purified by high-
resolution anion-
exchange chromatography. Biotinylated peptide/HLA-A*02:01 monomers were stored
in
PBS at -80 C.
[0610] SDS-PAGE of the purified HPV16-E7 peptide/MHC complex can be performed
to
determine protein purity. For example, li.tg of the protein complex is mixed
with 2.5i.tL of the
NuPAGE LDS Sample Buffer (Life Technologies, NP0008) and brought up to 10i.tL
with
deionized water. The sample is heated at 70 C for 10 minutes, and then loaded
onto the gel.
Gel electrophoresis is performed at 180V for 1 hour.
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Example 2. Selection and Characterization of scFv Specific for HPV16-E7/HLA-
A*02:01 Complexes.
[0611] A collection of human scFv antibody phage display libraries (diversity
= 10x1010)
constructed by Eureka Therapeutics was used for the selection of human mAbs
specific to
HPV16-E7/HLA-A*02:01. 15 fully human phage scFv libraries were used to pan
against
HPV16-E7/HLA-A*02:01 complex. In order to reduce the conformational change of
MHCI
complex introduced by immobilizing the protein complex onto plastic surfaces,
solution
panning and cell panning were used in place of conventional plate panning. In
solution
panning, biotinylated antigens were first mixed with the human scFv phage
library after
extended washing with PBS buffer, and then antigen-scFv antibody phage
complexes were
pulled down by streptavidin-conjugated Dynabeads M-280 through a magnetic
rack. The
bound clones were then eluted and used to infect E.coli XL1-Blue cells. In
cell panning, T2
cells loaded with HPV16-E7 peptide were first mixed with the human scFv phage
library. T2
cells are a TAP-deficient, HLA-A*02:01+ lymphoblast cell line. To load
peptide, T2 cells
were pulsed with peptides (5Oug/m1) in serum-free RPMI1640 medium in the
presence of 20
i.t.g/m1 132M overnight. After extended washing with PBS, peptide-loaded T2
cells with bound
scFv antibody phage were spun down. The bound clones were then eluted and used
to infect
E.coli XL1-Blue cells. The phage clones expressed in bacteria were then
purified. The
panning was performed for 3-4 rounds with either solution panning, cell
panning or a
combination of solution and cell panning to enrich for scFv phage clones that
bound HPV16-
E7/ HLA-A*02:01 specifically.
[0612] Streptavidin ELISA plates were coated with biotinylated HPV16-E7 11-19
peptide/HLA-A*02:01 complex monomer or biotinylated C3 control peptide/HLA-
A*02:01
monomer respectively. C3 peptide is human nuclear protein p68-derived peptide
YLLPAIVHI (SEQ ID NO: 193). Individual phage clones from enriched phage
display
panning pools against HPV16-E7 11-19 peptide/HLA-A*02:01 complex were
incubated in
the coated plates. Binding of the phage clones was detected by HRP-conjugated
anti-M13
antibodies and developed using HRP substrate. The absorbance was read at
450nm. 145
positive clones were identified through ELISA screening of 1272 phage clones
enriched from
phage panning. FIG. 2 provides an example of phage clone binding to
biotinylated HPV16-
E7/HLA-A*02:01 monomer in an ELISA assay. 41 unique clones were identified by
DNA
sequencing of the 145 ELISA-positive phage clones. Specific and unique clones
were further
tested for their binding to HLA-A*02:01/peptide complexes on live cell surface
by flow
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cytometry (FACS analysis) using HPV16-E7-loaded live T2 cells. FIG. 3
demonstrates
binding of exemplar positive clones to HPV16-E7 11-19 peptide-loaded T2 cells
or control
peptide (C3 peptide)-loaded T2 cells. Controls included T2 cells without
peptide loading (cell
only) and R-PE conjugated horse anti-mouse IgG control (secondary antibody
only). In brief,
T2 cells loaded with HPV16-E7 11-19 peptide or C3 peptide were first stained
with purified
scFv phage clones, followed by a second staining with mouse anti-M13 mAb, and
a third
staining with R-PE conjugated horse anti-mouse IgG from Vector Labs. Each
staining step
was performed for between 30-60 minutes on ice and the cells were washed twice
between
stainings. Among the 41 clones tested, 24 recognized HPV16-E7-loaded T2 cells
specifically.
These 24 phage clones specifically bound to HPV16-E7-loaded T2 cells and did
not
recognize T2 cells loaded with C3 peptide in the context of HLA-A*02:01, or T2
cells
without peptide loaded.
Example 3. Characterization of FACS-positive HPV16-E7-specific phage clones
Cross-reactivity to HPV16-E7 peptide 11-19 variants
[0613] The HPV16-E7 11-19 peptide selected in this invention is highly
conserved among
various HPV16 strains. In one study, this peptide was found to be fully
conserved in 16 of 17
known HPV16-E7 protein sequences. A single amino acid mutation within E7 11-19
was
identified in the remaining sequence. This variant was then found in 1 of 35
HPV16 infected
cervical cancer or cervicitis patients analyzed (Zhang G.L. et al., Database
(Oxford).
2014:bau031, 2014). Clones selected from FACS binding analysis against HPV16-
E7 peptide
11-19-loaded T2 cells are characterized further for cross-reactivity towards
this HPV16-E7
peptide 11-19 variant/HLA-A*02:01 complex on live cell surfaces by FACS
analysis using
variant HPV16-E7 peptide-loaded live T2 cells. The variant peptide differs
from the
conserved HPV16-E7 11-19 peptide by a single amino acid at position 5. The
variant peptide
sequence is YMLDVQPET (SEQ ID NO: 11), while the conserved HPV16-E7 11-19
sequence is YMLDLQPET (SEQ ID NO: 4).
[0614] In brief, T2 cells are loaded with the conserved HPV16-E7 11-19
peptide, the
variant HPV16-E7 11-19 peptide, or 132M. The peptide-loaded T2 cells are
stained with
purified scFv phage clones, followed by a second staining with a mouse anti-
M13 mAb and a
third staining with R-PE conjugated horse anti-mouse IgG from Vector Labs.
Each staining
step is performed on ice for 30-60 minutes and the cells are washed twice
between each
staining.
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Epitope mapping by alanine walking
[0615] To investigate with precision the epitope for the mAb recognition,
HPV16-E7
peptides with alanine substitutions at positions 1, 5 and 8 were pulsed onto
T2 cells.
Antibody phage clones were then tested for binding to these peptide-loaded T2
cells by
FACS analysis. FACS mean fluorescent intensity (MFI) values of each FACS assay
are
shown in table 6. K07 helper phage is a negative control showing that the
phage alone
without scFv presentation on the phage particle surface does not bind to any
of the peptide-
loaded T2 cell groups. Antibody BB7.2 recognizes the HLA-A02 alpha chain.
Peptide
binding to MHC complex stabilizes cell-surface MHC complexes. Therefore, T2
cells loaded
with MHC-binding peptide have enhanced BB7.2 binding signal compared to T2
cells
without peptide loading (as shown in the first row of table 6). BB7.2 binding
data indicate
that the alanine-substituted peptides are still able to bind HLA-A*02:01
molecules on the T2
cell surface. Although all the antibodies tested recognized the small
conformational epitope
formed by the HPV16-E7 peptide and its surrounding MHC alpha chain residues,
the key
peptide residues interacting with the various antibodies were quite different.
For example,
clone #4 is predicted to bind to the N-terminal half of the HPV16-E7 peptide
since alanine
substitution at position 1 or 5 dramatically reduced binding to the peptide-
loaded T2 cells. In
contrast, alanine substitutions at position 8 did not change the binding of
the same clone to
the HLA-A*02:01 complex. Clone #11, on the other hand, was insensitive to
alanine
substitution at position 1 and 8, but alanine substitution at position 5
completely abrogated its
binding. FIG. 4 provides an example of FACS analysis, showing the binding of
phage clone
#11 to T2 cells loaded with the various HPV16-E7 peptides. Controls included
T2 cells
without peptide loading (cell only) and R-PE conjugated horse anti-mouse IgG
control
(secondary antibody only). Antibody binding to T2 cells loaded with these
different peptides
were tested by FACS assays.
Table 6
7:7:7:7:7:7:7:7:7:7:7:7:7:7:7:7:7:71:7507: type ifipa g7:7=MT. :T.
77:7:7:7:7:7:7:7:7:7:7:7:7:7:7:7:7:7:7:7:7:7:7. :T. :T. :T. :T. :T. :T. :73
A
d
1 fff ilhe substitution 'at ' "Al:ffine substitution : W t Ai 1
lanine substitutiori:W
peptie ........ ......
.... ...
... ...... ....
...
ii A01:#k ii YMLDLCIPET (FAM:
MR position 1 position 5 Po
.:.:.:AlyILDLCIPET (FACS MR) . . YMLDAQP ET (FACS MR) .YMLDLCIPsAitTio(Fn 8
172 *1"
ACS ::1
1_
...... "::.:.:.:. =....,::::::
Antibody
1456 1501 1205 1500 1040
1367.2 i
___.....................____ ____.................._....-
...................____
1 K07 Helper
9.76 9.71 , 10.3 10 NA
Phage
: .......
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,
clone #1 665 12.1 89.1 792 NA
.........................................................................
....õ"
,
clone #2 i 238 31.6 11.1 50.7 NA
clone #3 201 106 12.9 13.4 NA
;
clone #4 310 13 10.7 107 NA
clone #5 228 86.7 10.9 9.64 NA
:
,
clone #8 1 1528 287 11 2050 NA ¨
clone #9 1966 2511 607 19.4 NA
clone #11 2006 1423 10.8 2321 NA
. õ ---
,
clone #17 43 22.9 10.3 11.6 NA
clone #22 239 101 10.3 9.42 NA
..........................................
........................................... ............................._
,
clone #26 318 696 59.8 19.2 NA
.- ......................................................................
....õ"
clone #27 1777 2123 67.7 13.5 NA
.¨.1
,
clone #31 2280 1867306 17 NA
,
clone #40 i 724 653 L 10.5 559 NA
Antibody binding specificity evaluation against endogenous peptide
[0616] On average, each nucleated cell in the human body expresses about half
a million
different peptide/MHC Class I complexes. In order to develop anti-peptide/MHCI-
complex
antibodies into anti-cancer drugs with high specificity and therapeutic index,
it is essential for
the antibodies to specifically recognize the target peptide/MHCI complex, but
not the MHCI
molecule itself, or MHCI molecules bound to other peptides presented on cell
surfaces. For
the current study, the relevant MHCI molecule is HLA-A*02:01. During the early
stages of
our phage panning and screening, we eliminated antibodies that bound to the
HLA-A*02:01
molecule alone (see, for example, FIGs. 2 and 3). The top phage clones were
also screened
against 11 endogenous HLA-A*02:01 peptides, which were derived from proteins
normally
expressed in multiple types of nucleated human cells, such as globin alpha
chain, beta chain,
nuclear protein p68, and the like. Recombinant peptide/HLA-A*02:01 complexes
folded with
11 endogenous peptides (Table 7, SEQ ID NOs: 193-203) separately were coated
on
streptavidin plates and antibody binding was determined through ELISA
analysis. In brief,
individual phage clones were incubated on the peptide/HLA-A*02:01 complex-
coated plates.
Binding of the phage clones was detected by HRP-conjugated anti-M13 antibodies
and
developed using HRP substrate. The absorbance was read at 450nm. As shown in
FIG. 5, the
HPV16-E7 peptide/HLA-A*02:01-specific antibody phage clones bound HPV16-E7
peptide/HLA-A*02:01 complex, but not HLA-A*02:01 complexes folded with
endogenous
peptides. We conclude that the identified antibodies are specific to HPV16-E7
peptide/HLA-
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A*02:01 complexes, and do not recognize HLA-A*02:01 molecules bound to other
HLA-
A*02:01-restricted peptides.
Table 7
Peptide Code Peptide Sequence GenelD Symbol
Description
A2E-1 LLDVPTAAV 10437 1F130 interferon, gamma-inducible
protein 30
A2E-2 TLWVDPYEV 7832 BTG2 BTG family, member 2
A2E-3 FLLDHLKRV 613 BCR breakpoint cluster region
A2E-4 LLLDVPTAAV 10437 1F130 interferon, gamma-inducible
protein 30
A2E-5 VLFRGGPRGLLAV 6745 SSR1 signal sequence receptor,
alpha
A2E-6 SLLPAIVEL 5519 PPP2R1B protein phosphatase 2, regulatory
subunit
A, beta
A2E-7 (C3 YLLPAIVHI 1655 DDX5 DEAD (Asp-Glu-Ala-Asp)
box helicase 5
peptide)
A2E-8 FLLPTGAEA 1511 CTSG cathepsin G
A2E-9 LLDPKLCYLL 919 CD247 CD247 molecule
A2E-11 MLLSVPLLLG 811 CALR calreticulin
A2E-17 MVDGTLLLL 3133 HLA-E major histocompatibility complex,
class I, E
Example 4. Engineering bispecific antibodies
[0617] Bispecific antibodies (BsAbs) were generated using scFv sequences of
the HPV16-
E7/HLA-A*02:01-specific phage clones. The BsAbs are single-chain bispecific
antibodies
comprising the scFv sequence of an HPV16-E7/HLA-A*02:01-specific phage clone
at the N-
terminal end and an anti-human CD3E mouse monoclonal scFv at the C-terminal
end
(Brischwein, K. et al., Molecular Immunology 43:1129-1143, 2006). DNA
fragments coding
for the HPV16-E7 scFv and the anti-human CD3E scFv were synthesized by Genewiz
and
subcloned into Eureka's mammalian expression vector pGSN-Hyg using standard
DNA
techniques. A hexhistamine tag was inserted at the C-terminal end for antibody
purification
and detection. Chinese hamster ovary (CHO) cells were transfected with the
BsAb expression
vector, and then cultured for 7 days for BsAb antibody production. CHO cell
supernatants
containing secreted HPV16-E7 BsAb molecules were collected. BsAbs were
purified using
HisTrap HP column (GE healthcare) by FPLC AKTA system. Briefly, CHO cell
culture was
clarified and loaded onto the column with low imidazole concentration (20 mM),
and then an
isocratic high imidazole concentration elution buffer (500 mM) was used to
elute the bound
BsAb proteins. Purity and molecular weight of the purified HPV16-E7 BsAbs was
determined under reducing conditions by gel electrophoresis. 4i.tg of the
protein was mixed
with 2.5i.tL of the NuPAGE LDS Sample Buffer (Life Technologies, NP0008) and
brought
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up to 10i.tL with deionized water. The sample was heated at 70 C for 10
minutes, and then
loaded onto the gel. Gel electrophoresis was performed at 180V for 1 hour. ¨50
KD bands are
observed as the major bands on the gel (FIG. 6).
[0618] Antibody aggregation can be assessed by size-exclusion chromatography
(SEC).
For example, 50i.tL of the sample is injected into a SEC column (for example
Agilent, BioSEC-3,300A, 4.6x300mm) while flowing a buffer consisting of
Dulbecco's
Phosphate Buffered Saline (Fisher Scientific, 5H30028.FS) and 0.2M arginine
adjusted to pH
7Ø BsAbs with high molecular weight aggregation less than 10% are selected
for further
characterization.
Example 5. Characterization of HPV16-E7 BsAb antibodies
Binding affinity of HPV16-E7 BsAb antibodies
[0619] The binding affinity of HPV16-E7 BsAb antibodies to recombinant HPV16-
E7/HLA-A*02:01 complex is measured, for example, by Surface Plasma Resonance
(BiaCore). The binding parameters between the HPV16-E7 BsAb and the HPV16-
E7/HLA-
A*02:01 complex are measured, for example, using a His Capture Kit (GE
Healthcare, Cat#
28995056) on a Biacore X100 (GE Healthcare) according to the manufacturer's
protocol for
multi-cycle kinetics measurement. All of the proteins used in the assay are
diluted using
HBS-E buffer. For example, 1 i.t.g/mL of the HPV16-E7 BsAb is immobilized onto
a Sensor
Chip pre-functionalized with the anti-histidine antibody by flowing the
solution through the
flow cell 2 at 2 lL/min for 2 minutes. Binding towards the HPV16-E7/A*02:01
complex is
analyzed at, for example, 0.19, 0.38, 7.5, 15, and 30 i.t.g/mL, each run
consisting of a 3 minute
association and 3 minute dissociation at 30 lL/min. At the end of cycle, the
surface is
regenerated using the regeneration buffer from the His Capture kit. Following
the kinetics
measurement, the surface is regenerated using the regeneration solution from
the kit. The data
are analyzed using 1:1 binding site mode with the BiaCore X-100 evaluation
software. The
binding parameters (association on rate constant ka, dissociation constant kd,
and equilibrium
dissociation constant Kd) are then calculated.
T-cell killing assay with peptide-pulsed T2 cells
[0620] Tumor cytotoxicity was assayed by LDH Cytotoxicity Assay (Promega).
Human T
cells purchased from AllCells were activated and expanded with CD3/CD28
Dynabeads
(Invitrogen) according to manufacturer's protocol. Activated T cells (ATC)
were cultured and
maintained in RPMI1640 medium with 10% FBS plus 30 U/ml IL-2, and used at day
7-14.
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The T cells were > 99% CD3+ by FACS analysis. Activated T cells (effector
cells) and target
peptide-loaded T2 cells were co-cultured at a 5:1 ratio with 1 t.g/m1 or 0.2
t.g/m1 of BsAbs
for 16 hours. Peptide-loaded T2 cells were prepared by incubating T2 cells
overnight with 50
i.t.g/m1 of either the target HPV16-E7 11-19 peptide (YMLDLQPET, SEQ ID NO: 4)
or
negative control AFP158 peptide (FMNKFIYEI, SEQ ID NO: 192). A negative
control
AFP158/HLA-A*02:01 specific BsAb was also included. Cytotoxicities were
determined by
measuring LDH activities in culture supernatants. As shown in FIG. 7, clones
#2, #5, #17 and
#40 killed the HPV16-E7 peptide-loaded T2 cells in a selective and dose-
dependent manner.
The negative control anti-AFP158/HLA-A*02:01 BsAb specifically directed
killing of the
AFP158 peptide-loaded T2 cells.
T-cell killing assay with cancer cell lines
[0621] Tumor cytotoxicity was assayed by LDH Cytotoxicity Assay (Promega).
Human T
cells were purchased from AllCells and activated and expanded with CD3/CD28
Dynabeads
(Invitrogen) according to manufacturer's protocol. Activated T cells (ATC)
were cultured and
maintained in RPMI1640 medium with 10% FBS plus 30 U/ml IL-2, and used at day
7-14.
Activated T cells (effector cells) and target cancer cells were co-cultured at
a 5:1 ratio with
different concentrations of BsAbs (including for example 0.2, 0.04, 0.008, and
0.0016 t.g/m1
BsAbs) for 16 hours. Cytotoxicities were then determined by measuring LDH
activities in
culture supernatants.
[0622] Target cancer cells tested included cervical cancer cell line CaSki
(ATCC CRL-
1550; HLA-A2+, HPV16+) and cervical carcinoma cell line MS-751 (ATCC HTB-34;
HLA-
A2+, HPV16-). As shown in FIG. 8A, multiple clones (e.g. 2, 41 and US-7)
mediated
selective killing of HPV-16+ cell line CaSki vs HPV-16- cell line MS-751 at
0.2 i.t.g/m1
BsAb. The BsAb-mediated killing of target-positive CaSki is dose-dependent as
shown in
FIG. 8B.
[0623] Additional target cancer cells that may be tested include head and neck
squamous
cell carcinoma cell line UM-SCC-104 (Millipore; HLA-A2+, HPV16+), liver
adenocarcinoma
cell line SK-HEP-1 (ATCC HTB-52; HLA-A2+, HPV16-), cervical squamous cell
carcinoma
cell line SiHa (ATCC HTB-35; HLA-A2-, HPV16+), cervical cancer cell line C33A
(ATCC
HTB-31; HLA-A2+, HPV16-), cervical adenocarcinoma cell line HeLa (ATCC CCL-2;
HLA-
A2-, HPV16-), hepatocellular carcinoma cell line Hep3B (ATCC HB-8064; HLA-A2-,
HPV16-), hepatocellular carcinoma cell line HepG2 (ATCC HB-8065; HLA-A2-,
HPV16-),
lymphoma cell line Raji (ATCC CCL-86; HLA-A2-, HPV16-), T cell leukemia cell
line
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Jurkat (ATCC TIB-152; HLA-A2-, HPV16-), lymphoma cell line Daudi (ATCC CCL-
213;
HPA-A2-, HPV16-), and leukemia cell line K562 (ATCC CCL-243; HLA-A2-, HPV16-).
Cross-reactivity of HPV16-E7 BsAb antibodies against multiple HLA-A02 alleles
[0624] Human MHCI molecules consist of 6 class isoforms, HLA-A, -B, -C, -E, -F
and G.
The HLA-A, -B and-C heavy chain genes are highly polymorphic. For each
isoform, the
HLA genes are further grouped according to the similarity of heavy chain
sequences. For
example, HLA-A is divided into different alleles such as HLA-A01, -A02, -A03,
etc. For the
HLA-A02 allele, there are multiple subtypes, such as HLA-A*02:01, A*02:02,
etc. Between
the different subtypes of HLA-A02 group, the sequence differences are limited
to only
several amino acids. So in many cases, peptides that bind to HLA-A*02:01
molecule can also
form complexes with multiple subtypes of the HLA-A02 allele. As shown in Table
8
(http://www.allelefrequencies.net/), although HLA-A*02:01 is the dominant HLA-
A02
subtype among Caucasian populations, in Asia, A*02:05, A*02:06, A*02:07 and
A*02:11
are also common HLA-A02 subtypes. The ability of HPV16-E7 antibodies to
recognize not
only HPV16-E7 peptide in the context of HLA-A*02:01, but also other subtypes
of HLA-
A02, will greatly broaden the patient population that might be able to benefit
from HPV16-
E7 antibody drug treatment. To determine cross-reactivity, HPV16-E7/MHC class
I
complexes with other subtypes of the HLA-A02 allele are generated and the
binding affinity
of the HPV16-E7/HLA-A*02:01-specific antibodies for these other complexes is
tested.
Binding affinity is determined, for example, using a ForteBio Octet QK.
Briefly, 5 i.t.g/mL
biotinylated HPV16-E7 peptide/HLA-A02 MHC complex having varying subtypes is
loaded
onto a streptavidin biosensor. After washing off excess antigen, BsAbs are
tested at, for
example, 10 i.t.g/mL for association and dissociation kinetics. Binding
parameters are
calculated using a 1:1 binding site, partial fit model.
Table 8
australia china europe india north sub- taiwan us
africa saharan
africa
A*02:01
97.8% 39.5% 94.0% 53.9% 73.3% 56.3% 35.1% 79.4%
A*02:02 0.0% 0.1% 0.3% 0.9% 9.7% 24.1%
0.0% 3.6%
A*02:03
0.0% 15.3% 0.2% 4.9% 0.0% 0.4% 19.3% 2.2%
A*02:04 0.0% 0.1% 0.0% 0.3% 2.6% 0.4%
0.0% 0.2%
A*02:05
1.1% 0.9% 3.2% 5.8% 13.8% 15.9% 0.1% 4.5%
A*02:06
0.0% 16.0% 0.9% 10.6% 0.0% 0.7% 12.8% 5.5%
A*02:07
1.1% 26.1% 0.4% 0.4% 0.0% 0.0% 32.7% 2.4%
A*02:08 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
0.0% 0.0%
A*02:09 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
0.0% 0.0%
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A*02:10 0.0% 1.1% 0.0% 0.0% 0.0% 0.2%
0.0% 0.1%
A*02:11
0.0% 0.1% 0.1% 22.3% 0.0% 1.5% 0.0% 1.7%
other A02 subtypes 0.0% 0.7% 0.8% 0.9% 0.5% 0.5%
0.0% 0.6%
(A*02:12 - A*02:93)
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
Example 6. Generation of HPV16-E7/HLA-A*02:01 specific chimeric antigen
receptor-
presenting T cells (CAR-T)
[0625] Chimeric antigen receptor therapy (CAR-T therapy) is a new form of
targeted
immunotherapy. It merges the exquisite targeting specificity of monoclonal
antibodies with
the potent cytotoxicity and long-term persistence provided by cytotoxic T
cells. This
technology enables T cells to acquire long-term novel antigenic specificity
independent of the
endogenous TCR. Clinical trials have shown clinically significant antitumor
activity of CAR-
T therapy in neuroblastoma (Louis C.U. et al., Blood 118(23):6050-6056), B-ALL
(Maude
S.L. et al., N. Engl. J. Med. 371(16):1507-1517, 2014), CLL (Brentjens R.J. et
al., Blood
118(18):4817-4828, 2011), and B cell lymphoma (Kochenderfer J.N. et al.,
Blood.
116(20):4099-4102, 2010). In one study, a 90% complete remission rate in 30
patients with
B-ALL treated with CD19-CAR T therapy was reported (Maude S.L. et al., supra).
[0626] To further explore the potency of the HPV16-E7/HLA-A*02:01 specific
antibodies,
HPV16-E7 scFv expressing CARs are constructed and transduced into T cells. For
example,
HPV16-E7/HLA-A*02:01 specific CARs are constructed using a lentiviral CAR
expression
vector. Anti-HPV16-E7/HLA-A*02:01 scFvs are grafted onto a second generation
CAR
(Mackall C.L. et al., Nat. Rev. Clin. Oncol. 11(12):693-703, 2014) with CD28
signaling
domain and TCK engineered in cis to provide intracellular T cell stimulation
signals and to
activate T cells. FIG. 9 provides a schematic illustration of an HPV16-E7 CAR
construct.
Example 7. Characterization of HPV16-E7 CAR-T cells
In vitro Cytotoxicity study of HPV16-E7 CAR-T cells
[0627] Lentiviruses containing HPV16-E7/HLA-A*02:01 specific chimeric antigen
receptors are produced, for example, by transfection of 293T cells with CAR
vectors. Human
T-cells are used for transduction after 2-day stimulation with CD3/CD28 beads
(Dynabeads , Invitrogen) in the presence of interleukin-2 at 30 U/ml.
Concentrated
lentiviruses are applied to T-cells in Retronectin (Takara) coated 6-well
plates for 72 hours.
Transduction efficiency is assessed by FACS using biotinylated HPV16-E7
tetramer and PE-
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conjugated streptavidin. Repeat FACS analyses are done at 72 hours and every 3-
4 days
thereafter.
[0628] Functional assessment of the transduced T cells (HPV16-E7 CAR-T cells)
is
performed using LDH Cytotoxicity Assay. Effector-to-target ratios used
include, for
example, 5:1 and 10:1. The target cell lines may include, for example,
cervical cancer cell
line CaSki (ATCC CRL-1550; HLA-A2+, HPV16 ), head and neck squamous cell
carcinoma
cell line UM-SCC-104 (Millipore; HLA-A2+, HPV16 ), liver adenocarcinoma cell
line SK-
HEP-1 (ATCC HTB-52; HLA-A2+, HPV16-), cervical squamous cell carcinoma cell
line
SiHa (ATCC HTB-35; HLA-A2-, HPV16 ), cervical cancer cell line C33A (ATCC HTB-
31;
HLA-A2+, HPV16-), cervical carcinoma cell line MS-751 (ATCC HTB-34; HLA-A2+,
HPV16-), cervical adenocarcinoma cell line HeLa (ATCC CCL-2; HLA-A2-, HPV16-),
hepatocellular carcinoma cell line Hep3B (ATCC HB-8064; HLA-A2-, HPV16-),
hepatocellular carcinoma cell line HepG2 (ATCC HB-8065; HLA-A2-, HPV16-),
lymphoma
cell line Raji (ATCC CCL-86; HLA-A2-, HPV16-), T cell leukemia cell line
Jurkat (ATCC
TIB-152; HLA-A2-, HPV16-), lymphoma cell line Daudi (ATCC CCL-213; HPA-A2-,
HPV16-), and leukemia cell line K562 (ATCC CCL-243; HLA-A2-, HPV16-). As a
control,
SK-HEP-1-MiniG is generated by transducing SK-HEP-1 with an HPV16-E7 peptide
expressing minigene cassette, which results in a high level of cell surface
expression of
HPV16-E7/HLA-A*02:01 complex. The specificity and efficiency of the HPV16-E7
CAR
expressing T cells to kill the target-positive cancer cells is determined.
Example 8. Generation and Characterization of the full-length IgG1 HPV16-E7
antibodies
[0629] Full-length human IgG1 of the selected phage clones are produced, for
example, in
HEK293 and Chinese hamster ovary (CHO) cell lines, as described (Tomimatsu K.
et al.,
Biosci. Biotechnol. Biochem. 73(7):1465-1469, 2009). In brief, antibody
variable regions are
subcloned into mammalian expression vectors, with matching human lambda or
kappa light
chain constant region and human IgG1 constant region sequences. Applying the
same cloning
strategy, chimeric HPV16-E7 full-length antibodies with mouse IgG1 heavy chain
and light
chain constant regions are generated. Molecular weight of the purified full
length IgG
antibodies is measured under both reducing and non-reducing conditions by
electrophoresis.
SDS-PAGE of purified HPV16-E7 mouse chimeric IgG1 antibodies is performed to
determine protein purity. In brief, 2i.tg of the protein is mixed with 2.5i.tL
of NuPAGE LDS
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Sample Buffer (Life Technologies, NP0008) and brought up to 10i.tL with
deionized water.
The sample is heated at 70 C for 10 minutes, and then loaded onto the gel. Gel
electrophoresis is performed at 180V for 1 hour.
[0630] HPV16-E7 chimeric IgG1 antibody is tested for binding towards HPV16-E7
presenting SK-HEP-1 cells by flow cytometry. SK-HEP-1 is an HLA-A*02:01
positive and
HPV16-E7 negative cell line. An HPV16-E7 minigene cassette is transfected into
SK-HEP-1
cells to generate the HPV16-E7-presenting SK-HEP-1-miniG cells. 10 i.t.g/mL of
antibody is
added to cells on ice for 1 hour. After washing, R-PE conjugated anti-mouse
IgG(H+L)
(Vector Labs#EI-2007) is added to detect antibody binding. Binding affinity of
the mouse
chimeric IgG1 HPV16-E7 antibodies is determined by ForteBio Octet QK. 5
i.t.g/mL
biotinylated HPV16-E7 peptide/HLA-A*02:01 complex is loaded onto a
streptavidin
biosensor. After washing off excess antigen, mouse chimeric full-length
antibodies are tested
at 10 i.t.g/mL for association and dissociation kinetics. Binding parameters
are calculated
using a 1:1 binding site, partial fit model.
[0631] HPV16-E7-specific and negative control (such as ET901) mouse chimeric
IgG1 are
tested for binding towards HPV16-E7/HLA-A*02:01, HPV16-E7 recombinant protein
and
free HPV16-E7 peptide in an ELISA assay. Antibodies are tested, for example,
at 3x serial
dilution, starting from 100 ng/mL for a total of 8 concentrations.
Biotinlyated HPV16-
E7/A*02:01 MHC is coated onto streptavidin plates at 2 i.t.g/mL, HPV16-E7
protein is coated
at 2 i.t.g/mL and HPV16-E7 peptide is coated at 40 ng/mL. The ability of full-
length anti-
HPV16-E7/HLA-A*02:01 antibodies to recognize the HPV16-E7 peptide only in the
context
of HLA-A02, and not bind recombinant HPV16-E7 protein or free HPV16-E7 peptide
is
determined.
Example 9. In vivo efficacy studies
HPV16-E7 CAR-T cell treatment in mice
[0632] HLA-A02 /HPV16-E7+ cancer cell line (such as CaSki or UM-SCC-104) sub-
cutaneous (s.c.) xenograft models are generated in SCID-beige (no functional T-
, B-, NK-
cells) mice. Animals are randomized when average s.c. tumor volume reaches 200
mm3. 24
hours prior to CART administration, animals are treated (via intraperitoneal
route) with 60
mg/kg cyclophosphamide. Mice are divided into 4 groups (n=8-10 mice/group)
that receive
one of the following: (i) no treatment (ii) 107 mock transduced CAR T cells,
lx/week for 4
weeks (iii) 107 anti-E7MC CAR T cells, lx/week for 4 weeks, or (iv) 2x106 anti-
E7MC CAR
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T cells, lx/week for 4 weeks. The animals in each group are monitored for
tumor volume,
adverse response, human cytokine profile, histopathology of tumor for human
CD3+ cells in
tumor and organs for CAR T cell infiltration, serum HPV16-E7, body weight and
general
health condition (eating, walking, daily activities).
Example 10. Affinity Maturation of anti-HPV16-E7 antibody agents
[0633] This example demonstrates the affinity maturation of anti-HPV16-E7
antibody
agents. In particular, this example specifically demonstrates the generation
of a series of
antibody variants by incorporation of random mutations into a representative
anti-HPV16-E7
antibody agent (clone US-7) followed by screening and characterization of the
antibody
variants.
Generation of variant phage libraries
[0634] DNA encoding anti-HPV16-E7 clone US-7 scFv was subjected to random
mutagenesis using GeneMorph II Random Mutagenesis kit (Agilent Technologies)
according
to the manufacturer's specifications. After mutagenesis, DNA sequences were
cloned into an
scFv-expressing phagemid vector to build a variant human antibody phage
library which
contained about 5x108 unique phage clones. On average, variant clones have two
nucleotide
mutations compared with the parental anti-HPV16-E7 clone, ranging from 1 to 4
nucleotide
mutations, per scFv sequence.
Cell panning
[0635] The human phage scFv library with mutants generated from clone US-7 was
used to
pan against HPV16-E7 11-19 peptide/HLA-A*02:01 complex as described in Example
2. In
particular, cell panning was used. Human scFv phage library was first mixed
with T2 cells
loaded with 50 ug/ml of a pool of 20 different endogenous peptides (P20, SEQ
ID NOs: 193-
212) as negative control panning. The negative control-depleted human scFv
phage library
was then mixed with T2 cells loaded with HPV16-E7 11-19 peptide (1.5 ug/ml
first round,
0.8 ug/ml second round, 0.4 ug/ml third round). To load peptide, T2 cells were
pulsed with
peptides in serum-free RPMI1640 medium in the presence of 20 t.g/m1 132M
overnight. After
extended washing with PBS, peptide-loaded T2 cells with bound scFv antibody
phage were
spun down. The bound clones were then eluted and used to infect E.coli XL1-
Blue cells. The
phage clones expressed in bacteria were then purified. The panning was
performed for 3
rounds to enrich for scFv phage clones that bound HPV16-E7 11-19 peptide/HLA-
A*02:01
specifically.
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[0636] Streptavidin ELISA plates were coated with biotinylated HPV16-E7 11-19
peptide/HLA-A*02:01 complex monomer or biotinylated P20 control peptides/HLA-
A*02:01 monomer. Individual phage clones from enriched phage display panning
pools
against HPV16-E7 11-19 peptide/HLA-A*02:01 complex were incubated in the
coated
plates. Binding of the phage clones was detected by HRP-conjugated anti-M13
antibodies
and developed using HRP substrate. The absorbance was read at 450nm. 15
positive clones
were identified through ELISA screening of 135 phage clones enriched from
phage panning.
11 unique clones were identified by DNA sequencing of the 15 ELISA-positive
phage clones.
Specific and unique clones were further tested for their binding to HLA-
A*02:01/peptide
complexes on live cell surface by flow cytometry (FACS analysis) using HPV16-
E7 11-19
peptide-loaded live T2 cells. Controls included T2 cells without peptide
loading (cells only)
and R-PE conjugated horse anti-mouse IgG control (secondary antibody only).
Briefly, T2
cells loaded with HPV16-E7 11-19 peptide or P20 peptide pool were first
stained with
purified scFv phage clones, followed by a second staining with mouse anti-M13
mAb, and a
third staining with R-PE conjugated horse anti-mouse IgG from Vector Labs.
Each staining
step was performed for 30-60 minutes on ice and the cells were washed twice
between
staining steps. Among the 10 unique clones tested, 8 recognized HPV16-E7 11-19-
loaded T2
cells specifically (SEQ ID NOs: 223-230). These 8 phage clones specifically
bound to
HPV16-E7 11-19-loaded T2 cells and did not recognize T2 cells loaded with P20
peptide
pool in the context of HLA-A*02:01, or T2 cells without peptide loaded.
Example 11. Characterization of bi-specific antibody molecules based on anti-
HPV16-
E7 affinity maturation variants
Generation of bispecific antibodies
[0637] Bispecific antibodies (BsAbs) were generated using scFv sequences of
the affinity
matured HPV16-E7/HLA-A*02:01-specific phage clones isolated in Example 10 (SEQ
ID
NOs: 223-232) using the method described in Example 4. The resulting single-
chain
bispecific antibodies comprised the scFv sequence of an HPV16-E7/HLA-A*02:01-
specific
phage clone at the N-terminal end and an anti-human CD3E mouse monoclonal scFv
at the C-
terminal end.
Determination of binding affinities of bispecific antibodies to HPV16-E7/HLA-
A*02:01
[0638] The binding affinity of 7 HPV16-E7 BsAb antibodies (derived from
affinity
matured clones 7-1, 7-3, 7-6, 7-7, 7-8, and 7-9, corresponding to SEQ ID NOs:
223, 225, and
227-230, respectively, and parental clone US-7) to recombinant HPV16-E7/HLA-
A*02:01
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complex was measured by Surface Plasma Resonance (BiaCore). The binding
parameters
between the HPV16-E7 BsAbs and HPV16-E7/HLA-A*02:01 complex were measured
using
a Biotin CAPture Kit (GE Healthcare, Cat# 28920233) on a Biacore X100 (GE
Healthcare)
according to the manufacturer's protocol for multi-cycle kinetics measurement.
All of the
proteins used in the assay were diluted using HBS-EP running buffer. 5
i.t.g/mL of
biotinylated HPV16-E7 11-19/HLA-A*02:01/02M complex was immobilized onto a
Sensor
Chip CAP pre-functionalized with streptavidin (-3,800 RU of streptavidin
captured) by
flowing the solution through the flow cell at 5 lL/min for 75 seconds (-120 RU
of MHC
complex was captured per cycle). Binding towards the HPV16-E7 BsAbs was
analyzed at
150 nM, 75 nM, 37.5 nM, 18.8 nM, and 9.4 nM, each run consisting of a 2 minute
association
and 10 minute dissociation at 30 lL/min. At the end of cycle, the surface was
regenerated
using the regeneration buffer from the Biotin CAPture kit. The data was
analyzed using 1:1
binding site mode with the BiaCore X-100 evaluation software. The binding
parameters
(association on rate constant ka, dissociation constant kd, and equilibrium
dissociation
constant Kd) were then calculated, and are shown in Table 9. BsAbs derived
from clones 7-1,
7-3, 7-6, 7-7, and 7-9 showed increased binding affinity compared to the BsAb
derived from
parental clone US-7, while the BsAb derived from clone 7-8 showed decreased
binding
affinity.
Table 9
Protein ka [1/Ms] kd [1/s] KD [nM]
4 -3
Clone # US-7 BsAb 3.46x10 1.93x10 55.6
-3
Clone # 7-1 BsAb 7.29x10 5.14x10 7.0
5 -3
Clone # 7-3 BsAb 1.67x10 1.37x10 8.2
5 -3
Clone # 7-6 BsAb 1.30x10 1.60x10 12.1
5 -3
Clone #7-7 BsAb 1.04x10 1.22x10 11.7
4 -3
Clone # 7-8 BsAb 4.58x10 2.66x10 58.0
5 -4
Clone # 7-9 BsAb 1.37x10 8.23x10 6.0
Cross-reactivity and binding affinities of HPV]6-E7 bispecific antibodies
against multiple
HIA-A02 alleles
[0639] As described above in Example 5, the different subtypes of the HLA-A02
group are
quite conserved, and cross-reactivity against multiple HLA-A02 subtypes is
highly desired.
Therefore, experiments were performed to determine whether the bi-specific
antibodies
(BsAb) generated from the parental clone US-7 and the affinity maturation
variants cross-
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react with non-HLA-A*02:01 subtypes of the HLA-A02 group. Specifically, HPV16-
E7 11-
19/MHC class I complexes with various subtypes of the HLA-A02 allele were
generated, and
their binding affinities to the HPV16-E7 11-19/HLA-A*02:01-specific antibodies
were
determined using the Octet QKe System by Pall ForteBio LLC (Menlo Park, CA),
which
utilizes the Biolayer Interferometry (BLI) technology. The BsAbs tested
include the parental
clone US-7 and affinity maturation variant clones 7-1, 7-3, 7-6, 7-7, 7-8, and
7-9. Five i.t.g/mL
biotinylated HPV16-E7 11-19 peptide/HLA-A02 complex having varying subtypes of
HLA-
A02 was loaded onto a streptavidin biosensor. After washing off excess antigen
(the HPV16-
E7 peptide/HLA-A02 complex), BsAbs were tested at 10 i.t.g/mL for association
and
dissociation kinetics. Binding parameters were calculated using a 1:1 binding
site, partial fit
model. The results of the BLI assay are shown below in Table 10.
Table 10. HLA-A02 subtype binding parameters
Binding Affinity (KanMil
BsAb
A0201 A0202 A0203 A0205 A0206 A0207 A0211
Clone #US-7 51.5 92.1 25.4 80.4
Clone #7-1 45.3 13.5 43 14.9 39.3
Clone #7-3 42.4 14.4 41.4 14.6 37
Clone #7-6 58.5 12 47.6 10.7 43.1
Clone #7-7 32.8 14.8 53.2 17.7 63.4
Clone #7-8 99.7 62.1 22 90.7
Clone #7-9 21.7 15.3 38.6 10.6 38.2
"-" indicates no detectable specific binding
[0640] The results show that each of the tested antibody clones specifically
bound to at
least four out of the seven HLA-A02 subtypes tested. Several affinity
maturation variant
antibodies bound to more HLA-A02 subtypes than the parental clone did, and
many of the
affinity maturation variant antibodies bound to HPV16-E7 11-19/HLA-A02 with
higher
affinities than the parental clone did, indicated by lower KD values.
Peptide binding specificity assay
[0641] In order to confirm the specificity of the peptide recognized by the
affinity
maturation variant antibodies, a FACS analysis was performed with T2 cells
loaded with
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HPV16-E7 11-19 peptide, the P20 peptide pool, or no peptide. The result showed
that all the
BsAbs tested, including the parental clone US-7 and affinity maturation
variant clones 7-1, 7-
3, 7-6, 7-7, 7-8, and 7-9, specifically bound to T2 cells loaded with HPV16-E7
11-19 peptide,
but did not bind to T2 cells loaded with the P20 peptide pool or not loaded
with any peptide.
The FACS data is shown herein in Table 12.
Epitope mapping by alanine walking
[0642] To investigate with precision the sensitive residues of the HPV16-E7 11-
19 peptide
for recognition by BsAbs Clone # US-7 and its affinity maturation variants, a
series of mutant
peptides were designed and synthesized to have each of the nine residues
substituted with
alanine (shown in Table 11).
Table 11. HPV16-E7 11-19 wild-type and mutant peptides
Ala Substitution SEQ
ID NO
Peptide ID Peptide sequence
Position
HPV16-E7 11-19 YMLDLQPET 4
HPV16-E7 11-19mutl AMLDLQPET 1 12
HPV16-E7 11-19mut2 YALDLQPET 2 258
HPV16-E7 11-19mut3 YMADLQPET 3 259
HPV16-E7 11-19mut4 YMLALQPET 4 260
HPV16-E7 11-19mut5 YMLDAQPET 5 13
HPV16-E7 11-19mut6 YMLDLAPET 6 261
HPV16-E7 11-19mut7 YMLDLQAET 7 262
HPV16-E7 11-19mut8 YMLDLQPAT 8 14
HPV16-E7 11-19mut9 YMLDLQPEA 9 263
[0643] BsAb clones were then tested by FACS analysis for binding to T2 cells
loaded with
the peptides from Table 11, T2 cells loaded with the negative control P20
peptide pool, or T2
cells with no peptide loaded. Peptide binding to MHC on the T2 cells was
assessed by BB7.2
mouse antibody staining. All peptides except for HPV16-E7 11-19mut8 showed
greater
BB7.2 antibody binding compared to control T2 cels without peptide (data not
shown),
indicated that all peptides except for HPV16-E7 11-19mut8 were successfully
able to bind
MHC on the T2 cells. For BsAb analysis, cells were stained with 10 i.t.g/mL
BsAb followed
by 20X dilution of APC anti-His antibody (R&D System #IC050A). FACS mean
fluorescent
intensity (MFI) values of each FACS assay are shown in Table 12. The parental
US-7 and
affinity matured scFvs are predicted to bind to the C-terminal half of the
HPV16-E7 peptide
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since alanine substitution at positions 5-8 generally reduced binding to the
peptide-loaded T2
cells. The individual FACS binding assay results are shown in FIGS. 10A and
10B.
Table 12. Alanine walking of HPV16-E7 11-19 (FACS, mean fluorescent intensity)
BsAb Sample Clone # Clone # Clone # Clone # Clone # Clone # Clone #
US-7 7-1 7-3 7-6 7-7 7-8 7-9
T2withnapeptue 14 14 201
P20 160 180 156 163 195 164 247
HPV16E7 1149 (wild- 24000 27000 25700 24100 20S00 22400 17600
HPV16-E7 11-19mut1 20700 23100 19700 21000 18900 19100
15500
HPV16-E7 11-19mut3 18100 24100 20300 20600 18200 17700
16100
HPV16-E7 11-19mut5 6274 13400 11900 11300 8452 8242 9660
HPV16-E7 11-19-mut7 7575 14000 13900 11600 9650 9199
10100
HPV16-E7 11-19-mut8 237 733 703 211 740 293 1135
HPV16-E7 11-19-mut9 19200 20900 19900 22900 18500 19700
16100
...............................................................................
...............................................................................
...............................................................................
....
...............................................................................
...............................................................................
...............................................................................
....
Sensitive Position* 5,6,7,8 5,8 5,8 5,7,8 5,7,8 5,6,7,8
5,8
...............................................................................
...............................................................................
...............................................................................
....
Reduced binding 6,7 6.7 6 6 6.7
position**
*: Sensitive position: <50% MFI (T2 loaded with wild-type HPV16-E7 11-19)
**: Reduced binding: 50-75% MFI (T2 loaded with wild-type HPV16-E7 11-19)
Example 12. Generation of chimeric antigen receptor-presenting T cells (CAR-T)
with
anti-HPV16-E7 affinity maturation scFvs variants
[0644] Chimeric antigen receptors expressing affinity matured HPV16-E7 scFvs
were
constructed and transduced into T cells. CAR vectors for production of
lentivirus encoding
the HPV16-E7/HLA-A*02:01 specific CARs were constructed by fusing an affinity
matured
scFv isolated in Example 10 (7-1, 7-3, 7-6, 7-7, 7-8, and 7-9, corresponding
to SEQ ID NOs:
223, 225, and 227-230, respectively) or the parental US-7 scFv with either a
polypeptide
having CD28 and CD3t signaling domains (SEQ ID NO: 256) or a polypeptide
having 4-
1BB and CD3t signaling domains (SEQ ID NO: 257).
[0645] Lentiviruses containing the HPV16-E7/HLA-A*02:01 specific chimeric
antigen
receptors were produced by transfection of 293T cells with the CAR vectors.
Human T-cells
were used for transduction after 1-day stimulation with CD3/CD28 beads
(Dynabeads ,
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Invitrogen) in the presence of interleukin-2 at 100 U/ml. Concentrated
lentiviruses were
applied to T-cells in Retronectin (Takara) coated 6-well plates for 72 hours.
Five days after
transduction, HPV16-E7 CAR-T cells and mock-transduced cells were co-stained
with
biotinylated HPV16-E7 11-19 peptide/HLA-A*02:01 tetramers and CD4 or CD8
antibodies
and analyzed by flow cytometry. FIGS. 11A-11C show flow cytometry results for
HPV16-E7
CAR-T cells and mock-transduced cells. Results are summarized in Table 13.
Each of the
tested CARs with an affinity matured scFv had higher transduction efficiency
than the CAR
with the parental scFv.
Table 13
Clone % Tetramer+
Mock 0.12
US-7 4-1BB/CD3 39.8
US-7 CD28/CD3 43.0
7-1 4-1BB/CD3 69.6
7-1 CD28/CD3 67.9
7-3 4-1BB/CD3 74.4
7-3 CD28/CD3 70.5
7-6 4-1BB/CD3 64.3
7-6 CD28/CD3 68.3
7-7 4-1BB/CD3 68.4
7-7 CD28/CD3 62.9
7-8 4-1BB/CD3 71.2
7-8 CD28/CD3 63.6
7-9 4-1BB/CD3 77.3
7-9 CD28/CD3 66.1
Example 13. T-cell killing assay using CAR-T with anti-HPV16-E7 affinity
maturation
variants with cancer cell lines
In vitro Cytotoxicity study of HPV16-E7 CAR-T cells
[0646] Tumor cytotoxicity of T cells expressing a CAR having an affinity
matured scFv (7-
1, 7-3, 7-6, 7-7, 7-8, and 7-9, corresponding to SEQ ID NOs: 223, 225, and 227-
230,
respectively) or the parental US-7 scFv was assayed by LDH Cytotoxicity Assay
(Promega).
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Human T cells purchased from AllCells were activated, then transduced with the
CAR and
expanded with CD3/CD28 Dynabeads (Invitrogen) according to manufacturer's
protocol.
Activated CAR-T cells were cultured and maintained in RPMI1640 medium with 10%
FBS
plus 100 U/ml IL-2, and used at day 7-14. Activated CAR-T cells (effector
cells) and target
cancer cells were co-cultured at a 5:1 ratio for 16 hours. Cytotoxicities were
then determined
by measuring LDH activities in culture supernatants.
[0647] Target cancer cells tested included cervical cancer cell line CaSki
(ATCC CRL-
1550; HLA-A2+, HPV16 ), cervical carcinoma cell line MS-751 (ATCC HTB-34; HLA-
A2+,
HPV16-), and mantle cell lymphoma cell line JeKo-1 (ATCC CRL-3006; HLA-A2+,
HPV16-
). As shown in FIG. 12, the affinity matured clones (7-1, 7-3, 7-6, 7-7, 7-8,
and 7-9)
demonstrated significantly increased killing activity in both CD28/CD3 and 4-
1BB/CD3
formats compared to the parental clone US-7.
[0648] Additional target cancer cells that may be tested include head and neck
squamous
cell carcinoma cell line UM-SCC-104 (Millipore; HLA-A2+, HPV16 ), liver
adenocarcinoma
cell line SK-HEP-1 (ATCC HTB-52; HLA-A2+, HPV16-), cervical squamous cell
carcinoma
cell line SiHa (ATCC HTB-35; HLA-A2-, HPV16 ), cervical cancer cell line C33A
(ATCC
HTB-31; HLA-A2+, HPV16-), cervical adenocarcinoma cell line HeLa (ATCC CCL-2;
HLA-
A2-, HPV16-), hepatocellular carcinoma cell line Hep3B (ATCC HB-8064; HLA-A2-,
HPV16-), hepatocellular carcinoma cell line HepG2 (ATCC HB-8065; HLA-A2-,
HPV16-),
lymphoma cell line Raji (ATCC CCL-86; HLA-A2-, HPV16-), T cell leukemia cell
line
Jurkat (ATCC TIB-152; HLA-A2-, HPV16-), lymphoma cell line Daudi (ATCC CCL-
213;
HPA-A2-, HPV16-), and leukemia cell line K562 (ATCC CCL-243; HLA-A2-, HPV16-).
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Sequence Listing
SEQ ID NO: 1, HPV16 E7 protein
MHGDTPTLHEYMLDLQPETTDLYCYEQLNDS SEEEDEIDGPAGQAEPDRAHYNIVTF
CCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP
SEQ ID NO: 2, HPV16 E7 CDS
ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGA
CAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATG
AAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAATATTG
TAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACA
CGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGC
CCCATCTGTTCTCAGAAACCATAA
SEQ ID NO: 3, HPV16 E7 7-15
TLHEYMLDL
SEQ ID NO: 4, HPV16 E7 11-19
YMLDLQPET
SEQ ID NO: 5, HPV16 E7 16-25
QPETTDLYCY
SEQ ID NO: 6, HPV16-E7 44-52
QAEPDRAHY
SEQ ID NO: 7, HPV16-E7 46-55
EPDRAHYNIV
SEQ ID NO: 8, HPV16-E7 49-57
RAHYNIVTF
SEQ ID NO: 9, HPV16 E7 82-90
LLMGTLGIV
SEQ ID NO: 10, HPV16 E7 86-93
TLGIVCPI
SEQ ID NO: 11, HPV16 E7 11-19 variant
YMLDVQPET
SEQ ID NO: 12, HPV16 E7 11-19 Al
AMLDLQPET
SEQ ID NO: 13, HPV16 E7 11-19 AS
YMLDAQPET
SEQ ID NO: 14, HPV16 E7 11-19 A8
212
TZ
CI 11A3H '17Z :ON III OHS
SSAIAIIDODAk
HCIDIVAASDIADNVDAAAVIGHSNISNIHIAIAVISISICILLIIANDNANOVICIIND
NAISDADIA&HIDODdIVONAA&SIDACISAIADSINDSANASVDdNNAHVDSONIOAO
H 11A3H 'CZ :ON III OHS
SSAIA1
IDODA&ACIACIVAANONVDAAAVIGHVNISNIAIMAISNNVNCINSILDIDNASCIVAM
ISSSSSIASAAkTIONDdlOINAA&NIAISASSAdADSVVDSINOSNOcIOAADDDSONIOAO
OI 11A3H `ZZ :ON III OHS
SSAIAIIDO
DAOCRAIIAIDA&ASANVDAAAVIGHVNISNIAIOIXISNNVNCINSILDIDNASCIAAANHS
DCIONINVAAkTIONDdIVONAA&SIAMASSAIADSVIVDSINISDOdOAIDDDSHAIOAH
6 11A3H 'IZ :ON III OHS
SSAIAIIDODAU
CRAIMAIXADVAASNVDAAAVICICISNISNIHIAIAVISISICILLIALLANDOINOVANIND
NSVSIA&DIAIA&HIDODdIVONAA&NIDANVAIADSVNDSANASVD(INNAHVDSONIOAH
8 11A3H 'OZ :ON III OHS
SSAIAIID
Opmaansx0AwsPIVDAAAVIGHSNIDSIMAIHVISISVCIVILLANDOANOVACIdi
DdIdIRIDIAMTIDODdVONAA&SAVASNAIDDSVNDSANASSD(INNAHVDSONIOAO
S 11A3H '61 :ON III OHS
SSAIAIIDO
DA&NCHASDASDHIAINVDAAAVIGHVNISNIAIMAIINNSNCINSILDIDNASCIVAANN
IDGASIAVAAkTIONDdIVONAA&HIVASSAIADSVIVDSINISNWOAADDDSONIOAO
1711A3H '81 :ON III OHS
SSAIAII
DODA&SCISHAkAkAldONVDAAAVICICISNISSIHIAIAVISISNCIVIAIANDOAHOVACId
IDIIAMIRIDIAMTIDODdIVONAA&SIVVIAISAIDDSVNDSANASSD(INNAHVDSONIOAH
C 11A3H `LI :ON III OHS
SSAIAIIDO
DA&HCIAdVSOSADAADNVDAAAVICESNISSIHIAIAVISISHCIVILLANDOANOVANV
IDdIdIIDDIAIA&HIDODdIVONAA&SIVASSAIDDSVNDSANASSD(DINAHVDSONIOAO
Z 11A3H '91 :ON III OHS
SSAIAIIDO
DA&ACINADDAXASNVDAAAVICICISIISSIHIAIAVIIISICIRLIALLANDOANOVANIDD
SMINDADIAMTIDODdIVONAAMIXACRAIADSVNDSANASVDdNNAHVDSOAIOAO
1 11A3H `SI :ON III OHS
IIMOICIHATA
179I0/9IOZSI1LIDcl Li6Z81/910Z OM
SO-ZT-LTOZ L6886Z0 VD
17TZ
SSAINI
IDODMSGAADMAD2121VDAAAVIGHVNISNIAIMAISN)IVNG2ISIIANDNASGVAAI
ASSSSSISSAMTIMIDdIVONAMNIAISASSAIADSVVDSINISOD(DINIDDDSONIOVO
IV 11A311 'CC :ON CR OHS
SSAINIIDODM
S (IMAM SANAANNVDAAAVICICI S21121S1HIAIAAINI SICIRLIALLANDOAN OVAHIAD
INdNIMDIAIMTIDOWDONIMAIAAGSASADSVIDSANASVDd)DIAHVDSONIOAH
0.17 11A311 `Z :ON CR OHS
SSAINI
IDODM)IGASDAWIVDAAAVIGHVNISNIAIMAIIN)ISNMISIALLANDHASGSAAIN
DDSOSIVSAMTIMIDdVONAMNIAIVI-121SASADSVIVDSINISDOdONIDDDSHNIOAO
6 11A311 'IC :ON CR OHS
SSAINI
IDODMIGSDMIAJDNVDAAAVIGHSNISSIHIAIAVISIS)IGIVIIIANDOANOVANV
IDIMIRIDIAIMTIDODdIVONAMSIVASSAIDDSVMDSANASSOd)DIAHVDSONIOAH
Z 11A311 '0 :ON CR OHS
SSAINIID
ODMAGII-IDMAD2121VDAAAVIGHVNINNIAMAASN)IVNG2ISIIDIMIASGAAANAS
DGMIINDAMTIMIDdIVONAMSIAIMASSAIADSVVDSINISODdONIDDDSONIOAO
1 11A311 '6Z :ON III OHS
SSAINII
DODMSGAVSMAS2121VDAAAVIGHVNISNIAIMAIIN)ISNMISIIDIMIASGVAAIS
DDSOSIVSAMTIMIDdIVONAMSIAIVASSAIADSVVDSINISDWONIDDDSHNIOAH
LZ 11A311 '8Z :ON III OHS
SSAINIIDOD
MAGIAIIDAOHMONVDAAAVIGGS21121SIHIAIAVISISIGNIIALLANDOANOVANIDDS
NdNIMDIAIMTIDODdIVONAMHIAIAADIAIADSVMDSANASVD(DDIAHVDSONIOAO
9Z 11A311 'LZ :ON III OHS
SSAINI
IDODMAGHDASASHMIVDAAAVIGHVNISNIAIMAISN)IVNGNSIIANDHASVIVAA
IIDDdSIASIMTIMIDdIVONIMIIAIAAGSANADSVIVDSINISOD(DINIDDDSHNIOAH
ZZ 11A311 '9Z :ON III OHS
SSAINII
DOomsamoSMOXISDNVDAAAVICESNISSITIAAISISNCIVSTLANDOANOdNELL
IGINdIRIDIAIMadDODdIVONAMSAVA)RIAIGDMIDSANASSOd)RIAHVDSONIOAO
LI 11A311 `SZ :ON III OHS
SSAIA
IIDODMAGNAMIVDAAAVIGGS211S2MIAIAVISISIGIIIALLANDOMIOVANIND
NAdSIMDIAIMTIDODdIVONAMSIDASIAIADMIDSANASVDd)DIAHVDSONIOAO
179I0/9IOZSI1LIDcl Li6Z81/910Z OM
SO-ZT-LTOZ L6886Z0 VD
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SEQ ID NO: 34, HCVR US-5
EVQLVQS GAEVKKPGS SVKVSCKAS GGTFS SYGISWVRQAPGQGLEWMGGIIPIFGT
TNYAQKFQGRVTITADESTRTAYMELSSLRSEDTAVYYCARVYGS YFYDDWGQGTL
VTVSS
SEQ ID NO: 35, HCVR US-7
QVQLQQWGAGLLKPSETLS LTCAVYGGSFS GYYWSWIRQPPGKGLEWIGEINHS GST
NYNPSLKSRVTIS VDTS KNQFSLKLS S VTAADTAVYYCARAPQSWYVGDVWGQGTL
VTVSS
SEQ ID NO: 36, LCVR 1
QAVLTQPPS AS GTPGQRVTISCS GS S SNIGTNPVTWYQHLPGTAPKLLIYGNYQRPS G
VPDRFS GS KS GTS AS LAIS GLQSEDEADYYCAAWDDS LTGYVFGTGTKVTVLG
SEQ ID NO: 37, LCVR 2
QS VLTQPPS LS VAPGKTASITCGGNNIGMKS VHWYQQRPGQAPVLVIYYDSDRPS GIP
GRFS GSNS GNTATLTISRVEAGDEADYYCQVWDTTGDHPGYVFGTGTKVTVLG
SEQ ID NO: 38, LCVR 3
QS VVTQPPS VS AAPGQKVTISCS GS S SNIGNNYVSWYQHFPGAAPKLLIYDNYKRPS G
IPDRFS GS KS GTS ATLGITGLQTGDEAEYYCGTWDS SLS AYVFGTGTKVTVLG
SEQ ID NO: 39, LCVR 4
SYELTQPPSVSVAPGKTARITCGGNNIGS KSVHWYQQKPGQAPVLVIYYNSDRPS GIP
ERFS GSNS GNTATLTISRVEAGDEADYYCQVWDS S SDHWVFGGGTKLTVLG
SEQ ID NO: 40, LCVR 5
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSG
VPSRFS GS GS GTDFTLTIS S LQPEDFATYYCQQANSFPLTFGGGTKVEIKR
SEQ ID NO: 41, LCVR 8
QSVLTQPPSASGTPGQRVTISCSGHNSNIETNPVSWYQQVPGTTPKWYSYYYRPSG
VPDRFS GS KS GTSAS LAIS GLQSEDEADYFCASWDDSVQGYVFGS GTKVTVLG
SEQ ID NO: 42, LCVR 9
S SELTQDPAVS VALGQTVRITCQGDS LRS YYASWYQQKPGQAPVLVIYGKNNRPS GI
PDRFS GS S S GNTASLTITGAQAEDEADYYCNSRDS S GNHQVFGTGTKVTVLG
SEQ ID NO: 43, LCVR 10
QSVLTQPPSVSVAPGKTARITCGGNNIGS KNVHWYQQKPGQAPVLVVYDDHDGPS G
IPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLG
SEQ ID NO: 44, LCVR 11
QAVLTQPPS AS GTPGQRVTISCS GS S SNIGINTVNWYQQLPGTAPKLLIYGNSHRPS G
VPDRFS GS KS GASASLAITGLQADDEGAYFCQSYDTS LTVIFGGGTKLTVLG
SEQ ID NO: 45, LCVR 13
QTVVTQEPS LTVSPGGTVTLTCAS S TGAVTS GYYPNWFQQKPGQAPRALIYS TNNKH
SWTPARFSGSLLGGKAALTLSGVQPEDEAEYYCLLYYGGAQLWVFGGGTKLTVLG
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SEQ ID NO: 46, LCVR 17
DIQMTQSPS S LSAS VGDRVTITCRAS QS IS SYLNWYQQKPGQAPNLLIYGASSFQS GV
PSRFS GS GS GTDFTLTIS SLQPEDFATYYCQQSYSAPLTFGGGTKVDIKR
SEQ ID NO: 47, LCVR 22
QAVLTQPPSAS GTPGQRVTISCS GS S SNIGSNS VNWYQQVPGTAPKLLIYSNNQRPS G
VPDRFS GS KS GTSAS LAITKLQSEDEADYYCAAWDDS LNGYVFGTGTKVTVLG
SEQ ID NO: 48, LCVR 26
QSVLTQPPSVS GAPGQRVTISCTGS S SNIGAGYDVHWYQQLPGTAPKLLIYESNNRPS
GVPDRFS GS KS GTSASLAITGLQAEDEADYYCQSYDTSLKGVFGPGTKVTVLG
SEQ ID NO: 49, LCVR 27
S SELTQDPAVS VALGQTVTITCQGDILRNYYATWYQQKPGQAPSLVLYGQNNRPS GI
PDRFS GSTS GNTASLTITGAQAEDEGDYYCDSRDS S GNHWVFGGGTKLTVLG
SEQ ID NO: 50, LCVR 31
SSELTQDPALSVALGQTVTITCRGDNLRNS YASWYQQKPGQAPVVVLYGKNRRPS GI
PDRFS GS S S GNTASLTITGAQADDEADYYCNSRDS S GFPPYVFGPGTKVTVLG
SEQ ID NO: 51, LCVR 32
SYVLTQPPSVSVSPGQTASITCS GDKLGEKYVYWYQQRPGQSPVLVIFQNDKRPS GIP
ERLS GSRS GNTATLTIS GTQAMDEADYYCQSWAS S S VYVFGTGTKLTVLG
SEQ ID NO: 52, LCVR 39
S SELTQDPAVS VALGQTVRITCQGDS LRNYYASWYQQKPGQAPVLVLYGKDNRPS G
IPDRFS GS S S GNTAS LTITGAQAEDEADYYCNSRDS S GNHRVFGGGTKLTVLG
SEQ ID NO: 53, LCVR 40
QAVLTQPPS AS GTPGQRVTISCS GS S SNIGSNHVNWYQQLPGTAPKLLIS NNNQRPS G
VPDRFS GS KS GTSAS LAIS GLQSEDEADYYCAAWDDS LNVPVFGGGTQLTVLS
SEQ ID NO: 54, LCVR 41
S SELTQDPAVS VALGQTVRITCQGDTLRSFYASWYQQKPGQAPVLVFYGKNNRPS GI
PERFS GSNSENTAS LTIAGAQAEDEADYYCNSRDS GGDPVVFGGGTKLTVLG
SEQ ID NO: 55, LCVR US-5
QAVVTQEPSLTVSPGGTVTLTCAS STGAVTS SYYPNWFQQKPGQAPRALIYSTSNTH
SWTPARFSGSLLGGKAALTVSGVQPEDEAEYYCLLYYGGARVFGGGTKLTVLG
SEQ ID NO: 56, LCVR US-7
S YELTQPPS VS GTPGQRVAISCS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCAAWDDNLKS YVFGTGTKVTVLG
SEQ ID NO: 57, HC-CDR1 1
GYTFIDYY
SEQ ID NO: 58, HC-CDR1 2
216
CA 02988397 2017-12-05
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PCT/US2016/031364
GGTFSSYA
SEQ ID NO: 59, HC-CDR1 3
GGTFSMAA
SEQ ID NO: 60, HC-CDR1 4
GFTFSSYA
SEQ ID NO: 61, HC-CDR1 5
GGTFRSYA
SEQ ID NO: 62, HC-CDR1 8
GYTFANYG
SEQ ID NO: 63, HC-CDR1 9
GFTFSSYW
SEQ ID NO: 64, HC-CDR1 10
GFPFS SYS
SEQ ID NO: 65, HC-CDR1 11
GYTFSDYG
SEQ ID NO: 66, HC-CDR1 13
GYTFTSYG
SEQ ID NO: 67, HC-CDR1 17
GDIFRKFA
SEQ ID NO: 68, HC-CDR1 22
GFNFSDYY
SEQ ID NO: 69, HC-CDR1 26
GYTFTGYY
SEQ ID NO: 70, HC-CDR1 27
GFTFSSYA
SEQ ID NO: 71, HC-CDR1 31
GFTFSSYW
SEQ ID NO: 72, HC-CDR1 32
GGTFSSYA
SEQ ID NO: 73, HC-CDR1 39
GFSFSRHA
SEQ ID NO: 74, HC-CDR1 40
GYSFSDYY
217
CA 02988397 2017-12-05
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PCT/US2016/031364
SEQ ID NO: 75, HC-CDR1 41
GFTFSSYS
SEQ ID NO: 76, HC-CDR1 US-5
GGTFSSYG
SEQ ID NO: 77, HC-CDR1 US-7
GGSFSGYY
SEQ ID NO: 78, HC-CDR2 1
INPKSGGT
SEQ ID NO: 79, HC-CDR2 2
IIPIFGTA
SEQ ID NO: 80, HC-CDR2 3
IIPMIGIP
SEQ ID NO: 81, HC-CDR2 4
ISYDGTNK
SEQ ID NO: 82, HC-CDR2 5
IIPIFGTP
SEQ ID NO: 83, HC-CDR2 8
ISASNGNT
SEQ ID NO: 84, HC-CDR2 9
INQDGSEK
SEQ ID NO: 85, HC-CDR2 10
ISSSSSTI
SEQ ID NO: 86, HC-CDR2 11
ISTYNGNT
SEQ ID NO: 87, HC-CDR2 13
ISPYNGNT
SEQ ID NO: 88, HC-CDR2 17
IIPNLDIT
SEQ ID NO: 89, HC-CDR2 22
ISPGGTT
SEQ ID NO: 90, HC-CDR2 26
INPNSGGT
218
CA 02988397 2017-12-05
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SEQ ID NO: 91, HC-CDR2 27
ISGSGGST
SEQ ID NO: 92, HC-CDR2 31
IKEDGSVK
SEQ ID NO: 93, HC-CDR2 32
IIPILGIA
SEQ ID NO: 94, HC-CDR2 39
ISGSGGNT
SEQ ID NO: 95, HC-CDR2 40
INPNTGVT
SEQ ID NO: 96, HC-CDR2 41
ISSSSSYI
SEQ ID NO: 97, HC-CDR2 US-5
IIPIFGTT
SEQ ID NO: 98, HC-CDR2 US-7
INHSGST
SEQ ID NO: 99, HC-CDR3 1
ARSYYYGGVKDY
SEQ ID NO: 100, HC-CDR3 2
ARGVYGYSGSAPYDE
SEQ ID NO: 101, HC-CDR3 3
ARGPLYWWHSDS
SEQ ID NO: 102, HC-CDR3 4
ARMHGSYGSYFDK
SEQ ID NO: 103, HC-CDR3 5
ARSSMYQYSHDD
SEQ ID NO: 104, HC-CDR3 8
ARSYYAGYYMFMDF
SEQ ID NO: 105, HC-CDR3 9
ARYSVWGMMDE
SEQ ID NO: 106, HC-CDR3 10
ARQNYYADYDY
SEQ ID NO: 107, HC-CDR3 11
219
CA 02988397 2017-12-05
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ARGYTGSYYATGDE
SEQ ID NO: 108, HC-CDR3 13
ARGYKDY
SEQ ID NO: 109, HC-CDR3 17
ARGSIYQWSGWDS
SEQ ID NO: 110, HC-CDR3 22
ARGHSFSYGEDY
SEQ ID NO: 111, HC-CDR3 26
ARGWHQYGIMDY
SEQ ID NO: 112, HC-CDR3 27
ARRSVWSAYDS
SEQ ID NO: 113, HC-CDR3 31
ARRGVWGHIDF
SEQ ID NO: 114, HC-CDR3 32
ARGTYTWGSDT
SEQ ID NO: 115, HC-CDR3 39
ARYWGSYDK
SEQ ID NO: 116, HC-CDR3 40
ARNVYRYSMMHDS
SEQ ID NO: 117, HC-CDR3 41
ARRGVWGYFDS
SEQ ID NO: 118, HC-CDR3 US-5
ARVYGSYFYDD
SEQ ID NO: 119, HC-CDR3 US-7
ARAPQSWYVGDV
SEQ ID NO: 120, LC-CDR1 1
SSNIGTNP
SEQ ID NO: 121, LC-CDR1 2
NIGMKS
SEQ ID NO: 122, LC-CDR1 3
SSNIGNNY
SEQ ID NO: 123, LC-CDR1 4
NIGS KS
220
CA 02988397 2017-12-05
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SEO ID NO: 124, LC-CDR1 5
QGIS SW
SEO ID NO: 125, LC-CDR1 8
NSNIETNP
SEO ID NO: 126, LC-CDR1 9
SLRSYY
SEO ID NO: 127, LC-CDR1 10
NIGSKN
SEO ID NO: 128, LC-CDR1 11
SSNIGINT
SEO ID NO: 129, LC-CDR1 13
TGAVTSGYY
SEO ID NO: 130, LC-CDR1 17
QSISSY
SEO ID NO: 131, LC-CDR1 22
SSNIGSNS
SEO ID NO: 132, LC-CDR1 26
SSNIGAGYD
SEO ID NO: 133, LC-CDR1 27
ILRNYY
SEO ID NO: 134, LC-CDR1 31
NLRNSY
SEO ID NO: 135, LC-CDR1 32
KLGEKY
SEO ID NO: 136, LC-CDR1 39
SLRNYY
SEO ID NO: 137, LC-CDR1 40
SSNIGSNH
SEO ID NO: 138, LC-CDR1 41
TLRSFY
SEO ID NO: 139, LC-CDR1 US-5
TGAVTSSYY
221
CA 02988397 2017-12-05
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PCT/US2016/031364
SEQ ID NO: 140, LC-CDR1 US-7
SSNIGTRM
SEQ ID NO: 141, LC-CDR2 1
GNY
SEQ ID NO: 142, LC-CDR2 2
YDS
SEQ ID NO: 143, LC-CDR2 3
DNY
SEQ ID NO: 144, LC-CDR2 4
YNS
SEQ ID NO: 145, LC-CDR2 5
AAS
SEQ ID NO: 146, LC-CDR2 8
SYY
SEQ ID NO: 147, LC-CDR2 9
GKN
SEQ ID NO: 148, LC-CDR2 10
DDH
SEQ ID NO: 149, LC-CDR2 11
GNS
SEQ ID NO: 150, LC-CDR2 13
STN
SEQ ID NO: 151, LC-CDR2 17
GAS
SEQ ID NO: 152, LC-CDR2 22
SNN
SEQ ID NO: 153, LC-CDR2 26
ESN
SEQ ID NO: 154, LC-CDR2 27
GQN
SEQ ID NO: 155, LC-CDR2 31
GKN
SEQ ID NO: 156, LC-CDR2 32
222
CA 02988397 2017-12-05
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PCT/US2016/031364
QND
SEQ ID NO: 157, LC-CDR2 39
GKD
SEQ ID NO: 158, LC-CDR2 40
NNN
SEQ ID NO: 159, LC-CDR2 41
GKN
SEQ ID NO: 160, LC-CDR2 US-5
STS
SEQ ID NO: 161, LC-CDR2 US-7
NNN
SEQ ID NO: 162, LC-CDR3 1
AAWDDSLTGYV
SEQ ID NO: 163, LC-CDR3 2
QVWDTTGDHPGYV
SEQ ID NO: 164, LC-CDR3 3
GTWDSSLSAYV
SEQ ID NO: 165, LC-CDR3 4
QVWDSSSDHWV
SEQ ID NO: 166, LC-CDR3 5
QQANSFPLT
SEQ ID NO: 167, LC-CDR3 8
ASWDDSVQGYV
SEQ ID NO: 168, LC-CDR3 9
NSRDSSGNHQV
SEQ ID NO: 169, LC-CDR3 10
QVWDSSSDHVV
SEQ ID NO: 170, LC-CDR3 11
QSYDTSLTVI
SEQ ID NO: 171, LC-CDR3 13
LLYYGGAQLWV
SEQ ID NO: 172, LC-CDR3 17
QQSYSAPLT
223
CA 02988397 2017-12-05
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PCT/US2016/031364
SEQ ID NO: 173, LC-CDR3 22
AAWDDSLNGYV
SEQ ID NO: 174, LC-CDR3 26
QSYDTSLKGV
SEQ ID NO: 175, LC-CDR3 27
DSRDSSGNHWV
SEQ ID NO: 176, LC-CDR3 31
NSRDSSGFPPYV
SEQ ID NO: 177, LC-CDR3 32
QSWASSSVYV
SEQ ID NO: 178, LC-CDR3 39
NSRDSSGNHRV
SEQ ID NO: 179, LC-CDR3 40
AAWDDSLNVPV
SEQ ID NO: 180, LC-CDR3 41
NSRDSGGDPVV
SEQ ID NO: 181, LC-CDR3 US-5
LLYYGGARV
SEQ ID NO: 182, LC-CDR3 US-7
AAWDDNLKSYV
SEQ ID NO: 183, HC-CDR1 consensus
G-F/G/Y-S/T-F-S/T-S-Y-A/G
SEQ ID NO: 184, HC-CDR2 consensus 1
I-N/I P X X G G/T/I-T/A/P
SEQ ID NO: 185, HC-CDR2 consensus 2
I-S-X-S/D-G/N-G/S-N-T/I/K
SEQ ID NO: 186, HC-CDR3 consensus 1
A-R-S/R-Y/S/G-Y/V-Y/W-G-X-Y-D
SEQ ID NO: 187, HC-CDR3 consensus 2
ARGXXXYY/G/S
SEQ ID NO: 188, HC-CDR3 consensus 3
AR G X X Y Q/W-W-S-X-D-D
224
CA 02988397 2017-12-05
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PCT/US2016/031364
SEQ ID NO: 189, LC-CDR1 consensus 1
N-I-G-S-N/K
SEQ ID NO: 190, LC-CDR1 consensus 2
L-R-S/N-X-Y
SEQ ID NO: 191, LC-CDR3 consensus
A/Q/N-S/A/V-W/Y/R-D-S/D-S-L/S/G-X-X-X-V
SEQ ID NO: 192, AFP158 control peptide
FMNKFIYEI
SEQ ID NO: 193, C3 control peptide (A2E-7)
YLLPAIVHI
SEQ ID NO: 194, A2E-1
LLDVPTAAV
SEQ ID NO: 195, A2E-2
TLWVDPYEV
SEQ ID NO: 196, A2E-3
FLLDHLKRV
SEQ ID NO: 197, A2E-4
LLLDVPTAAV
SEQ ID NO: 198, A2E-5
VLFRGGPRGLLAV
SEQ ID NO: 199, A2E-6
SLLPAIVEL
SEQ ID NO: 200, A2E-8
FLLPTGAEA
SEQ ID NO: 201, A2E-9
LLDPKLCYLL
SEQ ID NO: 202, A2E-11
MLLSVPLLLG
SEQ ID NO: 203, A2E-17
MVDGTLLLL
SEQ ID NO: 204, DMTN control
SLPHFHHPET
SEQ ID NO: 205, PIM1 control
225
CA 02988397 2017-12-05
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LLYDMVCGDIP
SEQ ID NO: 206, IFI30 control
LLLDVPTAAVQ
SEQ ID NO: 207, IFI30 control
LLLDVPTAAVQA
SEQ ID NO: 208, SSR1 control
VLFRGGPRGLLAVA
SEQ ID NO: 209, RPS6KB1 control
YMAPEILMRS
SEQ ID NO: 210, CSF2RA control
FIYNADLMNC
SEQ ID NO: 211, IL7 control
KQYESVLMVSI
SEQ ID NO: 212, Beta globin control
KVNVDEVGGE
SEQ ID NO: 213, Clone 7-1 CDS
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCG
CCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTA
CCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCATCTTTAATAACAATCAGCGG
CCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCC
TGGCCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATG
GGATGACAACCTGAAAAGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCT
AGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATC
CCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCC
TTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACT
ACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAA
TCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCA
TATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGC
CGCGGACACGGCCGTGTATTACTGTGCGCGCGCTCCGCAGTCGTGGTACCGAGGT
GATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEQ ID NO: 214, Clone 7-2 CDS
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCG
CCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTA
CCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCATCTTTAATAACAATCAGCGG
CCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCC
TGGCCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATG
GGATGACAACCTGAAAAGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCT
AGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATC
CCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCC
TTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACT
226
CA 02988397 2017-12-05
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ACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAA
TCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCA
TATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGC
CGCGGACACGGCCGTGTATTACTGTTTGCGCGCGCCGCAGTCCTGGTACGTTGGT
GATGTATGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEO ID NO: 215, Clone 7-3 CDS
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCG
CCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTA
CCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCATCTTTAATAACAATCAGCGG
CCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCC
TGGCCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATG
GGATGACAACCTGAAAAGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCT
AGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATC
CCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCC
TTCGGAGACCATGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACT
ACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAA
TCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCA
TATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGC
CGCGGACACGGCCGTGTATTACTGTGCGCGCGCCCCGCAGTCTTGGTACCGTGGT
GATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEO ID NO: 216, Clone 7-5 CDS
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCG
CCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTA
CCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCATCTTTAATAACAATCAGCGG
CCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCC
TGGCCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAACATG
GGATGAAAACCCGAAAAGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCT
AGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATC
CCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCC
TTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACT
ACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAA
TCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCA
TATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGC
CGCGGACACGGCCGTGTATTACTGTGCGCGCGCTCCGCAGTCTTGGTACGTTGGT
GATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEO ID NO: 217, Clone 7-6 CDS
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCG
CCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTA
CCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCATCTTTAATAACAATCAGCGG
CCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCC
TGGCCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAACATG
GGAGGACAACCGGAAAAGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCT
AGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATC
CCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCC
TTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACT
ACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAA
227
CA 02988397 2017-12-05
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TCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCA
TATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGC
CGCGGACACGGCCGTGTATTACTGTGCGCGCGCTCCGCAGTCTTGGTACGTTGGT
GATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEO ID NO: 218, Clone 7-7 CDS
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCG
CCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGGACTCGTGTGGTGACCTGGTA
CCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCATCTTTAATAACAATCAGCGG
CCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCC
TGGCCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATG
GGATGACAACCTGAAAAGTTACGTCTTCGGAACTGGGACCAAGGTCACCGTCCT
AGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATC
CCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCC
TTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACT
ACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAA
TCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCA
TATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGC
CGCGGACACGGCCGTGTATTACTGTGCGCGCGCTCCGCAGTCTTGGTACGTTGGT
GATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEO ID NO: 219, Clone 7-8 CDS
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCG
CCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTA
CCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCATCTTTAATAACAATCAGCGG
CCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCC
TGGCCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAACATG
GGATGACAACGTCAAAAGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCT
AGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATC
CCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCC
TTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACT
ACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAA
TCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCA
TATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGC
CGCGGACACGGCCGTGTATTACTGTGCGCGCGCTCCGCAGTCTTGGTACGTTGGT
GATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEO ID NO: 220, Clone 7-9 CDS
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCG
CCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTA
CCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCATCTTTAATAACAATCAGCGG
CCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCC
TGGCCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATG
GGATGATAACCCGAAAAGTTATCACTTCGGAACTGGGACCAAGGTCACCGTCCT
AGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATC
CCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCC
TTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACT
ACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAA
TCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCA
228
CA 02988397 2017-12-05
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TATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGC
CGCGGACACGGCCGTGTATTACTGTGCGCGCGCTCCGCAGTCTTGGTACGTTGGT
GATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEQ ID NO: 221, Clone 7-10 CDS
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCG
CCATCTCATGTTCTGGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTA
CCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCATCTTTAATAACAATCAGCGG
CCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCC
TGACCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATG
GGATGACAACCTGAAAAGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCT
AGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATC
CCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCC
TTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACT
ACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAA
TCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCA
TGTCAGTAGACACGTCCAAGAGGCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGC
CGCGGACACGGCCGTGTATTACTGTGCGCGCGCTCCGCAGTCTTGGTACGTTGGT
GATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEQ ID NO: 222, Clone 7-11 CDS
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGGCAGAGGGTCG
CCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGGACTCGTATGGTGACCTGGTA
CCAGCATGTCCCAGGGACGGCCCCCAAACTCCTCATCTTTAATAACAATCAGCGG
CCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCC
TGGCCATCATTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATG
GGATGACAACCTGAAAAGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCT
AGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGATGGTGGTGGATC
CCTCGAGATGGCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCC
TTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACT
ACTGGAGCTGGATCCGCCAGCTCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAA
TCAATCATAGTGGAAGCACCAACTACAACCCGTACCTCAAGAGTCGAGTCACCA
TATCAGTAGACACGTCCAAGAGGCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGC
CGCGGACACGGCCGTGTATTACTGTGCGCGCGCTCCGCAGTCTTGGTACGTTGGT
GATGTTTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA
SEQ ID NO: 223, Clone 7-1 protein
SYELTQPPS VS GTPGQRVAISCS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS ASKS GTS ASLAIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLGSRG
GGGS GGGGS GGGGSLEMAQVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWI
RQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLS SVTAADTAVYYC
ARAPQSWYRGDVWGQGTLVTVSS
SEQ ID NO: 224, Clone 7-2 protein
SYELTQPPS VS GTPGQRVAISCS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS ASKS GTS ASLAIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLGSRG
GGGS GGGGS GGGGSLEMAQVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWI
RQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLS SVTAADTAVYYC
LRAPQSWYVGDVWGQGTLVTVSS
229
CA 02988397 2017-12-05
WO 2016/182957 PCT/US2016/031364
SEQ ID NO: 225, Clone 7-3protein
SYELTQPPS VS GTPGQRVAIS CS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCAAWDDNLKS YVFGTGTKVTVLGSRG
GGGS GGGGS GGGGSLEMAQVQLQQWGAGLLKPSETMSLTCAVYGGSFS GYYWSWI
RQPPGKGLEWIGEINHS GS TNYNPS LKSRVTIS VDTS KNQFS LKLS SVTAADTAVYYC
ARAPQSWYRGDVWGQGTLVTVSS
SEQ ID NO: 226, Clone 7-5 protein
SYELTQPPS VS GTPGQRVAIS CS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCATWDENPKS YVFGTGTKVTVLGSRGG
GGS GGGGS GGGGSLEMAQVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWIR
QPPGKGLEWIGEINHS GS TNYNPSLKSRVTIS VDTS KNQFSLKLS S VTAADTAVYYCA
RAPQSWYVGDVWGQGTLVTVSS
SEQ ID NO: 227, Clone 7-6 protein
SYELTQPPS VS GTPGQRVAIS CS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCATWEDNRKS YVFGTGTKVTVLGSRGG
GGS GGGGS GGGGSLEMAQVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWIR
QPPGKGLEWIGEINHS GS TNYNPSLKSRVTIS VDTS KNQFSLKLS S VTAADTAVYYCA
RAPQSWYVGDVWGQGTLVTVSS
SEQ ID NO: 228, Clone 7-7 protein
SYELTQPPS VS GTPGQRVAIS CS GS S SNIGTRVVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCAAWDDNLKS YVFGTGTKVTVLGSRG
GGGS GGGGS GGGGSLEMAQVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWI
RQPPGKGLEWIGEINHS GS TNYNPS LKSRVTIS VDTS KNQFS LKLS SVTAADTAVYYC
ARAPQSWYVGDVWGQGTLVTVSS
SEQ ID NO: 229, Clone 7-8 protein
SYELTQPPS VS GTPGQRVAIS CS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCATWDDNVKS YVFGTGTKVTVLGSRG
GGGS GGGGS GGGGSLEMAQVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWI
RQPPGKGLEWIGEINHS GS TNYNPS LKSRVTIS VDTS KNQFS LKLS SVTAADTAVYYC
ARAPQSWYVGDVWGQGTLVTVSS
SEQ ID NO: 230, Clone 7-9 protein
SYELTQPPS VS GTPGQRVAIS CS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCAAWDDNPKS YHFGTGTKVTVLGSRGG
GGS GGGGS GGGGSLEMAQVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWIR
QPPGKGLEWIGEINHS GS TNYNPSLKSRVTIS VDTS KNQFSLKLS S VTAADTAVYYCA
RAPQSWYVGDVWGQGTLVTVSS
SEQ ID NO: 231, Clone 7-10 protein
SYELTQPPS VS GTPGQRVAIS CS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFSAS KS GTSASLTIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLGSRGG
GGS GGGGS GGGGSLEMAQVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWIR
QPPGKGLEWIGEINHS GS TNYNPSLKSRVTMS VDTS KRQFS LKLS S VTAADTAVYYC
ARAPQSWYVGDVWGQGTLVTVSS
230
CA 02988397 2017-12-05
WO 2016/182957 PCT/US2016/031364
SEQ ID NO: 232, Clone 7-11 protein
S YELTQPPS VS GTPGQRVAISCS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCAAWDDNLKS YVFGTGTKVTVLGSRG
GGGS GGGGSDGGGSLEMAQVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWI
RQLPGKGLEWIGEINHS GS TNYNPYLKSRVTIS VDTS KRQFS LKLS SVTAADTAVYYC
ARAPQSWYVGDVWGQGTLVTVSS
SEQ ID NO: 233, HCVR 7-1
QVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWIRQPPGKGLEWIGEINHS GST
NYNPSLKSRVTIS VDTS KNQFSLKLS S VTAADTAVYYCARAPQSWYRGDVWGQGTL
VTVSS
SEQ ID NO: 234, HCVR 7-2
QVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWIRQPPGKGLEWIGEINHS GST
NYNPSLKSRVTIS VDTS KNQFSLKLS S VTAADTAVYYCLRAPQSWYVGDVWGQGTL
VTVSS
SEQ ID NO: 235, HCVR 7-3
QVQLQQWGAGLLKPSETMSLTCAVYGGSFS GYYWSWIRQPPGKGLEWIGEINHS GS
TNYNPS LKSRVTIS VDTS KNQFS LKLS S VTAADTAVYYCARAPQSWYRGDVWGQGT
LVTVSS
SEQ ID NO: 236, HCVR 7-10
QVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWIRQPPGKGLEWIGEINHS GST
NYNPSLKSRVTMS VDTS KRQFS LKLS S VTAADTAVYYCARAPQSWYVGDVWGQGT
LVTVSS
SEQ ID NO: 237, HCVR 7-11
QVQLQQWGAGLLKPSETLSLTCAVYGGSFS GYYWSWIRQLPGKGLEWIGEINHS GS
TNYNPYLKSRVTIS VDTS KRQFS LKLS S VTAADTAVYYCARAPQS WYVGDVWGQG
TLVTVSS
SEQ ID NO: 238, LCVR 7-5
S YELTQPPS VS GTPGQRVAISCS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCATWDENPKS YVFGTGTKVTVLG
SEQ ID NO: 239, LCVR 7-6
S YELTQPPS VS GTPGQRVAISCS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCATWEDNRKS YVFGTGTKVTVLG
SEQ ID NO: 240, LCVR 7-7
S YELTQPPS VS GTPGQRVAISCS GS S SNIGTRVVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCAAWDDNLKS YVFGTGTKVTVLG
SEQ ID NO: 241, LCVR 7-8
S YELTQPPS VS GTPGQRVAISCS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCATWDDNVKS YVFGTGTKVTVLG
231
CA 02988397 2017-12-05
WO 2016/182957 PCT/US2016/031364
SEQ ID NO: 242, LCVR 7-9
SYELTQPPS VS GTPGQRVAIS CS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFS AS KS GTS AS LAIIGLQSDDEADYYCAAWDDNPKS YHFGTGTKVTVLG
SEQ ID NO: 243, LCVR 7-10
SYELTQPPS VS GTPGQRVAIS CS GS S SNIGTRMVTWYQHVPGTAPKLLIFNNNQRPS G
VPDRFSAS KS GTSASLTIIGLQSDDEADYYCAAWDDNLKSYVFGTGTKVTVLG
SEQ ID NO: 244, HC-CDR3 7-1
ARAPQSWYRGDV
SEQ ID NO: 245, HC-CDR3 7-2
LRAPQSWYVGDV
SEQ ID NO: 246, LC-CDR1 7-7
SSNIGTRV
SEQ ID NO: 247, LC-CDR3 7-5
ATWDENPKSYV
SEQ ID NO: 248, LC-CDR3 7-6
ATWEDNRKSYV
SEQ ID NO: 249, LC-CDR3 7-8
ATWDDNVKSYV
SEQ ID NO: 250, LC-CDR3 7-9
AAWDDNPKSYH
SEQ ID NO: 251, HC-FR1 7-3
VQLQQWGAGLLKPSETMSLTCAVY
SEQ ID NO: 252, HC-FR2 7-11
WSWIRQLPGKGLEWIGE
SEQ ID NO: 253, HC-FR3 7-10
NYNPSLKSRVTMS VDTS KRQFS LKLS S VTAADTAVYYC
SEQ ID NO: 254, HC-FR3 7-11
NYNPYLKSRVTIS VDTS KRQFSLKLS S VTAADTAVYYC
SEQ ID NO: 255, LC-FR3 7-10
QRPS GVPDRFSAS KS GTSASLTIIGLQSDDEADYYC
SEQ ID NO: 256, CD28/CD3
AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPS KPFWVLVVVGGVLACYS
LLVTVAFIIFWVRS KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKF
SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY
NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR
232
CA 02988397 2017-12-05
WO 2016/182957 PCT/US2016/031364
SEQ ID NO: 257, 4-1BB/CD3
TGTTTPAPRPPTPAPTIAS QPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC
GVLLLS LVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC S CRFPEEEEGGCELRVK
FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL
YNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR
SEQ ID NO: 258, HPV16 E7 11-19 A2
YALDLQPET
SEQ ID NO: 259, HPV16 E7 11-19 A3
YMADLQPET
SEQ ID NO: 260, HPV16 E7 11-19 A4
YMLALQPET
SEQ ID NO: 261, HPV16 E7 11-19 A6
YMLDLAPET
SEQ ID NO: 262, HPV16 E7 11-19 A7
YMLDLQAET
SEQ ID NO: 263, HPV16 E7 11-19 A9
YMLDLQPEA
233
