Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ANTI-HER2 ANTIBODIES AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S. Provisional
Patent Application
No. 63/229,134, filed August 4, 2021, the entire contents of which are
incorporated herein by
.. reference.
TECHNICAL FIELD
[0002] The present technology relates generally to the preparation of
immunoglobulin-related
compositions (e.g., antibodies or antigen binding fragments thereof) that
specifically bind HER2
protein and uses of the same. In particular, the present technology relates to
the preparation of
.. HER2 binding antibodies and their use in detecting and treating HER2-
associated cancer.
BACKGROUND
[0003] The following description of the background of the present technology
is provided simply
as an aid in understanding the present technology and is not admitted to
describe or constitute
prior art to the present technology.
[0004] Monoclonal antibody (mAb)-based therapy for cancer is one of the most
successful
strategies for treating patients with both hematological and nonhematological
malignancies.
[0005] One of the main clinical accomplishments for mAbs was the discovery of
trastuzumab/Herceptin, a humanized monoclonal antibody that binds to the
extracellular domain
of HER2. In fact, for HER2-positive breast cancer the standard of care
treatment includes
chemotherapy in combination with trastuzumab. Trastuzumab was designed to
inhibit cell
growth and proliferation, and kills HER2-positive tumor cells through antibody-
dependent
cellular cytotoxicity (ADCC). Both the combination of trastuzumab with
conventional
chemotherapies in breast cancer and gastric cancer and the use of trastuzumab
as a single agent
have proven to prolong the progression-free survival of patients with
amplified HER2.
Importantly, standard HER2-targeted therapies are exclusively offered to
patients scoring HER2
immunohistochemistry (IHC) 3+ (i.e. strongly overexpressed), or SPoT-Light
HER2
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Chromogenic IHC (CISH) positive (i.e. ERBB2 gene-amplified). However, a
significant group
of patients do not respond to this targeted therapy and most of the patients
that initially respond
acquire resistance in response to trastuzumab treatment. There are multiple
mechanisms
contributing to trastuzumab resistance that include activation of the HER2
downstream signaling
pathways and parallel receptor tyrosine kinase pathways, all of which provide
potential targets to
combat trastuzumab resistance. A major mechanism of resistance involves the
activation of a
bypass-signaling pathway making inhibition of HER2-signaling irrelevant to
tumor progression.
Up to 70% of HER2+ breast cancers become resistant to a single-agent treatment
of trastuzumab.
Despite the drug resistance, these tumors generally still overexpress HER2.
[0006] Accordingly, there is an urgent need for developing alternative HER2-
targeted therapies
that overcome the resistance to signaling inhibition and benefit more
patients.
SUMMARY OF THE PRESENT TECHNOLOGY
[0007] In one aspect, the present disclosure provides an antibody or an
antigen binding fragment
thereof, comprising an antibody or antigen binding fragment thereof comprising
a heavy chain
immunoglobulin variable domain (VH) and a light chain immunoglobulin variable
domain (VL),
wherein (a) (i) the VH comprises a VH-CDR1 sequence of SEQ ID NO: 1, a VH-CDR2
sequence of SEQ ID NO: 2 or SEQ ID NO: 7 and a VH-CDR3 sequence of SEQ ID NO:
8; or (ii)
the VH comprises a VH-CDR1 sequence of SEQ ID NO: 1, a VH-CDR2 sequence of SEQ
ID NO:
7 and a VH-CDR3 sequence of SEQ ID NO: 3 or SEQ ID NO: 8; and/or (b) (i) the
VL comprises
a VL-CDR1 sequence of SEQ ID NO: 9, a VL-CDR2 sequence of SEQ ID NO: 5, SEQ ID
NO:
10, or SEQ ID NO: 11, and a VL-CDR3 sequence of SEQ ID NO: 6 or SEQ ID NO: 12;
or (ii)
the VL comprises a VL-CDR1 sequence of SEQ ID NO: 4 or SEQ ID NO: 9, a VL-CDR2
sequence of SEQ ID NO: 10, or SEQ ID NO: 11, and a VL-CDR3 sequence of SEQ ID
NO: 6 or
SEQ ID NO: 12; or (iii) the VL comprises a VL-CDR1 sequence of SEQ ID NO: 4 or
SEQ ID
NO: 9, a VL-CDR2 sequence of SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11,
and a VL-
CDR3 sequence of SEQ ID NO: 12.
[0008] In one aspect, the present disclosure provides an antibody or antigen
binding fragment
thereof comprising a heavy chain immunoglobulin variable domain (VH) and a
light chain
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immunoglobulin variable domain (VIA wherein: (a) the VH comprises an amino
acid sequence
selected from any one of SEQ ID NOs: 13, 15, or 17; and/or (b) the VL
comprises an amino acid
sequence selected from any one of SEQ ID NOs: 14, 16, 18, 19, or 20. In some
embodiments,
the antibody or antigen binding fragment thereof comprises heavy chain
immunoglobulin
variable domain (VH) and light chain immunoglobulin variable domain (VI) amino
acid
sequences selected from the group consisting of: SEQ ID NOs: 13 and 14, SEQ ID
NOs: 15 and
16, SEQ ID NOs: 17 and 14, SEQ ID NOs: 15 and 18, SEQ ID NOs: 15 and 19, and
SEQ ID
NOs: 15 and 20, respectively.
[0009] In another aspect, the present disclosure provides an antibody
comprising a first
polypeptide chain, a second polypeptide chain, a third polypeptide chain and a
fourth
polypeptide chain, wherein the first and second polypeptide chains are
covalently bonded to one
another, the second and third polypeptide chains are covalently bonded to one
another, and the
third and fourth polypeptide chain are covalently bonded to one another, and
wherein: (a) each of
the first polypeptide chain and the fourth polypeptide chain comprises in the
N-terminal to C-
terminal direction: (i) a light chain variable domain of a first
immunoglobulin that is capable of
specifically binding to a first epitope; (ii) a light chain constant domain of
the first
immunoglobulin; (iii) a flexible peptide linker comprising the amino acid
sequence (GGGGS)3;
and (iv) a light chain variable domain of a second immunoglobulin that is
linked to a
complementary heavy chain variable domain of the second immunoglobulin, or a
heavy chain
variable domain of a second immunoglobulin that is linked to a complementary
light chain
variable domain of the second immunoglobulin, wherein the light chain and
heavy chain variable
domains of the second immunoglobulin are capable of specifically binding to a
second epitope,
and are linked together via a flexible peptide linker comprising the amino
acid sequence
(GGGGS)6 to form a single-chain variable fragment; and (b) each of the second
polypeptide
chain and the third polypeptide chain comprises in the N-terminal to C-
terminal direction: (i) a
heavy chain variable domain of the first immunoglobulin that is capable of
specifically binding
to the first epitope; and (ii) a heavy chain constant domain of the first
immunoglobulin; and
wherein the heavy chain variable domain of the first immunoglobulin or the
heavy chain variable
domain of the second immunoglobulin comprises any one of SEQ ID NOs: 13, 15,
or 17, and/or
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the light chain variable domain of the first immunoglobulin or the light chain
variable domain of
the second immunoglobulin comprises any one of SEQ ID NOs: 14, 16, 18, 19, or
20.
[0010] In another aspect, the present disclosure provides an antibody or
antigen binding
fragment comprising a heavy chain (HC) and a light chain (LC) selected from
the group
consisting of SEQ ID NOs: 21 and 22, SEQ ID NOs: 21 and 23, SEQ ID NOs: 21 and
24, SEQ
ID NOs: 21 and 25, SEQ ID NOs: 21 and 26, SEQ ID NOs: 21 and 27, SEQ ID NOs:
21 and 28,
SEQ ID NOs: 21 and 29, SEQ ID NOs: 21 and 30, SEQ ID NOs: 21 and 31, SEQ ID
NOs: 21
and 32, SEQ ID NOs: 21 and 33, SEQ ID NOs: 34 and 33, SEQ ID NOs: 21 and 35,
SEQ ID
NOs: 36 and 33, SEQ ID NOs: 21 and 37, SEQ ID NOs: 21 and 38, SEQ ID NOs: 21
and 39,
SEQ ID NOs: 21 and 40, SEQ ID NOs: 21 and 41, SEQ ID NOs: 21 and 42, SEQ ID
NOs: 21
and 43, SEQ ID NOs: 21 and 44, SEQ ID NOs: 21 and 45, SEQ ID NOs: 21 and 46,
SEQ ID
NOs: 21 and 47, SEQ ID NOs: 21 and 48, SEQ ID NOs: 21 and 49, SEQ ID NOs: 21
and 50,
SEQ ID NOs: 21 and 51, SEQ ID NOs: 21 and 52, SEQ ID NOs: 21 and 53, SEQ ID
NOs: 21
and 54, SEQ ID NOs: 21 and 55, SEQ ID NOs: 21 and 56, SEQ ID NOs: 21 and 57,
SEQ ID
NOs: 21 and 58, SEQ ID NOs: 21 and 59, SEQ ID NOs: 21 and 60, SEQ ID NOs: 21
and 61,
SEQ ID NOs: 21 and 62, SEQ ID NOs: 21 and 63, SEQ ID NOs: 21 and 64, SEQ ID
NOs: 21
and 65, SEQ ID NOs: 21 and 66, SEQ ID NOs: 21 and 67, SEQ ID NOs: 21 and 68,
SEQ ID
NOs: 21 and 69, SEQ ID NOs: 21 and 70, SEQ ID NOs: 21 and 71, SEQ ID NOs: 21
and 72, and
SEQ ID NOs: 21 and 85, respectively.
100111 Additionally or alternatively, in some embodiments, the antibody or
antigen binding
fragment further comprises a Fc domain of an isotype selected from the group
consisting of
IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, and IgE. In certain embodiments,
the antibody
or antigen binding fragment comprises an IgG1 constant region comprising one
or more amino
acid substitutions selected from the group consisting of N297A, L234A, L235A,
and K322A. In
other embodiments, the antibody or antigen binding fragment comprises an IgG4
constant region
comprising a 5228P mutation.
[0012] In any and all embodiments of the antigen binding fragment disclosed
herein, the antigen
binding fragment is selected from the group consisting of Fab, F(ab')2, Fab',
scFv, and F.
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Additionally or alternatively, in some embodiments, the antibody or antigen
binding fragment of
the present technology is a monoclonal antibody, a chimeric antibody, a
humanized antibody, a
bispecific antibody, or multi-specific antibody, and/or lacks a-1,6-fucose
modifications.
[0013] In any and all embodiments of the antibody or antigen binding fragment
disclosed herein,
5 the multi-specific antibody or antigen binding fragment binds to T cells,
B-cells, myeloid cells,
plasma cells, or mast-cells. Additionally or alternatively, in some
embodiments of the antibody
or antigen binding fragment disclosed herein, the multi-specific antibody or
antigen binding
fragment binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR,
CD74,
CD22, CD14, CD15, CD16, CD123, TCR gamma/delta, NKp46, KIR, or a small
molecule
DOTA hapten.
[0014] Additionally or alternatively, in some embodiments, the multi-specific
antibody or
antigen binding fragment of the present technology also binds to T cells
and/or CD3. In one
aspect, the present disclosure provides a T cell that is armed ex vivo with a
multi-specific
antibody or antigen binding fragment of the present technology that also binds
to T cells and/or
CD3. In another aspect, the present disclosure provides an ex vivo method of
making a
therapeutic T cell, comprising arming a T cell ex vivo with a multi-specific
antibody or antigen
binding fragment of the present technology that is capable of binding to T
cells and/or CD3,
wherein the T cell is optionally a human T cell, and wherein the binding is
noncovalent. In
another aspect, the present disclosure provides a method for treating cancer
in a subject in need
thereof, comprising administering to the subject an effective amount of a T
cell that is armed ex
vivo with a multi-specific antibody or antigen binding fragment of the present
technology that
also binds to T cells and/or CD3.
[0015] In one aspect, the present disclosure provides a recombinant nucleic
acid sequence
encoding any of the antibodies or antigen binding fragments described herein.
In another aspect,
the present disclosure provides a host cell or vector comprising any of the
recombinant nucleic
acid sequences disclosed herein.
[0016] In another aspect, the present disclosure provides a pharmaceutical
composition
comprising any of the antibodies or antigen binding fragments described herein
and a
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pharmaceutically-acceptable carrier, wherein the antibody or antigen binding
fragment is
optionally conjugated to an agent selected from the group consisting of
isotopes, dyes,
chromagens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme
inhibitors, hormones,
hormone antagonists, growth factors, radionuclides, metals, liposomes,
nanoparticles, RNA,
DNA or any combination thereof. In some embodiments, the pharmaceutical
composition
further comprises an agent selected from the group consisting of isotopes,
dyes, chromagens,
contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors,
hormones, hormone
antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles,
RNA, DNA or any
combination thereof.
[0017] In one aspect, the present disclosure provides a method for treating
cancer in a subject in
need thereof, comprising administering to the subject an effective amount of
any of the
antibodies or antigen binding fragments described herein, or any of the
pharmaceutical
compositions disclosed herein, wherein the antibody or antigen binding
fragment specifically
binds to HER2. In some embodiments, the cancer is a solid tumor. Examples of
cancer include,
but are not limited to, breast cancer, gastric cancer, osteosarcoma,
desmoplastic small round cell
cancer, squamous cell carcinoma of head and neck cancer, ovarian cancer,
prostate cancer,
pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma,
gastroesophageal
junction adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue
sarcoma, leukemia,
melanoma, Ewing's sarcoma, rhabdomyosarcoma, and neuroblastoma.
[0018] Additionally or alternatively, in some embodiments of the method, the
antibody or
antigen binding fragment is administered to the subject separately,
sequentially or
simultaneously with an additional therapeutic agent. Examples of additional
therapeutic agents
include one or more of alkylating agents, platinum agents, taxanes, vinca
agents, anti-estrogen
drugs, aromatase inhibitors, ovarian suppression agents, VEGF/VEGFR
inhibitors, EGF/EGFR
inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics,
antimetabolites,
endocrine/hormonal agents, bisphosphonate therapy agents, T cells, and immuno-
modulating/stimulating antibodies (e.g., an anti-PD-1 antibody, an anti-PD-Li
antibody, an anti-
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PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4-1BB
antibody, an
anti-CD73 antibody, an anti-GITR antibody, or an anti-LAG-3 antibody).
[0019] In another aspect, the present disclosure provides a method for
detecting cancer in a
subject in vivo comprising (a) administering to the subject an effective
amount of the antibody or
antigen binding fragment of the present technology, wherein the antibody or
antigen binding
fragment is configured to localize to a cancer cell expressing HER2, and is
labeled with a
radioisotope; and (b) detecting the presence of a tumor in the subject by
detecting radioactive
levels emitted by the antibody or antigen binding fragment that are higher
than a reference value.
In certain embodiments, the cancer is a solid tumor. In some embodiments, the
subject is
diagnosed with or is suspected of having cancer. Examples of cancer include,
but are not limited
to, breast cancer, gastric cancer, osteosarcoma, desmoplastic small round cell
cancer, squamous
cell carcinoma of head and neck cancer, ovarian cancer, prostate cancer,
pancreatic cancer,
glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal
junction
adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma,
leukemia,
melanoma, Ewing's sarcoma, rhabdomyosarcoma, and neuroblastoma. Radioactive
levels
emitted by the antibody or antigen binding fragment may be detected using
positron emission
tomography or single photon emission computed tomography. Additionally or
alternatively, in
some embodiments, the method further comprises administering to the subject an
effective
amount of an immunoconjugate comprising the antibody or antigen binding
fragment of the
present technology conjugated to a radionuclide.
[0020] In any and all embodiments of the methods disclosed herein, the subject
is human.
[0021] In yet another aspect, the present disclosure provides a method for
detecting HER2
protein expression levels in a biological sample comprising contacting the
biological sample with
any of the antibodies or antigen binding fragments disclosed herein, and
detecting binding to
HER2 protein in the biological sample.
[0022] Also disclosed herein are kits for the detection and/or treatment of
HER2-associated
cancers comprising at least one immunoglobulin-related composition of the
present technology
(e.g., any antibody or antigen binding fragment described herein), and
instructions for use. In
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certain embodiments, the immunoglobulin-related composition is coupled to one
or more
detectable labels. In one embodiment, the one or more detectable labels
comprise a radioactive
label, a fluorescent label, or a chromogenic label. Additionally or
alternatively, in some
embodiments, the kit further comprises a secondary antibody that specifically
binds to an anti-
HER2 immunoglobulin-related composition described herein. In some embodiments,
the
secondary antibody is coupled to at least one detectable label selected from
the group consisting
of a radioactive label, a fluorescent label, or a chromogenic label.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows the amino acid sequences of VH CDR1 (SEQ ID NO: 1), VH
CDR2 (SEQ
ID NO: 2 or 7), VH CDR3 (SEQ ID NO: 3 or 8), VL CDR1 (SEQ ID NO: 4 or 9), VL
CDR2
(SEQ ID NO: 5, 10 or 11), and VL CDR3 (SEQ ID NO:, 6 or 12) of the anti-HER2
immunoglobulin-related compositions of the present technology. Mutations in
the CDR regions
of the trastuzumab variants compared to CDR regions (SEQ ID NOs: 1-6) of the
parental
trastuzumab antibody are underlined.
[0024] FIG. 2 shows the amino acid sequences of the variable heavy
immunoglobulin domain
(VH) and the variable light immunoglobulin domain (VL) of 6 of the anti-HER2
immunoglobulin-related compositions of the present technology: ABP100s.10.1
HER2 (SEQ ID
NOs: 13 and 14, respectively), ABP100s.10.2 HER2 (SEQ ID NOs: 15 and 16,
respectively),
ABP100s.10.3 HER2 (SEQ ID NOs: 17 and 14, respectively), ABP100s.10.4 HER2
(SEQ ID
NOs: 15 and 18, respectively), ABP100s.10.5 HER2 (SEQ ID NOs: 15 and 19,
respectively), and
ABP100s.10.6 HER2 (SEQ ID NO: 15 and 20, respectively). The VH CDR 1-3 and VL
CDR 1-3
amino acid sequences are underlined. Mutations in the CDR regions of the
trastuzumab variants
compared to CDR regions (SEQ ID NOs: 1-6) of the parental trastuzumab antibody
are double
underlined.
[0025] FIG. 3 shows the heavy chain (HC) and light chain (LC) amino acid
sequences of 40
exemplary HER2xCD3 bispecific antibodies of the present technology. The VH CDR
1-3 and VL
CDR 1-3 amino acid sequences are underlined. The linker sequences are
italicized. Point
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mutations and disulfide mutations are bold and double underlined. Fe
substitutions are bold and
underlined.
[0026] FIG. 4 shows an overview of two groups of BsAbs: the first targets both
HER2 and the T
cell co-receptor CD3, and the second selectively targets and kills HER2
amplified cancer cells.
[0027] FIG. 5A-5B shows individual mouse tumor responses in mouse xenograft
models using
BsAbs with varying interdomain spacing formats (FIG. 5A) administered
intravenously (IV) (10
pmol, twice per week) along with 20 million huATCs and subcutaneous human IL-2
(1000 U) or
(FIG. 5B) ex-vivo "armed" T-cell (EAT) administration of BsAbs and human IL-2
(1,000 U)
(subcutaneously, twice per week). Each line represents a single mouse, and the
dashed line
.. represents the group average. See Santich et at., Sci. Transl. Med. 12:
eaax1315 (2020), which is
incorporated herein by reference.
[0028] FIG. 6 summarizes the process for arming activated T-cells with the
anti-HER2 x CD3
bispecific antibodies of the present technology.
[0029] FIG. 7A shows CD3+ T cells that were stained in duplicate wells with
either SP34-
hIgG1 or bispecific antibodies for 30 minutes at 4 C, followed by washing and
incubation with
anti-human IgG Fe specific PE conjugate (Jackson 1:200) for 30 minutes at 4 C
followed by
washing and data collection on the BD FACSCelesta, with data analyzed in
FlowJo and
GraphPad PRISM. Notably, the constructs used in this experiment differed only
in their HER2-
binding arm, and all had the same CD3-reactive arm. FIGs. 7B-7C show
differential killing of
HER2-low expressing target cells through lowering the affinity of the HER2
arm. A TDCC
assay at an effector:target (E:T) ratio of 5:1 was performed with a dose range
for bispecific
antibodies, with CD3+ T cells used as effector cells (50k/well), while target
cells (10k/well)
expressed either high relative amounts of HER-2 (FIG. 7B, SKBR-3), or low
relative amounts of
HER2 (FIG. 7C, MCF-7). Cells were incubated with antibody in RPMI1640/10% FBS
for 28
hours at 37 C /5% CO2, with luminescence quantified on the SpectraMax iD3
plate reader and
data analysis in GraphPad PRISM constructs in this experiment differed only in
their HER2-
binding arm, and all had the same CD3-reactive arm.
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[0030] FIGs. 8A-8B shows characterization of activated T cells armed with the
anti-HER2 x
CD3 parent antibody (trastuzumab x huOKT3). FIG. 8A shows qualitative flow
cytometry data
demonstrating binding of trastuzumab x huOKT3 to activated T cells after
arming at various
concentrations. FIG. 8B shows trastuzumab x huOKT3-mediated killing of cell
lines expressing
5 different levels of HER2.
[0031] FIGs. 9A-9B show differential killing of HER2-high and HER2-low
expressing target
cells by TDCC assay. FIG. 9A and FIG. 9B show the effects of the HER2xCD3
bispecific
antibodies of the present technology (ABP100s.5, ABP100s.5.1, ABP100s.10.2,
ABP100s.10.4,
ABP100s.10.5, ABP100s.10.6) on SK-BR-3 cells and MCF-7 cells, respectively.
10 [0032] FIG. 10 shows affinities of the HER2xCD3 bispecific antibodies of
the present
technology to the CD3 and HER2 targets as disclosed in FIG. 3.
[0033] FIG. 11 shows the heavy chain (HC) and light chain (LC) amino acid
sequences of 12
additional exemplary HER2xCD3 bispecific antibodies of the present technology.
The VH CDR
1-3 and VL CDR 1-3 amino acid sequences are indicated in bold. The linker
sequences are
indicated in italics.
[0034] FIG. 12 shows affinities of the HER2xCD3 bispecific antibodies of the
present
technology to the CD3 and HER2 targets as disclosed in FIG. 11.
[0035] FIGs. 13A-13E show the results of CD3/TCR NFAT T cell activation
reporter assays. A
Jurkat CD3/TCR NFAT T cell activation reporter assay (Promega) was used to
assess bispecific
antibody (0.00004 - 40nM) activation of the CD3/TCR complex following
incubation with Her2-
high (FIG. 13A: SK-BR-3, FIG. 13B: HCC1954) and Her2-low (FIG. 13C: MCF-7,
FIG. 13D:
HT55) target cell lines or no target cells (FIG. 13E). The expression of
reporter activity was
detected and quantified as Relative Luminesence Units (RLU), which were
plotted after a 7-hour
incubation period.
.. [0036] FIGs. 14A-14D show the results of T cell dependent cellular
cytotoxicity (TDCC) on
Her2-high and Her2-low target cell lines with human CD3+ T cells. Bispecific
antibodies were
incubated with CD3+ T cells and target cells (Effector: Target ratio 5:1) for
40 hours at 37 C.
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Using a sensitive ATP quantification method (Cell Titer Glo 2.0),
%Cytotoxicity was quantified
in comparison to [Effector plus target]-only wells for Her2-high target cells
(FIG. 14A: SKBR-
3, FIG. 14B: HCC1954) and Her2-low cell lines (FIG. 14C: MCF-7, FIG. 14D:
HT55).
[0037] FIGs. 15A-151I show the results of in-vitro multiplex cytokine
detection assay in Her2-
high and Her2-low target cell lines with human PBMCs. Bispecific antibodies
(range: 30, 0.3,
0.003, 0.00003 nM) were incubated with human PBMCs and target cells (Effector
(100,000
cells): Target (10,000 cells), E:T ratio 10:1) for 24 hours at 37 C. Cytokine
release on SKBR-3
(Her2-high) target cells (FIGs. 15A-15D) and MCF-7 (Her2-low) target cells
(FIGs. 15E-1511)
were quantified by diluting supernatants 1:4 for use in a multiplex bead-based
assay for TNF-a
(FIG. 15A, FIG. 15E), IL-6 (FIG. 15B, FIG. 15F), IL-2 (FIG. 15C, FIG. 15G),
IFN-y (FIG.
15D, FIG. 1511), and are presented here in picograms/mL as quantified by
Luminex xMAP
software.
[0038] FIGs. 16A-16E show the results of flow cytometric analysis of
bispecific antibody
binding to activated T cells and Her-2 expressing target cells. Bispecific
antibodies were
incubated with Her2-high (FIG. 16A: SKBR-3, FIG. 16B: SKOV-3) or Her2-low
(FIG. 16C:
MCF-7, FIG. 16D: HT55) target cells or activated T cells (FIG. 16E). 100,000
cells/well were
incubated with primary bispecific antibodies followed by incubation with anti-
human IgG PE
secondary antibody. Results were quantified as Median Fluorescence Intensity
(MFI) of single
live cells in FlowJo software.
.. [0039] FIGs. 17A-17B show Biacore (SPR) affinity data for the Fab formats
(attached to a
human IgG1 scaffold) of the anti-HER2 x CD3 BsAbs of the present technology.
The anti-
HER2 x CD3 BsAbs of the present technology cross-react with both human and non-
human
primate (Cyno) HER2 and CD3 antigens.
DETAILED DESCRIPTION
.. [0040] It is to be appreciated that certain aspects, modes, embodiments,
variations and features
of the present methods are described below in various levels of detail in
order to provide a
substantial understanding of the present technology.
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[0041] The present disclosure generally provides immunoglobulin-related
compositions (e.g.,
antibodies or antigen binding fragments thereof), which can specifically bind
to HER2
polypeptides. The immunoglobulin-related compositions of the present
technology are useful in
methods for detecting or treating HER2-associated cancers in a subject in need
thereof.
Accordingly, the various aspects of the present methods relate to the
preparation,
characterization, and manipulation of anti-HER2 antibodies. The immunoglobulin-
related
compositions of the present technology are useful alone or in combination with
additional
therapeutic agents for treating cancer. In some embodiments, the
immunoglobulin-related
composition is a monoclonal antibody, a humanized antibody, a chimeric
antibody, a bispecific
antibody, or a multi-specific antibody.
[0042] In practicing the present methods, many conventional techniques in
molecular biology,
protein biochemistry, cell biology, immunology, microbiology and recombinant
DNA are used.
See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory
Manual, 3rd
edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular
Biology; the series
Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991)
PCR 1: A
Practical Approach (IRL Press at Oxford University Press); MacPherson et al.
(1995) PCR 2: A
Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory
Manual; Freshney
(2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait
ed. (1984)
Oligonucleotide Synthesis;U U.S. Patent No. 4,683,195; Hames and Higgins eds.
(1984) Nucleic
Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and
Higgins eds.
(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press
(1986));
Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Cabs eds.
(1987) Gene
Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides
ed. (2003)
Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987)
Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);
and
Herzenberg et al. eds (1996) Weir 's Handbook of Experimental Immunology.
Methods to detect
and measure levels of polypeptide gene expression products (i.e., gene
translation level) are well-
known in the art and include the use of polypeptide detection methods such as
antibody detection
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13
and quantification techniques. (See also, Strachan & Read, Human Molecular
Genetics, Second
Edition. (John Wiley and Sons, Inc., NY, 1999)).
[0043] The present disclosure provides HER2 bispecific antibodies with reduced
affinity that
take advantage of avidity interactions to selectively bind to and kill cells
with a high density of
Her2 (such as cells of cancerous tissues) and not bind and spare Her2-low
density cells. Without
wishing to be bound by theory, it is believed that lowering CD3 affinity in
HER2xCD3
bispecific antibodies would reduce T cell activation and cytokine production,
leading to lower
adverse events such as cytokine release syndrome in the clinic.
Definitions
[0044] Unless defined otherwise, all technical and scientific terms used
herein generally have the
same meaning as commonly understood by one of ordinary skill in the art to
which this
technology belongs. As used in this specification and the appended claims, the
singular forms
"a", "an" and "the" include plural referents unless the content clearly
dictates otherwise. For
example, reference to "a cell" includes a combination of two or more cells,
and the like.
Generally, the nomenclature used herein and the laboratory procedures in cell
culture, molecular
genetics, organic chemistry, analytical chemistry and nucleic acid chemistry
and hybridization
described below are those well-known and commonly employed in the art.
[0045] As used herein, the term "about" in reference to a number is generally
taken to include
numbers that fall within a range of 1%, 5%, or 10% in either direction
(greater than or less than)
of the number unless otherwise stated or otherwise evident from the context
(except where such
number would be less than 0% or exceed 100% of a possible value).
[0046] As used herein, the "administration" of an agent or drug to a subject
includes any route of
introducing or delivering to a subject a compound to perform its intended
function.
Administration can be carried out by any suitable route, including but not
limited to, orally,
intranasally, parenterally (intravenously, intramuscularly, intraperitoneally,
or subcutaneously),
rectally, intrathecally, intratumorally or topically. Administration includes
self-administration
and the administration by another.
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[0047] As used herein, the term "antibody" collectively refers to
immunoglobulins or
immunoglobulin-like molecules including by way of example and without
limitation, IgA, IgD,
IgE, IgG and IgM, combinations thereof, and similar molecules produced during
an immune
response in any vertebrate, for example, in mammals such as humans, goats,
rabbits and mice, as
well as non-mammalian species, such as shark immunoglobulins. As used herein,
"antibodies"
(includes intact immunoglobulins) and "antigen binding fragments" specifically
bind to a
molecule of interest (or a group of highly similar molecules of interest) to
the substantial
exclusion of binding to other molecules (for example, antibodies and antibody
fragments that
have a binding constant for the molecule of interest that is at least 103 M-1
greater, at least 104M-
1 greater or at least 105 M-1 greater than a binding constant for other
molecules in a biological
sample). The term "antibody" also includes genetically engineered forms such
as chimeric
antibodies (for example, humanized murine antibodies), heteroconjugate
antibodies (such as,
bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995
(Pierce Chemical
Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New
York, 1997.
[0048] More particularly, antibody refers to a polypeptide ligand comprising
at least a light chain
immunoglobulin variable region or heavy chain immunoglobulin variable region
which
specifically recognizes and binds an epitope of an antigen. Antibodies are
composed of a heavy
and a light chain, each of which has a variable region, termed the variable
heavy (VH) region and
the variable light (VI) region. Together, the VH region and the VL region are
responsible for
binding the antigen recognized by the antibody. Typically, an immunoglobulin
has heavy (H)
chains and light (L) chains interconnected by disulfide bonds. There are two
types of light chain,
lambda (X) and kappa (x). There are five main heavy chain classes (or
isotypes) which
determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA
and IgE. Each
heavy and light chain contains a constant region and a variable region, (the
regions are also
known as "domains"). In combination, the heavy and the light chain variable
regions
specifically bind the antigen. Light and heavy chain variable regions contain
a "framework"
region interrupted by three hypervariable regions, also called
"complementarity-determining
regions" or "CDRs". The extent of the framework region and CDRs have been
defined (see,
Kabat et at., Sequences of Proteins of Immunological Interest,U U.S.
Department of Health and
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Human Services, 1991, which is hereby incorporated by reference). The Kabat
database is now
maintained online. The sequences of the framework regions of different light
or heavy chains
are relatively conserved within a species. The framework region of an
antibody, that is the
combined framework regions of the constituent light and heavy chains, largely
adopt a 13-sheet
5 conformation and the CDRs form loops which connect, and in some cases
form part of, the 13-
sheet structure. Thus, framework regions act to form a scaffold that provides
for positioning the
CDRs in correct orientation by inter-chain, non-covalent interactions.
[0049] The CDRs are primarily responsible for binding to an epitope of an
antigen. The CDRs
of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered
sequentially
10 starting from the N-terminus, and are also typically identified by the
chain in which the particular
CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy
chain of the
antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable
domain of the
light chain of the antibody in which it is found. An antibody that binds HER2
protein will have a
specific VH region and the VL region sequence, and thus specific CDR
sequences. Antibodies
15 with different specificities (i.e. different combining sites for
different antigens) have different
CDRs. Although it is the CDRs that vary from antibody to antibody, only a
limited number of
amino acid positions within the CDRs are directly involved in antigen binding.
These positions
within the CDRs are called specificity determining residues (SDRs).
"Immunoglobulin-related
compositions" as used herein, refers to antibodies (including monoclonal
antibodies, polyclonal
antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies,
multi-specific
antibodies, bispecific antibodies, etc.,) as well as antibody fragments. An
antibody or antigen
binding fragment thereof specifically binds to an antigen.
[0050] As used herein, the term "antibody-related polypeptide" means antigen-
binding antibody
fragments, including single-chain antibodies, that can comprise the variable
region(s) alone, or in
combination, with all or part of the following polypeptide elements: hinge
region, CHi, CH2, and
CH3 domains of an antibody molecule. Also included in the technology are any
combinations of
variable region(s) and hinge region, CHi, CH2, and CH3 domains. Antibody-
related molecules
useful in the present methods, e.g., but are not limited to, Fab, Fab' and
F(a1302, Fd, single-chain
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Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising either
a VL or VH domain. Examples include: (i) a Fab fragment, a monovalent fragment
consisting of
the VL, VH, CL and CHi domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a Fd
fragment consisting of the
and CHi domains; (iv) a Fv fragment consisting of the VL and VH domains of a
single arm of
an antibody, (v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989),
which consists of a
VH domain; and (vi) an isolated complementarity determining region (CDR). As
such "antibody
fragments" or "antigen binding fragments" can comprise a portion of a full
length antibody,
generally the antigen binding or variable region thereof. Examples of antibody
fragments or
antigen binding fragments include Fab, Fab', F(ab')2, and Fv fragments;
diabodies; linear
antibodies; single-chain antibody molecules; and multi-specific antibodies
formed from antibody
fragments.
[0051] "Bispecific antibody" or "BsAb," as used herein, refers to an antibody
that can bind
simultaneously to two targets that have a distinct structure, e.g., two
different target antigens,
two different epitopes on the same target antigen, or a hapten and a target
antigen or epitope on a
target antigen. A variety of different bispecific antibody structures are
known in the art. In some
embodiments, each antigen binding moiety in a bispecific antibody includes VH
and/or VL
regions; in some such embodiments, the VH and/or VL regions are those found in
a particular
monoclonal antibody. In some embodiments, the bispecific antibody contains two
antigen
binding moieties, each including VH and/or VL regions from different
monoclonal antibodies. In
some embodiments, the bispecific antibody contains two antigen binding
moieties, wherein one
of the two antigen binding moieties includes an immunoglobulin molecule having
VH and/or VL
regions that contain CDRs from a first monoclonal antibody, and the other
antigen binding
moiety includes an antibody fragment (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB,
scFv, etc.) having
VH and/or VL regions that contain CDRs from a second monoclonal antibody.
[0052] As used herein, the term "antibody-dependent cell-mediated
cytotoxicity" or "ADCC",
refers to a mechanism of cell-mediated immune defense whereby an effector cell
of the immune
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system actively lyses a target cell, such as a tumor cell, whose membrane-
surface antigens have
been bound by antibodies, such as anti-HER2 antibodies.
[0053] As used herein, an "antigen" refers to a molecule to which an antibody
(or antigen
binding fragment thereof) can selectively bind. The target antigen may be a
protein,
carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or
synthetic compound. In
some embodiments, the target antigen may be a polypeptide (e.g., a HER2
polypeptide). An
antigen may also be administered to an animal to generate an immune response
in the animal.
[0054] The term "antigen binding fragment" refers to a fragment of the whole
immunoglobulin
structure which possesses a part of a polypeptide responsible for binding to
antigen. Examples
of the antigen binding fragment useful in the present technology include scFv,
(scFv)2, scFvFc,
Fab, Fab' and F(ab1)2, but are not limited thereto. Any of the above-noted
antibody fragments are
obtained using conventional techniques known to those of skill in the art, and
the fragments are
screened for binding specificity and neutralization activity in the same
manner as are intact
antibodies.
[0055] As used herein, "binding affinity" means the strength of the total
noncovalent interactions
between a single binding site of a molecule (e.g., an antibody) and its
binding partner (e.g., an
antigen or antigenic peptide). The affinity of a molecule X for its partner Y
can generally be
represented by the dissociation constant (KD). Affinity can be measured by
standard methods
known in the art, including those described herein. A low-affinity complex
contains an antibody
that generally tends to dissociate readily from the antigen, whereas a high-
affinity complex
contains an antibody that generally tends to remain bound to the antigen for a
longer duration.
[0056] As used herein, the term "biological sample" means sample material
derived from living
cells. Biological samples may include tissues, cells, protein or membrane
extracts of cells, and
biological fluids (e.g., ascites fluid or cerebrospinal fluid (C SF)) isolated
from a subject, as well
as tissues, cells and fluids present within a subject. Biological samples of
the present technology
include, but are not limited to, samples taken from breast tissue, renal
tissue, the uterine cervix,
the endometrium, the head or neck, the gallbladder, parotid tissue, the
prostate, the brain, the
pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small
intestine, the colon,
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the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung
tissue, the bladder, adipose
tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis
tissue, the tonsils,
thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid,
seminal fluid,
urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears.
Biological samples
can also be obtained from biopsies of internal organs or from cancers.
Biological samples can be
obtained from subjects for diagnosis or research or can be obtained from non-
diseased
individuals, as controls or for basic research. Samples may be obtained by
standard methods
including, e.g., venous puncture and surgical biopsy. In certain embodiments,
the biological
sample is a tissue sample obtained by needle biopsy.
[0057] As used herein, the term "CDR grafting" means replacing at least one
CDR of an
"acceptor" antibody with a CDR "graft" from a "donor" antibody possessing a
desirable antigen
specificity.
[0058] As used herein, the term "chimeric antibody" means an antibody in which
the Fc constant
region of a monoclonal antibody from one species (e.g., a mouse Fc constant
region) is replaced,
using recombinant DNA techniques, with an Fc constant region from an antibody
of another
species (e.g., a human Fc constant region). See generally, Robinson et at.,
PCT/U586/02269;
Akira et at., European Patent Application 184,187; Taniguchi, European Patent
Application
171,496; Morrison et al., European Patent Application 173,494; Neuberger et
al., WO 86/01533;
Cabilly et at. U.S. Patent No. 4,816,567; Cabilly et at., European Patent
Application 0125,023;
Better et al., Science 240: 1041-1043, 1988; Liu et al., Proc. Natl. Acad.
Sci. USA 84: 3439-
3443, 1987; Liu et al., I Immunol 139: 3521-3526, 1987; Sun et al., Proc.
Natl. Acad. Sci. USA
84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Wood et
al., Nature 314:
446-449, 1885; and Shaw et al., Natl. Cancer Inst. 80: 1553-1559, 1988.
[0059] As used herein, the term "complement-dependent cytotoxicity" or "CDC"
generally
refers to an effector function of IgG and IgM antibodies, which trigger
classical complement
pathway when bound to a surface antigen, inducing formation of a membrane
attack complex
and target cell lysis.
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[0060] As used herein, the term "conjugated" refers to the association of two
molecules by any
method known to those in the art. Suitable types of associations include
chemical bonds and
physical bonds. Chemical bonds include, for example, covalent bonds and
coordinate bonds.
Physical bonds include, for instance, hydrogen bonds, dipolar interactions,
van der Waal forces,
electrostatic interactions, hydrophobic interactions and aromatic stacking.
[0061] As used herein, the term "consensus FR" means a framework (FR) antibody
region in a
consensus immunoglobulin sequence. The FR regions of an antibody do not
contact the antigen.
[0062] As used herein, a "control" is an alternative sample used in an
experiment for comparison
purpose. A control can be "positive" or "negative." For example, where the
purpose of the
experiment is to determine a correlation of the efficacy of a therapeutic
agent for the treatment
for a particular type of disease, a positive control (a compound or
composition known to exhibit
the desired therapeutic effect) and a negative control (a subject or a sample
that does not receive
the therapy or receives a placebo) are typically employed.
[0063] As used herein, the term "diabodies" refers to small antibody fragments
with two antigen-
binding sites, which fragments comprise a heavy-chain variable domain (VH)
connected to a
light-chain variable domain (VI) in the same polypeptide chain (VH VL). By
using a linker that
is too short to allow pairing between the two domains on the same chain, the
domains are forced
to pair with the complementary domains of another chain and create two antigen
binding sites.
Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and
Hollinger et at.,
Proc Natl Acad Sci USA, 90: 6444-6448 (1993).
[0064] As used herein, the term "effective amount" refers to a quantity
sufficient to achieve a
desired therapeutic and/or prophylactic effect, e.g., an amount which results
in the prevention of,
or a decrease in a disease or condition described herein or one or more signs
or symptoms
associated with a disease or condition described herein. In the context of
therapeutic or
prophylactic applications, the amount of a composition administered to the
subject will vary
depending on the composition, the degree, type, and severity of the disease
and on the
characteristics of the individual, such as general health, age, sex, body
weight and tolerance to
drugs. The skilled artisan will be able to determine appropriate dosages
depending on these and
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other factors. The compositions can also be administered in combination with
one or more
additional therapeutic compounds. In the methods described herein, the
therapeutic
compositions may be administered to a subject having one or more signs or
symptoms of a
disease or condition described herein. As used herein, a "therapeutically
effective amount" of a
5 .. composition refers to composition levels in which the physiological
effects of a disease or
condition are ameliorated or eliminated. A therapeutically effective amount
can be given in one
or more administrations.
[0065] As used herein, the term "effector cell" means an immune cell which is
involved in the
effector phase of an immune response, as opposed to the cognitive and
activation phases of an
10 immune response. Exemplary immune cells include a cell of a myeloid or
lymphoid origin, e.g.,
lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)),
killer cells, natural
killer cells, macrophages, monocytes, eosinophils, neutrophils,
polymorphonuclear cells,
granulocytes, mast cells, and basophils. Effector cells express specific Fc
receptors and carry out
specific immune functions. An effector cell can induce antibody-dependent cell-
mediated
15 cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For
example, monocytes,
macrophages, neutrophils, eosinophils, and lymphocytes which express FcaR are
involved in
specific killing of target cells and presenting antigens to other components
of the immune
system, or binding to cells that present antigens.
[0066] As used herein, the term "epitope" means a protein determinant capable
of specific
20 .. binding to an antibody. Epitopes usually consist of chemically active
surface groupings of
molecules such as amino acids or sugar side chains and usually have specific
three dimensional
structural characteristics, as well as specific charge characteristics.
Conformational and non-
conformational epitopes are distinguished in that the binding to the former
but not the latter is
lost in the presence of denaturing solvents. In some embodiments, an "epitope"
of the HER2
.. protein is a region of the protein to which the anti-HER2 antibodies of the
present technology
specifically bind. In some embodiments, the epitope is a conformational
epitope or a non-
conformational epitope. To screen for anti-HER2 antibodies which bind to an
epitope, a routine
cross-blocking assay such as that described in Antibodies, A Laboratory
Manual, Cold Spring
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21
Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This
assay can be
used to determine if an anti-HER2 antibody binds the same site or epitope as
an anti-HER2
antibody of the present technology. Alternatively, or additionally, epitope
mapping can be
performed by methods known in the art. For example, the antibody sequence can
be
mutagenized such as by alanine scanning, to identify contact residues. In a
different method,
peptides corresponding to different regions of HER2 protein can be used in
competition assays
with the test antibodies or with a test antibody and an antibody with a
characterized or known
epitope.
[0067] As used herein, "expression" includes one or more of the following:
transcription of the
gene into precursor mRNA; splicing and other processing of the precursor mRNA
to produce
mature mRNA; mRNA stability; translation of the mature mRNA into protein
(including codon
usage and tRNA availability); and glycosylation and/or other modifications of
the translation
product, if required for proper expression and function.
[0068] As used herein, the term "gene" means a segment of DNA that contains
all the
information for the regulated biosynthesis of an RNA product, including
promoters, exons,
introns, and other untranslated regions that control expression.
[0069] As used herein, "homology" or "identity" or "similarity" refers to
sequence similarity
between two peptides or between two nucleic acid molecules. Homology can be
determined by
comparing a position in each sequence which may be aligned for purposes of
comparison. When
a position in the compared sequence is occupied by the same base or amino
acid, then the
molecules are homologous at that position. A degree of homology between
sequences is a
function of the number of matching or homologous positions shared by the
sequences. A
polynucleotide or polynucleotide region (or a polypeptide or polypeptide
region) has a certain
percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%
or 99%) of
"sequence identity" to another sequence means that, when aligned, that
percentage of bases (or
amino acids) are the same in comparing the two sequences. This alignment and
the percent
homology or sequence identity can be determined using software programs known
in the art. In
some embodiments, default parameters are used for alignment. One alignment
program is
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BLAST, using default parameters. In particular, programs are BLASTN and
BLASTP, using the
following default parameters: Genetic code=standard; filter=none; strand=both;
cutoff=60;
expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by =HIGH SCORE;
Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+SwissProtein+SPupdate+PIR. Details of these programs can be found
at the
National Center for Biotechnology Information. Biologically equivalent
polynucleotides are
those having the specified percent homology and encoding a polypeptide having
the same or
similar biological activity. Two sequences are deemed "unrelated" or "non-
homologous" if they
share less than 40% identity, or less than 25% identity, with each other.
[0070] As used herein, "humanized" forms of non-human (e.g., murine)
antibodies are chimeric
antibodies which contain minimal sequence derived from non-human
immunoglobulin. For the
most part, humanized antibodies are human immunoglobulins in which
hypervariable region
residues of the recipient are replaced by hypervariable region residues from a
non-human species
(donor antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity,
affinity, and capacity. In some embodiments, Fv framework region (FR) residues
of the human
immunoglobulin are replaced by corresponding non-human residues. Furthermore,
humanized
antibodies may comprise residues which are not found in the recipient antibody
or in the donor
antibody. These modifications are made to further refine antibody performance
such as binding
affinity. Generally, the humanized antibody will comprise substantially all of
at least one, and
typically two, variable domains (e.g., Fab, Fab', F(ab1)2, or Fv), 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 FR regions are those of a human immunoglobulin
consensus FR sequence
although the FR regions may include one or more amino acid substitutions that
improve binding
affinity. The number of these amino acid substitutions in the FR are typically
no more than 6 in
the H chain, and in the L chain, no more than 3. The humanized antibody
optionally may also
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a human
immunoglobulin. For further details, see Jones et al., Nature 321:522-525
(1986); Reichmann et
at., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596
(1992). See e.g.,
Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).
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[0071] As used herein, the term "hypervariable region" refers to the amino
acid residues of an
antibody which are responsible for antigen-binding. The hypervariable region
generally
comprises amino acid residues from a "complementarity determining region" or
"CDR" (e.g.,
around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and
around about 31-
35B (H1), 50-65 (H2) and 95-102 (H3) in the VH (Kabat et at., Sequences of
Proteins of
Immunological Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda,
MD. (1991)) and/or those residues from a "hypervariable loop" (e.g., residues
26-32 (L1), 50-52
(L2) and 91-96 (L3) in the VL, and 26-32 (H1), 52A-55 (H2) and 96-101 (H3) in
the VH (Chothia
and Leski Mol. Biol. 196:901-917 (1987)).
[0072] As used herein, the terms "identical" or percent "identity", when used
in the context of
two or more nucleic acids or polypeptide sequences, refer to two or more
sequences or
subsequences that are the same or have a specified percentage of amino acid
residues or
nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region
(e.g.,
nucleotide sequence encoding an antibody described herein or amino acid
sequence of an
antibody described herein)), when compared and aligned for maximum
correspondence over a
comparison window or designated region as measured using a BLAST or BLAST 2.0
sequence
comparison algorithms with default parameters described below, or by manual
alignment and
visual inspection (e.g., NCBI web site). Such sequences are then said to be
"substantially
identical." This term also refers to, or can be applied to, the complement of
a test sequence. The
term also includes sequences that have deletions and/or additions, as well as
those that have
substitutions. In some embodiments, identity exists over a region that is at
least about 25 amino
acids or nucleotides in length, or 50-100 amino acids or nucleotides in
length.
[0073] As used herein, the term "intact antibody" or "intact immunoglobulin"
means an antibody
that has at least two heavy (H) chain polypeptides and two light (L) chain
polypeptides
interconnected by disulfide bonds. Each heavy chain is comprised of a heavy
chain variable
region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
The heavy chain
constant region is comprised of three domains, CHi, CH2 and CH3. Each light
chain is
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comprised of a light chain variable region (abbreviated herein as LCVR or VL)
and a light chain
constant region. The light chain constant region is comprised of one domain,
CL. The VH and
VL regions can be further subdivided into regions of hypervariability, termed
complementarity
determining regions (CDR), interspersed with regions that are more conserved,
termed
framework regions (FR). Each VH and VL is composed of three CDRs and four FRs,
arranged
from amino-terminus to carboxyl-terminus in the following order: FRi, CDRi,
FR2, CDR2, FR3,
CDR3, FR4. The variable regions of the heavy and light chains contain a
binding domain that
interacts with an antigen. The constant regions of the antibodies can mediate
the binding of the
immunoglobulin to host tissues or factors, including various cells of the
immune system (e.g.,
effector cells) and the first component (Clq) of the classical complement
system.
[0074] As used herein, the term "linker" refers to a functional group (e.g.,
chemical or
polypeptide) that covalently attaches two or more polypeptides or nucleic
acids so that they are
connected to one another. As used herein, a "peptide linker" refers to one or
more amino acids
used to couple two proteins together (e.g., to couple VH and VL domains). In
certain
embodiments, the linker comprises amino acids sequence of (GGGGS)n, wherein n
is 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more. In certain embodiments, the
linker comprises amino
acids having the sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 73) or
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 74).
[0075] The term "monoclonal antibody" as used herein refers to an antibody
obtained from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies comprising
the population are identical except for possible naturally occurring mutations
that may be present
in minor amounts. For example, a monoclonal antibody can be an antibody that
is derived from
a single clone, including any eukaryotic, prokaryotic, or phage clone, and not
the method by
which it is produced. A monoclonal antibody composition displays a single
binding specificity
.. and affinity for a particular epitope. Monoclonal antibodies are highly
specific, being directed
against a single antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody
preparations which typically include different antibodies directed against
different determinants
(epitopes), each monoclonal antibody is directed against a single determinant
on the antigen.
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The modifier "monoclonal" indicates the character of the antibody as being
obtained from a
substantially homogeneous population of antibodies, and is not to be construed
as requiring
production of the antibody by any particular method. Monoclonal antibodies can
be prepared
using a wide variety of techniques known in the art including, e.g., but not
limited to, hybridoma,
5 recombinant, and phage display technologies. For example, the monoclonal
antibodies to be
used in accordance with the present methods may be made by the hybridoma
method first
described by Kohler et at., Nature 256:495 (1975), or may be made by
recombinant DNA
methods (See, e.g.,U U.S. Patent No. 4,816,567). The "monoclonal antibodies"
may also be
isolated from phage antibody libraries using the techniques described in
Clackson et at., Nature
10 352:624-628 (1991) and Marks et al., I Mot. Biol. 222:581-597 (1991),
for example.
[0076] As used herein, the term "nucleic acid" or "polynucleotide" means any
RNA or DNA,
which may be unmodified or modified RNA or DNA. Polynucleotides include,
without
limitation, single- and double-stranded DNA, DNA that is a mixture of single-
and double-
stranded regions, single- and double-stranded RNA, RNA that is mixture of
single- and double-
15 stranded regions, and hybrid molecules comprising DNA and RNA that may
be single-stranded
or, more typically, double-stranded or a mixture of single- and double-
stranded regions. In
addition, polynucleotide refers to triple-stranded regions comprising RNA or
DNA or both RNA
and DNA. The term polynucleotide also includes DNAs or RNAs containing one or
more
modified bases and DNAs or RNAs with backbones modified for stability or for
other reasons.
20 [0077] As used herein, the term "pharmaceutically acceptable carrier" is
intended to include any
and all solvents, dispersion media, coatings, antibacterial and antifungal
compounds, isotonic
and absorption delaying compounds, and the like, compatible with
pharmaceutical
administration. Pharmaceutically-acceptable carriers and their formulations
are known to one
skilled in the art and are described, for example, in Remington's
Pharmaceutical Sciences
25 (20th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins,
Philadelphia, Pa.).
[0078] As used herein, the term "polyclonal antibody" means a preparation of
antibodies derived
from at least two (2) different antibody-producing cell lines. The use of this
term includes
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preparations of at least two (2) antibodies that contain antibodies that
specifically bind to
different epitopes or regions of an antigen.
[0079] As used herein, the terms "polypeptide," "peptide" and "protein" are
used
interchangeably herein to mean a polymer comprising two or more amino acids
joined to each
other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
Polypeptide refers to
both short chains, commonly referred to as peptides, glycopeptides or
oligomers, and to longer
chains, generally referred to as proteins. Polypeptides may contain amino
acids other than the 20
gene-encoded amino acids. Polypeptides include amino acid sequences modified
either by
natural processes, such as post-translational processing, or by chemical
modification techniques
that are well known in the art. Such modifications are well described in basic
texts and in more
detailed monographs, as well as in a voluminous research literature.
[0080] As used herein, the term "recombinant" when used with reference, e.g.,
to a cell, or
nucleic acid, protein, or vector, indicates that the cell, nucleic acid,
protein or vector, has been
modified by the introduction of a heterologous nucleic acid or protein or the
alteration of a native
nucleic acid or protein, or that the material is derived from a cell so
modified. Thus, for
example, recombinant cells express genes that are not found within the native
(non-recombinant)
form of the cell or express native genes that are otherwise abnormally
expressed, under
expressed or not expressed at all.
[0081] As used herein, the term "separate" therapeutic use refers to an
administration of at least
two active ingredients at the same time or at substantially the same time by
different routes.
[0082] As used herein, the term "sequential" therapeutic use refers to
administration of at least
two active ingredients at different times, the administration route being
identical or different.
More particularly, sequential use refers to the whole administration of one of
the active
ingredients before administration of the other or others commences. It is thus
possible to
administer one of the active ingredients over several minutes, hours, or days
before
administering the other active ingredient or ingredients. There is no
simultaneous treatment in
this case.
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[0083] As used herein, the term "simultaneous" therapeutic use refers to the
administration of at
least two active ingredients by the same route and at the same time or at
substantially the same
time.
[0084] As used herein, the terms "single-chain antibodies" or "single-chain Fv
(scFv)" refer to
an antibody fusion molecule of the two domains of the Fv fragment, VL and VH.
Single-chain
antibody molecules may comprise a polymer with a number of individual
molecules, for
example, dimer, trimer or other polymers. Furthermore, although the two
domains of the F,
fragment, VL and VH, are coded for by separate genes, they can be joined,
using recombinant
methods, by a synthetic linker that enables them to be made as a single
protein chain in which
the VL and VH regions pair to form monovalent molecules (known as single-chain
F, (say)).
Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc Natl Acad
Sci 85:5879-
5883. Such single-chain antibodies can be prepared by recombinant techniques
or enzymatic or
chemical cleavage of intact antibodies.
[0085] As used herein, "specifically binds" refers to a molecule (e.g., an
antibody or antigen
binding fragment thereof) which recognizes and binds another molecule (e.g.,
an antigen), but
that does not substantially recognize and bind other molecules. The terms
"specific binding,"
"specifically binds to," or is "specific for" a particular molecule (e.g., a
polypeptide, or an
epitope on a polypeptide), as used herein, can be exhibited, for example, by a
molecule having a
KD for the molecule to which it binds to of about 10-4M, 10-5M, 10'M, 10-7M,
10-8M,
10-9M, 10-1 M, 10"M, or 10-12M. The term "specifically binds" may also refer
to binding
where a molecule (e.g., an antibody or antigen binding fragment thereof) binds
to a particular
polypeptide (e.g., a HER2 polypeptide), or an epitope on a particular
polypeptide, without
substantially binding to any other polypeptide, or polypeptide epitope.
[0086] As used herein, the terms "subject", "patient", or "individual" can be
an individual
organism, a vertebrate, a mammal, or a human. In some embodiments, the
subject, patient or
individual is a human.
[0087] As used herein, the term "therapeutic agent" is intended to mean a
compound that, when
present in an effective amount, produces a desired therapeutic effect on a
subject in need thereof
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[0088] "Treating" or "treatment" as used herein covers the treatment of a
disease or disorder
described herein, in a subject, such as a human, and includes: (i) inhibiting
a disease or disorder,
i.e., arresting its development; (ii) relieving a disease or disorder, i.e.,
causing regression of the
disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting,
relieving, or slowing
progression of one or more symptoms of the disease or disorder. In some
embodiments,
treatment means that the symptoms associated with the disease are, e.g.,
alleviated, reduced,
cured, or placed in a state of remission.
[0089] It is also to be appreciated that the various modes of treatment of
disorders as described
herein are intended to mean "substantial," which includes total but also less
than total treatment,
and wherein some biologically or medically relevant result is achieved. The
treatment may be a
continuous prolonged treatment for a chronic disease or a single, or few time
administrations for
the treatment of an acute condition.
[0090] Amino acid sequence modification(s) of the anti-HER2 antibodies
described herein are
contemplated. Such modifications may be performed to improve the binding
affinity and/or
other biological properties of the antibody, for example, to render the
encoded amino acid
glycosylated, or to destroy the antibody's ability to bind to C I q, Fc
receptor, or to activate the
complement system. Amino acid sequence variants of an anti-HER2 antibody are
prepared by
introducing appropriate nucleotide changes into the antibody nucleic acid, by
peptide synthesis,
or by chemical modifications. Such modifications include, for example,
deletions from, and/or
insertions into and/or substitutions of, residues within the amino acid
sequences of the antibody.
Any combination of deletion, insertion, and substitution is made to obtain the
antibody of
interest, as long as the obtained antibody possesses the desired properties.
The modification also
includes the change of the pattern of glycosylation of the protein. The sites
of greatest interest
for substitutional mutagenesis include the hypervariable regions, but FR
alterations are also
contemplated.
[0091] Conservative amino acid substitutions are amino acid substitutions that
change a given
amino acid to a different amino acid with similar biochemical properties
(e.g., charge,
hydrophobicity and size). "Conservative substitutions" are shown in the Table
below.
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Table 1. Amino Acid Substitutions
Original Conservative
Exemplary Substitutions
Residue 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
leu; val; met; ala; phe;
Ile (I) leu
norleucine
norleucine; ile; val; met;
Leu (L) ile
ala; phe
Lys (K) arg; gln; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr tyr
Pro (P) ala ala
Ser (S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
ile; leu; met; phe; ala;
Val (V) leu
norleucine
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[0092] One type of substitutional variant involves substituting one or more
hypervariable region
residues of a parent antibody. A convenient way for generating such
substitutional variants
involves affinity maturation using phage display. Specifically, several
hypervariable region sites
(e.g., 6-7 sites) are mutated to generate all possible amino acid
substitutions at each site. The
5 antibody variants thus generated are displayed in a monovalent fashion
from filamentous phage
particles as fusions to the gene III product of M13 packaged within each
particle. The phage-
displayed variants are then screened for their biological activity (e.g.,
binding affinity) as herein
disclosed. In order to identify candidate hypervariable region sites for
modification, alanine
scanning mutagenesis can be performed to identify hypervariable region
residues contributing
10 significantly to antigen binding. Alternatively, or additionally, it may
be beneficial to analyze a
crystal structure of the antigen-antibody complex to identify contact points
between the antibody
and the antigen. Such contact residues and neighboring residues are candidates
for substitution
according to the techniques elaborated herein. Once such variants are
generated, the panel of
variants is subjected to screening as described herein and antibodies with
similar or superior
15 properties in one or more relevant assays may be selected for further
development.
HER2
[0093] HER2 (GenBank: NP 004439.2 (SEQ ID NO: 84)) is a receptor tyrosine
kinase of the
epidermal growth factor receptor family. Amplification or overexpression of
HER2 has been
demonstrated in the development and progression of cancers. Hercepting
(trastuzumab) is an
20 anti-HER2 monoclonal antibody approved for treating HER2-positive
metastatic breast cancer
and HER2-positive gastric cancer (Trastuzumab [Highlights of Prescribing
Information]. South
San Francisco, CA: Genentech, Inc.; 2014). Ertumaxomab is a tri-specific HER2-
CD3 antibody
with intact Fc-receptor binding (see, for example, Kiewe et at. 2006, Clin
Cancer Res, 12(10):
3085-3091). Ertumaxomab is a rat-mouse antibody; therefore, upon
administration to humans, a
25 human anti-mouse antibody response and a human anti-rat antibody
response are expected.
2502A, the parental antibody of ertumaxomab, has low affinity for HER2 and low
avidity
(Diermeier-Daucher et al., MAbs, 2012, 4(5): 614-622).
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Immunoglobulin-related Compositions of the Present Technology
[0094] The present technology describes methods and compositions for the
generation and use of
anti-HER2 immunoglobulin-related compositions (e.g., anti-HER2 antibodies or
antigen binding
fragments thereof). The antibodies and antigen binding fragments of the
present technology
selectively bind to HER2 polypeptides. The anti-HER2 immunoglobulin-related
compositions of
the present disclosure may be useful in the diagnosis, or treatment of HER2-
associated cancers.
Anti-HER2 immunoglobulin-related compositions within the scope of the present
technology
include, e.g., but are not limited to, monoclonal, chimeric, humanized,
bispecific antibodies and
diabodies that specifically bind the target polypeptide, a homolog, derivative
or a fragment
thereof. The present disclosure also provides antigen binding fragments of any
of the anti-HER2
antibodies disclosed herein, wherein the antigen binding fragment is selected
from the group
consisting of Fab, F(ab)'2, Fab', scFv, and F. The amino acid sequences of the
anti-HER2
immunoglobulin-related compositions of the present technology are described in
Figures 1-3.
Region Sequence Fragment
CDR-Ell GFNIKDTYIH (SEQ ID NO: 1)
RIYPTNGYTRYADSVKG (SEQ ID NO: 2)
CDR-H2
RGYPTNGYTRYADSVKG (SEQ ID NO: 7)
CDR H3 WGGDGFYAMDY (SEQ ID NO: 3)
- WGDDGFYAMDY (SEQ ID NO: 8)
CDR Li RASQDVNTAVA (SEQ ID NO: 4)
- RASQDVPTAVA (SEQ ID NO: 9)
SASFLYS (SEQ ID NO: 5)
CDR-L2 SASDLYS (SEQ ID NO: 10)
DASFLYS (SEQ ID NO: 11)
QQHYTTPPT (SEQ ID NO: 6)
CDR-L3
QQHYTAPPT (SEQ ID NO: 12):
[0095] In one aspect, the present disclosure provides an antibody or antigen
binding fragment
thereof comprising a heavy chain immunoglobulin variable domain (VH) and a
light chain
immunoglobulin variable domain (VIA wherein (a) (i) the VH comprises a VH-CDR1
sequence of
SEQ ID NO: 1, a VH-CDR2 sequence of SEQ ID NO: 2 or SEQ ID NO: 7 and a VH-CDR3
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sequence of SEQ ID NO: 8; or (ii) the VH comprises a VH-CDR1 sequence of SEQ
ID NO: 1, a
VH-CDR2 sequence of SEQ ID NO: 7 and a VH-CDR3 sequence of SEQ ID NO: 3 or SEQ
ID
NO: 8; and/or (b) (i) the VL comprises a VL-CDR1 sequence of SEQ ID NO: 9, a
VL-CDR2
sequence of SEQ ID NO: 5, SEQ ID NO: 10, or SEQ ID NO: 11, and a VL-CDR3
sequence of
.. SEQ ID NO: 6 or SEQ ID NO: 12; or (ii) the VL comprises a VL-CDR1 sequence
of SEQ ID
NO: 4 or SEQ ID NO: 9, a VL-CDR2 sequence of SEQ ID NO: 10, or SEQ ID NO: 11,
and a VL-
CDR3 sequence of SEQ ID NO: 6 or SEQ ID NO: 12; or (iii) the VL comprises a VL-
CDR1
sequence of SEQ ID NO: 4 or SEQ ID NO: 9, a VL-CDR2 sequence of SEQ ID NO: 5,
SEQ ID
NO: 10, or SEQ ID NO: 11, and a VL-CDR3 sequence of SEQ ID NO: 12.
[0096] In one aspect, the present disclosure provides an antibody or
antigen binding
fragment thereof comprising a heavy chain immunoglobulin variable domain (VH)
and a light
chain immunoglobulin variable domain (VL), wherein: (a) the VH comprises an
amino acid
sequence selected from any one of SEQ ID NOs: 13, 15, or 17; and/or (b) the VL
comprises an
amino acid sequence selected from any one of SEQ ID NOs: 14, 16, 18, 19, or
20. In some
embodiments, the antibody or antigen binding fragment thereof comprises a VH
and a VL
selected from the group consisting of SEQ ID NOs: 13 and 14, SEQ ID NOs: 15
and 16, SEQ ID
NOs: 17 and 14, SEQ ID NOs: 15 and 18, SEQ ID NOs: 15 and 19, and SEQ ID NOs:
15 and 20,
respectively.
[0097] In any of the above embodiments, the antibody further comprises a
Fc domain of any
.. isotype, e.g., but are not limited to, IgG (including IgGl, IgG2, IgG3, and
IgG4), IgA (including
IgAi and IgA2), IgD, IgE, or IgM, and IgY. Non-limiting examples of constant
region sequences
include:
[0098] Human IgD constant region, Uniprot: P01880 (SEQ ID NO: 75)
APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRR
DSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQA
EGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV
QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLT
LPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLC
EVSGESPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYT
CVVSHEDSRTLLNASRSLEVSYVTDHGPMK
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[0099] Human IgG1 constant region, Uniprot: P01857 (SEQ ID NO: 76)
AS TKGP SVFPLAP S SK S T S GGTAALGCLVKDYFPEP VTV SWNS GALT SGVHTFPAVLQ SS
GLYSL S SVVTVP SS SLGTQTYICNVNHKP SNTKVDKKVEPK S CDK THT CPP CP APELL GG
P S VFLF PPKPKD TLMI SRTPEVT C VVVD V SHEDPEVKFNWYVD GVEVHNAK TKPREEQ Y
N S TYRVV S VL TVLHQDWLNGKEYK CKV SNK ALP AP IEK TI SKAK GQPREP Q VYTLPP SR
DEL TKNQVSLTCL VK GF YP SD IAVEWE SNGQPENNYKTTPPVLD SDGSFFLYSKLTVDK
SRWQQGNVF S C SVMHEALHNHYTQK SL SL SPGK
[00100] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO: 77)
AS TKGP SVFPLAPC SRST SES TAALGCLVKDYFPEPVTVS WNS GAL T S GVHTFPAVL Q S S
GLYSL S SVVTVP S SNFGTQTYTCNVDHKP SNTKVDKTVERKCCVECPP CP APP VAGP SV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEM
TKNQVSLTCLVKGFYP SDI S VEWE SNGQ PENNYKT TPPMLD SDGSFFLYSKLTVDKSRW
QQGNVF SC SVMHEALHNHYTQKSLSL SPGK
[00101] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO: 78)
AS TKGP SVFPLAPC SRST S GGTAALGCLVKDYFPEP VTVS WNS GAL T S GVHTFPAVLQ SS
GLYSL S SVVTVP SS SL GT Q TY T CNVNHKP SNTKVDKRVELKTPLGDTTHTCPRCPEPKSC
DTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVK
GFYP SDIAVEWES SGQPENNYNTTPPMLD SDGSFFLYSKLTVDKSRWQQGNIF SC SVMH
EALHNRF TQKSL SL SP GK
[00102] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 79)
GSASAPTLFPLVS CEN SP SDTS SVAVGCLAQDFLPD SITL SWKYKNNSDIS STRGFP SVLR
GGKYAAT SQVLLP SKD VMQ GTDEHVVCKVQHPNGNKEKNVPLP VIAELPPKV S VF VPP
RDGFF GNPRK SKLIC QAT GF SPRQIQVSWLREGKQVGS GVT TDQVQAEAKES GP TTYKV
T STLTIKESDWLGQ SMFTCRVDHRGLTFQQNAS SMCVPDQDTAIRVFAIPP SFASIFLTKS
TKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGE
RFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPAD
VFVQWMQRGQPLSPEKYVT S APMPEP Q AP GRYF AH S IL TV SEEEWNT GET YT C VAHEA
LPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY
[00103] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO: 80)
AS TKGP SVFPLAPC SRST SES TAALGCLVKDYFPEPVTVS WNS GAL T S GVHTFPAVL Q S S
GLYSL S SVVTVP SS SLGTKTYTCNVDHKP SNTKVDKRVE SKY GPPCP S CPAPEFLGGP S V
FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS S IEK T I SKAK GQPREP Q VYTLPP SQEEM
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TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW
QEGNVFSCSVMHEALHNHYTQKSLSLSLGK
[00104] Human IgAl constant region, Uniprot: P01876 (SEQ ID NO: 81)
ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDAS
GDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPS
CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLC
GCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEEL
ALNELVTLTCLARGF SPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILR
VAAEDWKKGDTF SCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY
[00105] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 82)
ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDAS
GDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPA
LEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGC
AQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVULLPPPSEELALNELVTLTCLA
RGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDT
FSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY
[00106] Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 83)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[00107] In some embodiments, the immunoglobulin-related compositions of the
present
technology comprise a heavy chain constant region that is at least 80%, at
least 85%, at least
90%, at least 95%, at least 99%, or is 100% identical to SEQ ID NOs: 75-82.
Additionally or
alternatively, in some embodiments, the immunoglobulin-related compositions of
the present
technology comprise a light chain constant region that is at least 80%, at
least 85%, at least 90%,
at least 95%, at least 99%, or is 100% identical to SEQ ID NO: 83.
[00108] Additionally or alternatively, in some embodiments, the antibody or
antigen binding
fragment binds to the extracellular region of a HER2 polypeptide. In certain
embodiments, the
epitope is a conformational epitope or non-conformational epitope.
[00109] In some embodiments, the heavy chain (HC) and light chain (LC)
immunoglobulin
variable domain sequences are components of the same polypeptide chain. In
other
embodiments, the HC and LC immunoglobulin variable domain sequences are
components of
different polypeptide chains. In certain embodiments, the antibody is a full-
length antibody.
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[00110] In some embodiments, the immunoglobulin-related compositions of the
present
technology bind specifically to at least one HER2 polypeptide. In some
embodiments, the
immunoglobulin-related compositions of the present technology bind at least
one HER2
polypeptide with a dissociation constant (KD) of about 10-3M, 10'M, 10-5M, 10-
6M, 10-7M,
5 1OM, 10-9M, 10-mM, 10"M, or 10-'2M. In certain embodiments, the
immunoglobulin-
related compositions are monoclonal antibodies, chimeric antibodies, humanized
antibodies,
bispecific antibodies, or multi-specific antibodies. In some embodiments, the
antibodies
comprise a human antibody framework region.
[00111] In certain embodiments, the immunoglobulin-related compositions
contain an IgG1
10 constant region comprising one or more amino acid substitutions selected
from the group
consisting of N297A, K322A, L234A and L235A. Additionally or alternatively, in
some
embodiments, the immunoglobulin-related compositions contain an IgG4 constant
region
comprising a S228P mutation.
[00112] In one aspect, the present disclosure provides a multi-specific
(e.g., bispecific)
15 antibody comprising a first polypeptide chain, a second polypeptide
chain, a third polypeptide
chain and a fourth polypeptide chain, wherein the first and second polypeptide
chains are
covalently bonded to one another, the second and third polypeptide chains are
covalently bonded
to one another, and the third and fourth polypeptide chain are covalently
bonded to one another,
and wherein: (a) each of the first polypeptide chain and the fourth
polypeptide chain comprises
20 in the N-terminal to C-terminal direction: (i) a light chain variable
domain of a first
immunoglobulin that is capable of specifically binding to a first epitope;
(ii) a light chain
constant domain of the first immunoglobulin; (iii) a flexible peptide linker
comprising the amino
acid sequence (GGGGS)3; and (iv) a light chain variable domain of a second
immunoglobulin
that is linked to a complementary heavy chain variable domain of the second
immunoglobulin, or
25 .. a heavy chain variable domain of a second immunoglobulin that is linked
to a complementary
light chain variable domain of the second immunoglobulin, wherein the light
chain and heavy
chain variable domains of the second immunoglobulin are capable of
specifically binding to a
second epitope, and are linked together via a flexible peptide linker
comprising the amino acid
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36
sequence (GGGGS)6 to form a single-chain variable fragment; and (b) each of
the second
polypeptide chain and the third polypeptide chain comprises in the N-terminal
to C-terminal
direction: (i) a heavy chain variable domain of the first immunoglobulin that
is capable of
specifically binding to the first epitope; and (ii) a heavy chain constant
domain of the first
immunoglobulin; and wherein the heavy chain variable domain of the first
immunoglobulin or
the heavy chain variable domain of the second immunoglobulin comprises any one
of SEQ ID
NOs: 13, 15, or 17, and/or the light chain variable domain of the first
immunoglobulin or the
light chain variable domain of the second immunoglobulin comprises any one of
SEQ ID NOs:
14, 16, 18, 19, or 20.
[00113] In one aspect, the immunoglobulin-related compositions of the present
technology
comprise a heavy chain (HC) and a light chain (LC) selected from the group
consisting of SEQ
ID NOs: 21 and 22, SEQ ID NOs: 21 and 23, SEQ ID NOs: 21 and 24, SEQ ID NOs:
21 and 25,
SEQ ID NOs: 21 and 26, SEQ ID NOs: 21 and 27, SEQ ID NOs: 21 and 28, SEQ ID
NOs: 21
and 29, SEQ ID NOs: 21 and 30, SEQ ID NOs: 21 and 31, SEQ ID NOs: 21 and 32,
SEQ ID
NOs: 21 and 33, SEQ ID NOs: 34 and 33, SEQ ID NOs: 21 and 35, SEQ ID NOs: 36
and 33,
SEQ ID NOs: 21 and 37, SEQ ID NOs: 21 and 38, SEQ ID NOs: 21 and 39, SEQ ID
NOs: 21
and 40, SEQ ID NOs: 21 and 41, SEQ ID NOs: 21 and 42, SEQ ID NOs: 21 and 43,
SEQ ID
NOs: 21 and 44, SEQ ID NOs: 21 and 45, SEQ ID NOs: 21 and 46, SEQ ID NOs: 21
and 47,
SEQ ID NOs: 21 and 48, SEQ ID NOs: 21 and 49, SEQ ID NOs: 21 and 50, SEQ ID
NOs: 21
and 51, SEQ ID NOs: 21 and 52, SEQ ID NOs: 21 and 53, SEQ ID NOs: 21 and 54,
SEQ ID
NOs: 21 and 55, SEQ ID NOs: 21 and 56, SEQ ID NOs: 21 and 57, SEQ ID NOs: 21
and 58,
SEQ ID NOs: 21 and 59, SEQ ID NOs: 21 and 60, SEQ ID NOs: 21 and 61, SEQ ID
NOs: 21
and 62, SEQ ID NOs: 21 and 63, SEQ ID NOs: 21 and 64, SEQ ID NOs: 21 and 65,
SEQ ID
NOs: 21 and 66, SEQ ID NOs: 21 and 67, SEQ ID NOs: 21 and 68, SEQ ID NOs: 21
and 69,
.. SEQ ID NOs: 21 and 70, SEQ ID NOs: 21 and 71, SEQ ID NOs: 21 and 72, and
SEQ ID NOs:
21 and 85, respectively.
[00114] In some aspects, the anti-HER2 immunoglobulin-related compositions
described
herein contain structural modifications to facilitate rapid binding and cell
uptake and/or slow
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release. In some aspects, the anti-HER2 immunoglobulin-related composition of
the present
technology (e.g., an antibody) may contain a deletion in the CH2 constant
heavy chain region to
facilitate rapid binding and cell uptake and/or slow release. In some aspects,
a Fab fragment is
used to facilitate rapid binding and cell uptake and/or slow release. In some
aspects, a F(ab)'2
fragment is used to facilitate rapid binding and cell uptake and/or slow
release.
[00115] In one aspect, the present technology provides a nucleic acid sequence
encoding any
of the immunoglobulin-related compositions described herein. Also disclosed
herein are
recombinant nucleic acid sequences encoding any of the antibodies described
herein.
[00116] In another aspect, the present technology provides a host cell
expressing any nucleic
acid sequence encoding any of the immunoglobulin-related compositions
described herein.
[00117] The immunoglobulin-related compositions of the present technology
(e.g., an anti-
HER2 antibody) can be monospecific, bispecific, trispecific or of greater
multi-specificity.
Multi-specific antibodies can be specific for different epitopes of one or
more HER2
polypeptides as well as for heterologous compositions such as a heterologous
polypeptide or
solid support material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO
92/05793;
Tutt et at., I Immunol. 147: 60-69 (1991); U.S. Pat. Nos. 5,573,920,
4,474,893, 5,601,819,
4,714,681, 4,925,648; 6,106,835; Kostelny etal.,I Immunol. 148: 1547-1553
(1992). In some
embodiments, the immunoglobulin-related compositions are chimeric. In certain
embodiments,
the immunoglobulin-related compositions are humanized.
[00118] The immunoglobulin-related compositions of the present technology can
further be
recombinantly fused to a heterologous polypeptide at the N- or C-terminus or
chemically
conjugated (including covalently and non-covalently conjugations) to
polypeptides or other
compositions. For example, the immunoglobulin-related compositions of the
present technology
can be recombinantly fused or conjugated to molecules useful as labels in
detection assays and
effector molecules such as heterologous polypeptides, drugs, or toxins. See,
e.g., WO 92/08495;
WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 0 396 387.
[00119] In any of the above embodiments of the immunoglobulin-related
compositions of the
present technology, the antibody or antigen binding fragment may be optionally
conjugated to an
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agent selected from the group consisting of isotopes, dyes, chromagens,
contrast agents, drugs,
toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists,
growth factors,
radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination
thereof. For a
chemical bond or physical bond, a functional group on the immunoglobulin-
related composition
typically associates with a functional group on the agent. Alternatively, a
functional group on
the agent associates with a functional group on the immunoglobulin-related
composition.
[00120] The functional groups on the agent and immunoglobulin-related
composition can
associate directly. For example, a functional group (e.g., a sulfhydryl group)
on an agent can
associate with a functional group (e.g., sulfhydryl group) on an
immunoglobulin-related
composition to form a disulfide. Alternatively, the functional groups can
associate through a
cross-linking agent (i.e., linker). Some examples of cross-linking agents are
described below.
The cross-linker can be attached to either the agent or the immunoglobulin-
related composition.
The number of agents or immunoglobulin-related compositions in a conjugate is
also limited by
the number of functional groups present on the other. For example, the maximum
number of
agents associated with a conjugate depends on the number of functional groups
present on the
immunoglobulin-related composition. Alternatively, the maximum number of
immunoglobulin-
related compositions associated with an agent depends on the number of
functional groups
present on the agent.
[00121] In yet another embodiment, the conjugate comprises one immunoglobulin-
related
composition associated to one agent. In one embodiment, a conjugate comprises
at least one
agent chemically bonded (e.g., conjugated) to at least one immunoglobulin-
related composition.
The agent can be chemically bonded to an immunoglobulin-related composition by
any method
known to those in the art. For example, a functional group on the agent may be
directly attached
to a functional group on the immunoglobulin-related composition. Some examples
of suitable
functional groups include, for example, amino, carboxyl, sulfhydryl,
maleimide, isocyanate,
isothiocyanate and hydroxyl.
[00122] The agent may also be chemically bonded to the immunoglobulin-related
composition
by means of cross-linking agents, such as dialdehydes, carbodiimides,
dimaleimides, and the
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like. Cross-linking agents can, for example, be obtained from Pierce
Biotechnology, Inc.,
Rockford, Ill. The Pierce Biotechnology, Inc. web-site can provide assistance.
Additional cross-
linking agents include the platinum cross-linking agents described in U.S.
Pat. Nos. 5,580,990;
5,985,566; and 6,133,038 of Kreatech Biotechnology, B.V., Amsterdam, The
Netherlands.
[00123] Alternatively, the functional group on the agent and immunoglobulin-
related
composition can be the same. Homobifunctional cross-linkers are typically used
to cross-link
identical functional groups. Examples of homobifunctional cross-linkers
include EGS (i.e.,
ethylene glycol bis[succinimidylsuccinate]), DSS (i.e., disuccinimidyl
suberate), DMA (i.e.,
dimethyl adipimidate.2HC1), DTSSP (i.e., 3,3'-
dithiobis[sulfosuccinimidylpropionate])), DPDPB
(i.e., 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane), and BMH (i.e., bis-
maleimidohexane).
Such homobifunctional cross-linkers are also available from Pierce
Biotechnology, Inc.
[00124] In other instances, it may be beneficial to cleave the agent from the
immunoglobulin-
related composition. The web-site of Pierce Biotechnology, Inc. described
above can also
provide assistance to one skilled in the art in choosing suitable cross-
linkers which can be
cleaved by, for example, enzymes in the cell. Thus the agent can be separated
from the
immunoglobulin-related composition. Examples of cleavable linkers include SMPT
(i.e., 4-
succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), Sulfo-LC-SPDP
(i.e.,
sulfosuccinimidyl 6-(342-pyridyldithio]-propionamido)hexanoate), LC- SPDP
(i.e., succinimidyl
6-(342-pyridyldithio]-propionamido)hexanoate), Sulfo-LC-SPDP (i.e.,
sulfosuccinimidyl 6-(3-
[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-
pyridyldithio]-
propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid
HC1).
[00125] In another embodiment, a conjugate comprises at least one agent
physically bonded
with at least one immunoglobulin-related composition. Any method known to
those in the art
can be employed to physically bond the agents with the immunoglobulin-related
compositions.
For example, the immunoglobulin-related compositions and agents can be mixed
together by any
method known to those in the art. The order of mixing is not important. For
instance, agents can
be physically mixed with immunoglobulin-related compositions by any method
known to those
in the art. For example, the immunoglobulin-related compositions and agents
can be placed in a
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container and agitated, by for example, shaking the container, to mix the
immunoglobulin-related
compositions and agents.
[00126] The immunoglobulin-related compositions can be modified by any method
known to
those in the art. For instance, the immunoglobulin-related composition may be
modified by
5 means of cross-linking agents or functional groups, as described above.
A. Methods of Preparing Anti-HER2 Antibodies of the Present Technology
[00127] General Overview. Initially, a target polypeptide is chosen to which
an antibody of
the present technology can be raised. For example, an antibody may be raised
against the full-
length HER2 protein, or to a portion of the extracellular domain of the HER2
protein.
10 Techniques for generating antibodies directed to such target
polypeptides are well known to
those skilled in the art. Examples of such techniques include, for example,
but are not limited to,
those involving display libraries, xeno or human mice, hybridomas, and the
like. Target
polypeptides within the scope of the present technology include any
polypeptide derived from
HER2 protein containing the extracellular domain which is capable of eliciting
an immune
15 response.
[00128] It should be understood that recombinantly engineered antibodies and
antibody
fragments, e.g., antibody-related polypeptides, which are directed to HER2
protein and
fragments thereof are suitable for use in accordance with the present
disclosure.
[00129] Anti-HER2 antibodies that can be subjected to the techniques set forth
herein include
20 monoclonal and polyclonal antibodies, and antibody fragments such as
Fab, Fab', F(ab1)2, Fd,
scFv, diabodies, antibody light chains, antibody heavy chains and/or antibody
fragments.
Methods useful for the high yield production of antibody Fv-containing
polypeptides, e.g., Fab'
and F(ab1)2 antibody fragments have been described. See U .S . Pat. No.
5,648,237.
[00130] Generally, an antibody is obtained from an originating species. More
particularly, the
25 nucleic acid or amino acid sequence of the variable portion of the light
chain, heavy chain or
both, of an originating species antibody having specificity for a target
polypeptide antigen is
obtained. An originating species is any species which was useful to generate
the antibody of the
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41
present technology or library of antibodies, e.g., rat, mouse, rabbit,
chicken, monkey, human, and
the like.
[00131] Phage or phagemid display technologies are useful techniques to derive
the antibodies
of the present technology. Techniques for generating and cloning monoclonal
antibodies are
well known to those skilled in the art. Expression of sequences encoding
antibodies of the
present technology, can be carried out in E. coil.
[00132] Due to the degeneracy of nucleic acid coding sequences, other
sequences which
encode substantially the same amino acid sequences as those of the naturally
occurring proteins
may be used in the practice of the present technology These include, but are
not limited to,
nucleic acid sequences including all or portions of the nucleic acid sequences
encoding the above
polypeptides, which are altered by the substitution of different codons that
encode a functionally
equivalent amino acid residue within the sequence, thus producing a silent
change. It is
appreciated that the nucleotide sequence of an immunoglobulin according to the
present
technology tolerates sequence homology variations of up to 25% as calculated
by standard
methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule
Sequencing
and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R.
Liss, Inc.) so long
as such a variant forms an operative antibody which recognizes HER2 proteins.
For example,
one or more amino acid residues within a polypeptide sequence can be
substituted by another
amino acid of a similar polarity which acts as a functional equivalent,
resulting in a silent
alteration. Substitutes for an amino acid within the sequence may be selected
from other
members of the class to which the amino acid belongs. For example, the
nonpolar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and
methionine. The polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine,
asparagine, and glutamine. The positively charged (basic) amino acids include
arginine, lysine
and histidine. The negatively charged (acidic) amino acids include aspartic
acid and glutamic
acid. Also included within the scope of the present technology are proteins or
fragments or
derivatives thereof which are differentially modified during or after
translation, e.g., by
glycosylation, proteolytic cleavage, linkage to an antibody molecule or other
cellular ligands, etc.
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Additionally, an immunoglobulin encoding nucleic acid sequence can be mutated
in vitro or in
vivo to create and/or destroy translation, initiation, and/or termination
sequences or to create
variations in coding regions and/or form new restriction endonuclease sites or
destroy pre-
existing ones, to facilitate further in vitro modification. Any technique for
mutagenesis known in
the art can be used, including but not limited to in vitro site directed
mutagenesis, I Biol. Chem.
253:6551, use of Tab linkers (Pharmacia), and the like.
[00133] Preparation of PolyclonalAntisera and Immunogens. Methods of
generating
antibodies or antibody fragments of the present technology typically include
immunizing a
subject (generally a non-human subject such as a mouse or rabbit) with a
purified HER2 protein
or fragment thereof, or with a cell expressing the HER2 protein or fragment
thereof. An
appropriate immunogenic preparation can contain, e.g., a recombinantly-
expressed HER2 protein
or a chemically-synthesized HER2 peptide. The extracellular domain of the HER2
protein, or a
portion or fragment thereof, can be used as an immunogen to generate an anti-
HER2 antibody
that binds to the HER2 protein, or a portion or fragment thereof using
standard techniques for
polyclonal and monoclonal antibody preparation.
[00134] In some embodiments, the antigenic HER2 peptide comprises at least 10,
at least 20,
at least 30, at least 40, at least 50, at least 60, at least 70, at least 80,
at least 90, or at least 100
amino acid residues. Longer antigenic peptides are sometimes desirable over
shorter antigenic
peptides, depending on use and according to methods well known to those
skilled in the art.
Multimers of a given epitope are sometimes more effective than a monomer.
[00135] If needed, the immunogenicity of the HER2 protein (or fragment
thereof) can be
increased by fusion or conjugation to a carrier protein such as keyhole limpet
hemocyanin (KLH)
or ovalbumin (OVA). Many such carrier proteins are known in the art. One can
also combine
the HER2 protein with a conventional adjuvant such as Freund's complete or
incomplete
adjuvant to increase the subject's immune reaction to the polypeptide. Various
adjuvants used to
increase the immunological response include, but are not limited to, Freund's
(complete and
incomplete), mineral gels (e.g., aluminum hydroxide), surface active
substances (e.g.,
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
dinitrophenol, etc.), human
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adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or
similar
immunostimulatory compounds. These techniques are standard in the art.
[00136] Alternatively, nanoparticles, for example, virus-like particles
(VLPs), can be used to
present antigens, e.g., HER2, to a host animal. Virus-like particles are
multiprotein structures
that mimic the organization and conformation of authentic native viruses
without being
infectious, since they do not carry any viral genetic material (Urakami A, et
at, Clin Vaccine
Immunol 24: e00090-17 (2017)) When introduced to a host immune system, VLPs
can evoke
effective immune responses, making them attractive carriers of foreign
antigens. An important
advantage of a VLP-based antigen presenting platform is that it can display
antigens in a dense,
.. repetitive manner. Thus, antigen-bearing VLPs are able to induce strong B-
cell responses by
effectively enabling the cross-linking of B cell receptors (BCRs). VLPs may be
genetically
manipulated to fine their properties, e.g., immunogenicity. These techniques
are standard in the
art.
[00137] The isolation of sufficient purified protein or polypeptide to
which an antibody is to
be raised may be time consuming and sometimes technically challenging.
Additional challenges
associated with conventional protein-based immunization include concerns over
safety, stability,
scalability and consistency of the protein antigen. Nucleic acid (DNA and RNA)
based
immunizations have emerged as promising alternatives. DNA vaccines are usually
based on
bacterial plasmids that encode the polypeptide sequence of candidate antigen,
e.g., HER2. With
a robust eukaryotic promoter, the encoded antigen can be expressed to yield
enough levels of
transgene expression once the host is inoculated with the plasmids (Galvin
T.A., et at.,
Vaccine 2000, 18:2566-2583). Modern DNA vaccine generation relies on DNA
synthesis,
possibly one-step cloning into the plasmid vector and subsequent isolation of
the plasmid, which
takes significantly less time and cost to manufacture. The resulting plasmid
DNA is also highly
stable at room temperature, avoiding cold transportation and leading to
substantially extended
shelf-life. These techniques are standard in the art.
[00138] Alternatively, nucleic acid sequences encoding the antigen of
interest, e.g., HER2,
can be synthetically introduced into a mRNA molecule. The mRNA is then
delivered into a host
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animal, whose cells would recognize and translate the mRNA sequence to the
polypeptide
sequence of the candidate antigen, e.g., HER2, thus triggering the immune
response to the
foreign antigen. An attractive feature of mRNA antigen or mRNA vaccine is that
mRNA is a
non-infectious, non-integrating platform. There is no potential risk of
infection or insertional
mutagenesis associated with DNA vaccines. In addition, mRNA is degraded by
normal cellular
processes and has a controllable in vivo half-life through modification of
design and delivery
methods (Kariko, K., et aL, Mal Ther 16: 1833-1840 (2008); Kauffman, K. J., et
aL õI Control
Release 240, 227-234 (2016); Guan, S. & Rosenecker. J.. Gene Ther 24, 133-143
(2017): Mess,
A., et aL, Mal Ther 23, 1456---1464 (2015)). These techniques are standard in
the art.
.. [00139] In describing the present technology, immune responses may be
described as either
"primary" or "secondary" immune responses. A primary immune response, which is
also
described as a "protective" immune response, refers to an immune response
produced in an
individual as a result of some initial exposure (e.g., the initial
"immunization" or "priming") to a
particular antigen, e.g., HER2 protein. In some embodiments, the immunization
can occur as a
result of vaccinating the individual with a vaccine containing the antigen.
For example, the
vaccine can be a HER2 vaccine comprising one or more HER2 protein-derived
antigens. A
primary immune response can become weakened or attenuated over time and can
even disappear
or at least become so attenuated that it cannot be detected. Accordingly, the
present technology
also relates to a "secondary" immune response, which is also described here as
a "memory
immune response." The term secondary immune response refers to an immune
response elicited
in an individual after a primary immune response has already been produced.
[00140] Thus, a secondary immune response can be elicited, e.g., to enhance an
existing
immune response that has become weakened or attenuated (e.g., boosting), or to
recreate a
previous immune response that has either disappeared or can no longer be
detected. The
secondary or memory immune response can be either a humoral (antibody)
response or a cellular
response. A secondary or memory humoral response occurs upon stimulation of
memory B cells
that were generated at the first presentation of the antigen. Delayed type
hypersensitivity (DTH)
reactions are a type of cellular secondary or memory immune response that are
mediated by
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CD4+ T cells. A first exposure to an antigen primes the immune system and
additional
exposure(s) results in a DTH.
[00141] Following appropriate immunization, the anti-HER2 antibody can be
prepared from
the subject's serum. If desired, the antibody molecules directed against the
HER2 protein can be
5 isolated from the mammal (e.g., from the blood) and further purified by
well-known techniques,
such as polypeptide A chromatography to obtain the IgG fraction.
[00142] Monoclonal Antibody. In one embodiment of the present technology, the
antibody is
an anti-HER2 monoclonal antibody. For example, in some embodiments, the anti-
HER2
monoclonal antibody may be a human or a mouse anti-HER2 monoclonal antibody.
For
10 .. preparation of monoclonal antibodies directed towards the HER2 protein,
or derivatives,
fragments, analogs or homologs thereof, any technique that provides for the
production of
antibody molecules by continuous cell line culture can be utilized. Such
techniques include, but
are not limited to, the hybridoma technique (See, e.g., Kohler & Milstein,
1975. Nature 256: 495-
497); the trioma technique; the human B-cell hybridoma technique (See, e.g.,
Kozbor, et at.,
15 1983. Immunol. Today 4: 72) and the EBV hybridoma technique to produce
human monoclonal
antibodies (See, e.g., Cole, et at., 1985. In: MONOCLONAL ANTIBODIES AND
CANCER
THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be
utilized in the
practice of the present technology and can be produced by using human
hybridomas (See, e.g.,
Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by
transforming human B-cells
20 with Epstein Barr Virus in vitro (See, e.g., Cole, et al., 1985. In:
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a
population of nucleic acids that encode regions of antibodies can be isolated.
PCR utilizing
primers derived from sequences encoding conserved regions of antibodies is
used to amplify
sequences encoding portions of antibodies from the population and then DNAs
encoding
25 antibodies or fragments thereof, such as variable domains, are
reconstructed from the amplified
sequences. Such amplified sequences also can be fused to DNAs encoding other
proteins ¨ e.g.,
a bacteriophage coat, or a bacterial cell surface protein ¨ for expression and
display of the fusion
polypeptides on phage or bacteria. Amplified sequences can then be expressed
and further
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selected or isolated based, e.g., on the affinity of the expressed antibody or
fragment thereof for
an antigen or epitope present on the HER2 protein. Alternatively, hybridomas
expressing anti-
HER2 monoclonal antibodies can be prepared by immunizing a subject and then
isolating
hybridomas from the subject's spleen using routine methods. See, e.g.,
Milstein et at., (Galfre
and Milstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomas using
standard
methods will produce monoclonal antibodies of varying specificity (i.e., for
different epitopes)
and affinity. A selected monoclonal antibody with the desired properties,
e.g., HER2 binding,
can be used as expressed by the hybridoma, it can be bound to a molecule such
as polyethylene
glycol (PEG) to alter its properties, or a cDNA encoding it can be isolated,
sequenced and
manipulated in various ways. Synthetic dendromeric trees can be added to
reactive amino acid
side chains, e.g., lysine, to enhance the immunogenic properties of HER2
protein. Also, CPG-
dinucleotide techniques can be used to enhance the immunogenic properties of
the HER2
protein. Other manipulations include substituting or deleting particular amino
acyl residues that
contribute to instability of the antibody during storage or after
administration to a subject, and
affinity maturation techniques to improve affinity of the antibody of the HER2
protein.
[00143] Hybridoma Technique. In some embodiments, the antibody of the present
technology
is an anti-HER2 monoclonal antibody produced by a hybridoma which includes a B
cell obtained
from a transgenic non-human animal, e.g., a transgenic mouse, having a genome
comprising a
human heavy chain transgene and a light chain transgene fused to an
immortalized cell.
Hybridoma techniques include those known in the art and taught in Harlow et
at., Antibodies: A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349
(1988);
Hammerling et at., Monoclonal Antibodies And T-Cell Hybridomas, 563-681(1981).
Other
methods for producing hybridomas and monoclonal antibodies are well known to
those of skill in
the art.
[00144] Phage Display Technique. As noted above, the antibodies of the present
technology
can be produced through the application of recombinant DNA and phage display
technology.
For example, anti-HER2 antibodies, can be prepared using various phage display
methods
known in the art. In phage display methods, functional antibody domains are
displayed on the
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surface of a phage particle which carries polynucleotide sequences encoding
them. Phages with
a desired binding property are selected from a repertoire or combinatorial
antibody library (e.g.,
human or murine) by selecting directly with an antigen, typically an antigen
bound or captured to
a solid surface or bead. Phages used in these methods are typically
filamentous phage including
fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains that are
recombinantly
fused to either the phage gene III or gene VIII protein. In addition, methods
can be adapted for
the construction of Fab expression libraries (See, e.g., Huse, et al., Science
246: 1275-1281,
1989) to allow rapid and effective identification of monoclonal Fab fragments
with the desired
specificity for a HER2 polypeptide, e.g., a polypeptide or derivatives,
fragments, analogs or
homologs thereof. Other examples of phage display methods that can be used to
make the
antibodies of the present technology include those disclosed in Huston et at.,
Proc. Natl. Acad.
Sci U.S.A., 85: 5879-5883, 1988; Chaudhary et at., Proc. Natl. Acad. Sci
U.S.A., 87: 1066-1070,
1990; Brinkman et al., I Immunol. Methods 182: 41-50, 1995; Ames et al., I
Immunol. Methods
184: 177-186, 1995; Kettleborough et al., Eur. I Immunol. 24: 952-958, 1994;
Persic et al.,
Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994;
PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236;
WO 95/15982; WO 95/20401; WO 96/06213; WO 92/01047 (Medical Research Council
et al.);
WO 97/08320 (Morphosys); WO 92/01047 (CAT/MRC); WO 91/17271 (Affymax); and
U.S.
Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753,
5,821,047,
5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743. Methods
useful for
displaying polypeptides on the surface of bacteriophage particles by attaching
the polypeptides
via disulfide bonds have been described by Lohning, U.S. Pat. No. 6,753,136.
As described in
the above references, after phage selection, the antibody coding regions from
the phage can be
isolated and used to generate whole antibodies, including human antibodies, or
any other desired
antigen binding fragment, and expressed in any desired host including
mammalian cells, insect
cells, plant cells, yeast, and bacteria. For example, techniques to
recombinantly produce Fab,
Fab' and F(a1302 fragments can also be employed using methods known in the art
such as those
disclosed in WO 92/22324; Mullinax et al., BioTechniques 12: 864-869, 1992;
and Sawai et al.,
AIRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.
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[00145] Generally, hybrid antibodies or hybrid antibody fragments that are
cloned into a
display vector can be selected against the appropriate antigen in order to
identify variants that
maintain good binding activity, because the antibody or antibody fragment will
be present on the
surface of the phage or phagemid particle. See, e.g., B arb as III et at.,
Phage Display, A
Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 2001).
However, other vector formats could be used for this process, such as cloning
the antibody
fragment library into a lytic phage vector (modified T7 or Lambda Zap systems)
for selection
and/or screening.
[00146] Expression of Recombinant Anti-HER2 Antibodies. As noted above, the
antibodies of
the present technology can be produced through the application of recombinant
DNA
technology. Recombinant polynucleotide constructs encoding an anti-HER2
antibody of the
present technology typically include an expression control sequence operably-
linked to the
coding sequences of anti-HER2 antibody chains, including naturally-associated
or heterologous
promoter regions. As such, another aspect of the technology includes vectors
containing one or
more nucleic acid sequences encoding an anti-HER2 antibody of the present
technology. For
recombinant expression of one or more of the polypeptides of the present
technology, the nucleic
acid containing all or a portion of the nucleotide sequence encoding the anti-
HER2 antibody is
inserted into an appropriate cloning vector, or an expression vector (i.e., a
vector that contains
the necessary elements for the transcription and translation of the inserted
polypeptide coding
.. sequence) by recombinant DNA techniques well known in the art and as
detailed below.
Methods for producing diverse populations of vectors have been described by
Lerner et al.,U U.S.
Pat. Nos. 6,291,160 and 6,680,192.
[00147] In general, expression vectors useful in recombinant DNA techniques
are often in the
form of plasmids. In the present disclosure, "plasmid" and "vector" can be
used interchangeably
as the plasmid is the most commonly used form of vector. However, the present
technology is
intended to include such other forms of expression vectors that are not
technically plasmids, such
as viral vectors (e.g., replication defective retroviruses, adenoviruses and
adeno-associated
viruses), which serve equivalent functions. Such viral vectors permit
infection of a subject and
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expression of a construct in that subject. In some embodiments, the expression
control
sequences are eukaryotic promoter systems in vectors capable of transforming
or transfecting
eukaryotic host cells. Once the vector has been incorporated into the
appropriate host, the host is
maintained under conditions suitable for high level expression of the
nucleotide sequences
encoding the anti-HER2 antibody, and the collection and purification of the
anti-HER2 antibody,
e.g., cross-reacting anti-HER2 antibodies. See generally,U .S . 2002/0199213.
These expression
vectors are typically replicable in the host organisms either as episomes or
as an integral part of
the host chromosomal DNA. Commonly, expression vectors contain selection
markers, e.g.,
ampicillin-resistance or hygromycin-resistance, to permit detection of those
cells transformed
with the desired DNA sequences. Vectors can also encode signal peptide, e.g.,
pectate lyase,
useful to direct the secretion of extracellular antibody fragments. See U.S.
Pat. No. 5,576,195.
[00148] The recombinant expression vectors of the present technology comprise
a nucleic
acid encoding a protein with HER2 binding properties in a form suitable for
expression of the
nucleic acid in a host cell, which means that the recombinant expression
vectors include one or
more regulatory sequences, selected on the basis of the host cells to be used
for expression that is
operably-linked to the nucleic acid sequence to be expressed. Within a
recombinant expression
vector, "operably-linked" is intended to mean that the nucleotide sequence of
interest is linked to
the regulatory sequence(s) in a manner that allows for expression of the
nucleotide sequence
(e.g., in an in vitro transcription/translation system or in a host cell when
the vector is introduced
into the host cell). The term "regulatory sequence" is intended to include
promoters, enhancers
and other expression control elements (e.g., polyadenylation signals). Such
regulatory sequences
are described, e.g., in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences
include
those that direct constitutive expression of a nucleotide sequence in many
types of host cell and
those that direct expression of the nucleotide sequence only in certain host
cells (e.g., tissue-
specific regulatory sequences). It will be appreciated by those skilled in the
art that the design of
the expression vector can depend on such factors as the choice of the host
cell to be transformed,
the level of expression of polypeptide desired, etc. Typical regulatory
sequences useful as
promoters of recombinant polypeptide expression (e.g., anti-HER2 antibody),
include, e.g., but
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are not limited to, promoters of 3-phosphoglycerate kinase and other
glycolytic enzymes.
Inducible yeast promoters include, among others, promoters from alcohol
dehydrogenase,
isocytochrome C, and enzymes responsible for maltose and galactose
utilization. In one
embodiment, a polynucleotide encoding an anti-HER2 antibody of the present
technology is
5 operably-linked to an ara B promoter and expressible in a host cell. See
U.S. Pat. 5,028,530.
The expression vectors of the present technology can be introduced into host
cells to thereby
produce polypeptides or peptides, including fusion polypeptides, encoded by
nucleic acids as
described herein (e.g., anti-HER2 antibody, etc.).
[00149] Another aspect of the present technology pertains to anti-HER2
antibody-expressing
10 host cells, which contain a nucleic acid encoding one or more anti-HER2
antibodies. The
recombinant expression vectors of the present technology can be designed for
expression of an
anti-HER2 antibody in prokaryotic or eukaryotic cells. For example, an anti-
HER2 antibody can
be expressed in bacterial cells such as Escherichia coil, insect cells (using
baculovirus expression
vectors), fungal cells, e.g., yeast, yeast cells or mammalian cells. Suitable
host cells are
15 discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the
recombinant expression vector can be transcribed and translated in vitro,
e.g., using T7 promoter
regulatory sequences and T7 polymerase. Methods useful for the preparation and
screening of
polypeptides having a predetermined property, e.g., anti-HER2 antibody, via
expression of
20 stochastically generated polynucleotide sequences has been previously
described. See U.S. Pat.
Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862;
6,492,107; 6,569,641.
[00150] Expression of polypeptides in prokaryotes is most often carried out in
E. coil with
vectors containing constitutive or inducible promoters directing the
expression of either fusion or
non-fusion polypeptides. Fusion vectors add a number of amino acids to a
polypeptide encoded
25 therein, usually to the amino terminus of the recombinant polypeptide.
Such fusion vectors
typically serve three purposes: (i) to increase expression of recombinant
polypeptide; (ii) to
increase the solubility of the recombinant polypeptide; and (iii) to aid in
the purification of the
recombinant polypeptide by acting as a ligand in affinity purification. Often,
in fusion
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expression vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety
and the recombinant polypeptide to enable separation of the recombinant
polypeptide from the
fusion moiety subsequent to purification of the fusion polypeptide. Such
enzymes, and their
cognate recognition sequences, include Factor Xa, thrombin and enterokinase.
Typical fusion
expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson,
1988. Gene 67:
31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.)
that fuse glutathione S-transferase (GST), maltose E binding polypeptide, or
polypeptide A,
respectively, to the target recombinant polypeptide.
[00151] Examples of suitable inducible non-fusion E. coil expression vectors
include pTrc
(Amrann et at., (1988) Gene 69: 301-315) and pET lid (Studier et at., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990) 60-89). Methods for targeted assembly of distinct active peptide or
protein domains to
yield multifunctional polypeptides via polypeptide fusion has been described
by Pack et al.,U U.S.
Pat. Nos. 6,294,353; 6,692,935. One strategy to maximize recombinant
polypeptide expression,
e.g., an anti-HER2 antibody, in E. coil is to express the polypeptide in host
bacteria with an
impaired capacity to proteolytically cleave the recombinant polypeptide. See,
e.g., Gottesman,
GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,
San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic
acid sequence of the
nucleic acid to be inserted into an expression vector so that the individual
codons for each amino
acid are those preferentially utilized in the expression host, e.g., E. coil
(See, e.g., Wada, et at.,
1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid
sequences of the present
technology can be carried out by standard DNA synthesis techniques.
[00152] In another embodiment, the anti-HER2 antibody expression vector is a
yeast
expression vector. Examples of vectors for expression in yeast Saccharomyces
cerevisiae
include pYepSecl (Baldari, et at., 1987. EMBO J. 6: 229-234), pMfa (Kurj an
and Herskowitz,
Cell 30: 933-943, 1982), pJRY88 (Schultz et at., Gene 54: 113-123, 1987),
pYES2 (Invitrogen
Corporation, San Diego, Calif), and picZ (Invitrogen Corp, San Diego, Calif).
Alternatively, an
anti-HER2 antibody can be expressed in insect cells using baculovirus
expression vectors.
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Baculovirus vectors available for expression of polypeptides, e.g., anti-HER2
antibody, in
cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al.,
Mol. Cell. Biol. 3:
2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170:
31-39).
[00153] In yet another embodiment, a nucleic acid encoding an anti-HER2
antibody of the
present technology is expressed in mammalian cells using a mammalian
expression vector.
Examples of mammalian expression vectors include, e.g., but are not limited
to, pCDM8 (Seed,
Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO 1 6: 187-195, 1987).
When used
in mammalian cells, the expression vector's control functions are often
provided by viral
regulatory elements. For example, commonly used promoters are derived from
polyoma,
adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable
expression systems for
both prokaryotic and eukaryotic cells that are useful for expression of the
anti-HER2 antibody of
the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al.,
MOLECULAR
CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[00154] In another embodiment, the recombinant mammalian expression vector is
capable of
directing expression of the nucleic acid in a particular cell type (e.g.,
tissue-specific regulatory
elements). Tissue-specific regulatory elements are known in the art. Non-
limiting examples of
suitable tissue-specific promoters include the albumin promoter (liver-
specific; Pinkert, et al.,
Genes Dev. . 1: 268-277, 1987), lymphoid-specific promoters (Calame and Eaton,
Adv. Immunol.
43: 235-275, 1988), promoters of T cell receptors (Winoto and Baltimore, EAJBO
J. 8: 729-733,
1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and
Baltimore, Cell
33: 741-748, 1983.), neuron-specific promoters (e.g., the neurofilament
promoter; Byrne and
Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1989), pancreas-specific
promoters (Edlund,
et al., 1985. Science 230: 912-916), and mammary gland-specific promoters
(e.g., milk whey
promoter; U.S. Pat. No. 4,873,316 and European Application Publication No.
264,166).
Developmentally-regulated promoters are also encompassed, e.g., the murine hox
promoters
(Kessel and Gruss, Science 249: 374-379, 1990) and the a-fetoprotein promoter
(Campes and
Tilghman, Genes Dev. 3: 537-546, 1989).
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[00155] Another aspect of the present methods pertains to host cells into
which a recombinant
expression vector of the present technology has been introduced. The terms
"host cell" and
"recombinant host cell" are used interchangeably herein. It is understood that
such terms refer
not only to the particular subject cell but also to the progeny or potential
progeny of such a cell.
.. Because certain modifications may occur in succeeding generations due to
either mutation or
environmental influences, such progeny may not, in fact, be identical to the
parent cell, but are
still included within the scope of the term as used herein.
[00156] A host cell can be any prokaryotic or eukaryotic cell. For example, an
anti-HER2
antibody can be expressed in bacterial cells such as E. coil, insect cells,
yeast or mammalian
cells. Mammalian cells are a suitable host for expressing nucleotide segments
encoding
immunoglobulins or fragments thereof See Winnacker, From Genes To Clones, (VCH
Publishers, NY, 1987). A number of suitable host cell lines capable of
secreting intact
heterologous proteins have been developed in the art, and include Chinese
hamster ovary (CHO)
cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell
lines. In some
embodiments, the cells are non-human. Expression vectors for these cells can
include expression
control sequences, such as an origin of replication, a promoter, an enhancer,
and necessary
processing information sites, such as ribosome binding sites, RNA splice
sites, polyadenylation
sites, and transcriptional terminator sequences. Queen et al., Immunol. Rev.
89: 49, 1986.
Illustrative expression control sequences are promoters derived from
endogenous genes,
cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. Co et
al., J Immunol.
148: 1149, 1992. Other suitable host cells are known to those skilled in the
art.
[00157] Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional
transformation or transfection techniques. As used herein, the terms
"transformation" and
"transfection" are intended to refer to a variety of art-recognized techniques
for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate
or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection,
electroporation,
biolistics or viral-based transfection. Other methods used to transform
mammalian cells include
the use of polybrene, protoplast fusion, liposomes, electroporation, and
microinjection (See
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generally, Sambrook et al., Molecular Cloning). Suitable methods for
transforming or
transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A
LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory
manuals. The vectors
containing the DNA segments of interest can be transferred into the host cell
by well-known
methods, depending on the type of cellular host.
[00158] Non-limiting examples of suitable vectors include those designed for
propagation and
expansion, or for expression or both. For example, a cloning vector can be
selected from the
group consisting of the pUC series, the pBluescript series (Stratagene,
LaJolla, Calif.), the pET
series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala,
Sweden), and
the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as
lamda-GT10,
lamda-GT11, lamda-ZapII (Stratagene), lamda-EMBL4, and lamda-NM1149, can also
be used.
Non-limiting examples of plant expression vectors include pBI110, pBI101.2,
pBI101.3, pBI121
and pBIN19 (Clontech). Non-limiting examples of animal expression vectors
include pEUK-C1,
pMAM and pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Calsbad, CA,
Carlsbad, CA) can also be used in accordance with the manufacturer's
recommendations.
[00159] In certain embodiments, the vector is a mammalian vector. In certain
embodiments,
the mammalian vector contains at least one promoter element, which mediates
the initiation of
transcription of mRNA, the antibody-coding sequence, and signals required for
the termination
of transcription and polyadenylation of the transcript. In certain
embodiments, the mammalian
vector contains additional elements, such as, for example, enhancers, Kozak
sequences and
intervening sequences flanked by donor and acceptor sites for RNA splicing. In
certain
embodiments, highly efficient transcription can be achieved with, for example,
the early and late
promoters from 5V40, the long terminal repeats (LTRS) from retroviruses, for
example, RSV,
HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). Cellular
elements can also
be used (e.g., the human actin promoter). Non-limiting examples of mammalian
expression
vectors include, vectors such as pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or
pLNCX
(Clonetech Labs, Palo Alto, Calif.), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or
pcDNA3.1/Hygro
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(+/-) (Invitrogen, Calsbad, CA), PSVL and PMSG (Pharmacia, Uppsala, Sweden),
pRSVcat
(ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Non-limiting
examples of mammalian host cells that can be used in combination with such
mammalian vectors
include human Hela 293, HEK 293, H9 and Jurkat cells, mouse 3T3, NIH3T3 and
C127 cells,
5 COS 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese
hamster ovary (CHO)
cells.
[00160] In certain embodiments, the vector is a viral vector, for
example, retroviral vectors,
parvovirus-based vectors, e.g., adeno-associated virus (AAV)-based vectors,
AAV-adenoviral
chimeric vectors, and adenovirus-based vectors, and lentiviral vectors, such
as Herpes simplex
10 (HSV)-based vectors. In certain embodiments, the viral vector is
manipulated to render the virus
replication deficient. In certain embodiments, the viral vector is manipulated
to eliminate
toxicity to the host. These viral vectors can be prepared using standard
recombinant DNA
techniques described in, for example, Sambrook et at., Molecular Cloning, a
Laboratory Manual,
2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); and
Ausubel et at.,
15 Current Protocols in Molecular Biology, Greene Publishing Associates and
John Wiley & Sons,
New York, N.Y. (1994).
[00161] In certain embodiments, a vector or polynucleotide described herein
can be
transferred to a cell (e.g., an ex vivo cell) by conventional techniques and
the resulting cell can be
cultured by conventional techniques to produce an anti-HER2 antibody or
antigen binding
20 fragment described herein. Accordingly, provided herein are cells
comprising a polynucleotide
encoding an anti-HER2 antibody or antigen binding fragment thereof operably
linked to a
regulatory expression element (e.g., promoter) for expression of such
sequences in the host cell.
In certain embodiments, a vector encoding the heavy chain operably linked to a
promoter and a
vector encoding the light chain operably linked to a promoter can be co-
expressed in the cell for
25 .. expression of the entire anti-HER2 antibody or antigen binding fragment.
In certain
embodiments, a cell comprises a vector comprising a polynucleotide encoding
both the heavy
chain and the light chain of an anti-HER2 antibody or antigen binding fragment
described herein
that are operably linked to a promoter. In certain embodiments, a cell
comprises two different
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vectors, a first vector comprising a polynucleotide encoding a heavy chain
operably linked to a
promoter, and a second vector comprising a polynucleotide encoding a light
chain operably
linked to a promoter. In certain embodiments, a first cell comprises a first
vector comprising a
polynucleotide encoding a heavy chain of an anti-HER2 antibody or antigen
binding fragment
described herein, and a second cell comprises a second vector comprising a
polynucleotide
encoding a light chain of an anti-HER2 antibody or antigen binding fragment
described herein.
In certain embodiments, provided herein is a mixture of cells comprising said
first cell and said
second cell. Examples of cells include, but are not limited to, a human cell,
a human cell line, E.
coil (e.g., E. coli TB-1, TG-2, DH5a, XL-Blue MRF' (Stratagene), SA2821 and
Y1090), B.
subtilis, P. aerugenosa, S. cerevisiae, N. crassa, insect cells (e.g., SP9,
Ea4) and the like.
[00162] For stable transfection of mammalian cells, it is known that,
depending upon the
expression vector and transfection technique used, only a small fraction of
cells may integrate
the foreign DNA into their genome. In order to identify and select these
integrants, a gene that
encodes a selectable marker (e.g., resistance to antibiotics) is generally
introduced into the host
cells along with the gene of interest. Various selectable markers include
those that confer
resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid
encoding a
selectable marker can be introduced into a host cell on the same vector as
that encoding the anti-
HER2 antibody or can be introduced on a separate vector. Cells stably
transfected with the
introduced nucleic acid can be identified by drug selection (e.g., cells that
have incorporated the
selectable marker gene will survive, while the other cells die).
[00163] A host cell that includes an anti-HER2 antibody of the present
technology, such as a
prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e.,
express) recombinant
anti-HER2 antibody. In one embodiment, the method comprises culturing the host
cell (into
which a recombinant expression vector encoding the anti-HER2 antibody has been
introduced) in
a suitable medium such that the anti-HER2 antibody is produced. In another
embodiment, the
method further comprises the step of isolating the anti-HER2 antibody from the
medium or the
host cell. Once expressed, collections of the anti-HER2 antibody, e.g., the
anti-HER2 antibodies
or the anti-HER2 antibody-related polypeptides are purified from culture media
and host cells.
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The anti-HER2 antibody can be purified according to standard procedures of the
art, including
HPLC purification, column chromatography, gel electrophoresis and the like. In
one
embodiment, the anti-HER2 antibody is produced in a host organism by the
method of Boss et
at., U.S. Pat. No. 4,816,397. Usually, anti-HER2 antibody chains are expressed
with signal
sequences and are thus released to the culture media. However, if the anti-
HER2 antibody
chains are not naturally secreted by host cells, the anti-HER2 antibody chains
can be released by
treatment with mild detergent. Purification of recombinant polypeptides is
well known in the art
and includes ammonium sulfate precipitation, affinity chromatography
purification technique,
column chromatography, ion exchange purification technique, gel
electrophoresis and the like
(See generally Scopes, Protein Purification (Springer-Verlag, N.Y., 1982).
[00164] Polynucleotides encoding anti-HER2 antibodies, e.g., the anti-HER2
antibody coding
sequences, can be incorporated in transgenes for introduction into the genome
of a transgenic
animal and subsequent expression in the milk of the transgenic animal. See,
e.g., U.S. Pat. Nos.
5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding
sequences for light
and/or heavy chains in operable linkage with a promoter and enhancer from a
mammary gland
specific gene, such as casein or P-lactoglobulin. For production of transgenic
animals,
transgenes can be microinjected into fertilized oocytes, or can be
incorporated into the genome
of embryonic stem cells, and the nuclei of such cells transferred into
enucleated oocytes.
[00165] Single-Chain Antibodies. In one embodiment, the anti-HER2 antibody of
the present
technology is a single-chain anti-HER2 antibody. According to the present
technology,
techniques can be adapted for the production of single-chain antibodies
specific to a HER2
protein (See, e.g.,U U.S. Pat. No. 4,946,778). Examples of techniques which
can be used to
produce single-chain Fvs and antibodies of the present technology include
those described in
U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology,
203: 46-88,
1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and
Skerra et al., Science
240: 1038-1040, 1988.
[00166] Chimeric and Humanized Antibodies. In one embodiment, the anti-HER2
antibody of
the present technology is a chimeric anti-HER2 antibody. In one embodiment,
the anti-HER2
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antibody of the present technology is a humanized anti-HER2 antibody. In one
embodiment of
the present technology, the donor and acceptor antibodies are monoclonal
antibodies from
different species. For example, the acceptor antibody is a human antibody (to
minimize its
antigenicity in a human), in which case the resulting CDR-grafted antibody is
termed a
"humanized" antibody.
[00167] Recombinant anti-HER2 antibodies, such as chimeric and humanized
monoclonal
antibodies, comprising both human and non-human portions, can be made using
standard
recombinant DNA techniques, and are within the scope of the present
technology. For some
uses, including in vivo use of the anti-HER2 antibody of the present
technology in humans as
well as use of these agents in in vitro detection assays, it is possible to
use chimeric or
humanized anti-HER2 antibodies. Such chimeric and humanized monoclonal
antibodies can be
produced by recombinant DNA techniques known in the art. Such useful methods
include, e.g.,
but are not limited to, methods described in International Application No.
PCT/U586/02269;
U.S. Pat. No. 5,225,539; European Patent No. 184187; European Patent No.
171496; European
Patent No. 173494; PCT International Publication No. WO 86/01533; U.S. Pat.
Nos. 4,816,567;
5,225,539; European Patent No. 125023; Better, etal., 1988. Science 240: 1041-
1043; Liu, etal.,
1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, etal., 1987.1 Immunol.
139: 3521-3526;
Sun, etal., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, etal.,
1987. Cancer Res.
47: 999-1005; Wood, etal., 1985. Nature 314: 446-449; Shaw, etal., 1988. Natl.
Cancer Inst.
80: 1553-1559; Morrison (1985) Science 229: 1202-1207; 0i, etal. (1986)
BioTechniques 4:
214; Jones, etal., 1986. Nature 321: 552-525; Verhoeyan, etal., 1988. Science
239: 1534;
Morrison, Science 229: 1202, 1985; Oi etal., BioTechniques 4: 214, 1986;
Gillies etal.,
Immunol. Methods, 125: 191-202, 1989; U.S. Pat. No. 5,807,715; and Beidler,
etal., 1988.
Immunol. 141: 4053-4060. For example, antibodies can be humanized using a
variety of
techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. No.
5,530,101;
5,585,089; 5,859,205; 6,248,516; EP460167), veneering or resurfacing (EP 0 592
106; EP 0 519
596; Padlan E. A., Molecular Immunology, 28: 489-498, 1991; Studnicka etal.,
Protein
Engineering 7: 805-814, 1994; Roguska etal., PNAS 91: 969-973, 1994), and
chain shuffling
(U.S. Pat. No. 5,565,332). In one embodiment, a cDNA encoding a murine anti-
HER2
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59
monoclonal antibody is digested with a restriction enzyme selected
specifically to remove the
sequence encoding the Fc constant region, and the equivalent portion of a cDNA
encoding a
human Fc constant region is substituted (See Robinson et al., PCT/US86/02269;
Akira et al.,
European Patent Application 184,187; Taniguchi, European Patent Application
171,496;
Morrison et al., European Patent Application 173,494; Neuberger et al., WO
86/01533; Cabilly
et at. U.S. Patent No. 4,816,567; Cabilly et at., European Patent Application
125,023; Better et
at. (1988) Science 240: 1041-1043; Liu et at. (1987) Proc. Natl. Acad. Sci.
USA 84: 3439-3443;
Liu et al. (1987)J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad.
Sci. USA 84:
214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985)
Nature 314:
446-449; and Shaw et al. (1988)1 Natl. Cancer Inst. 80: 1553-1559; U.S. Pat.
No. 6,180,370;
U.S. Pat. Nos. 6,300,064; 6,696,248; 6,706,484; 6,828,422.
[00168] In one embodiment, the present technology provides the construction of
humanized
anti-HER2 antibodies that are unlikely to induce a human anti-mouse antibody
(hereinafter
referred to as "HAMA") response, while still having an effective antibody
effector function. As
used herein, the terms "human" and "humanized", in relation to antibodies,
relate to any
antibody which is expected to elicit a therapeutically tolerable weak
immunogenic response in a
human subject. In one embodiment, the present technology provides for a
humanized anti-HER2
antibodies, heavy and light chain immunoglobulins.
[00169] CDR Antibodies. In some embodiments, the anti-HER2 antibody of the
present
technology is an anti-HER2 CDR antibody. Generally the donor and acceptor
antibodies used to
generate the anti-HER2 CDR antibody are monoclonal antibodies from different
species;
typically the acceptor antibody is a human antibody (to minimize its
antigenicity in a human), in
which case the resulting CDR-grafted antibody is termed a "humanized"
antibody. The graft
may be of a single CDR (or even a portion of a single CDR) within a single VH
or VL of the
acceptor antibody, or can be of multiple CDRs (or portions thereof) within one
or both of the VH
and VL. Frequently, all three CDRs in all variable domains of the acceptor
antibody will be
replaced with the corresponding donor CDRs, though one needs to replace only
as many as
necessary to permit adequate binding of the resulting CDR-grafted antibody to
HER2 protein.
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Methods for generating CDR-grafted and humanized antibodies are taught by
Queen et at. U.S.
Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; and
Winter U.S.
5,225,539; and EP 0682040. Methods useful to prepare VH and VL polypeptides
are taught by
Winter et al.,U.S. Pat. Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161;
6,545,142;
5 EP 0368684; EP0451216; and EP0120694.
[00170] After selecting suitable framework region candidates from the same
family and/or the
same family member, either or both the heavy and light chain variable regions
are produced by
grafting the CDRs from the originating species into the hybrid framework
regions. Assembly of
hybrid antibodies or hybrid antibody fragments having hybrid variable chain
regions with regard
10 to either of the above aspects can be accomplished using conventional
methods known to those
skilled in the art. For example, DNA sequences encoding the hybrid variable
domains described
herein (i.e., frameworks based on the target species and CDRs from the
originating species) can
be produced by oligonucleotide synthesis and/or PCR. The nucleic acid encoding
CDR regions
can also be isolated from the originating species antibodies using suitable
restriction enzymes
15 and ligated into the target species framework by ligating with suitable
ligation enzymes.
Alternatively, the framework regions of the variable chains of the originating
species antibody
can be changed by site-directed mutagenesis.
[00171] Since the hybrids are constructed from choices among multiple
candidates
corresponding to each framework region, there exist many combinations of
sequences which are
20 amenable to construction in accordance with the principles described
herein. Accordingly,
libraries of hybrids can be assembled having members with different
combinations of individual
framework regions. Such libraries can be electronic database collections of
sequences or
physical collections of hybrids.
[00172] This process typically does not alter the acceptor antibody's FRs
flanking the grafted
25 CDRs. However, one skilled in the art can sometimes improve antigen
binding affinity of the
resulting anti-HER2 CDR-grafted antibody by replacing certain residues of a
given FR to make
the FR more similar to the corresponding FR of the donor antibody. Suitable
locations of the
substitutions include amino acid residues adjacent to the CDR, or which are
capable of
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interacting with a CDR (See, e.g., US 5,585,089, especially columns 12-16). Or
one skilled in
the art can start with the donor FR and modify it to be more similar to the
acceptor FR or a
human consensus FR. Techniques for making these modifications are known in the
art.
Particularly if the resulting FR fits a human consensus FR for that position,
or is at least 90% or
more identical to such a consensus FR, doing so may not increase the
antigenicity of the
resulting modified anti-HER2 CDR-grafted antibody significantly compared to
the same
antibody with a fully human FR.
[00173] Bispecific Antibodies (BsAbs). A bispecific antibody is an antibody
that can bind
simultaneously to two targets that have a distinct structure, e.g., two
different target antigens,
.. two different epitopes on the same target antigen, or a hapten and a target
antigen or epitope on a
target antigen. BsAbs can be made, for example, by combining heavy chains
and/or light chains
that recognize different epitopes of the same or different antigen. In some
embodiments, by
molecular function, a bispecific binding agent binds one antigen (or epitope)
on one of its two
binding arms (one VHNL pair), and binds a different antigen (or epitope) on
its second arm (a
different VH/VL pair). By this definition, a bispecific binding agent has two
distinct antigen
binding arms (in both specificity and CDR sequences), and is monovalent for
each antigen to
which it binds.
[00174] Multi-specific antibodies, such as bispecific antibodies (BsAb)
and bispecific
antibody fragments (BsFab) have at least one arm that specifically binds to,
for example, HER2
and at least one other arm that specifically binds to a second target antigen.
In some
embodiments, the second target antigen is an antigen or epitope of a B-cell, a
T-cell, a myeloid
cell, a plasma cell, or a mast-cell. Additionally or alternatively, in certain
embodiments, the
second target antigen is selected from the group consisting of CD3, CD4, CD8,
CD20, CD19,
CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR
gamma/delta, NKp46 and KIR. Exemplary VH and VL sequences that bind to a
second target
antigen (e.g., CD3) are shown in FIG. 3 (included in the LC sequences). In
certain
embodiments, the BsAbs are capable of binding to tumor cells that express HER2
antigen on the
cell surface. In some embodiments, the BsAbs have been engineered to
facilitate killing of
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tumor cells by directing (or recruiting) cytotoxic T cells to a tumor site.
Other exemplary BsAbs
include those with a first antigen binding site specific for HER2 and a second
antigen binding
site specific for a small molecule hapten (e.g., DTP A, IMP288, DOTA, DOTA-Bn,
DOTA-
desferrioxamine, other DOTA-chelates described herein, Biotin, fluorescein, or
those disclosed
in Goodwin, D A. et al, 1994, Cancer Res. 54(22):5937-5946).
[00175] A variety of bispecific fusion proteins can be produced using
molecular engineering.
For example, BsAbs have been constructed that either utilize the full
immunoglobulin
framework (e.g., IgG), single chain variable fragment (scFv), or combinations
thereof. In some
embodiments, the bispecific fusion protein is divalent, comprising, for
example, a scFv with a
single binding site for one antigen and a Fab fragment with a single binding
site for a second
antigen. In some embodiments, the bispecific fusion protein is divalent,
comprising, for
example, an scFv with a single binding site for one antigen and another scFv
fragment with a
single binding site for a second antigen. In other embodiments, the bispecific
fusion protein is
tetravalent, comprising, for example, an immunoglobulin (e.g., IgG) with two
binding sites for
one antigen and two identical scFvs for a second antigen. BsAbs composed of
two scFv units in
tandem have been shown to be a clinically successful bispecific antibody
format. In some
embodiments, BsAbs comprise two single chain variable fragments (scFvs) in
tandem have been
designed such that an scFv that binds a tumor antigen (e.g., HER2) is linked
with an scFv that
engages T cells (e.g., by binding CD3). In this way, T cells are recruited to
a tumor site such that
they can mediate cytotoxic killing of the tumor cells. See e.g., Dreier et
at., I Immunol.
170:4397-4402 (2003); Bargou et al., Science 321 :974- 977 (2008)). In some
embodiments,
BsAbs of the present technology comprise two single chain variable fragments
(scFvs) in tandem
have been designed such that an scFv that binds a tumor antigen (e.g., HER2)
is linked with an
scFv that engages a small molecule DOTA hapten.
[00176] Recent methods for producing BsAbs include engineered recombinant
monoclonal
antibodies which have additional cysteine residues so that they crosslink more
strongly than the
more common immunoglobulin isotypes. See, e.g., FitzGerald et at., Protein
Eng. 10(10):1221-
1225 (1997). Another approach is to engineer recombinant fusion proteins
linking two or more
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different single-chain antibody or antibody fragment segments with the needed
dual specificities.
See, e.g., Coloma et at., Nature Biotech. 15:159-163 (1997). A variety of
bispecific fusion
proteins can be produced using molecular engineering.
[00177] Bispecific fusion proteins linking two or more different single-chain
antibodies or
antibody fragments are produced in a similar manner. Recombinant methods can
be used to
produce a variety of fusion proteins. In some certain embodiments, a BsAb
according to the
present technology comprises an immunoglobulin, which immunoglobulin comprises
a heavy
chain and a light chain, and an scFv. In some certain embodiments, the scFv is
linked to the C-
terminal end of the heavy chain of any HER2 immunoglobulin disclosed herein.
In some certain
embodiments, scFvs are linked to the C-terminal end of the light chain of any
HER2
immunoglobulin disclosed herein. In various embodiments, scFvs are linked to
heavy or light
chains via a linker sequence. Appropriate linker sequences necessary for the
in-frame
connection of the heavy chain Fd to the scFv are introduced into the \/1_, and
Vkappa domains
through PCR reactions. The DNA fragment encoding the scFv is then ligated into
a staging
.. vector containing a DNA sequence encoding the CH1 domain. The resulting
scFv-CH1
construct is excised and ligated into a vector containing a DNA sequence
encoding the \Tx region
of a HER2 antibody. The resulting vector can be used to transfect an
appropriate host cell, such
as a mammalian cell for the expression of the bispecific fusion protein.
[00178] In some embodiments, a linker is at least 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85,
90, 95, 100 or more amino acids in length. In some embodiments, a linker is
characterized in
that it tends not to adopt a rigid three-dimensional structure, but rather
provides flexibility to the
polypeptide (e.g., first and/or second antigen binding sites). In some
embodiments, a linker is
employed in a BsAb described herein based on specific properties imparted to
the BsAb such as,
for example, an increase in stability. In some embodiments, a BsAb of the
present technology
comprises a G45 linker. In some certain embodiments, a BsAb of the present
technology
comprises a (GIS), linker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15 or more.
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[00179] Fc Modifications. In some embodiments, the anti-HER2 antibodies of the
present
technology comprise a variant Fe region, wherein said variant Fe region
comprises at least one
amino acid modification relative to a wild-type Fe region (or the parental Fe
region), such that
said molecule has an altered affinity for an Fe receptor (e.g., an FcyR),
provided that said variant
Fe region does not have a substitution at positions that make a direct contact
with Fe receptor
based on crystallographic and structural analysis of Fe-Fe receptor
interactions such as those
disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of
positions within the
Fe region that make a direct contact with an Fe receptor such as an FcyR,
include amino acids
234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299
(C7E loop), and
amino acids 327-332 (F/G) loop.
[00180] In some embodiments, an anti-HER2 antibody of the present technology
has an
altered affinity for activating and/or inhibitory receptors, having a variant
Fe region with one or
more amino acid modifications, wherein said one or more amino acid
modification is a N297
substitution with alanine, or a K322 substitution with alanine. Additionally
or alternatively, in
some embodiments, the Fe regions of the HER2 antibodies disclosed herein
comprise two amino
acid substitutions, Leu234Ala and Leu235Ala (so called LALA mutations) to
eliminate FcyRIIa
binding. The LALA mutations are commonly used to alleviate the cytokine
induction from T
cells, thus reducing toxicity of the antibodies (Wines BD, et al., J Immunol
164:5313-5318
(2000)).
[00181] Glycosylation Modifications. In some embodiments, anti-HER2 antibodies
of the
present technology have an Fe region with variant glycosylation as compared to
a parent Fe
region. In some embodiments, variant glycosylation includes the absence of
fucose; in some
embodiments, variant glycosylation results from expression in GnTl-deficient
CHO cells.
[00182] In some embodiments, the antibodies of the present technology, may
have a modified
glycosylation site relative to an appropriate reference antibody that binds to
an antigen of interest
(e.g., HER2), without altering the functionality of the antibody, e.g.,
binding activity to the
antigen. As used herein, "glycosylation sites" include any specific amino acid
sequence in an
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antibody to which an oligosaccharide (i.e., carbohydrates containing two or
more simple sugars
linked together) will specifically and covalently attach.
[00183] Oligosaccharide side chains are typically linked to the backbone of an
antibody via
either N-or 0-linkages. N-linked glycosylation refers to the attachment of an
oligosaccharide
5 moiety to the side chain of an asparagine residue. 0-linked glycosylation
refers to the
attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine,
threonine. For
example, an Fc-glycoform (hHER2-IgGln) that lacks certain oligosaccharides
including fucose
and terminal N- acetylglucosamine may be produced in special CHO cells and
exhibit enhanced
ADCC effector function.
10 [00184] In some embodiments, the carbohydrate content of an
immunoglobulin-related
composition disclosed herein is modified by adding or deleting a glycosylation
site. Methods for
modifying the carbohydrate content of antibodies are well known in the art and
are included
within the present technology, see, e.g.,U U.S. Patent No. 6,218,149; EP
0359096B1; U.S. Patent
Publication No. US 2002/0028486; International Patent Application Publication
WO 03/035835;
15 U. S . Patent Publication No. 2003/0115614; U.S. Patent No. 6,218,149;
U.S. Patent No.
6,472,511; all of which are incorporated herein by reference in their
entirety. In some
embodiments, the carbohydrate content of an antibody (or relevant portion or
component
thereof) is modified by deleting one or more endogenous carbohydrate moieties
of the antibody.
In some certain embodiments, the present technology includes deleting the
glycosylation site of
20 the Fc region of an antibody, by modifying position 297 from asparagine
to alanine.
[00185] Engineered glycoforms may be useful for a variety of purposes,
including but not
limited to enhancing or reducing effector function. Engineered glycoforms may
be generated by
any method known to one skilled in the art, for example by using engineered or
variant
expression strains, by co-expression with one or more enzymes, for example N-
25 acetylglucosaminyltransferase III (GnTIII), by expressing a molecule
comprising an Fc region in
various organisms or cell lines from various organisms, or by modifying
carbohydrate(s) after
the molecule comprising Fc region has been expressed. Methods for generating
engineered
glycoforms are known in the art, and include but are not limited to those
described in Umana et
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at., 1999, Nat. Biotechnol. 17: 176-180; Davies et at., 2001, Biotechnol.
Bioeng. 74:288-294;
Shields et al., 2002,1 Biol. Chem. 277:26733-26740; Shinkawa et al., 2003,1
Biol. Chem.
278:3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application Serial No.
10/277,370; U.S.
Patent Application Serial No. 10/113,929; International Patent Application
Publications WO
00/61739A1 ; WO 01/292246A1; WO 02/311140A1; WO 02/30954A1; POTILLEGENTTm
technology (Biowa, Inc. Princeton, N.J.); GLYCOMABTm glycosylation engineering
technology
(GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated
herein by
reference in its entirety. See, e.g., International Patent Application
Publication WO 00/061739;
U.S. Patent Application Publication No. 2003/0115614; Okazaki et at., 2004,
.1114B, 336: 1239-
49.
[00186] Fusion Proteins. In one embodiment, the anti-HER2 antibody of the
present
technology is a fusion protein. The anti-HER2 antibodies of the present
technology, when fused
to a second protein, can be used as an antigenic tag. Examples of domains that
can be fused to
polypeptides include not only heterologous signal sequences, but also other
heterologous
functional regions. The fusion does not necessarily need to be direct, but can
occur through
linker sequences. Moreover, fusion proteins of the present technology can also
be engineered to
improve characteristics of the anti-HER2 antibodies. For instance, a region of
additional amino
acids, particularly charged amino acids, can be added to the N-terminus of the
anti-HER2
antibody to improve stability and persistence during purification from the
host cell or subsequent
handling and storage. Also, peptide moieties can be added to an anti-HER2
antibody to facilitate
purification. Such regions can be removed prior to final preparation of the
anti-HER2 antibody.
The addition of peptide moieties to facilitate handling of polypeptides are
familiar and routine
techniques in the art. The anti-HER2 antibody of the present technology can be
fused to marker
sequences, such as a peptide which facilitates purification of the fused
polypeptide. In select
embodiments, the marker amino acid sequence is a hexa-histidine peptide, such
as the tag
provided in a pQE vector (QIAGEN, Inc., Chatsworth, Calif), among others, many
of which are
commercially available. As described in Gentz et at., Proc. Natl. Acad. Sci.
USA 86: 821-824,
1989, for instance, hexa-histidine provides for convenient purification of the
fusion protein.
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Another peptide tag useful for purification, the "HA" tag, corresponds to an
epitope derived from
the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.
[00187] Thus, any of these above fusion proteins can be engineered using the
polynucleotides
or the polypeptides of the present technology. Also, in some embodiments, the
fusion proteins
described herein show an increased half-life in vivo.
[00188] Fusion proteins having disulfide-linked dimeric structures (due to the
IgG) can be
more efficient in binding and neutralizing other molecules compared to the
monomeric secreted
protein or protein fragment alone. Fountoulakis et al., I Biochem. 270: 3958-
3964, 1995.
[00189] Similarly, EP-A-0 464 533 (Canadian counterpart 2045869) discloses
fusion proteins
comprising various portions of constant region of immunoglobulin molecules
together with
another human protein or a fragment thereof. In many cases, the Fc part in a
fusion protein is
beneficial in therapy and diagnosis, and thus can result in, e.g., improved
pharmacokinetic
properties. See EP-A 0232 262. Alternatively, deleting or modifying the Fc
part after the fusion
protein has been expressed, detected, and purified, may be desired. For
example, the Fc portion
can hinder therapy and diagnosis if the fusion protein is used as an antigen
for immunizations. In
drug discovery, e.g., human proteins, such as hIL-5, have been fused with Fc
portions for the
purpose of high-throughput screening assays to identify antagonists of hIL-5.
Bennett et al.,
Molecular Recognition 8:52-58, 1995; Johanson et al., Biol. Chem., 270: 9459-
9471, 1995.
[00190] Labeled Anti-HER2 antibodies. In one embodiment, the anti-HER2
antibody of the
present technology is coupled with a label moiety, i.e., detectable group. The
particular label or
detectable group conjugated to the anti-HER2 antibody is not a critical aspect
of the technology,
so long as it does not significantly interfere with the specific binding of
the anti-HER2 antibody
of the present technology to the HER2 protein. The detectable group can be any
material having
a detectable physical or chemical property. Such detectable labels have been
well-developed in
the field of immunoassays and imaging. In general, almost any label useful in
such methods can
be applied to the present technology. Thus, a label is any composition
detectable by
spectroscopic, photochemical, biochemical, immunochemical, electrical, optical
or chemical
means. Labels useful in the practice of the present technology include
magnetic beads (e.g.,
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DynabeadsTm), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,
rhodamine, and the
like), radiolabels (e.g., 3H, 14C, 35s, 1251, 1211, 1311, 112=n,
1
99mTc), other imaging agents such as
microbubbles (for ultrasound imaging), 18F, nc, 150 89Zr (for Positron
emission tomography),
99mTc,
(for Single photon emission tomography), enzymes (e.g., horse radish
peroxidase,
alkaline phosphatase and others commonly used in an ELISA), and calorimetric
labels such as
colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene,
latex, and the like)
beads. Patents that describe the use of such labels include U.S. Pat. Nos.
3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated
herein by
reference in their entirety and for all purposes. See also Handbook of
Fluorescent Probes and
Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene OR.).
[00191] The label can be coupled directly or indirectly to the desired
component of an assay
according to methods well known in the art. As indicated above, a wide variety
of labels can be
used, with the choice of label depending on factors such as required
sensitivity, ease of
conjugation with the compound, stability requirements, available
instrumentation, and disposal
provisions.
[00192] Non-radioactive labels are often attached by indirect means.
Generally, a ligand
molecule (e.g., biotin) is covalently bound to the molecule. The ligand then
binds to an anti-
ligand (e.g., streptavidin) molecule which is either inherently detectable or
covalently bound to a
signal system, such as a detectable enzyme, a fluorescent compound, or a
chemiluminescent
compound. A number of ligands and anti-ligands can be used. Where a ligand has
a natural
anti-ligand, e.g., biotin, thyroxine, and cortisol, it can be used in
conjunction with the labeled,
naturally-occurring anti-ligands. Alternatively, any haptenic or antigenic
compound can be used
in combination with an antibody, e.g., an anti-HER2 antibody.
[00193] The molecules can also be conjugated directly to signal generating
compounds, e.g.,
by conjugation with an enzyme or fluorophore. Enzymes of interest as labels
will primarily be
hydrolases, particularly phosphatases, esterases and glycosidases, or
oxidoreductases,
particularly peroxidases. Fluorescent compounds useful as labeling moieties,
include, but are not
limited to, e.g., fluorescein and its derivatives, rhodamine and its
derivatives, dansyl,
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umbelliferone, and the like. Chemiluminescent compounds useful as labeling
moieties, include,
but are not limited to, e.g., luciferin, and 2,3-dihydrophthalazinediones,
e.g., luminol. For a
review of various labeling or signal-producing systems which can be used, see
U.S. Pat. No.
4,391,904.
.. [00194] Means of detecting labels are well known to those of skill in the
art. Thus, for
example, where the label is a radioactive label, means for detection include a
scintillation counter
or photographic film as in autoradiography. Where the label is a fluorescent
label, it can be
detected by exciting the fluorochrome with the appropriate wavelength of light
and detecting the
resulting fluorescence. The fluorescence can be detected visually, by means of
photographic
.. film, by the use of electronic detectors such as charge coupled devices
(CCDs) or
photomultipliers and the like. Similarly, enzymatic labels can be detected by
providing the
appropriate substrates for the enzyme and detecting the resulting reaction
product. Finally,
simple colorimetric labels can be detected simply by observing the color
associated with the
label. Thus, in various dipstick assays, conjugated gold often appears pink,
while various
.. conjugated beads appear the color of the bead.
[00195] Some assay formats do not require the use of labeled components. For
instance,
agglutination assays can be used to detect the presence of the target
antibodies, e.g., the anti-
HER2 antibodies. In this case, antigen-coated particles are agglutinated by
samples comprising
the target antibodies. In this format, none of the components need be labeled
and the presence of
.. the target antibody is detected by simple visual inspection.
B. Identifying and Characterizing the Anti-HER2 Antibodies of the Present
Technology
[00196] Methods for identifying and/or screening the anti-HER2 antibodies of
the present
technology. Methods useful to identify and screen antibodies against HER2
polypeptides for
those that possess the desired specificity to HER2 protein (e.g., those that
bind to the
.. extracellular domain of HER2 protein, such as polypeptides comprising the
amino acid sequence
of GenBank: NP 004439.2 (SEQ ID NO: 84) include any immunologically-mediated
techniques
known within the art. Components of an immune response can be detected in
vitro by various
methods that are well known to those of ordinary skill in the art. For
example, (1) cytotoxic T
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lymphocytes can be incubated with radioactively labeled target cells and the
lysis of these target
cells detected by the release of radioactivity; (2) helper T lymphocytes can
be incubated with
antigens and antigen presenting cells and the synthesis and secretion of
cytokines measured by
standard methods (Windhagen A et at., Immunity, 2: 373-80, 1995); (3) antigen
presenting cells
5 can be incubated with whole protein antigen and the presentation of that
antigen on MHC
detected by either T lymphocyte activation assays or biophysical methods
(Harding et at., Proc.
Natl. Acad. Sc., 86: 4230-4, 1989); (4) mast cells can be incubated with
reagents that cross-link
their Fc-epsilon receptors and histamine release measured by enzyme
immunoassay (Siraganian
et at., TIPS, 4: 432-437, 1983); and (5) enzyme-linked immunosorbent assay
(ELISA).
10 [00197] Similarly, products of an immune response in either a model
organism (e.g., mouse)
or a human subject can also be detected by various methods that are well known
to those of
ordinary skill in the art. For example, (1) the production of antibodies in
response to vaccination
can be readily detected by standard methods currently used in clinical
laboratories, e.g., an
ELISA; (2) the migration of immune cells to sites of inflammation can be
detected by scratching
15 the surface of skin and placing a sterile container to capture the
migrating cells over scratch site
(Peters et al., Blood, 72: 1310-5, 1988); (3) the proliferation of peripheral
blood mononuclear
cells (PBMCs) in response to mitogens or mixed lymphocyte reaction can be
measured using 41-
thymidine; (4) the phagocytic capacity of granulocytes, macrophages, and other
phagocytes in
PBMCs can be measured by placing PBMCs in wells together with labeled
particles (Peters et
20 at., Blood, 72: 1310-5, 1988); and (5) the differentiation of immune
system cells can be
measured by labeling PBMCs with antibodies to CD molecules such as CD4 and CD8
and
measuring the fraction of the PBMCs expressing these markers.
[00198] In one embodiment, anti-HER2 antibodies of the present technology are
selected
using display of HER2 peptides on the surface of replicable genetic packages.
See, e.g. ,U .S .
25 Pat. Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108;
6,225,447; 6,291,650;
6,492,160; EP 585 287; EP 605522; EP 616640; EP 1024191; EP 589 877; EP 774
511; EP 844
306. Methods useful for producing/selecting a filamentous bacteriophage
particle containing a
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phagemid genome encoding for a binding molecule with a desired specificity has
been described.
See, e.g., EP 774 511; US 5871907; US 5969108; US 6225447; US 6291650; US
6492160.
[00199] In some embodiments, anti-HER2 antibodies of the present technology
are selected
using display of HER2 peptides on the surface of a yeast host cell. Methods
useful for the
isolation of scFv polypeptides by yeast surface display have been described by
Kieke et at.,
Protein Eng. 1997 Nov; 10(11): 1303-10.
[00200] In some embodiments, anti-HER2 antibodies of the present technology
are selected
using ribosome display. Methods useful for identifying ligands in peptide
libraries using
ribosome display have been described by Mattheakis et at., Proc. Natl. Acad.
Sci. USA 91: 9022-
26, 1994; and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997.
[00201] In certain embodiments, anti-HER2 antibodies of the present technology
are selected
using tRNA display of HER2 peptides. Methods useful for in vitro selection of
ligands using
tRNA display have been described by Merryman et al., Chem. Biol., 9: 741-46,
2002.
[00202] In one embodiment, anti-HER2 antibodies of the present technology are
selected
using RNA display. Methods useful for selecting peptides and proteins using
RNA display
libraries have been described by Roberts et at. Proc. Natl. Acad. Sci. USA,
94: 12297-302, 1997;
and Nemoto et al., FEBS Lett., 414: 405-8, 1997. Methods useful for selecting
peptides and
proteins using unnatural RNA display libraries have been described by Frankel
et at., Curr.
Opin. Struct. Biol., 13: 506-12, 2003.
[00203] In some embodiments, anti-HER2 antibodies of the present technology
are expressed
in the periplasm of gram negative bacteria and mixed with labeled HER2
protein. See
WO 02/34886. In clones expressing recombinant polypeptides with affinity for
HER2 protein,
the concentration of the labeled HER2 protein bound to the anti-HER2
antibodies is increased
and allows the cells to be isolated from the rest of the library as described
in Harvey et at., Proc.
Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Pat. Publication No. 2004/0058403.
[00204] After selection of the desired anti-HER2 antibodies, it is
contemplated that said
antibodies can be produced in large volume by any technique known to those
skilled in the art,
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e.g., prokaryotic or eukaryotic cell expression and the like. The anti-HER2
antibodies which are,
e.g., but not limited to, anti-HER2 hybrid antibodies or fragments can be
produced by using
conventional techniques to construct an expression vector that encodes an
antibody heavy chain
in which the CDRs and, if necessary, a minimal portion of the variable region
framework, that
are required to retain original species antibody binding specificity (as
engineered according to
the techniques described herein) are derived from the originating species
antibody and the
remainder of the antibody is derived from a target species immunoglobulin
which can be
manipulated as described herein, thereby producing a vector for the expression
of a hybrid
antibody heavy chain.
[00205] Measurement of HER2 Binding. In some embodiments, a HER2 binding assay
refers
to an assay format wherein HER2 protein and an anti-HER2 antibody are mixed
under conditions
suitable for binding between the HER2 protein and the anti-HER2 antibody and
assessing the
amount of binding between the HER2 protein and the anti-HER2 antibody. The
amount of
binding is compared with a suitable control, which can be the amount of
binding in the absence
of the HER2 protein, the amount of the binding in the presence of a non-
specific
immunoglobulin composition, or both. The amount of binding can be assessed by
any suitable
method. Binding assay methods include, e.g., ELISA, radioimmunoassays,
scintillation
proximity assays, fluorescence energy transfer assays, liquid chromatography,
membrane
filtration assays, and the like. Biophysical assays for the direct measurement
of HER2 protein
binding to anti-HER2 antibody are, e.g., nuclear magnetic resonance,
fluorescence, fluorescence
polarization, surface plasmon resonance (BIACORE chips) and the like. Specific
binding is
determined by standard assays known in the art, e.g., radioligand binding
assays, ELISA, FRET,
immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopy and the like. If the
specific
binding of a candidate anti-HER2 antibody is at least 1 percent greater than
the binding observed
in the absence of the candidate anti-HER2 antibody, the candidate anti-HER2
antibody is useful
as an anti-HER2 antibody of the present technology.
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Uses of the Anti-HER2 Antibodies of the Present Technology
[00206] General. The anti-HER2 antibodies of the present technology are useful
in methods
known in the art relating to the localization and/or quantitation of HER2
protein (e.g., for use in
measuring levels of the HER2 protein within appropriate physiological samples,
for use in
diagnostic methods, for use in imaging the polypeptide, and the like).
Antibodies of the present
technology are useful to isolate a HER2 protein by standard techniques, such
as affinity
chromatography or immunoprecipitation. An anti-HER2 antibody of the present
technology can
facilitate the purification of natural immunoreactive HER2 proteins from
biological samples,
e.g., mammalian sera or cells as well as recombinantly-produced immunoreactive
HER2 proteins
expressed in a host system. Moreover, anti-HER2 antibodies can be used to
detect an
immunoreactive HER2 protein (e.g., in plasma, a cellular lysate or cell
supernatant) in order to
evaluate the abundance and pattern of expression of the immunoreactive
polypeptide. The anti-
HER2 antibodies of the present technology can be used diagnostically to
monitor
immunoreactive HER2 protein levels in tissue as part of a clinical testing
procedure, e.g., to
determine the efficacy of a given treatment regimen. As noted above, the
detection can be
facilitated by coupling (i.e., physically linking) the anti-HER2 antibodies of
the present
technology to a detectable substance.
[00207] Detection of HER2 protein. An exemplary method for detecting the
presence or
absence of an immunoreactive HER2 protein in a biological sample involves
obtaining a
biological sample from a test subject and contacting the biological sample
with an anti-HER2
antibody of the present technology capable of detecting an immunoreactive HER2
protein such
that the presence of an immunoreactive HER2 protein is detected in the
biological sample.
Detection may be accomplished by means of a detectable label attached to the
antibody.
[00208] The term "labeled" with regard to the anti-HER2 antibody is intended
to encompass
direct labeling of the antibody by coupling (i.e., physically linking) a
detectable substance to the
antibody, as well as indirect labeling of the antibody by reactivity with
another compound that is
directly labeled, such as a secondary antibody. Examples of indirect labeling
include detection
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of a primary antibody using a fluorescently-labeled secondary antibody and end-
labeling of a
DNA probe with biotin such that it can be detected with fluorescently-labeled
streptavidin.
[00209] In some embodiments, the anti-HER2 antibodies disclosed herein are
conjugated to
one or more detectable labels. For such uses, anti-HER2 antibodies may be
detectably labeled
.. by covalent or non-covalent attachment of a chromogenic, enzymatic,
radioisotopic, isotopic,
fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast
agent or other label.
[00210] Examples of suitable chromogenic labels include diaminobenzidine and 4-
hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels
include malate
dehydrogenase, staphylococcal nuclease, A-5-steroid isomerase, yeast-alcohol
dehydrogenase, a-
l() glycerol phosphate dehydrogenase, triose phosphate isomerase,
peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, 0-galactosidase, ribonuclease, urease,
catalase, glucose-6-
phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.
[00211] Examples of suitable radioisotopic labels include 3H, "In, 1251,
1311, 32p, 35s, 14C,
51Cr, 57To, 58Co, 59Fe, 75Se, 152Eu, 90y, 67cti, 2170, 211At, 212pb, 47se,
109pd, etc. "In is an
exemplary isotope where in vivo imaging is used since its avoids the problem
of dehalogenation
of the 125I or 131I-labeled HER2-binding antibodies by the liver. In addition,
this isotope has a
more favorable gamma emission energy for imaging (Perkins et at, Eur. I Nucl.
Med. 70:296-
301 (1985); Carasquillo et al., I Nucl. Med. 25:281-287 (1987)). For example,
"In coupled to
monoclonal antibodies with 1-(P-isothiocyanatobenzy1)-DPTA exhibits little
uptake in non-
tumorous tissues, particularly the liver, and enhances specificity of tumor
localization (Esteban et
at., I Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioactive
isotopic labels
include 157Gd, 55M11, 162"y,
I) 52Tr, and 56Fe.
[00212] Examples of suitable fluorescent labels include an 152Eu label, a
fluorescein label, an
isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin
label, an
allophycocyanin label, a Green Fluorescent Protein (GFP) label, an o-
phthaldehyde label, and a
fluorescamine label. Examples of suitable toxin labels include diphtheria
toxin, ricin, and
cholera toxin.
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[00213] Examples of chemiluminescent labels include a luminol label, an
isoluminol label, an
aromatic acridinium ester label, an imidazole label, an acridinium salt label,
an oxalate ester
label, a luciferin label, a luciferase label, and an aequorin label. Examples
of nuclear magnetic
resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and
iron.
5 [00214] The detection method of the present technology can be used to
detect an
immunoreactive HER2 protein in a biological sample in vitro as well as in
vivo. In vitro
techniques for detection of an immunoreactive HER2 protein include enzyme
linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations,
radioimmunoassay, and
immunofluorescence. Furthermore, in vivo techniques for detection of an
immunoreactive HER2
10 protein include introducing into a subject a labeled anti-HER2 antibody.
For example, the anti-
HER2 antibody can be labeled with a radioactive marker whose presence and
location in a
subject can be detected by standard imaging techniques. In one embodiment, the
biological
sample contains HER2 protein molecules from the test subject.
[00215] Immunoassay and Imaging. An anti-HER2 antibody of the present
technology can be
15 used to assay immunoreactive HER2 protein levels in a biological sample
(e.g., human plasma)
using antibody-based techniques. For example, protein expression in tissues
can be studied with
classical immunohistological methods. Jalkanen, M. et al., I Cell. Biol. 101:
976-985, 1985;
Jalkanen, M. et al., I Cell. Biol. 105: 3087-3096, 1987. Other antibody-based
methods useful
for detecting protein gene expression include immunoassays, such as the enzyme
linked
20 immunosorbent assay (ELISA) and the radioimmunoassay (MA). Suitable
antibody assay labels
are known in the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes or
other radioactive agent, such as iodine (1251, 1211, 131-.-1),
carbon ("C), sulfur (35S), tritium (3H),
indium ("2In), and technetium (99mTc), and fluorescent labels, such as
fluorescein, rhodamine,
and green fluorescent protein (GFP), as well as biotin.
25 [00216] In addition to assaying immunoreactive HER2 protein levels in a
biological sample,
anti-HER2 antibodies of the present technology may be used for in vivo imaging
of HER2.
Antibodies useful for this method include those detectable by X-radiography,
NMR or ESR. For
X-radiography, suitable labels include radioisotopes such as barium or cesium,
which emit
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detectable radiation but are not overtly harmful to the subject. Suitable
markers for NMR and
ESR include those with a detectable characteristic spin, such as deuterium,
which can be
incorporated into the anti-HER2 antibodies by labeling of nutrients for the
relevant scFv clone.
[00217] An anti-HER2 antibody which has been labeled with an appropriate
detectable
imaging moiety, such as a radioisotope (e.g., 1311, 112=n,
1 99mTc), a radio-opaque substance, or a
material detectable by nuclear magnetic resonance, is introduced (e.g.,
parenterally,
subcutaneously, or intraperitoneally) into the subject. It will be understood
in the art that the size
of the subject and the imaging system used will determine the quantity of
imaging moiety needed
to produce diagnostic images. In the case of a radioisotope moiety, for a
human subject, the
quantity of radioactivity injected will normally range from about 5 to 20
millicuries of 99mTc.
The labeled anti-HER2 antibody will then accumulate at the location of cells
which contain the
specific target polypeptide. For example, labeled anti-HER2 antibodies of the
present
technology will accumulate within the subject in cells and tissues in which
the HER2 protein has
localized.
[00218] Thus, the present technology provides a diagnostic method of a medical
condition,
which involves: (a) assaying the expression of immunoreactive HER2 protein by
measuring
binding of an anti-HER2 antibody of the present technology in cells or body
fluid of an
individual; (b) comparing the amount of immunoreactive HER2 protein present in
the sample
with a standard reference, wherein an increase or decrease in immunoreactive
HER2 protein
levels compared to the standard is indicative of a medical condition.
[00219] Affinity Purification. The anti-HER2 antibodies of the present
technology may be
used to purify immunoreactive HER2 protein from a sample. In some embodiments,
the
antibodies are immobilized on a solid support. Examples of such solid supports
include plastics
such as polycarbonate, complex carbohydrates such as agarose and sepharose,
acrylic resins and
such as polyacrylamide and latex beads. Techniques for coupling antibodies to
such solid
supports are well known in the art (Weir et at., "Handbook of Experimental
Immunology" 4th
Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986);
Jacoby et at., Meth.
Enzym. 34 Academic Press, N.Y. (1974)).
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[00220] The simplest method to bind the antigen to the antibody-support matrix
is to collect
the beads in a column and pass the antigen solution down the column. The
efficiency of this
method depends on the contact time between the immobilized antibody and the
antigen, which
can be extended by using low flow rates. The immobilized antibody captures the
antigen as it
.. flows past. Alternatively, an antigen can be contacted with the antibody-
support matrix by
mixing the antigen solution with the support (e.g., beads) and rotating or
rocking the slurry,
allowing maximum contact between the antigen and the immobilized antibody.
After the
binding reaction has been completed, the slurry is passed into a column for
collection of the
beads. The beads are washed using a suitable washing buffer and then the pure
or substantially
pure antigen is eluted.
[00221] An antibody or polypeptide of interest can be conjugated to a solid
support, such as a
bead. In addition, a first solid support such as a bead can also be
conjugated, if desired, to a
second solid support, which can be a second bead or other support, by any
suitable means,
including those disclosed herein for conjugation of a polypeptide to a
support. Accordingly, any
of the conjugation methods and means disclosed herein with reference to
conjugation of a
polypeptide to a solid support can also be applied for conjugation of a first
support to a second
support, where the first and second solid support can be the same or
different.
[00222] Appropriate linkers, which can be cross-linking agents, for use for
conjugating a
polypeptide to a solid support include a variety of agents that can react with
a functional group
present on a surface of the support, or with the polypeptide, or both.
Reagents useful as cross-
linking agents include homo-bi-functional and, in particular, hetero-bi-
functional reagents.
Useful bi-functional cross-linking agents include, but are not limited to, N-
STAB, dimaleimide,
DTNB, N-SATA, N-SPDP, SMCC and 6-HYNIC. A cross-linking agent can be selected
to
provide a selectively cleavable bond between a polypeptide and the solid
support. For example,
a photolabile cross-linker, such as 3-amino-(2-nitrophenyl)propionic acid can
be employed as a
means for cleaving a polypeptide from a solid support. (Brown et at., Mol.
Divers, pp, 4-12
(1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996); and US. Pat.
No. 5,643,722).
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Other cross-linking reagents are well-known in the art. (See, e.g., Wong
(1991), supra; and
Hermanson (1996), supra).
[00223] An antibody or polypeptide can be immobilized on a solid support, such
as a bead,
through a covalent amide bond formed between a carboxyl group functionalized
bead and the
amino terminus of the polypeptide or, conversely, through a covalent amide
bond formed
between an amino group functionalized bead and the carboxyl terminus of the
polypeptide. In
addition, a bi-functional trityl linker can be attached to the support, e.g.,
to the 4-nitrophenyl
active ester on a resin, such as a Wang resin, through an amino group or a
carboxyl group on the
resin via an amino resin. Using a bi-functional trityl approach, the solid
support can require
treatment with a volatile acid, such as formic acid or trifluoroacetic acid to
ensure that the
polypeptide is cleaved and can be removed. In such a case, the polypeptide can
be deposited as a
beadless patch at the bottom of a well of a solid support or on the flat
surface of a solid support.
After addition of a matrix solution, the polypeptide can be desorbed into a
MS.
[00224] Hydrophobic trityl linkers can also be exploited as acid-labile
linkers by using a
volatile acid or an appropriate matrix solution, e.g., a matrix solution
containing 3-HPA, to
cleave an amino linked trityl group from the polypeptide. Acid lability can
also be changed. For
example, trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl can be
changed to the
appropriate p-substituted, or more acid-labile tritylamine derivatives, of the
polypeptide, i.e.,
trityl ether and tritylamine bonds can be made to the polypeptide.
Accordingly, a polypeptide
can be removed from a hydrophobic linker, e.g., by disrupting the hydrophobic
attraction or by
cleaving tritylether or tritylamine bonds under acidic conditions, including,
if desired, under
typical MS conditions, where a matrix, such as 3-HPA acts as an acid.
[00225]
Orthogonally cleavable linkers can also be useful for binding a first solid
support,
e.g., a bead to a second solid support, or for binding a polypeptide of
interest to a solid support.
Using such linkers, a first solid support, e.g., a bead, can be selectively
cleaved from a second
solid support, without cleaving the polypeptide from the support; the
polypeptide then can be
cleaved from the bead at a later time. For example, a disulfide linker, which
can be cleaved
using a reducing agent, such as DTT, can be employed to bind a bead to a
second solid support,
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and an acid cleavable bi-functional trityl group could be used to immobilize a
polypeptide to the
support. As desired, the linkage of the polypeptide to the solid support can
be cleaved first, e.g.,
leaving the linkage between the first and second support intact. Trityl
linkers can provide a
covalent or hydrophobic conjugation and, regardless of the nature of the
conjugation, the trityl
group is readily cleaved in acidic conditions.
[00226] For example, a bead can be bound to a second support through a linking
group which
can be selected to have a length and a chemical nature such that high density
binding of the
beads to the solid support, or high density binding of the polypeptides to the
beads, is promoted.
Such a linking group can have, e.g., "tree-like" structure, thereby providing
a multiplicity of
functional groups per attachment site on a solid support. Examples of such
linking group;
include polylysine, polyglutamic acid, penta-erythrole and tris-hydroxy-
aminomethane.
[00227] Noncovalent Binding Association. An antibody or polypeptide can be
conjugated to a
solid support, or a first solid support can also be conjugated to a second
solid support, through a
noncovalent interaction. For example, a magnetic bead made of a ferromagnetic
material, which
is capable of being magnetized, can be attracted to a magnetic solid support,
and can be released
from the support by removal of the magnetic field. Alternatively, the solid
support can be
provided with an ionic or hydrophobic moiety, which can allow the interaction
of an ionic or
hydrophobic moiety, respectively, with a polypeptide, e.g., a polypeptide
containing an attached
trityl group or with a second solid support having hydrophobic character.
[00228] A solid support can also be provided with a member of a specific
binding pair and,
therefore, can be conjugated to a polypeptide or a second solid support
containing a
complementary binding moiety. For example, a bead coated with avidin or with
streptavidin can
be bound to a polypeptide having a biotin moiety incorporated therein, or to a
second solid
support coated with biotin or derivative of biotin, such as iminobiotin.
[00229] It should be recognized that any of the binding members disclosed
herein or
otherwise known in the art can be reversed. Thus, biotin, e.g., can be
incorporated into either a
polypeptide or a solid support and, conversely, avidin or other biotin binding
moiety would be
incorporated into the support or the polypeptide, respectively. Other specific
binding pairs
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contemplated for use herein include, but are not limited to, hormones and
their receptors,
enzyme, and their substrates, a nucleotide sequence and its complementary
sequence, an
antibody and the antigen to which it interacts specifically, and other such
pairs knows to those
skilled in the art.
5 A. Diagnostic Uses of Anti-HER2 Antibodies of the Present Technology
[00230] General. The anti-HER2 antibodies of the present technology are useful
in diagnostic
methods. As such, the present technology provides methods using the antibodies
in the diagnosis
of HER2 activity in a subject. Anti-HER2 antibodies of the present technology
may be selected
such that they have any level of epitope binding specificity and very high
binding affinity to a
10 HER2 protein. In general, the higher the binding affinity of an antibody
the more stringent wash
conditions can be performed in an immunoassay to remove nonspecifically bound
material
without removing target polypeptide. Accordingly, anti-HER2 antibodies of the
present
technology useful in diagnostic assays usually have binding affinities of
about 108M-1, 109M-1,
1010 M-1, 1011 M-1 or 1012 M-1. Further, it is desirable that anti-HER2
antibodies used as
15 diagnostic reagents have a sufficient kinetic on-rate to reach
equilibrium under standard
conditions in at least 12 h, at least five (5) h, or at least one (1) hour.
[00231] Anti-HER2 antibodies can be used to detect an immunoreactive HER2
protein in a
variety of standard assay formats. Such formats include immunoprecipitation,
Western blotting,
ELISA, radioimmunoassay, and immunometric assays. See Harlow & Lane,
Antibodies, A
20 Laboratory Manual (Cold Spring Harbor Publications, New York, 1988);
U.S. Pat. Nos.
3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932; 3,817,837;
3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074;
3,984,533;
3,996,345; 4,034,074; and 4,098,876. Biological samples can be obtained from
any tissue or
body fluid of a subject. In certain embodiments, the subject is at an early
stage of cancer. In one
25 embodiment, the early stage of cancer is determined by the level or
expression pattern of HER2
protein in a sample obtained from the subject. In certain embodiments, the
sample is selected
from the group consisting of urine, blood, serum, plasma, saliva, amniotic
fluid, cerebrospinal
fluid (C SF), and biopsied body tissue.
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[00232] Immunometric or sandwich assays are one format for the diagnostic
methods of the
present technology. See U.S. Pat. No. 4,376,110, 4,486,530, 5,914,241, and
5,965,375. Such
assays use one antibody, e.g., an anti-HER2 antibody or a population of anti-
HER2 antibodies
immobilized to a solid phase, and another anti-HER2 antibody or a population
of anti-HER2
antibodies in solution. Typically, the solution anti-HER2 antibody or
population of anti-HER2
antibodies is labeled. If an antibody population is used, the population can
contain antibodies
binding to different epitope specificities within the target polypeptide.
Accordingly, the same
population can be used for both solid phase and solution antibody. If anti-
HER2 monoclonal
antibodies are used, first and second HER2 monoclonal antibodies having
different binding
specificities are used for the solid and solution phase. Solid phase (also
referred to as "capture")
and solution (also referred to as "detection") antibodies can be contacted
with target antigen in
either order or simultaneously. If the solid phase antibody is contacted
first, the assay is referred
to as being a forward assay. Conversely, if the solution antibody is contacted
first, the assay is
referred to as being a reverse assay. If the target is contacted with both
antibodies
simultaneously, the assay is referred to as a simultaneous assay. After
contacting the HER2
protein with the anti-HER2 antibody, a sample is incubated for a period that
usually varies from
about 10 min to about 24 hr and is usually about 1 hr. A wash step is then
performed to remove
components of the sample not specifically bound to the anti-HER2 antibody
being used as a
diagnostic reagent. When solid phase and solution antibodies are bound in
separate steps, a wash
can be performed after either or both binding steps. After washing, binding is
quantified,
typically by detecting a label linked to the solid phase through binding of
labeled solution
antibody. Usually for a given pair of antibodies or populations of antibodies
and given reaction
conditions, a calibration curve is prepared from samples containing known
concentrations of
target antigen. Concentrations of the immunoreactive HER2 protein in samples
being tested are
then read by interpolation from the calibration curve (i.e., standard curve).
Analyte can be
measured either from the amount of labeled solution antibody bound at
equilibrium or by kinetic
measurements of bound labeled solution antibody at a series of time points
before equilibrium is
reached. The slope of such a curve is a measure of the concentration of the
HER2 protein in a
sample.
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[00233] Suitable supports for use in the above methods include, e.g.,
nitrocellulose
membranes, nylon membranes, and derivatized nylon membranes, and also
particles, such as
agarose, a dextran-based gel, dipsticks, particulates, microspheres, magnetic
particles, test tubes,
microtiter wells, SEPHADEXTM (Amersham Pharmacia Biotech, Piscataway N.J.),
and the like.
Immobilization can be by absorption or by covalent attachment. Optionally,
anti-HER2
antibodies can be joined to a linker molecule, such as biotin for attachment
to a surface bound
linker, such as avidin.
[00234] In some embodiments, the present disclosure provides an anti-HER2
antibody of the
present technology conjugated to a diagnostic agent. The diagnostic agent may
comprise a
radioactive or non-radioactive label, a contrast agent (such as for magnetic
resonance imaging,
computed tomography or ultrasound), and the radioactive label can be a gamma-,
beta-, alpha-,
Auger electron-, or positron-emitting isotope. A diagnostic agent is a
molecule which is
administered conjugated to an antibody moiety, i.e., antibody or antibody
fragment, or
subfragment, and is useful in diagnosing or detecting a disease by locating
the cells containing
the antigen.
[00235] Useful diagnostic agents include, but are not limited to,
radioisotopes, dyes (such as
with the biotin-streptavidin complex), contrast agents, fluorescent compounds
or molecules and
enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging
(MM). U.S. Pat.
No. 6,331,175 describes MM technique and the preparation of antibodies
conjugated to a MM
.. enhancing agent and is incorporated in its entirety by reference. In some
embodiments, the
diagnostic agents are selected from the group consisting of radioisotopes,
enhancing agents for
use in magnetic resonance imaging, and fluorescent compounds. In order to load
an antibody
component with radioactive metals or paramagnetic ions, it may be necessary to
react it with a
reagent having a long tail to which are attached a multiplicity of chelating
groups for binding the
.. ions. Such a tail can be a polymer such as a polylysine, polysaccharide, or
other derivatized or
derivatizable chain having pendant groups to which can be bound chelating
groups such as, e.g.,
ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid
(DTPA),
porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and
like groups
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known to be useful for this purpose. Chelates may be coupled to the antibodies
of the present
technology using standard chemistries. The chelate is normally linked to the
antibody by a group
which enables formation of a bond to the molecule with minimal loss of
immunoreactivity and
minimal aggregation and/or internal cross-linking. Other methods and reagents
for conjugating
chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659. Particularly
useful metal-chelate
combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs,
used with
diagnostic isotopes for radio-imaging. The same chelates, when complexed with
non-radioactive
metals, such as manganese, iron and gadolinium are useful for MRI, when used
along with the
HER2 antibodies of the present technology. Macrocyclic chelates such as NOTA
(1,4,7-triaza-
cyclononane-N,N,N"-triacetic acid), DOTA, and TETA (p-bromoacetamido-benzyl-
tetraethylaminetetraacetic acid) are of use with a variety of metals and
radiometals, such as
radionuclides of gallium, yttrium and copper, respectively. Such metal-chelate
complexes can be
stabilized by tailoring the ring size to the metal of interest. Examples of
other DOTA chelates
include (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii) Ac-Lys(HSG)D-Tyr-
Lys(HSG)-
.. Lys(Tscg-Cys)-NH2; (iii) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (iv)
DOTA-D-
Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (v) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-
Lys(HSG)-NH2; (vi) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii) DOTA-D-
Phe-
D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (viii) Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-
NH2; (ix) Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (x) Ac-D-Phe-D-Lys(Bz-
DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2; (xi) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-
Cys)-NH2; (xii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-N}{2;
(xiii)
(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xiv) Tscg-D-Cys-
D-
Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xv) (Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-
Lys(HSG)-NH2; (xvi) Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2;
(xvii) Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xviii) Ac-D-Lys(DTPA)-D-
Tyr-D-
Lys(DTPA)-D-Lys(Tscg-Cys)-NH2; and (xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-
Lys(Tscg-Cys)-NH2.
[00236] Other ring-type chelates such as macrocyclic polyethers, which are of
interest for
stably binding nuclides, such as 223Ra for RAIT are also contemplated.
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B. Therapeutic Use of Anti-HER2 Antibodies of the Present Technology
[00237] In one aspect, the immunoglobulin-related compositions (e.g.,
antibodies or antigen
binding fragments thereof) of the present technology are useful for the
treatment of HER2-
associated cancers. Examples of HER2-associated cancers include, but are not
limited to, breast
cancer, gastric cancer, osteosarcoma, desmoplastic small round cell cancer,
squamous cell
carcinoma of head and neck cancer, ovarian cancer, prostate cancer, pancreatic
cancer,
glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal
junction
adenocarcinoma, cervical cancer, salivary gland cancer, soft tissue sarcoma,
leukemia,
melanoma, Ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or any other
neoplastic tissue
that expresses the HER2 receptor. In some embodiments, the HER2-associated
cancer is a solid
tumor. Such treatment can be used in patients identified as having
pathologically high levels of
the HER2 (e.g., those diagnosed by the methods described herein) or in
patients diagnosed with a
disease known to be associated with such pathological levels.
[00238] The compositions of the present technology may be employed in
conjunction with
other therapeutic agents useful in the treatment of HER2-associated cancers.
For example, the
antibodies or antigen binding fragments of the present technology may be
separately,
sequentially or simultaneously administered with at least one additional
therapeutic agent
selected from the group consisting of alkylating agents, platinum agents,
taxanes, vinca agents,
anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents,
VEGF/VEGFR inhibitors,
EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic
antibiotics,
antimetabolites, endocrine/hormonal agents, T cells, bisphosphonate therapy
agents and targeted
biological therapy agents (e.g., therapeutic peptides described in US 6306832,
WO 2012007137,
WO 2005000889, WO 2010096603 etc.). In some embodiments, the at least one
additional
therapeutic agent is a chemotherapeutic agent. Specific chemotherapeutic
agents include, but are
not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU),
methotrexate,
edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin,
taxanes, paclitaxel,
protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene,
toremifene, fulvestrant,
gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine,
vinblastine, eribulin,
mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide,
abarelix, buserlin,
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goserelin, megestrol acetate, risedronate, pamidronate, ibandronate,
alendronate, denosumab,
zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and
doxorubicin),
bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin,
microtubule
poisons, annonaceous acetogenins, or combinations thereof.
5 [00239] Additionally or alternatively, in some embodiments, the
antibodies or antigen binding
fragments of the present technology may be separately, sequentially or
simultaneously
administered with at least one additional immuno-modulating/stimulating
antibody including but
not limited to anti-PD-1 antibody, anti-PD-Li antibody, anti-PD-L2 antibody,
anti-CTLA-4
antibody, anti-TIM3 antibody, anti-4-1BB antibody, anti-CD73 antibody, anti-
GITR antibody,
10 and anti-LAG-3 antibody.
[00240] The compositions of the present technology may optionally be
administered as a
single bolus to a subject in need thereof. Alternatively, the dosing regimen
may comprise
multiple administrations performed at various times after the appearance of
tumors.
[00241] Administration can be carried out by any suitable route,
including orally, intranasally,
15 parenterally (intravenously, intramuscularly, intraperitoneally, or
subcutaneously), rectally,
intracranially, intratumorally, intrathecally, or topically. Administration
includes self-
administration and the administration by another. It is also to be appreciated
that the various
modes of treatment of medical conditions as described are intended to mean
"substantial", which
includes total but also less than total treatment, and wherein some
biologically or medically
20 relevant result is achieved.
[00242] In some embodiments, the antibodies of the present technology comprise
pharmaceutical formulations which may be administered to subjects in need
thereof in one or
more doses. Dosage regimens can be adjusted to provide the desired response
(e.g., a therapeutic
response).
25 [00243] Typically, an effective amount of the antibody compositions of
the present
technology, sufficient for achieving a therapeutic effect, range from about
0.000001 mg per
kilogram body weight per day to about 10,000 mg per kilogram body weight per
day. Typically,
the dosage ranges are from about 0.0001 mg per kilogram body weight per day to
about 100 mg
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per kilogram body weight per day. For administration of anti-HER2 antibodies,
the dosage
ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg every
week, every
two weeks or every three weeks, of the subject body weight. For example,
dosages can be 1
mg/kg body weight or 10 mg/kg body weight every week, every two weeks or every
three weeks
or within the range of 1-10 mg/kg every week, every two weeks or every three
weeks. In one
embodiment, a single dosage of antibody ranges from 0.1-10,000 micrograms per
kg body
weight. In one embodiment, antibody concentrations in a carrier range from 0.2
to 2000
micrograms per delivered milliliter. An exemplary treatment regime entails
administration once
per every two weeks or once a month or once every 3 to 6 months. Anti-HER2
antibodies may
be administered on multiple occasions. Intervals between single dosages can be
hourly, daily,
weekly, monthly or yearly. Intervals can also be irregular as indicated by
measuring blood levels
of the antibody in the subject. In some methods, dosage is adjusted to achieve
a serum antibody
concentration in the subject of from about 75 1.tg/mL to about 125 1.tg/mL,
10011g/mL to about
15011g/mL, from about 12511g/mL to about 17511g/mL, or from about 15011g/mL to
about 200
1.tg/mL. Alternatively, anti-HER2 antibodies can be administered as a
sustained release
formulation, in which case less frequent administration is required. Dosage
and frequency vary
depending on the half-life of the antibody in the subject. The dosage and
frequency of
administration can vary depending on whether the treatment is prophylactic or
therapeutic. In
prophylactic applications, a relatively low dosage is administered at
relatively infrequent
intervals over a long period of time. In therapeutic applications, a
relatively high dosage at
relatively short intervals is sometimes required until progression of the
disease is reduced or
terminated, or until the subject shows partial or complete amelioration of
symptoms of disease.
Thereafter, the patient can be administered a prophylactic regime.
[00244] In another aspect, the present disclosure provides a method for
detecting cancer in a
subject in vivo comprising (a) administering to the subject an effective
amount of an antibody (or
antigen binding fragment thereof) of the present technology, wherein the
antibody is configured
to localize to a cancer cell expressing HER2 and is labeled with a
radioisotope; and (b) detecting
the presence of a tumor in the subject by detecting radioactive levels emitted
by the antibody that
are higher than a reference value. In some embodiments, the reference value is
expressed as
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injected dose per gram (%ID/g). The reference value may be calculated by
measuring the
radioactive levels present in non-tumor (normal) tissues, and computing the
average radioactive
levels present in non-tumor (normal) tissues standard deviation. In some
embodiments, the
ratio of radioactive levels between a tumor and normal tissue is about 2:1,
3:1, 4:1, 5:1, 6:1, 7:1,
8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1,
65:1, 70:1, 75:1, 80:1,
85:1, 90:1, 95:1 or 100:1.
[00245] In some embodiments, the subject is diagnosed with or is suspected of
having cancer.
Radioactive levels emitted by the antibody may be detected using positron
emission tomography
or single photon emission computed tomography.
[00246] Additionally or alternatively, in some embodiments, the method further
comprises
administering to the subject an effective amount of an immunoconjugate
comprising an antibody
of the present technology conjugated to a radionuclide. In some embodiments,
the radionuclide
is an alpha particle-emitting isotope, a beta particle-emitting isotope, an
Auger-emitter, or any
combination thereof Examples of beta particle-emitting isotopes include 86Y,
90Y, 89Sr, 165Dy,
186Re, 188Re, 177A1_,n,
and 67Cu. Examples of alpha particle-emitting isotopes include 213Bi, 211At,
225Ac, 152Dy, 212Bi, 223Ra, 219Rn, 215po, 211Bi, 221Fr, 217 At, At and 255FM.
Examples of Auger-
emitters include "In, 67Ga, 51Cr, 58Co, 99mTc, 1 3mRh, 'Pt, iosb, 161Ho,
189mos, 1921r, 201T1,
and 203Pb. In some embodiments of the method, nonspecific FcR-dependent
binding in normal
tissues is eliminated or reduced (e.g., via N297A mutation in Fc region, which
results in
aglycosylation). The therapeutic effectiveness of such an immunoconjugate may
be determined
by computing the area under the curve (AUC) tumor: AUC normal tissue ratio. In
some
embodiments, the immunoconjugate has a AUC tumor: AUC normal tissue ratio of
about 2:1,
3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1,
45:1, 50:1, 55:1, 60:1,
65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.
[00247] Toxicity. Optimally, an effective amount (e.g., dose) of an anti-HER2
antibody
described herein will provide therapeutic benefit without causing substantial
toxicity to the
subject. Toxicity of the anti-HER2 antibody described herein can be determined
by standard
pharmaceutical procedures in cell cultures or experimental animals, e.g., by
determining the
LD5o (the dose lethal to 50% of the population) or the LDioo (the dose lethal
to 100% of the
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population). The dose ratio between toxic and therapeutic effect is the
therapeutic index. The
data obtained from these cell culture assays and animal studies can be used in
formulating a
dosage range that is not toxic for use in human. The dosage of the anti-HER2
antibody described
herein lies within a range of circulating concentrations that include the
effective dose with little
or no toxicity. The dosage can vary within this range depending upon the
dosage form employed
and the route of administration utilized. The exact formulation, route of
administration and
dosage can be chosen by the individual physician in view of the subject's
condition. See, e.g.,
Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).
[00248] Formulations of Pharmaceutical Compositions. According to the methods
of the
.. present technology, the anti-HER2 antibody can be incorporated into
pharmaceutical
compositions suitable for administration. The pharmaceutical compositions
generally comprise
recombinant or substantially purified antibody and a pharmaceutically-
acceptable carrier in a
form suitable for administration to a subject. Pharmaceutically-acceptable
carriers are
determined in part by the particular composition being administered, as well
as by the particular
method used to administer the composition. Accordingly, there is a wide
variety of suitable
formulations of pharmaceutical compositions for administering the antibody
compositions (See,
e.g., Remington' s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA
18th ed., 1990).
The pharmaceutical compositions are generally formulated as sterile,
substantially isotonic and
in full compliance with all Good Manufacturing Practice (GMP) regulations of
the U.S. Food
and Drug Administration. The pharmaceutical composition may further comprise
an agent
selected from the group consisting of isotopes, dyes, chromagens, contrast
agents, drugs, toxins,
cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth
factors,
radionuclides, metals, liposomes, nanoparticles, RNA, DNA or any combination
thereof
[00249] The terms "pharmaceutically-acceptable," "physiologically-
tolerable," and
grammatical variations thereof, as they refer to compositions, carriers,
diluents and reagents, are
used interchangeably and represent that the materials are capable of
administration to or upon a
subject without the production of undesirable physiological effects to a
degree that would
prohibit administration of the composition. For example, "pharmaceutically-
acceptable
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excipient" means an excipient that is useful in preparing a pharmaceutical
composition that is
generally safe, non-toxic, and desirable, and includes excipients that are
acceptable for veterinary
use as well as for human pharmaceutical use. Such excipients can be solid,
liquid, semisolid, or,
in the case of an aerosol composition, gaseous. "Pharmaceutically-acceptable
salts and esters"
means salts and esters that are pharmaceutically-acceptable and have the
desired
pharmacological properties. Such salts include salts that can be formed where
acidic protons
present in the composition are capable of reacting with inorganic or organic
bases. Suitable
inorganic salts include those formed with the alkali metals, e.g., sodium and
potassium,
magnesium, calcium, and aluminum. Suitable organic salts include those formed
with organic
bases such as the amine bases, e.g., ethanolamine, diethanolamine,
triethanolamine,
tromethamine, N-methylglucamine, and the like. Such salts also include acid
addition salts
formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and
organic acids (e.g.,
acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic
acids such as
methanesulfonic acid and benzenesulfonic acid). Pharmaceutically-acceptable
esters include
esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the
anti-HER2
antibody, e.g., C1-6 alkyl esters. When there are two acidic groups present, a
pharmaceutically-
acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or
ester; and similarly
where there are more than two acidic groups present, some or all of such
groups can be salified
or esterified. An anti-HER2 antibody named in this technology can be present
in unsalified or
unesterified form, or in salified and/or esterified form, and the naming of
such anti-HER2
antibody is intended to include both the original (unsalified and
unesterified) compound and its
pharmaceutically-acceptable salts and esters. Also, certain embodiments of the
present
technology can be present in more than one stereoisomeric form, and the naming
of such anti-
HER2 antibody is intended to include all single stereoisomers and all mixtures
(whether racemic
or otherwise) of such stereoisomers. A person of ordinary skill in the art,
would have no
difficulty determining the appropriate timing, sequence and dosages of
administration for
particular drugs and compositions of the present technology.
[00250] Examples of such carriers or diluents include, but are not limited to,
water, saline,
Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes
and non-
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aqueous vehicles such as fixed oils may also be used. The use of such media
and compounds for
pharmaceutically active substances is well known in the art. Except insofar as
any conventional
media or compound is incompatible with the anti-HER2 antibody, use thereof in
the
compositions is contemplated. Supplementary active compounds can also be
incorporated into
5 the compositions.
[00251] A pharmaceutical composition of the present technology is formulated
to be
compatible with its intended route of administration. The anti-HER2 antibody
compositions of
the present technology can be administered by parenteral, topical,
intravenous, oral,
subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial,
intrathecal,
10 intraperitoneal, intranasal; or intramuscular routes, or as inhalants.
The anti-HER2 antibody can
optionally be administered in combination with other agents that are at least
partly effective in
treating various HER2-associated cancers.
[00252] Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application
can include the following components: a sterile diluent such as water for
injection, saline
15 solution, fixed oils, polyethylene glycols, glycerine, propylene glycol
or other synthetic solvents;
antibacterial compounds such as benzyl alcohol or methyl parabens;
antioxidants such as
ascorbic acid or sodium bisulfite; chelating compounds such as
ethylenediaminetetraacetic acid
(EDTA); buffers such as acetates, citrates or phosphates, and compounds for
the adjustment of
tonicity such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such
20 as hydrochloric acid or sodium hydroxide. The parenteral preparation can
be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[00253] Pharmaceutical compositions suitable for injectable use include
sterile aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. For intravenous
administration, suitable
25 carriers include physiological saline, bacteriostatic water, Cremophor
EL (BASF, Parsippany,
N.J.) or phosphate buffered saline (PBS). In all cases, the composition must
be sterile and
should be fluid to the extent that easy syringeability exists. It must be
stable under the conditions
of manufacture and storage and must be preserved against the contaminating
action of
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microorganisms such as bacteria and fungi. The carrier can be a solvent or
dispersion medium
containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol,
and liquid polyethylene
glycol, and the like), and suitable mixtures thereof The proper fluidity can
be maintained, e.g.,
by the use of a coating such as lecithin, by the maintenance of the required
particle size in the
case of dispersion and by the use of surfactants. Prevention of the action of
microorganisms can
be achieved by various antibacterial and antifungal compounds, e.g., parabens,
chlorobutanol,
phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be
desirable to include
isotonic compounds, e.g., sugars, polyalcohols such as manitol, sorbitol,
sodium chloride in the
composition. Prolonged absorption of the injectable compositions can be
brought about by
including in the composition a compound which delays absorption, e.g.,
aluminum monostearate
and gelatin.
[00254] Sterile injectable solutions can be prepared by incorporating an anti-
HER2 antibody
of the present technology in the required amount in an appropriate solvent
with one or a
combination of ingredients enumerated above, as required, followed by filtered
sterilization.
Generally, dispersions are prepared by incorporating the anti-HER2 antibody
into a sterile
vehicle that contains a basic dispersion medium and the required other
ingredients from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, methods of preparation are vacuum drying and freeze-drying that
yields a powder of
the active ingredient plus any additional desired ingredient from a previously
sterile-filtered
solution thereof The antibodies of the present technology can be administered
in the form of a
depot injection or implant preparation which can be formulated in such a
manner as to permit a
sustained or pulsatile release of the active ingredient.
[00255] Oral compositions generally include an inert diluent or an edible
carrier. They can be
enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic
administration, the anti-HER2 antibody can be incorporated with excipients and
used in the form
of tablets, troches, or capsules. Oral compositions can also be prepared using
a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is applied
orally and swished and
expectorated or swallowed. Pharmaceutically compatible binding compounds,
and/or adjuvant
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materials can be included as part of the composition. The tablets, pills,
capsules, troches and the
like can contain any of the following ingredients, or compounds of a similar
nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient
such as starch or
lactose, a disintegrating compound such as alginic acid, Primogel, or corn
starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening
compound such as sucrose or saccharin; or a flavoring compound such as
peppermint, methyl
salicylate, or orange flavoring.
[00256] For administration by inhalation, the anti-HER2 antibody is delivered
in the form of
an aerosol spray from pressured container or dispenser which contains a
suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
[00257] Systemic administration can also be by transmucosal or transdermal
means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be permeated
are used in the formulation. Such penetrants are generally known in the art,
and include, e.g., for
transmucosal administration, detergents, bile salts, and fusidic acid
derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays or
suppositories. For
transdermal administration, the anti-HER2 antibody is formulated into
ointments, salves, gels, or
creams as generally known in the art.
[00258] The anti-HER2 antibody can also be prepared as pharmaceutical
compositions in the
form of suppositories (e.g., with conventional suppository bases such as cocoa
butter and other
glycerides) or retention enemas for rectal delivery.
[00259] In one embodiment, the anti-HER2 antibody is prepared with carriers
that will protect
the anti-HER2 antibody against rapid elimination from the body, such as a
controlled release
formulation, including implants and microencapsulated delivery systems.
Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of
such formulations will be apparent to those skilled in the art. The materials
can also be obtained
commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions
(including liposomes targeted to infected cells with monoclonal antibodies to
viral antigens) can
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also be used as pharmaceutically-acceptable carriers. These can be prepared
according to
methods known to those skilled in the art, e.g., as described in U.S. Pat. No.
4,522,811.
[00260] T Cells Bound to HER2 Multi-specific Binding Molecules Disclosed
Herein. Without
being bound by any theory, it is believed that when the anti-CD3 multi-
specific binding
molecules provided herein (e.g., HER2 x CD3) are bound to T cells, by, for
example, procedures
such as those described herein, an anti-CD3 scEv of the multi-specific binding
molecule binds to
CD3 on the surface of the T cell. Without being bound by any theory, it is
believed that binding
of the multi-specific binding molecule to the T cell (i.e., binding of an anti-
CD3 scEv to CD3
expressed on the T cell) activates the T cell, and consequently, allows for
the T cell receptor-
based cytotoxicity to be redirected to desired tumor targets, bypassing MHC
restrictions.
[00261] Thus, the present disclosure also provides T cells which are
bound to a multi-specific
binding molecule of the present technology. In specific embodiments, the T
cells are bound to
the multi-specific binding molecule noncovalently. In specific embodiments,
the T cells are
autologous to a subject to whom the T cells are to be administered. In
specific embodiments, the
T cells are allogeneic to a subject to whom the T cells are to be
administered. In specific
embodiments, the T cells are human T cells.
[00262] In specific embodiments, the T cells which are bound to multi-specific
binding
molecules of the invention are used in accordance with the therapeutic methods
described herein.
In specific embodiments, the T cells which are bound to multi-specific binding
molecules of the
present disclosure are used as part of a combination therapy as described
below.
[00263] In specific embodiments involving combination therapy with infusion of
T cells,
provided herein is a pharmaceutical composition comprising (a) a multi-
specific binding
molecule described herein; (b) T cells; and/or (c) a pharmaceutically
effective carrier. In specific
embodiments, the T cells are autologous to the subject to whom the T cells are
administered. In
certain embodiments, the T cells are allogeneic to the subject to whom the T
cells are
administered. In specific embodiments, the T cells are either bound or not
bound to the multi-
specific binding molecule. In specific embodiments, the binding of the T cells
to the multi-
specific binding molecule is noncovalently. In specific embodiments, the T
cells are human T
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cells. Methods that can be used to bind multi-specific binding molecules to T
cells are known in
the art. See, e.g., Lum et at., 2013, Blot Blood Marrow Transplant, 19:925-33,
Janeway et at.,
Immunobiology: The Immune System in Health and Disease, 5th edition, New York:
Garland
Science; Vaishampayan et al., 2015, Prostate Cancer, 2015:285193, and Stromnes
et al., 2014,
Immunol Rev. 257(1):145-164.
[00264] In a specific embodiment, the administering of a multi-specific
binding molecule
provided herein, polynucleotide, vector, or cell encoding the multi-specific
binding molecule, or
a pharmaceutical composition comprising the multi-specific binding molecule is
performed after
treating the patient with T cell infusion. In specific embodiments the T cell
infusion is
performed with T cells that are autologous to the subject to whom the T cells
are administered.
In specific embodiments, the T cell infusion is performed with T cells that
are allogeneic to the
subject to whom the T cells are administered. In specific embodiments, the T
cells can be bound
to molecules identical to a multi-specific binding molecule as described
herein. In specific
embodiments, the binding of the T cells to molecules identical to the multi-
specific binding
molecule is noncovalently. In specific embodiments, the T cells are human T
cells.
C. Kits
[00265] The present technology provides kits for the detection of HER2 and/or
treatment of
HER2-associated cancers, comprising at least one immunoglobulin-related
composition of the
present technology (e.g., any antibody or antigen binding fragment described
herein), or a
functional variant (e.g., substitutional variant) thereof. Optionally, the
above described
components of the kits of the present technology are packed in suitable
containers and labeled
for diagnosis and/or treatment of HER2-associated cancers. The above-mentioned
components
may be stored in unit or multi-dose containers, for example, sealed ampoules,
vials, bottles,
syringes, and test tubes, as an aqueous, preferably sterile, solution or as a
lyophilized, preferably
sterile, formulation for reconstitution. The kit may further comprise a second
container which
holds a diluent suitable for diluting the pharmaceutical composition towards a
higher volume.
Suitable diluents include, but are not limited to, the pharmaceutically
acceptable excipient of the
pharmaceutical composition and a saline solution. Furthermore, the kit may
comprise
instructions for diluting the pharmaceutical composition and/or instructions
for administering the
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pharmaceutical composition, whether diluted or not. The containers may be
formed from a
variety of materials such as glass or plastic and may have a sterile access
port (for example, the
container may be an intravenous solution bag or a vial having a stopper which
may be pierced by
a hypodermic injection needle). The kit may further comprise more containers
comprising a
5 pharmaceutically acceptable buffer, such as 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, syringes,
culture medium for one or
more of the suitable hosts. The kits may optionally include instructions
customarily included in
commercial packages of therapeutic or diagnostic products, that contain
information about, for
10 example, the indications, usage, dosage, manufacture, administration,
contraindications and/or
warnings concerning the use of such therapeutic or diagnostic products.
[00266] The kits are useful for detecting the presence of an immunoreactive
HER2 protein in
a biological sample, e.g., any body fluid including, but not limited to, e.g.,
serum, plasma,
lymph, cystic fluid, urine, stool, cerebrospinal fluid, ascitic fluid or blood
and including biopsy
15 samples of body tissue. For example, the kit can comprise: one or more
humanized, chimeric,
bispecific, or multi-specific anti-HER2 antibodies of the present technology
(or antigen binding
fragments thereof) capable of binding a HER2 protein in a biological sample;
means for
determining the amount of the HER2 protein in the sample; and means for
comparing the amount
of the immunoreactive HER2 protein in the sample with a standard. One or more
of the anti-
20 HER2 antibodies may be labeled. The kit components, (e.g., reagents) can
be packaged in a
suitable container. The kit can further comprise instructions for using the
kit to detect the
immunoreactive HER2 protein.
[00267] For antibody-based kits, the kit can comprise, e.g., 1) a first
antibody, e.g. a
humanized, chimeric, bispecific, or multi-specific HER2 antibody of the
present technology (or
25 an antigen binding fragment thereof), attached to a solid support, which
binds to a HER2 protein;
and, optionally; 2) a second, different antibody which binds to either the
HER2 protein or to the
first antibody, and is conjugated to a detectable label.
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[00268] The kit can also comprise, e.g., a buffering agent, a
preservative or a protein-
stabilizing agent. The kit can further comprise components necessary for
detecting the
detectable-label, e.g., an enzyme or a substrate. The kit can also contain a
control sample or a
series of control samples, which can be assayed and compared to the test
sample. Each
component of the kit can be enclosed within an individual container and all of
the various
containers can be within a single package, along with instructions for
interpreting the results of
the assays performed using the kit. The kits of the present technology may
contain a written
product on or in the kit container. The written product describes how to use
the reagents
contained in the kit, e.g., for detection of a HER2 protein in vitro or in
vivo, or for treatment of
HER2-associated cancers in a subject in need thereof In certain embodiments,
the use of the
reagents can be according to the methods of the present technology.
EXAMPLES
[00269] The present technology is further illustrated by the following
Examples, which should
not be construed as limiting in any way. The following Examples demonstrate
the preparation,
characterization, and use of illustrative anti-HER2 antibodies of the present
technology.
Example 1: Introduction and Preliminary Experiments
[00270] Early bispecific T cell engager (TCE) efforts have mainly focused on
maximizing
cytotoxic activity based on in vitro cell-based assays without anticipating
the biological
consequences of high potency on cytokine release and T-cell exhaustion or
depletion in the
patient. These safety concerns were summarized at a recent FDA-sponsored
workshop focused
on CD3 TCE safety assessment (Kamperschroer et at., J Immunotoxicol. 17(1):67-
85 (2020)).
Later generations of TCEs include Fcs or other similar domains for the purpose
of extending
half-life, but adverse events and clinical holds suggest that extending half-
life with a high
potency TCE could exacerbate serious adverse events associated with
neurotoxicity and cytokine
release syndrome (CRS) (Vafa et at., Front. Oncol. 10: 446 (2020)).
[00271] One potential strategy for overcoming resistance to current targeted
therapies is to
harness the killing activity of the T cell to defeat cancer by employing
BsAbs. These T-cell-
engaging antibodies are designed to simultaneously bind antigens on tumor
cells and T-cell
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activators such as the co-receptor CD3. BsAb engagement of the T cell mediates
the killing of
tumor cells by activating T cells through binding of CD3 and forming a
cytolytic synapse,
redirecting the killing activity toward the antigen-expressing tumor cells in
a major
histocompatibility complex (MHC)-independent manner.
[00272] Trastuzumab x huOKT3 (ABP100) is a bispecific antibody developed to
treat patients
affected by HER2+ types of cancer, including but not limited to breast,
gastric and colorectal.
This molecule binds HER2+ tumors and brings natural immune T cells to the
tumor to reduce it.
This BsAb is based on two well-known molecules: trastuzumab, a fully humanized
HER2-
targeting Immunoglobulin G1 (IgG1), and humanized muromonab-CD3 (huOKT3), a
CD3-
targeting IgGl. A key feature of ABP100 is that it was built using a symmetric
bivalent BsAb
platform IgG-[L]-scFv, in which a single-chain variable fragment (scFv)
recognizing human
CD3 is fused to the C terminus of each anti-tumor IgG antibody light chain
(FIG. 4). The
symmetric IgG-[L]-scFv design has provided potent in vitro and in vivo anti-
tumor activity
against multiple tumor antigens (GD2, CD3, GPA33, and HER2), and recent
reports demonstrate
that the IgG-L-scFv platform valency and spatial configuration drive
substantially more robust
anti-tumor responses than many other BsAb formats (Santich et at., Sci.
Transl. Med. 12:
eaax1315 (2020), FIGs. 5A-5B). To reduce the risk of CRS, the Fc domain
function of ABP100
was silenced to eliminate potential antibody-dependent cellular cytotoxicity
(ADCC) and
complement-dependent cytotoxicity (CDC) activity by introducing two well-
characterized
mutations: N297A to remove glycosylation, and K322A to reduce complement
activation.
[00273] Starting with ABP100 in the full format described above as a template,
a dual strategy
for the CD3xHER2 bispecific antibody program was proposed to limit the toxic
effects that are
commonly associated with first-generation TCEs and CAR T-cell therapeutics.
The two
products of this dual strategy are 1) ABP100a, a HER2 affinity-tuned BsAb with
selective killing
of HER2-high expressing cells designed for ex-vivo loading of patient T cells
for reinfusion, and
2) ABP102, a precisely redesigned BsAb for intravenous delivery with dual
affinity-tuned arms
for CD3 and HER2 binding (FIG. 4).
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[00274] ABP100a was built by replacing the huOKT3 portion of ABP100 with a
novel
humanized huSP34 CD3 binding arm and introducing novel affinity-tuned HER2
binding arms
(FIG. 4). The humanized huSP34 CD3 binding arm is cross-reactive with non-
human primate
(Cyno) CD3, which makes it a suitable model for assessing and predicting
toxicities in humans.
The affinity-tuned HER2 binding arms allows ABP100a to selectively kill HER2
high-
expressing cells, while sparing low, endogenous-level HER2-expressing cells.
The potential of
directly arming ABP100a onto patient-derived and activated T cells, a
technology that has shown
great potential in preclinical models, will be explored (FIG. 6). The approach
is to be explored
by using a relatively high-affinity CD3-binding arm with a low off-rate,
bearing in mind that
CRS concerns should be minimal by directly arming pre-activated T cells.
[00275] In parallel, ABP102 was developed by additionally affinity tuning the
CD3 T-cell
binding arm to select for CD3 affinity with potent killing of HER2-amplified
cancer cells with
the limited T-cell generation of cytokines such as interferon gamma (IFNy) and
tumour necrosis
factor alpha (TNFa). This approach invokes the key understanding that
individually tuning each
binding arm of the BsAb will not necessarily improve the overall efficacy or
safety profile of the
molecule as a whole, so the dual affinity-tuned BsAb in the context of the
full BsAb with all
modifications fully implemented was evaluated. This dual modification of the
HER2 and CD3
binding affinities on ABP102 yielded novel BsAbs for systemic delivery with
selective killing of
HER2-amplified cancer cells and is anticipated to show reduced cytokine
production for better
safety in the clinic.
Example 2: Antibody humanization, affinity tuning, and characterization
[00276] Antibody humanization and back-mutation by rational design
[00277] The complementarity-determining regions (CDRs), hypervariable loops,
and
framework regions (FRs) of a mouse anti-CD3 antibody were analyzed within the
variable
sequence and identified according to the KABAT delineation system. The CDRs of
the mouse
antibody were directly grafted to the human acceptor framework using the heavy
chain variable
(VH) and light chain variable domain (VI) that share the highest sequence
identities to the mouse
counterparts. Homology modeling was then performed to obtain the modeled
structure of the
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mouse antibody, and the solvent accessible surface area of framework residues
was calculated to
identify framework residues that are buried. Then, critical residues in the
sequences of the \Tx
and \/1_, that are different in the grafted and mouse antibody framework
sequences were identified
and back-mutated. Finally, the grafted sequence were inspected for potential
liability like N-
glycosylation sites, post-translational modifications and unpaired cysteine
residues which may
affect the binding activity of the grafted antibody.
[00278] Design and Construction of NNK Libraries, FASEBA Screening, and
Affinity Ranking
[00279] Initially, the affinity measurement of HER2 and the parental BsAb was
determined
using a Surface Plasmon Resonance (SPR) biosensor, Biacore 8K (GE Healthcare,
Marlborough,
MA). The equilibrium dissociation constant (KD) was calculated from the ratio
of kd over ka.
To determine the contribution of a specific residue to antibody affinity and
expression, paratope
mapping was performed by screening of NNK libraries. In brief, the VH and
\/1_, of the parental
antibody were searched by using NCBI Ig-Blast
(www.ncbi.nlm.nih.gov/projects/igblast/) and
CDRs defined by the KABAT delineation system. All residues within CDRs were
defined and
mutated by NNK method. Each individual NNK library was generated per residue
based on the
FASEBA platform with a theoretical diversity at 20. Over 48 clones were
randomly selected
from each NNK library for expression in E. coli in 96-deep-well plates. All
clones were
sequenced, and the unique clones selected. The crude selected protein secreted
in medium was
analyzed by ELISA against bovine serum albumin (BSA) and human and cyno
antigen protein
for the assessment of expression and binding specificity, respectively. The
"beneficial mutants"
that decrease antibody affinity, without compromising antibody expression,
were confirmed by
GenScript's FAst Screening for Expression level, Biophysical properties, and
Affinities
(FASEBA) platform via screening and affinity ranking.
[00280] Selection and synthesis of affinity-tuned bispecific antibodies
[00281] From the mutant library, a series of antibodies with varying
affinities for human and
cyno HER2 were constructed in a bispecific antibody format by subcloning into
an expression
vector for expression in Expi-CHO-S cells. The bispecific antibodies were
purified using protein
A columns.
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[00282] Binding between the parental and affinity-tuned bispecific antibodies
and human and
cyno target proteins (HER2 and CD3) were validated by ELISA, and a Biacore 8K
was used to
study the kinetics of the interaction between the antigen and the bispecific
antibodies. See FIGs.
10, 12 and 17A-17B.
Example 3: In vitro Characterization of Anti-HER2 Antibodies of the Present
Technology
[00283] T cell and target-cell binding by flow cytometry
[00284] Purified CD3+ T cells and whole human PBMCs were stained in bulk with
Live/Dead
stain, then incubated with a range of concentrations of bispecific antibodies
to assess for binding.
Controls included appropriate monoclonal antibodies (humanized 51334-hIgG1)
and isotype
control (hIgG1). A secondary antibody, anti-human IgG Fc specific PE
conjugate, was used for
detection.
[00285] Cell lines were grown per recommendations of the source of the cell
line, followed by
Accutase treatment to lift cells. Cells were resuspended in media, washed, and
stained in bulk
with Live/Dead stain, followed by staining with bispecific antibodies and a
control monoclonal
antibody (trastuzumab hIgG1). A secondary antibody, anti-human IgG Fc specific
PE conjugate,
was used for detection. Data collection was performed using the HTS platform
for the BD
FACSCelesta, with data analysis performed in FlowJo software. FIG. 7A shows
that the anti-
HER2xCD3 BsAbs of the present technology show reduced binding to T cells
compared to the
humanized 5P34-hIgG1 control.
[00286] T cell-dependent cellular cytotoxicity (TDCC) in vitro assay
[00287] To assess for biologic activity, established in-house TDCC assays
(Cell Titer Glo 2.0
(Promega) and Caspase3/7 green apoptosis assay (IncuCyte)) using effector
human CD3+ T cells
were performed to test bispecific HER2 x CD3 antibodies with differing CD3
affinities against
different target cell lines. Anti-HER2 x CD3 BsAbs showed selective killing of
high HER2-
expressing target cells (SKBR-3 cell line) while sparing target cells
expressing near endogenous
HER2 levels (MCF-7 cell line) (FIGs. 7B-7C, and FIGs. 9A-9B). In this
experiment,
ABP100s.10.0 had WT-trastuzumab-like affinity, and ABP100s.10.5 and
ABP100s.10.6 had
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lower HER2 affinity (-64-fold and ¨108-fold, respectively), whereas all
bispecific constructs
contained the same high-affinity CD3 scFv arm. Notably, ABP100s.10.5 and
ABP100s.10.6
constructs with low HER2 affinity allowed for differential and selective
killing of low HER2-
expressing target cells (MCF-7), in contrast to the antibody with WT-like
affinity
.. (ABP100s.10.0).
[00288] In contrast, the trastuzumab x huOKT3 parent antibody showed non-
selective killing
for both SKBR-3 and MCF-7 cell lines. See FIGs. 8A-8B. It is observed that
killing of the
HER2-high cell line is achieved at a lower concentration of trastuzumab x
huOKT3 than the
HER2-low cell line, but killing of the HER2-low cell line cannot be completely
eliminated (FIG.
.. 8B).
[00289] The CTG2.0 assay readout was on the Spectramax iD3, a standard
luminescence 96-
well plate reader, using white-colored well plates. The IncuCyte S3 platform
and software for
image-based analyses allowed determination of total green area/image from the
Caspase3/7
green reagent using black clear-bottom 96-well tissue-culture treated plates.
This assay provided
.. estimated relative frequency of apoptotic cells within each well.
[00290] These results demonstrate that reduction in CD3 scFv affinity has
minimal impact on
the killing of high HER2-expressing target cells (SKBR-3), while continuing to
exhibit no killing
of low HER2-expressing cells (MCF-7).
[00291] The effect of the various Her2 and CD3 affinities of each HER2xCD3
BsAbs of the
.. present technology on antibody binding, T cell activation, T cell-mediated
killing, and cytokine
production in the presence or absence of tumor cell lines expressing various
levels of Her2 was
investigated. SKBR3 and HCC1954 were selected as they represent high-density
Her-2
expressing cell lines with similar Her2-expression levels (Ram et at., MAbs.
2014 6(5): 1211-
1219) which correspond to HercepTest results of 3+ for SKBR3 (Dako HercepTest
Interpretation
.. Manual, WWW agilent.comlcs/librarvlusermanuals/public/28630 herceptest
interpretation manua
1-breast ihc row.pdf), and MCF-7 and HT55 as low-density Her2-expressing cells
with similar
expression levels to each other and to that of endogenous non-cancerous cells
expressing Her2,
with MCF-7 representing HercepTest scores of 0-1+ (Rhodes et at., Am J Clin
Pathol 2002
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118(3):408-17; Subik etal., Breast Cancer (Auckl). 2010; 4: 35-41; Slaga
etal., Sci Transl Med
2018 Oct 17;10(463):eaat5775). All p values from the data below for the NFAT T
cell receptor
activation assay, cytotoxicity assays, and cytokine assays were derived from a
two-Way
ANOVA with Tukey's multiple comparisons test ran on Graphpad Prism Version
9.4Ø
[00292] CD3/TCR NFAT T cell activation reporter assay: To assess for T cell
activation
by bispecific and monoclonal antibodies, the I cell activation Bioassay kit
(Promega J1621 /
J1625) was used with TCR1CD3 Jurkat effector cells (NEAT reporter), with
detection using Bio-
Glo Luciferase assay system (Promega G7941). Briefly, using White
bottom/chimney (solid
white) TC-treated plates (Corning 3917), 40,000 target cells (Her2-high: SK-BR-
3, HCC1954;
Her2-low: N1CF-7, H155) were plated overnight in 100uL media, and also used a
condition
without target cells. A.ssay was then performed in accordance with detailed
instructions provided
with the assay kit, with 7 hours of incubation followed by luminescence
readout.
[00293] Generally higher activation was seen with target cells expressing
higher amounts of
:Her2 (F1Gs. 13A43B). Notably, 10.5.1 and 10.6.1 were only slightly decreased
from the
parental construct (10.0) on fier2-high targets (SK-BR-3, HCC1954) with all
activation readouts
for all constructs being within 300,000-600,000 RLUs at the highest dose
examined (40 nM)
(F1Gs. 13.A43B). On Her2-low targets, the 10.0 construct exhibited similar, if
slightly lower
activation (Approximately 300,000 RLU at 40 nIVI on both MCF-7 and HT-55 cell
lines) while
the 10.5 and 10.6 constructs showed significantly diminished activation
(approximately 200,000
RUT) compared to the 10.0 on both cell lines (p <00001 for both comparisons)
(FIGs. 13C
13D). Furthermore, the 10.5.1 and 10.6.1 constructs exhibited significantly
lower activation at
40 nM (approximately 100,000 RLU) than 10.5 and 10.6 (p < 0.0001 for both
comparisons) on
Her2-low target cells (HT55, MCF-7; FIGs, 13C43D) and were statistically not
different from
the isotype control and were similar to the background activation seen in the
absence of target
cells (FIG. 13E). Taken together, these data are consistent with the lack of
10.5.1 and 10.6.1-
induced T cell activation in low-Her2-expressing cells.
[00294] T cell dependent cellular cytotoxicity (MC(7'): To assess in-
vitro functional
capacity of the bispecific antibodies to mediate T cell-mediated killing of
Her2-expressing target
cells, T cell dependent cellular cytotoxicity (TDCC) assays were performed
with CD3+ T cells.
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Target cells were plated to white bottom/chimney tissue culture treated plates
(Corning 3917) at
10,000 cells/well in 1001AL media and incubated overnight (Her2-high: SK-BR-3,
HCC1954;
Her2-low: MCF-7, HT55). Bispecific antibodies were diluted (range: 30, 0.3,
0.003, 0.00003
nM final concentrations) in RPM11640/10% heat-inactivated FBS. Culture media
was removed
from target cells and bispecific antibodies were added at 1004õ followed by
addition of purified
human cryopreserved T cells (StemCellTechnologies 70024) at an effector:target
ratio of 5 to 1
(T cells : target cells). Detection was performed at 40 hours with
CellTiterGlo2.0 (Promega) and
luminescence detection on a SpectraMax iD3 plate reader. Results in the figure
displayed are
representative of experiments done with three distinct donor CD3+ T cell
samples.
[00295] With SK-BR-3 target cells, 10.5.1 and 10.6.1 had only exhibited
modest, yet
significant (10.0 vs. 10.5.1: p = 0.0042; 10.0 vs 10.6.1: p = 0.0002)
reductions in killing versus
the parental construct (10.0) at 40 nM (FIG. 14A). There was no statistically
significant
difference between the Her2-mutated, CD3-unmutated 10.5 and 10.6 and the
parental 10.0 (FIG.
14A) in the presence of SKBR3 cells, consistent with the intended targeting of
high Her2-
expressing cells by 10.5.1 and 10.6.1. On HCC1954 target cells, while there
was significantly
reduced killing with 10.5 and 10.6 compared to the 10.0 parent (FIG. 14B),
this reduction was
slight (approximately 10-20% reduction from the 10.0 killing levels for the
SKBR3 and
HCC1954) in comparison to the reduction seen in the Her2-low expressing cells
(approximately
67-75% reduction from the 10.0 killing levels for the MCF-7 and HT55). On Her2-
low (HT55,
MCF-7) target cells at 40 nM, there was no significant difference between the
isotype control
killing levels and that of 10.5, 10.6, 10.5.1, and 10.6.1 (FIGs. 14C-14D)
(with the exception of
isotype vs. 10.5 for the :MCF-7 cell line, FIG. 14C). In contrast, at the same
dose in both MCF-7
and HT-55 cell lines, all of these constructs displayed significantly reduced
killing compared to
the 10.0 parent (p < 0.0001 for: 10.0 vs 10.5, 10.0 vs 10.6, 10.0 vs 10.5.1
and 10.0 vs 10.6.1)
(FIGs. 14C-14D) indicating that there was little to no killing of the low Her2-
expressing cell
lines by the Her2- and CD3-mutated constructs. The significantly increased
killing resulting
from 10.5 on the MCF-7 cell line was consistently observed in multiple
experiments indicating
that it may likely be due to the slightly stronger affinity of 10.5 for Her2
(KD is approximately
45 nM) than 10.6 (KD is approximately 67 nM).
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[00296] Multiplex Cytokine detection assay and associated cytotoxicity on Her2-
high and
11er24ow target cell lines with human PBMCs. To analyze cytokine release
mediated by
bispecific antibody constructs, human PBMCs were contacted with soluble
antibody with SKBR-
3 (Her2-high) and MCF-7 (Her2-low), as well as a "no target cell" condition.
Monoclonal anti-
CD3/anti-CD28 antibodies served as a control. Briefly, SKBR-3 (Her2-high) and
MCF-7 (Her2-
low) target cell lines were plated the night before assay. Bispecific
antibodies were diluted
(range: 30, 0.3, 0.003, 0.00003 ni\I final concentrations) in RPMI1640/10%
heat-inactivated
FBS. Culture media was removed from target cells and bispecific antibodies
were added at
100nL, followed by addition of human PBMCs (1 donor, Stem Cell Technologies
PBMCs ¨5 x
107 cells/vial) (10:1 E:T ratio; 100,000 PBMC : 10,000 target cells) in white-
chimney/bottom
plates in RPMI/10% HI FBS. Culture supernatants were harvested at 24 hours and
frozen -80 C
for multiplexed bead-based cytokine release assay (R&D Systems Human High
Sensitivity
Cytokine Base Kit B: IFN-y, IL-2, TNF-a, IL-6, GM-CSF) in conjunction with
signal detection
using a Magpix (Luminex) and quantification of cytokine in picograms/mL in
comparison to
standard wells using Luminex xMAP software. CellTiterGlo2.0 (Promega) was used
to develop
the assays for TDCC %cytotoxicity assessment with luminescence detection on a
SpectraMax
iD3 plate reader. Results in the figure displayed are representative of
experiments done with
three distinct donor PBMC samples. Cytotoxicity and cytokine release results
were compiled in
Excel and graphed in GraphPAD PRISM.
[00297] Since the excessive cytokine production associated with T cell
engager administration
can result in the primary toxicity of T cell engagers, cytokine release
syndrome (CRS), the effect
of weakened affinities for Her2 and CD3 on cytokine production was examined in
the presence
of tumor cell lines expressing high (SKBR3) or low (MCF-7) Her2 levels and
humans PBMCs.
The 10.0 parent, the Her2-reduced affinity 10.5 and 10.6 constructs, and the
Her2- and CD3-
weakened affinity 10.5.1 and 10.6.1 constructs all stimulated cytokine
production that was
significantly higher than the isotype control (control representing background
cytokine
production) in the presence of SKBR3 cells (FIGs. 15A-15D). However, in the
presence of
MCF-7 cells, only 10.0 displayed significantly increased cytokine production
compared to the
isotype control, indicating the reduced ability of the Her2- and/or CD3-
weakened constructs to
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stimulate cytokine production (FIGs. 15E-1511). At 30nM, all affinity-weakened
constructs
displayed significantly reduced IL-2, IFN-y, and TNF-a production in the
presence of MCF-7
cells (p < 0.0001 for: 10.0 vs 10.5, 10.0 vs 10.6, 10.0 vs 10.5.1 and 10.0 vs
10,6.1). Importantly,
the production of IL-6, a cytokine that is a crucial mediator of CRS
associated with
immunomodulatory agents (Morris et at., Nat Rev Immunol 2022;22(2):85-96), was
significantly
reduced with the Her2 and CD3-dually-weakened 10.5.1 and 10.6.1 constructs as
compared to
10.0 (p= 0.0432 and p= 0.029, respectively) in the presence of MCF-7 cells
(FIG. 15F). The
lack of significant difference in IL-6 production observed with the Her2
affinity-weakened 10.5
and 10.6 constructs compared to the 10.0 parent highlights the importance of
both Her2 and CD3
affinity weakening to achieve lowered IL-6 levels. The "no target cell"
condition showed that
PBMCs with bispecific antibodies did not alone promote cytokine release. For
SKBR-3 (Her2-
high) target cells, cytotoxicity was comparable for 10.5.1 and 10.6.1 relative
to parental construct
(10.0), with only minor differences observed.
[00298] These results demonstrate that these agents are useful for reducing
the incidence of
CRS when treating patients with high Her2 expressing (e.g. 3+ Herceptest
score) cancers by
selectively killing and producing cytokine in response to high Her2-expressing
cells while
sparing low Her2 expressing tissues with endogenous, noncancerous tissue
levels of Her2 (0-1+
Herceptest scores), thus reducing on-target, off-tumor toxicities in the
clinic.
[00299] Flow cytometric analysis of bispecific antibody binding to
activated T cells and
Her-2 expressing target cells. To assess flow cytometric binding of bispecific
antibodies to
activated T cells, human PBMCs (StemCell Technologies 70025.2) were stimulated
with an
OKT3/IL-2 stimulation protocol over 12 days. Briefly, PBMCs were activated
with 100 IU/m1_,
of recombinant human 11,2 (Stemcell Technologies, cat #78145.1) and 20 ng/mL
of OK T3
(Biolegend, mouse IgG2a. Cat#317326) in soluble format for 3 days, then
expanded/maintained
thereafter by using fresh media and IL-2 only by normalizing cells to lx106
cells/mi. in
RPIVII I 640/10%FBS. Activated T cells were cryopreseryed and stored in liquid
nitrogen freezer.
On the day of assay, T cells were thawed and washed, followed by staining with
diluted
bispecific antibodies (initial working stock was 240nM (2x) for a final
concentration of 120nM,
serially diluted 1:10 in FACS buffer for a total of seven serial dilutions).
Cells were stained for
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30 minutes at 4 C in cold PBS/1%BSA with primary antibodies, followed by
washing and
addition of 1:250 secondary antibody (anti-human IgG-PE, Thermo Fisher
Scientific) for 30
minutes at 4 C in cold PBS/1%BSA. Following wash step, resuspended cells in
1504,
PBS/1%BSA and detect PE signal on a FACSCelesta HTS system with 96 well V
bottom plate.
Live/dead was assessed using BV421 channel (Zombie violet, Thermo Fisher
Scientific) or
Trypan Blue prior to assay.
[00300] To assess flow cytometric binding of bispecific antibodies to
cell lines, including
Her2-high (SKBR-3, SKOV-3) and Her2-low (MCF-7, HT55), cells were grown in
their
respective culture media according to ATCC protocols to 70-80% confluence.
Cell lines were
.. treated with Accutase to preserve cell surface epitopes for flow cytometry.
Cells were
resuspended in 5mL PBS (no BSA) and stained with 1:1000 dilution of Live/Dead
Zombie dye
(Biolegend) at room temperature for 20 minutes. Cells were then washed and
normalized to add
100,000 cells/well on a 96-well V-bottom plate with staining for 30 minutes at
4 C in cold
PBS/1%BSA with primary antibodies. This was followed by washing and addition
of 1:250
secondary antibody (anti-human IgG-PE, Thermo Fisher Scientific) for 30
minutes at 4 C in cold
PBS/1%BSA. Following wash step, resuspended cells in 1504, PBS/1%BSA and
detect PE
signal on a FACSCelesta HTS system with 96 well V bottom plate. Live/dead was
assessed
using BV421 channel (Zombie violet, Thermo Fisher Scientific) or Trypan Blue
prior to assay.
Analysis was performed in FlowJo software to obtain PE (anti-human IgG-PE)
Median (MFT)
.. values, then data was organized and plotted in Microsoft Excel, and
GraphPad PRISM software
in GraphPAD Prism software.
[00301] Results: Activated T cell binding was reduced for 10.5.1 and 10.6.1
compared to the
parental construct (10.0) (FIG. 16E). The reduced T cell binding observed in
10.5.1 and 10.6.1
can be attributed at least in part to having a CD3 arm exhibiting reduced
affinity.
[00302] Target cell line binding to Her2-high (SK-BR-3, SK-OV-3) cell lines
was slightly
reduced with 10.5.1 and 10.6.1 constructs compared to the parental construct
(10.0)
(approximately 33% reduction in MFI for both 10.5.1 and 10.6.1 compared to
10.0 at about 100
nM concentration) (FIGs. 16A-16B). Target cell line binding to Her2-low (MCF-
7, HT55)
target cell lines was greatly reduced for the 10.5.1 and 10.6.1 constructs
compared to the parental
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construct (10.0) (approximately 84% reduction in MFI for both constructs
compared to 10.0 at
about 100 nM concentration) (FIGs. 16C-16D). Together, these results
demonstrate that the
dually affinity-weakened HER2 x CD3 constructs (10.5.1, 10.6.1) have
properties that contribute
to selectivity for Her2-high target cell lines and maintain the capability to
bind to CD3+ T cells
in order to potentiate cytotoxicity. Overall, the affinity-weakened constructs
10.5.1 and 10.6.1
show reduced cytotoxicity and cytokine release on Her2-low target cells when
compared to the
parental construct.
[00303] Taken together, the HER2 and CD3 reduced affinity bispecific antibody
constructs
described in FIG. 10 having comparable affinities to 10.5.1 and 10.6.1 are
expected to show
similar behavior as 10.5.1 and 10.6.1 in at least one of NFAT activation,
TDCC, and/or FACS.
Example 4: In Vivo Efficacy of the HER2 Affinity-tuned Bispecific Antibodies
of the Present
Technology
[00304] In order to evaluate the in vivo efficacy of the affinity tuned
bispecific antibodies of
the present technology, human tumor and peripheral blood mononuclear cell
(PBMC) co-
xenografts in mice will be performed. Given that low-affinity binding to HER2
results in
selective killing of high-HER2 expressing tumors, sparing low-HER2-expressing
tumors (used
as a surrogate for endogenous HER2 expression of normal, non-cancerous
tissues) in vitro (FIG.
14) we intend to evaluate if this selective killing would also be observed in
vivo. NSG mice will
be implanted subcutaneously with mixture of 1-5 million HER2-expressing tumors
and human
PBMCs (1:2 or 1:3 PBMC:tumor (E:T) ratio). A high-Her2-expressing tumor cell
line, such as
HCC1954 cells (representing expression levels of tumors expected in the clinic
(e.g. those with
2+ or 3+ HecepTest scores) or a low Her-2-expressing tumor cell line, such as
HT55 cells
(representing Her2-levels in noncancerous tissues) will be implanted into
sufficiently
immunocompromised mice, such as Nod-Scid-gamma (NSG) mice or similar.
Different dose
levels (at least a range that includes from 5 mg/kg to 0.005 mg/kg) of various
Her2- and CD3-
affinity weakened constructs will be administered to mice after implantation
of tumor cells and
PBMCs. Doses will be administered parenterally (e.g., i.v. or i.p.) once
weekly or more for one
or more weeks. Tumor volumes will be measured for the duration of the study.
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[00305] It is expected that the 10.0 parent will non-selectively inhibit the
growth of both high
and low Her2-expressing tumor cells at doses where 10.5.1 and 10.6.1 will
selectively inhibit the
growth of the high-Her2 tumors but have little or no growth inhibiting
activity on low Her2-
expressing tumors. It is also expected that the levels of cytokines such as IL-
2, IL-6, IFN-y, and
TNF-a will be reduced in the animals dosed with 10.5.1 and 10.6.1 clones as
compared to the
10.0 parent.
[00306] Accordingly, the immunoglobulin-related compositions of the present
technology are
useful to treat a HER2-associated cancer in a subject in need thereof.
EQUIVALENTS
[00307] The present technology is not to be limited in terms of the particular
embodiments
described in this application, which are intended as single illustrations of
individual aspects of
the present technology. Many modifications and variations of this present
technology can be
made without departing from its spirit and scope, as will be apparent to those
skilled in the art.
Functionally equivalent methods and apparatuses within the scope of the
present technology, in
addition to those enumerated herein, will be apparent to those skilled in the
art from the
foregoing descriptions. Such modifications and variations are intended to fall
within the scope
of the present technology. It is to be understood that this present technology
is not limited to
particular methods, reagents, compounds compositions or biological systems,
which can, of
course, vary. It is also to be understood that the terminology used herein is
for the purpose of
describing particular embodiments only, and is not intended to be limiting.
[00308] In addition, where features or aspects of the disclosure are described
in terms of
Markush groups, those skilled in the art will recognize that the disclosure is
also thereby
described in terms of any individual member or subgroup of members of the
Markush group.
[00309] As will be understood by one skilled in the art, for any and all
purposes, particularly
in terms of providing a written description, all ranges disclosed herein also
encompass any and
all possible subranges and combinations of subranges thereof. Any listed range
can be easily
recognized as sufficiently describing and enabling the same range being broken
down into at
least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range
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discussed herein can be readily broken down into a lower third, middle third
and upper third, etc.
As will also be understood by one skilled in the art all language such as "up
to," "at least,"
"greater than," "less than," and the like, include the number recited and
refer to ranges which can
be subsequently broken down into subranges as discussed above. Finally, as
will be understood
by one skilled in the art, a range includes each individual member. Thus, for
example, a group
having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group
having 1-5 cells refers
to groups having 1, 2, 3, 4, or 5 cells, and so forth.
[00310] All patents, patent applications, provisional applications, and
publications referred to
or cited herein are incorporated by reference in their entirety, including all
figures and tables, to
.. the extent they are not inconsistent with the explicit teachings of this
specification.
