U.S. patent application number 15/346439 was filed with the patent office on 2017-10-12 for use of erbb3 inhibitors in the treatment of triple negative and basal-like breast cancers.
The applicant listed for this patent is Merrimack Pharmaceuticals, Inc.. Invention is credited to Gabriela GARCIA, Victor MOYO.
Application Number | 20170291957 15/346439 |
Document ID | / |
Family ID | 44513103 |
Filed Date | 2017-10-12 |
United States Patent
Application |
20170291957 |
Kind Code |
A1 |
MOYO; Victor ; et
al. |
October 12, 2017 |
USE OF ERBB3 INHIBITORS IN THE TREATMENT OF TRIPLE NEGATIVE AND
BASAL-LIKE BREAST CANCERS
Abstract
Provided are methods of suppressing growth of triple negative
breast tumors and basal-like breast tumors by contacting tumor
cells with an ErbB3 inhibitor, e.g., an anti-ErbB3 antibody. Also
provided are methods for treating triple negative breast cancer or
basal-like breast cancer in a patient by administering to the
patient an ErbB3 inhibitor, e.g., an anti-ErbB3 antibody. The
treatment methods can further comprise selecting a patient having a
triple negative breast cancer or basal-like breast cancer and then
administering an ErbB3 inhibitor to the patient. The treatment
methods also can further comprise administering at least one
additional anti-cancer agent to the patient in combination with the
ErbB3 inhibitor.
Inventors: |
MOYO; Victor; (Ringoes,
NJ) ; GARCIA; Gabriela; (Roslindale, MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Merrimack Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
44513103 |
Appl. No.: |
15/346439 |
Filed: |
November 8, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14518900 |
Oct 20, 2014 |
9518130 |
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15346439 |
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13583949 |
Sep 11, 2012 |
8895001 |
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PCT/US11/28129 |
Mar 11, 2011 |
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14518900 |
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61312895 |
Mar 11, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/517 20130101;
A61K 39/39558 20130101; A61K 39/3955 20130101; C07K 16/40 20130101;
A61P 15/00 20180101; C07K 2317/92 20130101; C07K 16/22 20130101;
A61K 45/06 20130101; A61P 35/00 20180101; C07K 2317/24 20130101;
A61K 2039/507 20130101; A61K 31/337 20130101; A61K 31/517 20130101;
A61P 43/00 20180101; C07K 2317/565 20130101; C07K 16/3015 20130101;
A61K 2300/00 20130101; C07K 2317/73 20130101; C07K 2317/76
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; C07K
2317/56 20130101; C07K 16/2863 20130101; A61K 39/39558 20130101;
A61K 31/337 20130101; C07K 16/32 20130101; C07K 2317/21 20130101;
A61K 2039/505 20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32; A61K 45/06 20060101 A61K045/06; A61K 31/517 20060101
A61K031/517; C07K 16/28 20060101 C07K016/28; C07K 16/22 20060101
C07K016/22; A61K 39/395 20060101 A61K039/395; A61K 31/337 20060101
A61K031/337 |
Claims
1-23. (canceled)
24. A method of suppressing growth of triple negative breast cancer
cells in a patient having a tumor that is identified as triple
negative breast cancer, the method comprising administering to the
patient an effective amount of an anti-ErbB3 antibody, or
antigen-binding fragment thereof, comprising a V.sub.H CDR1
sequence of SEQ ID NO:3, a V.sub.H CDR2 sequence of SEQ ID NO:4 and
a V.sub.H CDR3 sequence of SEQ ID NO:5, and a V.sub.L CDR1 sequence
of SEQ ID NO:6, a V.sub.L CDR2 sequence of SEQ ID NO:7 and a
V.sub.L CDR3 sequence of SEQ ID NO:8.
25. The method of claim 24, wherein the anti-ErbB3 antibody
comprises a V.sub.H sequence as shown in SEQ ID NO:1 and a VL
sequence as shown in SEQ ID NO:2.
26. The method of claim 24, wherein the triple negative breast
cancer tumor is histopathologically characterized as having a
basal-like phenotype.
27. The method of claim 24, wherein the triple negative breast
cancer tumor is histopathologically characterized as having a
phenotype other than basal-like.
28. The method of claim 24, which further comprises administering
to the patient an effective amount of at least one additional
anti-cancer agent.
29. The method of claim 28, wherein the at least one additional
anti-cancer agent is selected from the group consisting of
platinum-based chemotherapy drugs, taxanes, tyrosine kinase
inhibitors, anti-EGFR antibodies, and combinations thereof.
30. The method of claim 29, wherein the at least one additional
anti-cancer agent is a taxane.
31. The method of claim 28, wherein the at least one additional
anti-cancer agent comprises an EGFR inhibitor.
32. The method of claim 31, wherein the EGFR inhibitor comprises an
anti-EGFR antibody.
33. The method of claim 32, wherein the anti-EGFR antibody is
selected from the group consisting of cetuximab, matuzumab,
panitumumab, nimotuzumab and mAb 806.
34. The method of claim 31, wherein the EGFR inhibitor is a small
molecule inhibitor of EGFR signaling selected from the group
consisting of gefitinib, lapatinib, and erlotinib HCL.
35. The method of claim 28, wherein the at least one additional
anti-cancer agent comprises a VEGF inhibitor.
36. The method of claim 35, wherein the VEGF inhibitor comprises
bevacizumab.
37. The method of claim 24, wherein the triple negative breast
cancer tumor scores negative for estrogen receptor (ER) and
progesterone receptor and yields a test result of 0, 1+, or 2+
using a semi-quantitative immunohistochemical assay using a
polyclonal anti-HER2 primary antibody.
38. The method of claim 37, wherein the tumor is FISH negative for
HER2 gene amplification.
39. A method of suppressing growth of triple negative breast cancer
cells in a patient, the method comprising: 1) identifying a patient
with a triple negative breast cancer tumor, and 2) administering to
the patient an effective amount of an anti-ErbB3 antibody, or
antigen-binding_fragment thereof, comprising a V.sub.H CDR1
sequence of SEQ ID NO:3, a V.sub.H CDR2 sequence of SEQ ID NO:4 and
a V.sub.H CDR3 sequence of SEQ ID NO:5, and a V.sub.L CDR1 sequence
of SEQ ID NO:6, a V.sub.L CDR2 sequence of SEQ ID NO:7 and a
V.sub.L CDR3 sequence of SEQ ID NO:8.
40. The method of claim 39, wherein the anti-ErbB3 antibody
comprises a V.sub.H sequence as shown in SEQ ID NO:1 and a V.sub.L
sequence as shown in SEQ ID NO:2.
41. The method of claim 39, wherein the triple negative breast
cancer tumor scores negative for estrogen receptor (ER) and
progesterone receptor and yields a test result of 0, 1+, or 2+
using a semi-quantitative immunohistochemical assay using a
polyclonal anti-HER2 primary antibody.
42. The method of claim 39, wherein the triple negative breast
cancer tumor is histopathologically characterized as having a
basal-like phenotype.
43. The method of claim 39, wherein the triple negative breast
cancer tumor is histopathologically characterized as having a
phenotype other than basal-like.
44. The method of claim 39, which further comprises administering
to the patient an effective amount of at least one additional
anti-cancer agent.
45. The method of claim 44, wherein the at least one additional
anti-cancer agent is a taxane.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 14/518,900, filed Oct. 20, 2014, which is a continuation of
U.S. Pat. No. 8,895,001, issued Feb. 7, 2013, which is a 35 U.S.C.
371 national stage filing of International Application No.
PCT/US2011/028129, filed Mar. 11, 2011, which claims priority to
U.S. Provisional Patent Application No. 61/312,895, filed Mar. 11,
2010. The entire contents of the aforementioned and any patents,
patent applications, or references cited throughout this
specification are hereby incorporated by reference in their
entireties.
BACKGROUND
[0002] In women, breast cancer is among the most common cancers and
is the fifth most common cause of cancer deaths. Due to the
heterogeneity of breast cancers, 10-year progression free survival
can vary widely with stage and type, from 98% to 10%. Different
forms of breast cancers can have remarkably different biological
characteristics and clinical behavior. Thus, classification of a
patient's breast cancer has become a critical component for
determining a treatment regimen. For example, along with
classification of histological type and grade, breast cancers now
are routinely evaluated for expression of hormone receptors
(estrogen receptor (ER) and progesterone receptor (PR)) and for
expression of HER2 (ErbB2), since a number of treatment modalities
are currently available that target hormone receptors or the HER2
receptor. ER and PR are both nuclear receptors (they are
predominantly located at cell nuclei, although they can also be
found at the cell membrane) and small molecular inhibitors that
target ER and/or PR have been developed. HER2, or human epidermal
growth factor receptor type 2, is a receptor normally located on
the cell surface and antibodies that target HER2 have been
developed as therapeutics. HER2 is the only member of the EGFR
family (which also includes HER1 (EGFR), HER3 (ErbB3) and HER4
(ErbB4) that is not capable of binding to an activating ligand on
its own. Thus HER2 is only functional as a receptor when
incorporated into a heterodimeric receptor complex with another
EGFR family member, such as HER3. Cancers classified as expressing
the estrogen receptor (estrogen receptor positive, or ER.sup.+
tumors) may be treated with an ER antagonist such as tamoxifen.
Similarly, breast cancers classified as expressing high levels the
HER2 receptor may be treated with an anti-HER2 antibody, such as
trastuzumab, or with a HER2-active receptor tyrosine kinase
inhibitor such as lapatinib.
[0003] Triple negative (TN) breast cancer is a term used to
designate a well-defined clinically relevant subtype of breast
carcinomas that account for approximately 15% of all breast cancer
cases. TN tumors score negative (i.e., using conventional
histopathology methods and criteria) for expression of ER and PR
and do not express amplified levels of HER2 (i.e., they are
ER.sup.-, PR.sup.-, HER2.sup.-). TN breast cancer comprises
primarily, but not exclusively, a molecularly and
histopathologically distinct subtype of breast cancer known as the
basal-like (BL) subtype. The BL subtype also is characterized by
the expression of cytokeratins (e.g., CK, CK5/6, CK14, CK17) and
other proteins found in normal basal/myoepithelial cells of the
breast. However, in addition to the BL subtype, certain other types
of breast cancers, including some "normal breast-like", metaplastic
carcinomas, medullary carcinomas and salivary gland-like tumors can
also exhibit the triple negative (TN) phenotype. Furthermore, TN
breast cancers occur more frequently in the presence of BRCA1
mutations and in pre-menopausal females of African-American or
Hispanic descent. TN tumors typically display very aggressive
behavior, with shorter post-relapse survival and poor overall
survival rates relative to other breast cancer types.
[0004] Not all BL breast cancers are TN. Basal-like breast tumors
are a heterogeneous tumor type that account for up to 15% of all
breast cancers and exhibit aggressive clinical behavior that makes
them particularly difficult to treat successfully. A majority of BL
breast cancers are ER-, PR-, and HER2 low (HER2.sup.1+ or HER2
negative). In addition, they typically express proteins usually
found in normal breast basal (myoepithelial) cells. These include
high molecular weight cytokeratins (e.g., 5/6, 8, 14, 17 and 18),
p-cadherin, caveolins 1 and 2, nestin, .alpha.B crystalline, and
EGFR. Furthermore, BL tumor cells typically lack the capacity for
competent homologous recombination DNA repair.
[0005] Histologically, most BL breast cancers are of IDC-NST type,
high histological grade, and exhibit very high mitotic indices.
They also typically have central necrotic or fibrotic zones,
pushing borders, conspicuous lymphocytic infiltrates, and
typical/atypical medullary features, and generally exhibit features
similar to those of human papilloma virus-induced squamous cell
carcinoma of the head and neck.
[0006] A great majority of medullary and atypical medullary,
metaplastic, secretory, myoepithelial, and adenoid cystic
carcinomas of the breast also exhibit BL characteristics.
[0007] Given the lack of expression of hormone receptors or of
significant amounts of HER2 in TN breast cancer cells, treatment
options have been very limited as the tumors are not responsive to
treatments that target ER (e.g., tamoxifen, aromatase inhibitors)
or HER2 (e.g., trastuzumab). Instead these tumors are treated with
conventional neoadjuvant and adjuvant chemotherapy regimens, which
have limited efficacy and many cytotoxic side effects. Furthermore,
such chemotherapy regimens can lead to drug resistance in tumors,
and the risk of recurrence of disease in TN breast cancers is
higher within the first three years of treatment than for other
types of breast cancers.
[0008] Basal-like breast cancers are also difficult to treat and
are associated with poor prognoses, though BL adenoid cystic
carcinomas generally are associated with better clinical
outcomes.
[0009] In view of the foregoing, a need remains for additional
treatment options and methods for treating triple negative breast
cancers and BL breast cancers.
SUMMARY
[0010] Provided herein are methods for treating triple negative
breast cancers (e.g., tumors) and basal-like breast cancers (e.g.,
tumors), as well as pharmaceutical compositions that can be used in
such methods. The methods and compositions are based, at least in
part, on the discovery that ErbB3 inhibition can suppress the
growth of TN breast cancer cells and BL breast cancer cells. In
particular, administration of anti-ErbB3 antibody is demonstrated
to suppress the growth of TN breast cancer cells in vivo.
[0011] Accordingly, use of an ErbB3 inhibitor (e.g., use thereof
for the manufacture of a medicament) for the treatment of TN or BL
breast cancer is provided. In another aspect, a method of
suppressing growth of a TN breast cancer tumor or a BL breast
cancer tumor is provided, the method comprising contacting the
tumor with an effective amount of an ErbB3 inhibitor. In another
aspect, a method of suppressing growth of a TN breast cancer tumor
or BL breast cancer tumor in a patient is provided, the method
comprising administering to the patient an effective amount of an
ErbB3 inhibitor. In yet another aspect, a method of treating a
patient for a TN breast cancer tumor or BL breast cancer tumor is
provided, the method comprising administering to the patient an
effective amount of an ErbB3 inhibitor. In still another aspect, a
method of treating a breast cancer tumor or BL breast cancer tumor
in a patient is provided, the method comprising: selecting a
patient with a triple negative breast cancer tumor or a BL breast
cancer tumor; and administering to the patient an effective amount
of an ErbB3 inhibitor.
[0012] In an exemplary embodiment, the ErbB3 inhibitor is an
anti-ErbB3 antibody. An exemplary anti-ErbB3 antibody is MM-121 (Ab
#6), comprising V.sub.H and V.sub.L sequences as shown in SEQ ID
NOs: 1 and 2, respectively. Another exemplary anti-ErbB3 antibody
is an antibody comprising, optionally in amino terminal to carboxy
terminal order, V.sub.H CDR1, 2 and 3 sequences as shown in SEQ ID
NOs: 3-5, respectively, and, optionally in amino terminal to
carboxy terminal order, V.sub.L CDR1, 2 and 3 sequences as shown in
SEQ ID NOs: 6-8, respectively. In other embodiments, the anti-ErbB3
antibody is Ab #3 (comprising V.sub.H and V.sub.L sequences as
shown in SEQ ID NOs: 9 and 10, respectively), Ab #14 (comprising
V.sub.H and V.sub.L sequences as shown in SEQ ID NOs: 17 and 18,
respectively), Ab #17 (comprising V.sub.H and V.sub.L sequences as
shown in SEQ ID NOs: 25 and 26, respectively) or Ab #19 (comprising
V.sub.H and V.sub.L sequences as shown in SEQ ID NOs: 33 and 34,
respectively). In still other embodiments, the anti-ErbB3 antibody
is selected from the group consisting of mAb 1B4C3, mAb 2D1D12,
AMG-888 and humanized mAb 8B8. In another embodiment,
administration of the anti-ErbB3 antibody inhibits growth or
invasiveness or metastasis of the tumor.
[0013] The methods provided herein can be used in the treatment of
TN breast cancers of various different histopathological
phenotypes. For example, in one embodiment, the triple negative
breast cancer tumor is histopathologically characterized as having
a basal-like phenotype. In another embodiment, the TN breast cancer
tumor is histopathologically characterized as having a phenotype
other than BL.
[0014] In each of the foregoing methods and compositions, the ErbB3
inhibitor may be comprised in a formulation comprising a
pharmaceutically acceptable carrier.
[0015] In another aspect, the treatment methods provided herein
further comprise administering to the patient at least one
additional anti-cancer agent that is not an ErbB3 inhibitor. In one
embodiment, the at least one additional anti-cancer agent comprises
at least one chemotherapeutic drug, such as a drug(s) selected from
the group consisting of platinum-based chemotherapy drugs, taxanes,
tyrosine kinase inhibitors, and combinations thereof. It has now
been observed that in the subset of TN breast cancers that test
HER2.sup.2+, treatment with anti-HER2 agents such as trastuzumab,
pertuzumab or lapatinib may provide benefits when used in
combination with anti-ErbB3 antibodies. Thus in another aspect the
treatment methods provided herein further comprise administering to
the patient an effective amount of at least one additional
anti-cancer agent that is an anti-HER2 agent. Such anti-HER2 agents
are well known and may include one or more of anti-ErbB2 antibodies
such as C6.5 (and the numerous derivatives thereof) described in
U.S. Pat. No. 5,977,322, trastuzumab, as described in U.S. Pat. No.
6,054,297, and pertuzumab, as described in U.S. Pat. No. 6,949,245;
as well as small molecule anti-HER2 agents such as lapatinib (which
also inhibits EGFR tyrosine kinase) and AG879.
[0016] In another embodiment, the at least one additional
anti-cancer agent comprises an EGFR inhibitor, such as an anti-EGFR
antibody or a small molecule inhibitor of EGFR signaling. An
exemplary anti-EGFR antibody comprises cetuximab. Other examples of
anti-EGFR antibodies include matuzumab, panitumumab, nimotuzumab
and mAb 806. An exemplary small molecule inhibitor of EGFR
signaling comprises gefitinib. Other examples of useful small
molecule inhibitors of EGFR signaling include lapatinib,
canertinib, erlotinib HCL, pelitinib, PKI-166, PD158780, and AG
1478.
[0017] In yet another embodiment, the at least one additional
anti-cancer agent comprises a VEGF inhibitor. An exemplary VEGF
inhibitor comprises an anti-VEGF antibody, such as the bevacizumab
antibody.
[0018] In another embodiment, administration of the anti-ErbB3
antibody and the at least one additional anti-cancer agent inhibits
growth or invasiveness or metastasis of the tumor.
[0019] In another aspect, methods of treating TN breast cancer or
BL breast cancer in a patient comprise administering to said
patient a combination comprising MM-121 and paclitaxel. In one
embodiment the combination exhibits therapeutic synergy in the
treatment of TN or BL breast cancers. In some examples, the
combination effects a log.sub.10 cell kill of at least 2.8, at
least 2.9 or at least 3.0. In other aspects, the combination
provides an improvement in tumor growth inhibition that is at least
about additive as compared to improvement obtained with each of the
single agents of the combination.
[0020] In another embodiment, there is provided a composition
comprising a combination of MM-121 and paclitaxel, wherein the
combination exhibits therapeutic synergy in the treatment of TN or
BL breast cancers. In some examples, the composition effects a
log.sub.10 cell kill of at least 2.8, at least 2.9 or at least
3.0.
[0021] Kits containing the combination pharmaceutical compositions
also are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a graph showing relative MAXF449 xenograft tumor
volume (%) (Y axis-normalized to initial tumor volume) plotted
against time in days following randomization (X axis) in NMRI nude
mice treated with MM-121 or vehicle control. TGI=200%.
[0023] FIG. 2 is a graph showing the percent change in MDA-MB-231
xenograft tumor volume (Y axis - normalized to initial tumor
volume) plotted against time in days following injection of
MDA-MB-231 cells (X axis) in Balb/c nude mice treated with MM-121
or vehicle control. Curves with open timepoint squares or circles
indicate mice treated with MM-121. Curves with filled timepoint
squares or circles indicate vehicle controls. In the inset, "mp"
indicates that the MDA-MB-231 cells were injected into the mammary
fat pad, while "sc" indicates that the MDA-MB-231 cells were
injected subcutaneously in the flank.
[0024] FIG. 3 is a graph showing MDA-MB-231 tumor volume in
mm.sup.3 (Y axis) plotted against time in days (X-axis) starting at
28 days following injection of MDA-MB-231 cells into the mammary
fat pads of Balb/c nude mice. Treatment was with MM-121 (150
.mu.g/mouse), paclitaxel (5 mg/kg), a combination of MM-121 (150
.mu.g/mouse) and paclitaxel (5 mg/kg), or vehicle control. Where
used in the figures, "mpk"=mg/kg.
[0025] FIGS. 4A and 4B present graphs showing MDA-MB-231 tumor
volume in mm.sup.3 (Y axis) plotted against time in days (X-axis)
starting at 28 days following injection of MDA-MB-231 cells into
the mammary fat pads of Balb/c nude mice. FIG. 4A depicts treatment
with MM-121, cetuximab, or paclitaxel; MM-121 and cetuximab; and
the triple combination MM-121 and cetuximab and paclitaxel. FIG. 4B
depicts treatment with MM-121, erlotinib, MM-121 and erlotinib, or
the triple combination of MM-121 and erlotinib and paclitaxel.
DETAILED DESCRIPTION
[0026] Provided herein are methods for treating triple negative and
basal-like breast cancers, as well as pharmaceutical compositions
that can be used in practicing such methods. As described further
in the Examples, it has now been demonstrated that an ErbB3
inhibitor, in particular an anti-ErbB3 antibody, is able to
suppress the growth of TN breast cancer cells in vivo. Accordingly,
methods for suppressing the growth of TN breast cancers and BL
breast cancers, as well as methods of treating such breast cancers
in patients, using an ErbB3 inhibitor are provided herein.
Definitions:
[0027] As used herein, the term "triple negative" or "TN" refers to
tumors (e.g., carcinomas), typically breast tumors, in which the
tumor cells score negative (i.e., using conventional histopathology
methods) for estrogen receptor (ER) and progesterone receptor (PR),
both of which are nuclear receptors (i.e., they are predominantly
located at cell nuclei), and the tumor cells are not amplified for
epidermal growth factor receptor type 2 (HER2 or ErbB2), a receptor
normally located on the cell surface. Tumor cells are considered
negative for expression of ER and PR if less than 5% of the tumor
cell nuclei are stained for ER and PR expression using standard
immunohistochemical techniques. Tumor cells are considered highly
amplified for HER2 ("HER2.sup.3+") if, when tested with a
HercepTest.TM. Kit (Code K5204, Dako North America, Inc.,
Carpinteria, Calif.), a semi-quantitative immunohistochemical assay
using a polyclonal anti-HER2 primary antibody, they yield a test
result score of 3+, or, the test HER2 positive by fluorescence
in-situ hybridization (FISH). As used herein, tumor cells are
considered negative for HER2 overexpression if they yield a test
result score of 0 or 1+, or 2+, or if they are HER2 FISH
negative.
[0028] Furthermore, the term "triple negative breast cancer(s)" or
"TN breast cancer(s)" encompasses carcinomas of differing
histopathological phenotypes. For example, certain TN breast
cancers are classified as "basal-like" ("BL"), in which the
neoplastic cells express genes usually found in normal
basal/myoepithelial cells of the breast, such as high molecular
weight basal cytokeratins (CK, CK5/6, CK14, CK17), vimentin,
p-cadherin, .alpha.B crystallin, fascin and caveolins 1 and 2.
Certain other TN breast cancers, however, have a different
histopathological phenotype, examples of which include high grade
invasive ductal carcinoma of no special type, metaplastic
carcinomas, medullary carcinomas and salivary gland-like tumors of
the breast.
[0029] The terms "ErbB3," "HER3," "ErbB3 receptor," and "HER3
receptor," as used interchangeably herein, refer to human ErbB3
protein, as described in U.S. Pat. No. 5,480,968.
[0030] As used herein, the term "ErbB3 inhibitor" is intended to
include therapeutic agents that inhibit, downmodulate, suppress or
downregulate activity of ErbB3. The term is intended to include
chemical compounds, such as small molecule inhibitors, and biologic
agents, such as antibodies, interfering RNA (shRNA, siRNA), soluble
receptors and the like. An exemplary ErbB3 inhibitor is an
anti-ErbB3 antibody.
[0031] An "antibody," as used herein is a protein consisting of one
or more polypeptides comprising binding domains substantially
encoded by immunoglobulin genes or fragments of immunoglobulin
genes, wherein the protein immunospecifically binds to an antigen.
The recognized immunoglobulin genes include the kappa, lambda,
alpha, gamma, delta, epsilon and mu constant region genes, as well
as myriad immunoglobulin variable region genes. Light chains are
classified as either kappa or lambda. Heavy chains are classified
as gamma, mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A
typical immunoglobulin structural unit comprises a tetramer that is
composed of two identical pairs of polypeptide chains, each pair
having one "light" (about 25 kD) and one "heavy" chain (about 50-70
kD). "V.sub.L" and V.sub.H'' refer to these light and heavy chains
respectively.
[0032] Antibodies include intact immunoglobulins as well as
antigen-binding fragments thereof, which may be produced by
digestion with various peptidases, or synthesized de novo either
chemically or using recombinant DNA expression technology. Such
fragments include, for example, F(ab).sub.2 dimers and Fab
monomers. Useful antibodies include single chain antibodies
(antibodies that exist as a single polypeptide chain), e.g., single
chain Fv antibodies (scFv) in which a V.sub.H and a V.sub.L chain
are joined together (directly or through a peptide linker) to form
a continuous polypeptide.
[0033] "Immunospecific" or "immunospecifically" refer to antibodies
that bind via domains substantially encoded by immunoglobulin genes
or fragments of immunoglobulin genes to one or more epitopes of a
protein of interest, but which do not substantially recognize and
bind other molecules in a sample containing a mixed population of
antigenic molecules. Typically, an antibody binds
immunospecifically to a cognate antigen with a K.sub.d with a value
of no greater than 50 nM, as measured by a surface plasmon
resonance assay or a cell binding assay. The use of such assays is
well known in the art, and is described in Example 3, below.
[0034] An "anti-ErbB3 antibody" is an antibody that
immunospecifically binds to the ectodomain of ErbB3 and an
"anti-ErbB2 antibody" is an antibody that immunospecifically binds
to the ectodomain of ErbB2. The antibody may be an isolated
antibody. Such binding to ErbB3 or ErB2 exhibits a K.sub.d with a
value of no greater than 50 nM as measured by a surface plasmon
resonance assay or a cell binding assay. An anti-ErbB3 antibody may
be an isolated antibody. Exemplary anti-ErbB3 antibodies inhibit
EGF-like ligand mediated phosphorylation of ErbB3. EGF-like ligands
include EGF, TGF.alpha., betacellulin, heparin-binding epidermal
growth factor, biregulin, epigen, epiregulin, and amphiregulin,
which typically bind to ErbB1 and induce heterodimerization of
ErbB1 with ErbB3.
[0035] As used herein, the term "EGFR inhibitor" is intended to
include therapeutic agents that inhibit, downmodulate, suppress or
downregulate EGFR signaling activity. The term is intended to
include chemical compounds, such as small molecule inhibitors
(e.g., small molecule tyrosine kinase inhibitors) and biologic
agents, such as antibodies, interfering RNA (shRNA, siRNA), soluble
receptors and the like.
[0036] As used herein, the term "VEGF inhibitor" is intended to
include therapeutic agents that inhibit, downmodulate, suppress or
downregulate VEGF signaling activity. The term is intended to
include chemical compounds, such as small molecule inhibitors
(e.g., small molecule tyrosine kinase inhibitors) and biologic
agents, such as antibodies, interfering RNA (shRNA, siRNA), soluble
receptors and the like.
[0037] The terms "suppress", "suppression", "inhibit" and
"inhibition" as used interchangeably herein, refer to any
statistically significant decrease in biological activity (e.g.,
tumor cell growth), including full blocking of the activity. For
example, "inhibition" can refer to a decrease of about 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological
activity.
[0038] The term "patient" includes a human or other mammalian
animal that receives either prophylactic or therapeutic
treatment.
[0039] The terms "treat," "treating," and "treatment," as used
herein, refer to therapeutic or preventative measures such as those
described herein. The methods of "treatment" employ administration
to a patient of an ErbB3 inhibitor provided herein, for example, a
patient having a TN or BL breast cancer tumor, in order to prevent,
cure, delay, reduce the severity of, or ameliorate one or more
symptoms of the disease or disorder or recurring disease or
disorder, or in order to prolong the survival of a patient beyond
that expected in the absence of such treatment.
[0040] The term "effective amount," as used herein, refers to that
amount of an agent, such as an ErbB3 inhibitor, for example an
anti-ErbB3 antibody, which is sufficient to effect treatment,
prognosis or diagnosis of a TN or BL breast cancer, when
administered to a patient. A therapeutically effective amount will
vary depending upon the patient and disease condition being
treated, the weight and age of the patient, the severity of the
disease condition, the manner of administration and the like, which
can readily be determined by one of ordinary skill in the art. The
dosages for administration can range from, for example, about 1 ng
to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to
about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about
7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500
mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg,
about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg,
about 500 ng to about 4,000 mg, about 1 .mu.g to about 3,500 mg,
about 5 .mu.g to about 3,000 mg, about 10 .mu.g to about 2,600 mg,
about 20 .mu.g to about 2,575 mg, about 30 .mu.g to about 2,550 mg,
about 40 .mu.g to about 2,500 mg, about 50 .mu.g to about 2,475 mg,
about 100 .mu.g to about 2,450 mg, about 200 .mu.g to about 2,425
mg, about 300 .mu.g to about 2,000, about 400 .mu.g to about 1,175
mg, about 500 .mu.g to about 1,150 mg, about 0.5 mg to about 1,125
mg, about 1 mg to about 1,100 mg, about 1.25 mg to about 1,075 mg,
about 1.5 mg to about 1,050 mg, about 2.0 mg to about 1,025 mg,
about 2.5 mg to about 1,000 mg, about 3.0 mg to about 975 mg, about
3.5 mg to about 950 mg, about 4.0 mg to about 925 mg, about 4.5 mg
to about 900 mg, about 5 mg to about 875 mg, about 10 mg to about
850 mg, about 20 mg to about 825 mg, about 30 mg to about 800 mg,
about 40 mg to about 775 mg, about 50 mg to about 750 mg, about 100
mg to about 725 mg, about 200 mg to about 700 mg, about 300 mg to
about 675 mg, about 400 mg to about 650 mg, about 500 mg, or about
525 mg to about 625 mg, of an antibody or antigen binding portion
thereof, as provided herein. Dosing may be, e.g., every week, every
2 weeks, every three weeks, every 4 weeks, every 5 weeks or every 6
weeks. Dosage regimens may be adjusted to provide the optimum
therapeutic response. An effective amount is also one in which any
toxic or detrimental effects (side effects) of the agent are
minimized and/or outweighed by the beneficial effects. For MM-121,
administration may be intravenous at exactly or about 6 mg/kg or 12
mg/kg weekly, or 12 mg/kg or 24 mg/kg biweekly. Additional dosing
regimens are described below.
[0041] The terms "anti-cancer agent" and "antineoplastic agent"
refer to drugs used to treat malignancies, such as cancerous
growths. Drug therapy may be used alone, or in combination with
other treatments such as surgery or radiation therapy.
[0042] Various aspects and embodiments are described in further
detail in the following subsections.
[0043] I. ErbB3 Inhibitors
[0044] As described in further detail herein, the methods and
compositions provided herein involve the use of one or more ErbB3
inhibitors.
[0045] In one embodiment, the ErbB3 inhibitor is an anti-ErbB3
antibody, e.g., a monoclonal antibody. An exemplary anti-ErbB3
monoclonal antibody is MM-121, described further in WO 2008/100624
and U.S. Pat. No. 7,846,440, and having V.sub.H and V.sub.L
sequences as shown in SEQ ID NOs: 1 and 2, respectively.
Alternately, the anti-ErbB3 monoclonal antibody is an antibody that
competes with MM-121 for binding to ErbB3. In another embodiment,
the anti-ErbB3 antibody is an antibody comprising the V.sub.H and
V.sub.L CDR sequences of MM-121, which are shown in SEQ ID NOs: 3-5
(V.sub.H CDR1, 2, 3) and 6-8 (V.sub.L CDR1, 2, 3), respectively.
Other examples of anti-ErbB3 antibodies include Ab #3, Ab #14, Ab
#17 and Ab #19, also described further in WO 2008/100624 and having
V.sub.H and V.sub.L sequences as shown in SEQ ID NOs: 9 and 10, 17
and 18, 25 and 26, and 33 and 34 respectively. In another
embodiment, the anti-ErbB3 antibody is an antibody comprising the
V.sub.H and V.sub.L CDR sequences of Ab # 3 (shown in SEQ ID NOs:
11-13 and 14-18, respectively) or antibody comprising the V.sub.H
and V.sub.L CDR sequences of Ab # 14 (shown in SEQ ID NOs: 19-21
and 22-24, respectively) or an antibody comprising the V.sub.H and
V.sub.L CDR sequences of Ab # 17 (shown in SEQ ID NOs: 27-29 and
30-32, respectively) or an antibody comprising the V.sub.H and
V.sub.L CDR sequences of Ab # 19 (shown in SEQ ID NOs: 35-37 and
38-40, respectively).
[0046] Alternately, the anti-ErbB3 antibody is a monoclonal
antibody or antigen binding portion thereof which binds an epitope
of human ErbB3 comprising residues 92-104 of SEQ ID NO:41 and is
characterized by inhibition of proliferation of a cancer cell
expressing ErbB3. The cancer cell may be a MALME-3M cell, an AdrR
cell, or an ACHN cell and the proliferation may be reduced by at
least 10% relative to control. In an additional embodiment this
isolated monoclonal antibody or antigen binding portion thereof
binds an epitope comprising residues 92-104 and 129 of SEQ ID
NO:41.
[0047] Other examples of useful anti-ErbB3 antibodies include the
antibodies 1B4C3 and 2D1D12 (U3 Pharma AG), both of which are
described in US Patent Application Publication No. 20040197332 by
Ullrich et al., and monoclonal antibodies (including humanized
versions thereof), such as AMG-888 (U3 Pharma AG and Amgen) and
8B8, as described in U.S. Pat. No. 5,968,511 by Akita et al.
[0048] In yet another embodiment, the anti-ErbB3 antibody can
comprise a mixture, or cocktail, of two or more anti-ErbB3
antibodies, each of which binds to a different epitope on ErbB3. In
one embodiment, the mixture, or cocktail, comprises three
anti-ErbB3 antibodies, each of which binds to a different epitope
on ErbB3.
[0049] In another embodiment, the ErbB3 inhibitor comprises a
nucleic acid molecule, such as an RNA molecule, that inhibits the
expression or activity of ErbB3. RNA antagonists of ErbB3 have been
described in the art (see e.g., US Patent Application Publication
No. 20080318894). Moreover, interfering RNAs specific for ErbB3,
such as shRNAs or siRNAs that specifically inhibits the expression
and/or activity of ErbB3, have been described in the art.
[0050] In yet another embodiment, the ErbB3 inhibitor comprises a
soluble form of the ErbB3 receptor that inhibits signaling through
the ErbB3 pathway. Such soluble ErbB3 molecules have been described
in the art (see e.g., U.S. Pat. No. 7,390,632, U.S. Patent
Application Publication No. 20080274504 and U.S. Patent Application
Publication No. 20080261270, each by Maihle et al., and U.S. Patent
Application Publication No. 20080057064 by Zhou).
[0051] II. Methods
[0052] In one aspect, use of an ErbB3 inhibitor for the manufacture
of a medicament for the treatment of TN breast cancer or BL breast
cancer is provided.
[0053] In another aspect, a method of suppressing growth of a
triple negative breast cancer cell is provided, the method
comprising contacting the cell with an effective amount of an ErbB3
inhibitor.
[0054] In another aspect, a method of suppressing growth of a TN or
BL breast cancer tumor in a patient is provided, the method
comprising administering to the patient an effective amount of an
ErbB3 inhibitor.
[0055] In yet another aspect, a method of treating a patient for a
TNor BL breast cancer tumor is provided, the method comprising
administering to the patient an effective amount of an ErbB3
inhibitor.
[0056] In still another aspect, a method of treating a breast
cancer tumor in a patient is provided, the method comprising:
[0057] selecting a patient with a TN or BL breast cancer tumor;
and
[0058] administering to the patient an effective amount of an ErbB3
inhibitor.
[0059] In another aspect, the patient with a TN or BL breast cancer
tumor is a patient further selected by use of the selection methods
disclosed in pending international application
PCT/US2009/054051.
[0060] Identification of a triple negative breast cancer cells, or
a patient having a triple negative breast cancer tumor, can be
achieved through standard methods well known in the art. For
example, immunohistochemical (IHC) staining is routinely used in
biopsy analysis and permits the detection, localization and
relative quantification of ER, PR, and HER2 within sections from
formalin-fixed, paraffin-embedded tissues (e.g., breast cancer
tissues routinely processed for histological evaluation). In the
context of identifying TN tumors, staining of less than 5% of tumor
cell nuclei is considered negative for each of for ER and PR. The
primary antibody used for IHC staining of ER is e.g., 1D5
(Chemicon, Temecula Calif., catalog # IHC2055). The primary
antibody used for IHC staining of PR is e.g., PgR636 (Thermo Fisher
Scientific, Fremont, Calif., catalog # MS-1882-R7) or PgR 1294
(Dako North America, Inc., Carpinteria, Calif., Code M3568). The
ErbB2 IHC assay used is e.g., the HercepTest.TM. Kit (Dako North
America, Inc., Carpinteria, Calif., Code K5204), a
semi-quantitative IHC assay using a polyclonal anti-HER2 primary
antibody to determine HER2 protein overexpression in breast cancer
tissues routinely processed for histological evaluation, which is
used according to the manufacturer's directions. In the context of
identifying TN tumors, a test result of 0 to 1+ is considered Her2
negative.
[0061] In one embodiment, the triple negative breast cancer tumor
is histopathologically characterized as having a basal-like
phenotype. In another embodiment, the triple negative breast cancer
tumor is histopathologically characterized as having a phenotype
other than basal-like. Examples of TN breast cancer
histopathological phenotypes that are other than BL include high
grade invasive ductal carcinoma of no special type, metaplastic
carcinomas, medullary carcinomas and salivary gland-like tumors of
the breast.
[0062] In one aspect, the TN or BL breast cancer to be treated with
ErbB3 inhibitor coexpresses ErbB1 (EGFR), ErbB3, and heregulin
(HRG). Expression of EGFR and HRG can be identified by RT-PCR or by
standard immunoassay techniques, such as ELISA assay or
immunohistochemical staining of formalin-fixed, paraffin-embedded
tissues (e.g., breast cancer tissues routinely processed for
histological evaluation), using an anti-EGFR antibody, anti-ErbB3
antibody or an anti-HRG antibody. Additional characteristics of
tumors for treatment in accordance with the disclosure herein are
set forth in pending U.S. Patent Publication No. 20110027291, which
claims priority to PCT application No. PCT/US2009/054051.
[0063] In one embodiment, the ErbB3 inhibitor administered to the
patient is an anti-ErbB3 antibody. An exemplary anti-ErbB3 antibody
is MM-121, comprising V.sub.H and V.sub.L sequences as shown in SEQ
ID NOs: 1 and 2, respectively, or an antibody comprising V.sub.H
CDR1, 2 and 3 sequences as shown in SEQ ID NOs: 3-5, respectively,
and V.sub.L CDR1, 2 and 3 sequences as shown in SEQ ID NOs: 6-8,
respectively (i.e., the V.sub.H and V.sub.L CDRs of MM-121).
Additional non-limiting exemplary anti-ErbB3 antibodies and other
forms of ErbB3 inhibitors are described in detail in Subsection I
above.
[0064] The ErbB3 inhibitor can be administered to the patient by
any route suitable for the effective delivery of the inhibitor to
the patient. For example, many small molecule inhibitors are
suitable for oral administration. Antibodies and other biologic
agents typically are administered parenterally, e.g.,
intravenously, intraperitoneally, subcutaneously or
intramuscularly. Various routes of administration, dosages and
pharmaceutical formulations suitable for use in the methods
provided herein are described in further detail below.
[0065] III. Pharmaceutical Compositions
[0066] In another aspect, pharmaceutical compositions are provided
that can be used in the methods disclosed herein, i.e.,
pharmaceutical compositions for treating TN or BL breast cancer
tumors.
[0067] In one embodiment, the pharmaceutical composition for
treating TN breast cancer comprises an ErbB3 inhibitor and a
pharmaceutical carrier. The ErbB3 inhibitor can be formulated with
the pharmaceutical carrier into a pharmaceutical composition.
Additionally, the pharmaceutical composition can include, for
example, instructions for use of the composition for the treatment
of patients for TN or BL breast cancer tumors.
[0068] In one embodiment, the ErbB3 inhibitor in the composition is
an anti-ErbB3 antibody, e.g., MM-121 or an antibody comprising the
V.sub.H and V.sub.L CDRs of MM-121 positioned in the antibody in
the same relative order as they are present in MM-121 so as to
provide immunospecific binding of ErbB3. Additional non-limiting
exemplary anti-ErbB3 antibodies and other forms of ErbB3 inhibitors
are described in detail in Subsection I above.
[0069] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, buffers, and other excipients that are
physiologically compatible. Preferably, the carrier is suitable for
parenteral, oral, or topical administration. Depending on the route
of administration, the active compound, e.g., small molecule or
biologic agent, may be coated in a material to protect the compound
from the action of acids and other natural conditions that may
inactivate the compound.
[0070] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion, as well
as conventional excipients for the preparation of tablets, pills,
capsules and the like. The use of such media and agents for the
formulation of pharmaceutically active substances is known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
pharmaceutical compositions provided herein is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0071] A pharmaceutically acceptable carrier can include a
pharmaceutically acceptable antioxidant. Examples of
pharmaceutically-acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0072] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions provided herein
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, and injectable organic esters, such as ethyl oleate. When
required, proper fluidity can be maintained, for example, by the
use of coating materials, such as lecithin, by the maintenance of
the required particle size in the case of dispersions, and by the
use of surfactants. In many cases, it will be useful to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought
about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0073] These compositions may also contain fuctional excipients
such as preservatives, wetting agents, emulsifying agents and
dispersing agents.
[0074] Therapeutic compositions typically must be sterile,
non-pyrogenic, and stable under the conditions of manufacture and
storage. The composition can be formulated as a solution,
microemulsion, liposome, or other ordered structure suitable to
high drug concentration.
[0075] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization, e.g., by
microfiltration. Generally, dispersions are prepared by
incorporating the active compound 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 include vacuum drying and freeze-drying
(lyophilization) that yield a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof. The active agent(s) may be mixed
under sterile conditions with additional pharmaceutically
acceptable carrier(s), and with any preservatives, buffers, or
propellants which may be required.
[0076] Prevention of presence of microorganisms may be ensured both
by sterilization procedures, supra, and by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0077] Pharmaceutical compositions comprising an ErbB3 inhibitor
can be administered alone or in combination therapy. For example,
the combination therapy can include a composition provided herein
comprising an ErbB3 inhibitor and at least one or more additional
therapeutic agents, such as one or more chemotherapeutic agents
known in the art, discussed in further detail in Subsection IV
below. Pharmaceutical compositions can also be administered in
conjunction with radiation therapy and/or surgery.
[0078] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation.
[0079] Exemplary dosage ranges for administration of an antibody
include: 10-1000 mg (antibody)/kg (body weight of the patient),
10-800 mg/kg, 10-600 mg/kg, 10-400 mg/kg, 10-200 mg/kg, 30-1000
mg/kg, 30-800 mg/kg, 30-600 mg/kg, 30-400 mg/kg, 30-200 mg/kg,
50-1000 mg/kg, 50-800 mg/kg, 50-600 mg/kg, 50-400 mg/kg, 50-200
mg/kg, 100-1000 mg/kg, 100-900 mg/kg, 100-800 mg/kg, 100-700 mg/kg,
100-600 mg/kg, 100-500 mg/kg, 100-400 mg/kg, 100-300 mg/kg and
100-200 mg/kg. Exemplary dosage schedules include once every three
days, once every five days, once every seven days (i.e., once a
week), once every 10 days, once every 14 days (i.e., once every two
weeks), once every 21 days (i.e., once every three weeks), once
every 28 days (i.e., once every four weeks) and once a month.
[0080] It may be advantageous to formulate parenteral compositions
in unit dosage form for ease of administration and uniformity of
dosage. Unit dosage form as used herein refers to physically
discrete units suited as unitary dosages for the patients to be
treated; each unit contains a predetermined quantity of active
agent calculated to produce the desired therapeutic effect in
association with any required pharmaceutical carrier. The
specification for unit dosage forms are dictated by and directly
dependent on (a) the unique characteristics of the active compound
and the particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
compound for the treatment of sensitivity in individuals.
[0081] Actual dosage levels of the active ingredients in the
pharmaceutical compositions disclosed herein may be varied so as to
obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient. "Parenteral" as used herein in the context of
administration means modes of administration other than enteral and
topical administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion.
[0082] The phrases "parenteral administration" and "administered
parenterally" as used herein refer to modes of administration other
than enteral (i.e., via the digestive tract) and topical
administration, usually by injection or infusion, and includes,
without limitation, intravenous, intramuscular, intraarterial,
intrathecal, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous,
subcuticular, intraarticular, subcapsular, subarachnoid,
intraspinal, epidural and intrasternal injection and infusion.
Intravenous injection and infusion are often (but not exclusively)
used for antibody administration.
[0083] When agents provided herein are administered as
pharmaceuticals, to humans or animals, they can be given alone or
as a pharmaceutical composition containing, for example, 0.001 to
90% (e.g., 0.005 to 70%, e.g., 0.01 to 30%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0084] IV. Combination Therapy
[0085] In certain embodiments, the methods and uses provided herein
for suppressing growth of TN breast cancer cells or for treating a
patient with a TN breast tumor or BL breast tumor can comprise
administration of an ErbB3 inhibitor and at least one additional
anti-cancer agent that is not an ErbB3 inhibitor.
[0086] In one embodiment, the at least one additional anti-cancer
agent comprises at least one chemotherapeutic drug. Non-limiting
examples of such chemotherapeutic drugs include platinum-based
chemotherapy drugs (e.g., cisplatin, carboplatin), taxanes (e.g.,
paclitaxel (Taxol.RTM.), docetaxel (Taxotere.RTM.), EndoTAG-1.TM.
(a formulation of paclitaxel encapsulated in positively charged
lipid-based complexes; MediGene), Abraxane.RTM. (a formulation of
paclitaxel bound to albumin)), tyrosine kinase inhibitors (e.g.,
imatinib/Gleevec.RTM., sunitinib/Sutent.RTM.,
dasatinib/Sprycel.RTM.), and combinations thereof.
[0087] In another embodiment, the at least one additional
anti-cancer agent comprises an EGFR inhibitor, such as an anti-EGFR
antibody or a small molecule inhibitor of EGFR signaling. An
exemplary anti-EGFR antibody is cetuximab (Erbitux.RTM.). Cetuximab
is commercially available from ImClone Systems Incorporated. Other
examples of anti-EGFR antibodies include matuzumab (EMD72000),
panitumumab (Vectibix.RTM.; Amgen); nimotuzumab (TheraCIM.TM.) and
mAb 806. An exemplary small molecule inhibitor of the EGFR
signaling pathway is gefitinib (Iressa.RTM.), which is commercially
available from AstraZeneca and Teva. Other examples of small
molecule inhibitors of the EGFR signaling pathway include erlotinib
HCL (OSI-774; Tarceva.RTM., OSI Pharma); lapatinib (Tykerb.RTM.,
GlaxoSmithKline); canertinib (canertinib dihydrochloride, Pfizer);
pelitinib (Pfizer); PKI-166 (Novartis); PD158780; and AG 1478
(4-(3-Chloroanillino)-6,7-dimethoxyquinazoline).
[0088] In yet another embodiment, the at least one additional
anti-cancer agent comprises a VEGF inhibitor. An exemplary VEGF
inhibitor comprises an anti-VEGF antibody, such as bevacizumab
(Avastatin.RTM.; Genentech).
[0089] In still another embodiment, the at least one additional
anti-cancer agent comprises an anti-ErbB2 antibody. Suitable
anti-ErbB2 antibodies include trastuzumab and pertuzumab.
[0090] In one aspect, the improved effectiveness of a combination
according to the invention can be demonstrated by achieving
therapeutic synergy.
[0091] The term "therapeutic synergy" is used when the combination
of two products at given doses is more efficacious than the best of
each of the two products alone at the same doses. In one example,
therapeutic synergy can be evaluated by comparing a combination to
the best single agent using estimates obtained from a two-way
analysis of variance with repeated measurements (e.g., time factor)
on parameter tumor volume.
[0092] The term "additive" refers to when the combination of two or
more products at given doses is equally efficacious than the sum of
the efficacies obtained with of each of the two or more products,
whilst the term "superadditive" refers to when the combination is
more efficacious than the sum of the efficacies obtained with of
each of the two or more products.
[0093] Another measure by which effectiveness (including
effectiveness of combinations) can be quantified is by calculating
the log.sub.10 cell kill, which is determined according to the
following equation:
log.sub.10 cell kill=T-C (days)/3.32.times.T.sub.d
in which T-C represents the delay in growth of the cells, which is
the average time, in days, for the tumors of the treated group (T)
and the tumors of the control group (C) to have reached a
predetermined value (1 g, or 10 mL, for example), and T.sub.d
represents the time, in days necessary for the volume of the tumor
to double in the control animals. When applying this measure, a
product is considered to be active if log.sub.10 cell kill is
greater than or equal to 0.7 and a product is considered to be very
active if log.sub.10 cell kill is greater than 2.8.
[0094] Using this measure, a combination, used at its own maximum
tolerated dose, in which each of the constituents is present at a
dose generally less than or equal to its maximum tolerated dose,
exhibits therapeutic synergy when the log.sub.10 cell kill is
greater than the value of the log.sub.10 cell kill of the best
constituent when it is administered alone. In an exemplary case,
the log.sub.10 cell kill of the combination exceeds the value of
the log.sub.10 cell kill of the best constituent of the combination
by at least one log cell kill.
EXAMPLES
Example 1
MM-121 Effects on Triple Negative Human Breast Cancer Xenograft
MAXF449
[0095] An analysis of the anti-tumor efficacy and tolerability of
MM-121 treatment of tumor-bearing mice is carried out using triple
negative human mammary carcinoma xenograft MAXF449 (ONCOTEST GmbH,
Frieburg, Germany) in NMRI nude mice. MAXF449 is a Human tumor
explant (histologically described upon explant as solid invasive
ductal, and poorly defined) established via subcutaneous injection
in serial passages in nude mice. The MAXF449 cells used in these
experiments have been passaged 22 times. NMRI nude mice are
obtained from Taconic farms, Charles River Laboratories
International, or Harlan Laboratories. The mice are housed in
Tecniplast Individually Ventilated polycarbonate (Macrolon) Cages
(IVC) set in climate-controlled rooms and have free access to food
and acidified water.
[0096] To investigate anti-tumor efficacy in monotherapy, MM-121 or
vehicle control (100 .mu.L) is given to tumor-bearing mice at 600
.mu.g per mouse (MM-121 as a 6 mg/mL solution in PBS) by IP
injection every three days. Control mice receive the PBS vehicle
only. Efficacy is determined by comparing tumor growth between the
antibody-treated mice and the vehicle control mice and is expressed
as the experimental to control ratio of median relative tumor
volumes (T/C value). A minimum T/C value below 50% is a
prerequisite for rating a treatment as effective. The control and
experimental groups each contain 10 mice bearing one tumor each. To
obtain 30 mice bearing tumors of similar sizes for randomization,
40 mice per tumor are implanted unilaterally.
[0097] Mice are randomized and therapy begins when a sufficient
number of individual tumors have grown to a volume of approximately
200 mm3. Tumors are measured (L.times.W) by digital caliper
measurement and the tumor volume is calculated using the formula
Pi/6 (W2.times.L). The first dose is administered either on Day 0
(day of randomization) or one day later.
[0098] Approximately 24 hours after administration of the final
dose all mice are bled to prepare serum; in addition, tumors are
collected from the same mice for flash-freezing and FFPE (1/2 tumor
each).
[0099] According to regulations for animal experiments, mice are
sacrificed if the tumor volume exceeds 1800 mm.sup.3 (one tumor per
mouse). Mice are monitored and dosed until their tumors have grown
to that size but no longer than 60 days. Thereafter, they are
sacrificed for sample collection.
[0100] At the end of the study, approximately 24 hours after
administration of the final dose, all mice on study are bled
sublingually to obtain a maximum amount of blood for the
preparation of serum. Serum is aliquoted in 2 tubes with
approximately 250 .mu.L in each.
[0101] In addition, tumors from all mice are excised without delay
for snap-freezing in liquid nitrogen (1/2 tumor, COVARIS bags for
the storage of samples are provided) and for fixation in 10%
buffered formalin for <24 hours, subsequent dehydration and
paraffin embedding (FFPE, 1/2 tumor).
[0102] Animal weights and tumor diameters (W and L) are measured
twice weekly and tumor volumes are calculated using the formula
Pi/6 (W2.times.L). Tumor growth curves are plotted. Tumor
inhibition and absolute growth delay for 2 and 4 doubling times are
calculated.
[0103] Results of experiments that were carried out substantially
as described are presented in FIG. 1. MM-121 treatment inhibited or
stopped tumor growth, and in some cases reduced tumor size. TGI
(tumor growth inhibition) in these human triple negative tumor
xenografts was calculated to be approximately 200%.
Example 2
MM-121 Effects on Triple-Negative Human Breast Cancer Xenograft
MDA-MB-231
[0104] Balb/c nude mice are injected under general anesthesia with
10.sup.7 MDA-MB-231 human triple negative breast cancer cells
(ATCC) either subcutaneously in the flank or into the mammary fat
pad. Mice with established tumors (i.e., after 7-10 days of tumor
growth following injection of cells) are then treated IP with
either PBS or MM-121 every 3 days with 600 ug MM-121 per mouse as
described in Example 1. Tumor volume is measured twice a week as
described in Example 1.
[0105] Results of experiments carried out substantially as
described are presented in FIG. 2. MM-121 treatment stopped human
triple negative breast cancer tumor growth essentially completely
in these experiments.
Example 3
Measurement of Binding Affinity (K.sub.D)
[0106] The dissociation constants of anti-ErbB antibodies may be
measured using either or both of two independent techniques, a
Surface Plasmon Resonance Assay and a cell binding assay.
Surface Plasmon Resonance Assay
[0107] The Surface Plasmon Resonance Assay is performed as
described in Wassaf et al. (2006) Analytical Biochem., 351:
241-253. One implementation uses a BIACORE 3000 instrument (GE
Healthcare) using a recombinant ErbB protein as the analyte and the
anti-ErbB antibody as the ligand The K.sub.D value is calculated
based on the formula K.sub.D=K.sub.d/K.sub.a.
Cell Binding Assay
[0108] A cell binding assay is performed using MALME-3M cells
(ATCC) for ErbB3 binding. The assay is performed substantially as
follows.
[0109] Cells are detached with 2 mLs trypsin-EDTA+2 mLs RMPI+5 mM
EDTA at room temperature for 5 minutes. Complete RPMI (10 mLs) is
added immediately to the trypsinized cells, resuspended gently and
spun down in a Beckman tabletop centrifuge at 1100 rpm for 5
minutes. Cells are resuspended in BD stain buffer (PBS+2% FBS+0.1%
sodium azide, Becton Dickinson) at a concentration of
2.times.10.sup.6 cells per ml and 50 .mu.l (1.times.10.sup.5 cells)
aliquots are plated in a 96-well titer plate.
[0110] A 150 .mu.l solution of 200 nM anti-ErbB antibody in BD
stain buffer is prepared and serially diluted 2-fold into 75 .mu.l
BD stain buffer. The concentrations of the diluted antibody ranged
from 200 nM to 0.4 nM. 50 .mu.l aliquots of the different protein
dilutions are then added directly to the 50 ul cell suspension
giving the final concentrations of 100 nM, 50 nM, 25 nM, 12 nM, 6
nM, 3 nM, 1.5 nM, 0.8 nM, 0.4 nM and 0.2 nM of the antibody.
[0111] Aliquoted cells in the 96-well plate are incubated with the
protein dilutions for 30 minutes at room temperature on a platform
shaker and washed 3 times with 300 .mu.l BD stain buffer. Cells are
then incubated with 100 .mu.l of secondary antibody (e.g., a 1:750
dilution of Alexa 647-labeled goat anti-human IgG in BD stain
buffer) for 45 minutes on a platform shaker in the cold room.
Finally, cells are washed twice, pelleted and resuspended in 250
.mu.l BD stain buffer+0.5 .mu.g/ml propidium iodide. Analysis of
10,000 cells is done in a FACSCALIBUR flow cytometer using the FL4
channel. MFI values and the corresponding concentrations of the
anti-ErbB-antibody are plotted on the y-axis and x-axis,
respectively. The K.sub.D of the molecule is determined using
GraphPad PRISM software using the one-site binding model for a
non-linear regression curve.
[0112] The K.sub.D value is calculated based on the formula Y=Bmax*
X/K.sub.D+X (Bmax=fluorescence at saturation. X=antibody
concentration. Y=degree of binding).
Example 4
Inhibition of Tumor Growth in Vivo by Combination Treatment with
MM-121 and Paclitaxel
Methods:
[0113] Balb/c nude mice (female, 4-5 weeks old from Charles River
lab) are implanted orthotopically with 10.times.106 cells in
mammary pad. Tumors are allowed to reach average of 100 mm.sup.3 in
size before randomization into 4 groups of 10 mice, containing mice
with a similar size distribution of tumors. Each group of mice is
treated with 1) MM-121 (150 ug/mouse, ip., Q3D) or 2) vehicle
control (PBS, ip.) or 3) paclitaxel (5 mg/kg LC Labs) or 4)
paclitaxel (5 mg/kg) and MM-121 (150 ug/mouse). Treatment is
continued for 4 weeks. Tumors are measured twice weekly and tumor
volume is calculated as p/6.times.length.times.width.sup.2, where
the width is the shorter measurement.
Results:
[0114] The combination of MM-121 with paclitaxel was investigated
in vivo in the MDA-MB-231 triple negative breast cancer xenograft
model using the methods described above or minor variations
thereof. Mice were treated with sub-optimal doses of MM-121,
paclitaxel, a combination of MM-121 and paclitaxel, or vehicle
control (FIG. 3). While both MM-121 and paclitaxel each inhibited
tumor growth in vivo, mice receiving a combination therapy of
MM-121 and paclitaxel exhibited an improvement of tumor growth
inhibition when compared to that obtained with each of the
individual treatments. The improvement in tumor growth inhibition
exhibited therapeutic synergy and was at least about additive as
compared to the improvement obtained with each of the single agents
of the combination.
[0115] Table 1 shows data used to generate FIG. 3. Table 2 shows
the mean % change in tumor volumes using data from the same
experiments shown in FIG. 3, normalized to initial tumor
volume.
TABLE-US-00001 TABLE 1 data used to generate FIG. 3 - mean tumor
volumes in mm.sup.3 Vehicle Mean 104.4 137.1 144.5 229.5 253.7
291.0 MM121 150 .mu.g Mean 99.4 115.5 137.5 180.4 187.2 242.7
paclitaxel 5 mg/kg Mean 97.9 113.5 144.6 166.2 178.8 202.2 MM121
150 .mu.g + Mean 96.2 100.8 98.3 104.1 113.0 121.6 paclitaxel 5
mg/kg
Example 5
MM-121 Combination with Targeted and Chemotherapies in Vivo
Methods:
[0116] Balb/c nude mice (female, 4-5 weeks old from Charles River
lab) are implanted orthotopically with 10.times.106 cells in
mammary pad. Tumors are allowed to reach average of 150 mm.sup.3 in
size before randomization into 9 groups of 8 mice, containing mice
with a similar size distribution of tumors. Each group of mice is
treated with a dose of 1) MM-121(300 ug/mouse, ip., Q3D) or 2)
vehicle control (PBS, ip.) or 3) paclitaxel (10 mg/kg LC Labs) or
4) erlotinib (50 mg/kg PO 5XQD) or 5) cetuximab (2 mg/kg Q3D) or
combination therapy with: 6) erlotinib (50 mg/kg) and MM-121 (300
ug/mouse), or 7) cetuximab (2 mg/kg) and MM-121 (300 ug/mouse), or
8) erlotinib (50 mg/kg) and MM-121 (300 ug/mouse) and paclitaxel
(10 mg/kg), or 9) cetuximab (2 mg/kg) and MM121 (300 ug/mouse) and
paclitaxel (10 mg/kg). Treatment is continued for 4 weeks. Tumors
are measured twice weekly and tumor volume is calculated as
p/6.times.length.times.width.sup.2, where the width is the shorter
measurement.
[0117] Results: In order to test the efficacy of MM-121 to inhibit
tumor growth when used in combination with other agents, these
combinations were tested in vivo in the MDA-MB-231 triple negative
breast cancer xenograft model using the methods described above or
minor variations thereof. Mice were treated with MM-121
(administered at sub-optimal doses in the combinations), cetuximab,
paclitaxel, MM-121 and cetuximab, and the triple combination MM-121
and cetuximab and paclitaxel. As shown in FIG. 4A, combination
therapy with MM-121 and cetuximab inhibited tumor growth to a
greater extent than either agent alone and essentially stopped
tumor growth until at least day 39. The decreased rate of growth
showed therapeutic synergy and, in certain cases represented at
least about an additive decrease in growth compared to the
decreased rates obtained with any of the single therapies. Addition
of paclitaxel did not enhance the effect of MM-121 and cetuximab.
Mice were then treated with MM-121, erlotinib, MM-121 and
erlotinib, or the triple combination of MM-121 and erlotinib and
paclitaxel. As shown in FIG. 4B, MM-121 in combination with
erlotinib did not have a statistically significant effect on the
rate of tumor growth compared with treatment with either agent
alone. Conversely, treatment with the triple combination of MM-121,
erlotinib, and paclitaxel resulted in a clearly decreased rate of
tumor growth and essentially stopped tumor growth until at least
day 39. The decreased rate of growth showed therapeutic synergy
and, in certain cases represented at least about an additive
decrease in growth compared to the decreased rates obtained with
any of the single or double therapies. Table 3 shows data used to
generate FIGS. 4A and 4B. Table 4 shows the mean % change in tumor
volume using data from the same experiments shown in FIGS. 4A and
4B, normalized to initial tumor volume.
TABLE-US-00002 TABLE 2 Data used to generate FIG. 4A and 4B - mean
tumor volumes in mm.sup.3. Day 28 32 36 39 43 46 49 53 PBS 163.7
199.0 242.8 304.5 369.4 423.4 458.4 490.7 MM121 300ug 178.6 197.3
219.1 257.8 269.4 291.4 351.3 425.0 erlotinib 50 mg/kg 172.1 182.1
216.1 273.2 252.8 245.6 303.1 327.4 cetuximab 2 mg/kg 172.4 210.6
245.0 269.2 296.3 279.7 283.5 358.1 MM121 + erlotinib 170.6 215.5
221.8 261.7 272.5 255.3 305.2 378.3 paclitaxel 10 mg/kg 155.2 167.0
182.4 216.6 228.1 247.0 292.6 383.5 MM121 + cetuximab 152.5 149.6
171.6 169.1 196.6 171.2 182.9 241.2 MM121 + erlotinib + 164.8 149.3
139.8 146.5 156.7 163.4 202.9 264.5 paclitaxel MM121 + cetuximab +
176.3 158.5 147.8 160.4 154.4 163.4 203.4 247.7 paclitaxel
Equivalents
[0118] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments described herein. Such
equivalents are intended to be encompassed by the following claims.
Any combinations of the embodiments disclosed in the dependent
claims are contemplated to be within the scope of the
invention.
INCORPORATION BY REFERENCE
[0119] Each and every, issued patent, patent application and
publication referred to herein is hereby incorporated herein by
reference in its entirety.
TABLE-US-00003 SUMMARY OF SEQUENCE LISTING MM-121 V.sub.H amino
acid sequence (SEQ ID NO: 1)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPGKGLEWVSS
ISSSGGWTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGL
KMATIFDYWGQGTLVTVSS MM-121 V.sub.L amino acid sequence (SEQ ID NO:
2) QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVVSWYQQHPGKAPKLII
YEVSQRPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYCCSYAGSSIFV IFGGGTKVTVL
MM-121 V.sub.H CDR1 (SEQ ID NO: 3) HYVMA MM-121 V.sub.H CDR2 (SEQ
ID NO: 4) SISSSGGWTLYADSVKG MM-121 V.sub.H CDR3 (SEQ ID NO: 5)
GLKMATIFDY MM-121 V.sub.L CDR1 (SEQ ID NO: 6) TGTSSDVGSYNVVS MM-121
V.sub.L CDR2 (SEQ ID NO: 7) EVSQRPS MM-121 V.sub.L CDR3 (SEQ ID NO:
8) CSYAGSSTVI Ab #3 V.sub.H amino acid sequence (SEQ ID NO: 9)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYNMRWVRQAPGKGLEWVSV
IYPSGGATRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGY
YYYGMDVWGQGTLVTVSS Ab #3 V.sub.L amino acid sequence (SEQ ID NO:
10) QSVLTQPPSASGTPGQRVTISCSGSDSNIGRNYIYWYQQFPGTAPKLLIY
RNNQRPSGVPDRISGSKSGTSASLAISGLRSEDEAEYHCGTWDDSLSGPV FGGGTKLTVL Ab #3
V.sub.H CDR1 (SEQ ID NO: 11) AYNMR Ab #3 V.sub.H CDR2 (SEQ ID NO:
12) VIYPSGGATRYADSVKG Ab #3 V.sub.H CDR3 (SEQ ID NO: 13) GYYYYGMDV
Ab #3 V.sub.L CDR1 (SEQ ID NO: 14) SGSDSNIGRNYIY Ab #3 V.sub.L CDR2
(SEQ ID NO: 15) RNNQRPS Ab #3 V.sub.L CDR3 (SEQ ID NO: 16)
GTWDDSLSGPV Ab #14 V.sub.H amino acid sequence (SEQ ID NO: 17)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYGMGWVRQAPGKGLEWVSY
ISPSGGHTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVL
ETGLLVDAFDIWGQGTMVTVSS Ab #14 V.sub.L amino acid sequence (SEQ ID
NO: 18) QYELTQPPSVSVYPGQTASITCSGDQLGSKFVSWYQQRPGQSPVLVMYKD
KRRPSEIPERFSGSNSGNTATLTISGTQAIDEADYYCQAWDSSTYVFGTG TKVTVL Ab #14
V.sub.H CDR1 (SEQ ID NO: 19) AYGMG Ab #14 V.sub.H CDR2 (SEQ ID NO:
20) YISPSGGHTKYADSVKG Ab #14 V.sub.H CDR3 (SEQ ID NO: 21)
VLETGLLVDAFDI Ab #14 V.sub.L CDR1 (SEQ ID NO: 22) SGDQLGSKFVS Ab
#14 V.sub.L CDR2 (SEQ ID NO: 23) YKDKRRPS Ab #14 V.sub.L CDR3 (SEQ
ID NO: 24) QAWDSSTYV Ab #17 V.sub.H amino acid sequence (SEQ ID NO:
25) EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYGMGWVRQAPGKGLEWVSY
ISPSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLN
YYYGLDVWGQGTTVTVSS Ab #17 V.sub.L amino acid sequence (SEQ ID NO:
26) QDIQMTQSPSSLSASVGDRITITCQASQDIGDSLNWYQQKPGKAPRLLIY
DASNLETGVPPRFSGSGSGTDFTFTFRSLQPEDIATYFCQQSANAPFTFG PGTKVDIK Ab #17
V.sub.H CDR1 (SEQ ID NO: 27) WYGMG Ab #17 V.sub.H CDR2 (SEQ ID NO:
28) YISPSGGITVYADSVKG Ab #17 V.sub.H CDR3 (SEQ ID NO: 29) LNYYYGLDV
Ab #17 V.sub.L CDR1 (SEQ ID NO: 30) QASQDIGDSLN Ab #17 V.sub.L CDR2
(SEQ ID NO: 31) DASNLET Ab #17 V.sub.L CDR3 (SEQ ID NO: 32)
QQSANAPFT Ab #19 VH amino acid sequence (SEQ ID NO: 33)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMWWVRQAPGKGLEWVSYI
GSSGGPTYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGRGT
PYYFDSWGQGTLVTVSS Ab #19 V.sub.L amino acid sequence (SEQ ID NO:
34) QYELTQPASVSGSPGQSITISCTGTSSDIGRWNIVSWYQQHPGKAPKLMIY
DVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTWVFG GGTKLTVL Ab #19
V.sub.H CDR1 (SEQ ID NO: 35) RYGMW Ab #19 V.sub.H CDR2 (SEQ ID NO:
36) YIGSSGGPTYYVDSVKG Ab #19 V.sub.H CDR3 (SEQ ID NO: 37)
GRGTPYYFDS Ab #19 V.sub.L CDR1 (SEQ ID NO: 38 TGTSSDIGRWNIVS Ab #19
V.sub.L CDR2 (SEQ ID NO: 39) DVSNRPS Ab #19 V.sub.L CDR3 (SEQ ID
NO: 40) SSYTSSSTWV ErbB3 (SEQ ID NO: 41)
SEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTG
HNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFV
MLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRD
RDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCF
GPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLT
FQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKM
CEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLN
GDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIG
GRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLN
WTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGPGQCLS
CRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSG
SDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQ
GCKGPELQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGTFLYWRGR
RIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELRKLKVLGSGV
FGTVHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHA
HIVRLLGLCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIA
KGMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYS
EAKTPIKWMALESIHFGKYTHQSDVWSYGVTVWELMTFGAEPYAGLRLAE
VPDLLEKGERLAQPQICTIDVYMVMVKCWMIDENIRPTFKELANEFTRMA
RDPPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEE
DNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQES
AVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRS
RSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGT
PSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPPRPSSLEE
LGYEYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDG
GGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEAT
DSAFDNPDYWHSRLFPKANAQRT
Sequence CWU 1
1
411119PRTHomo sapiens 1Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser His Tyr 20 25 30 Val Met Ala Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser
Ser Gly Gly Trp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Thr Arg Gly Leu Lys Met Ala Thr Ile Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 2111PRTHomo sapiens
2Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1
5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser
Tyr 20 25 30 Asn Val Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Ile Ile Tyr Glu Val Ser Gln Arg Pro Ser Gly
Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Thr Glu Asp Glu Ala Asp
Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser 85 90 95 Ser Ile Phe Val Ile
Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 35PRTHomo
sapiens 3His Tyr Val Met Ala 1 5 417PRTHomo sapiens 4Ser Ile Ser
Ser Ser Gly Gly Trp Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
510PRTHomo sapiens 5Gly Leu Lys Met Ala Thr Ile Phe Asp Tyr 1 5 10
614PRTHomo sapiens 6Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn Val
Val Ser 1 5 10 77PRTHomo sapiens 7Glu Val Ser Gln Arg Pro Ser 1 5
811PRTHomo sapiens 8Cys Ser Tyr Ala Gly Ser Ser Ile Phe Val Ile 1 5
10 9118PRTHomo sapiens 9Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Asn Met Arg Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Pro
Ser Gly Gly Ala Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser 115 10110PRTHomo sapiens 10Gln
Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10
15 Arg Val Thr Ile Ser Cys Ser Gly Ser Asp Ser Asn Ile Gly Arg Asn
20 25 30 Tyr Ile Tyr Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro
Asp Arg Ile Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu
Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Glu Tyr His
Cys Gly Thr Trp Asp Asp Ser Leu 85 90 95 Ser Gly Pro Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 115PRTHomo sapiens
11Ala Tyr Asn Met Arg 1 5 1217PRTHomo sapiens 12Val Ile Tyr Pro Ser
Gly Gly Ala Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
139PRTHomo sapiens 13Gly Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5
1413PRTHomo sapiens 14Ser Gly Ser Asp Ser Asn Ile Gly Arg Asn Tyr
Ile Tyr 1 5 10 157PRTHomo sapiens 15Arg Asn Asn Gln Arg Pro Ser 1 5
1611PRTHomo sapiens 16Gly Thr Trp Asp Asp Ser Leu Ser Gly Pro Val 1
5 10 17122PRTHomo sapiens 17Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Gly Met Gly Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser
Pro Ser Gly Gly His Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Val Leu Glu Thr Gly Leu Leu Val Asp Ala Phe Asp Ile
Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
18106PRTHomo sapiens 18Gln Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser
Val Tyr Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp
Gln Leu Gly Ser Lys Phe Val 20 25 30 Ser Trp Tyr Gln Gln Arg Pro
Gly Gln Ser Pro Val Leu Val Met Tyr 35 40 45 Lys Asp Lys Arg Arg
Pro Ser Glu Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Ile 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Tyr Val 85 90
95 Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 195PRTHomo
sapiens 19Ala Tyr Gly Met Gly 1 5 2017PRTHomo sapiens 20Tyr Ile Ser
Pro Ser Gly Gly His Thr Lys Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
2113PRTHomo sapiens 21Val Leu Glu Thr Gly Leu Leu Val Asp Ala Phe
Asp Ile 1 5 10 2211PRTHomo sapiens 22Ser Gly Asp Gln Leu Gly Ser
Lys Phe Val Ser 1 5 10 238PRTHomo sapiens 23Tyr Lys Asp Lys Arg Arg
Pro Ser 1 5 249PRTHomo sapiens 24Gln Ala Trp Asp Ser Ser Thr Tyr
Val 1 5 25118PRTHomo sapiens 25Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Trp Tyr 20 25 30 Gly Met Gly Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile
Ser Pro Ser Gly Gly Ile Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Leu Asn Tyr Tyr Tyr Gly Leu Asp Val Trp Gly
Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 26108PRTHomo
sapiens 26Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val 1 5 10 15 Gly Asp Arg Ile Thr Ile Thr Cys Gln Ala Ser Gln
Asp Ile Gly Asp 20 25 30 Ser Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Asp Ala Ser Asn Leu Glu
Thr Gly Val Pro Pro Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Phe Arg Ser Leu Gln 65 70 75 80 Pro Glu Asp Ile
Ala Thr Tyr Phe Cys Gln Gln Ser Ala Asn Ala Pro 85 90 95 Phe Thr
Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 275PRTHomo sapiens
27Trp Tyr Gly Met Gly 1 5 2817PRTHomo sapiens 28Tyr Ile Ser Pro Ser
Gly Gly Ile Thr Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
299PRTHomo sapiens 29Leu Asn Tyr Tyr Tyr Gly Leu Asp Val 1 5
3011PRTHomo sapiens 30Gln Ala Ser Gln Asp Ile Gly Asp Ser Leu Asn 1
5 10 317PRTHomo sapiens 31Asp Ala Ser Asn Leu Glu Thr 1 5
329PRTHomo sapiens 32Gln Gln Ser Ala Asn Ala Pro Phe Thr 1 5
33119PRTHomo sapiens 33Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Gly Met Trp Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Gly Ser
Ser Gly Gly Pro Thr Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Gly Gly Arg Gly Thr Pro Tyr Tyr Phe Asp Ser Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 34110PRTHomo sapiens
34Gln Tyr Glu Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1
5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Arg
Trp 20 25 30 Asn Ile Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly
Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 355PRTHomo sapiens
35Arg Tyr Gly Met Trp 1 5 3617PRTHomo sapiens 36Tyr Ile Gly Ser Ser
Gly Gly Pro Thr Tyr Tyr Val Asp Ser Val Lys 1 5 10 15 Gly
3710PRTHomo sapiens 37Gly Arg Gly Thr Pro Tyr Tyr Phe Asp Ser 1 5
10 3814PRTHomo sapiens 38Thr Gly Thr Ser Ser Asp Ile Gly Arg Trp
Asn Ile Val Ser 1 5 10 397PRTHomo sapiens 39Asp Val Ser Asn Arg Pro
Ser 1 5 4010PRTHomo sapiens 40Ser Ser Tyr Thr Ser Ser Ser Thr Trp
Val 1 5 10 411323PRTHomo sapiens 41Ser Glu Val Gly Asn Ser Gln Ala
Val Cys Pro Gly Thr Leu Asn Gly 1 5 10 15 Leu Ser Val Thr Gly Asp
Ala Glu Asn Gln Tyr Gln Thr Leu Tyr Lys 20 25 30 Leu Tyr Glu Arg
Cys Glu Val Val Met Gly Asn Leu Glu Ile Val Leu 35 40 45 Thr Gly
His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val 50 55 60
Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu 65
70 75 80 Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly
Lys Phe 85 90 95 Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser
Ser His Ala Leu 100 105 110 Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu
Ile Leu Ser Gly Gly Val 115 120 125 Tyr Ile Glu Lys Asn Asp Lys Leu
Cys His Met Asp Thr Ile Asp Trp 130 135 140 Arg Asp Ile Val Arg Asp
Arg Asp Ala Glu Ile Val Val Lys Asp Asn 145 150 155 160 Gly Arg Ser
Cys Pro Pro Cys His Glu Val Cys Lys Gly Arg Cys Trp 165 170 175 Gly
Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala 180 185
190 Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys
195 200 205 His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr
Asp Cys 210 215 220 Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys
Val Pro Arg Cys 225 230 235 240 Pro Gln Pro Leu Val Tyr Asn Lys Leu
Thr Phe Gln Leu Glu Pro Asn 245 250 255 Pro His Thr Lys Tyr Gln Tyr
Gly Gly Val Cys Val Ala Ser Cys Pro 260 265 270 His Asn Phe Val Val
Asp Gln Thr Ser Cys Val Arg Ala Cys Pro Pro 275 280 285 Asp Lys Met
Glu Val Asp Lys Asn Gly Leu Lys Met Cys Glu Pro Cys 290 295 300 Gly
Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser Gly Ser Arg 305 310
315 320 Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val Asn Cys
Thr 325 330 335 Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu
Asn Gly Asp 340 345 350 Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu
Lys Leu Asn Val Phe 355 360 365 Arg Thr Val Arg Glu Ile Thr Gly Tyr
Leu Asn Ile Gln Ser Trp Pro 370 375 380 Pro His Met His Asn Phe Ser
Val Phe Ser Asn Leu Thr Thr Ile Gly 385 390 395 400 Gly Arg Ser Leu
Tyr Asn Arg Gly Phe Ser Leu Leu Ile Met Lys Asn 405 410 415 Leu Asn
Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu Ile Ser Ala 420 425 430
Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr His His Ser 435
440 445 Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu
Asp 450 455 460 Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu
Gly Lys Val 465 470 475 480 Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys
Trp Gly Pro Gly Pro Gly 485 490 495 Gln Cys Leu Ser Cys Arg Asn Tyr
Ser Arg Gly Gly Val Cys Val Thr 500 505 510 His Cys Asn Phe Leu Asn
Gly Glu Pro Arg Glu Phe Ala His Glu Ala 515 520 525 Glu Cys Phe Ser
Cys His Pro Glu Cys Gln Pro Met Glu Gly Thr Ala 530 535 540 Thr Cys
Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys Ala His Phe 545 550 555
560 Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly Val Leu Gly
565 570 575 Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn Glu
Cys Arg 580 585 590 Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly
Pro Glu Leu Gln 595 600 605 Asp Cys Leu Gly Gln Thr Leu Val Leu Ile
Gly Lys Thr His Leu Thr 610 615 620 Met Ala Leu Thr Val Ile Ala Gly
Leu Val Val Ile Phe Met Met Leu 625 630 635 640 Gly Gly Thr Phe Leu
Tyr Trp Arg Gly Arg Arg Ile Gln Asn Lys Arg 645 650 655 Ala Met Arg
Arg Tyr Leu Glu Arg Gly Glu Ser Ile Glu Pro Leu Asp 660 665 670 Pro
Ser Glu Lys Ala Asn Lys Val Leu Ala Arg Ile Phe Lys Glu Thr 675 680
685 Glu Leu Arg Lys Leu Lys Val Leu Gly Ser Gly Val Phe Gly Thr Val
690 695 700 His Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys Ile
Pro Val 705 710 715 720 Cys Ile Lys Val Ile Glu Asp Lys Ser Gly Arg
Gln Ser Phe Gln Ala 725 730
735 Val Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His Ala His Ile
740 745 750 Val Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu Gln Leu
Val Thr 755 760 765 Gln Tyr Leu Pro Leu Gly Ser Leu Leu Asp His Val
Arg Gln His Arg 770 775 780 Gly Ala Leu Gly Pro Gln Leu Leu Leu Asn
Trp Gly Val Gln Ile Ala 785 790 795 800 Lys Gly Met Tyr Tyr Leu Glu
Glu His Gly Met Val His Arg Asn Leu 805 810 815 Ala Ala Arg Asn Val
Leu Leu Lys Ser Pro Ser Gln Val Gln Val Ala 820 825 830 Asp Phe Gly
Val Ala Asp Leu Leu Pro Pro Asp Asp Lys Gln Leu Leu 835 840 845 Tyr
Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu Glu Ser Ile 850 855
860 His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val
865 870 875 880 Thr Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr
Ala Gly Leu 885 890 895 Arg Leu Ala Glu Val Pro Asp Leu Leu Glu Lys
Gly Glu Arg Leu Ala 900 905 910 Gln Pro Gln Ile Cys Thr Ile Asp Val
Tyr Met Val Met Val Lys Cys 915 920 925 Trp Met Ile Asp Glu Asn Ile
Arg Pro Thr Phe Lys Glu Leu Ala Asn 930 935 940 Glu Phe Thr Arg Met
Ala Arg Asp Pro Pro Arg Tyr Leu Val Ile Lys 945 950 955 960 Arg Glu
Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro His Gly Leu 965 970 975
Thr Asn Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu Leu Asp Leu 980
985 990 Asp Leu Asp Leu Glu Ala Glu Glu Asp Asn Leu Ala Thr Thr Thr
Leu 995 1000 1005 Gly Ser Ala Leu Ser Leu Pro Val Gly Thr Leu Asn
Arg Pro Arg 1010 1015 1020 Gly Ser Gln Ser Leu Leu Ser Pro Ser Ser
Gly Tyr Met Pro Met 1025 1030 1035 Asn Gln Gly Asn Leu Gly Glu Ser
Cys Gln Glu Ser Ala Val Ser 1040 1045 1050 Gly Ser Ser Glu Arg Cys
Pro Arg Pro Val Ser Leu His Pro Met 1055 1060 1065 Pro Arg Gly Cys
Leu Ala Ser Glu Ser Ser Glu Gly His Val Thr 1070 1075 1080 Gly Ser
Glu Ala Glu Leu Gln Glu Lys Val Ser Met Cys Arg Ser 1085 1090 1095
Arg Ser Arg Ser Arg Ser Pro Arg Pro Arg Gly Asp Ser Ala Tyr 1100
1105 1110 His Ser Gln Arg His Ser Leu Leu Thr Pro Val Thr Pro Leu
Ser 1115 1120 1125 Pro Pro Gly Leu Glu Glu Glu Asp Val Asn Gly Tyr
Val Met Pro 1130 1135 1140 Asp Thr His Leu Lys Gly Thr Pro Ser Ser
Arg Glu Gly Thr Leu 1145 1150 1155 Ser Ser Val Gly Leu Ser Ser Val
Leu Gly Thr Glu Glu Glu Asp 1160 1165 1170 Glu Asp Glu Glu Tyr Glu
Tyr Met Asn Arg Arg Arg Arg His Ser 1175 1180 1185 Pro Pro His Pro
Pro Arg Pro Ser Ser Leu Glu Glu Leu Gly Tyr 1190 1195 1200 Glu Tyr
Met Asp Val Gly Ser Asp Leu Ser Ala Ser Leu Gly Ser 1205 1210 1215
Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile Met Pro Thr Ala 1220
1225 1230 Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr Met Asn Arg Gln
Arg 1235 1240 1245 Asp Gly Gly Gly Pro Gly Gly Asp Tyr Ala Ala Met
Gly Ala Cys 1250 1255 1260 Pro Ala Ser Glu Gln Gly Tyr Glu Glu Met
Arg Ala Phe Gln Gly 1265 1270 1275 Pro Gly His Gln Ala Pro His Val
His Tyr Ala Arg Leu Lys Thr 1280 1285 1290 Leu Arg Ser Leu Glu Ala
Thr Asp Ser Ala Phe Asp Asn Pro Asp 1295 1300 1305 Tyr Trp His Ser
Arg Leu Phe Pro Lys Ala Asn Ala Gln Arg Thr 1310 1315 1320
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