U.S. patent application number 14/574292 was filed with the patent office on 2015-06-18 for methods and compositions for the diagnosis and treatment of cancer.
The applicant listed for this patent is GENENTECH, INC.. Invention is credited to John Chant, Anthony S. Guerrero, Peter Haverty, Cynthia Honchell, Kenneth Jung, Thomas Wu.
Application Number | 20150167101 14/574292 |
Document ID | / |
Family ID | 38694721 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150167101 |
Kind Code |
A1 |
Chant; John ; et
al. |
June 18, 2015 |
METHODS AND COMPOSITIONS FOR THE DIAGNOSIS AND TREATMENT OF
CANCER
Abstract
Methods and compositions are provided for the diagnosis and
treatment of colorectal cancers associated with amplification or
overexpression of the FGFR2 gene.
Inventors: |
Chant; John; (Millbrae,
CA) ; Guerrero; Anthony S.; (San Francisco, CA)
; Haverty; Peter; (San Francisco, CA) ; Honchell;
Cynthia; (San Francisco, CA) ; Jung; Kenneth;
(San Francisco, CA) ; Wu; Thomas; (San Francisco,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC. |
South San Francisco |
CA |
US |
|
|
Family ID: |
38694721 |
Appl. No.: |
14/574292 |
Filed: |
December 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12300156 |
Mar 17, 2009 |
8945572 |
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PCT/US07/68737 |
May 11, 2007 |
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14574292 |
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60799772 |
May 12, 2006 |
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Current U.S.
Class: |
424/133.1 ;
424/174.1; 435/6.11; 514/19.3; 514/44A |
Current CPC
Class: |
A61P 43/00 20180101;
C12Q 1/6886 20130101; C12Q 2600/106 20130101; C12Q 2600/156
20130101; A61P 35/00 20180101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of diagnosing the presence of a colorectal cancer in a
mammal, the method comprising a) obtaining a test colorectal sample
or a colorectal cancer sample from the mammal; b) detecting the
copy number of the FGFR2 gene in the test colorectal sample or
colorectal cancer sample; c) comparing said copy number of the
FGFR2 gene detected in step b) with the copy number of the FGFR2
gene in a control normal sample; and d) identifying the mammal as
having colorectal cancer when a higher copy number of the FGFR2 is
detected relative to a control normal sample.
2. The method of claim 1, wherein detecting whether the FGFR2 gene
is amplified comprises detecting whether the copy number of the
FGFR2 gene is increased by at least 5-fold.
3-22. (canceled)
23. A method of treating a colorectal cancer associated with
amplification of the FGFR2 gene in an individual having the
colorectral cancer, the method comprising a) obtaining a test
colorectal sample or a colorectal cancer sample from the mammal; b)
detecting the copy number of the FGFR2 gene in the test colorectal
sample or colorectal cancer sample; c) comparing said copy number
of the FGFR2 gene detected in step b) with the copy number of the
FGFR2 gene in a control normal sample; d) identifying that the
individual having a colorectal cancer will respond to a therapeutic
that targets FGFR2 when a higher copy number of the FGFR2 gene is
detected relative to a control normal sample; and e) administering
to said individual having the colorectal cancer associated with
amplification of the FGFR2 gene, an effective amount of a
pharmaceutical formulation comprising a therapeutic that targets
FGFR2 or the FGFR2 gene.
24. The method of claim 23, wherein the FGFR2 antagonist is an
anti-FGFR2 antibody.
25. The method of claim 24, wherein the anti-FGFR2 antibody binds
to the extracellular domain of FGFR2.
26. The method of claim 24, wherein the anti-FGFR2 antibody is an
antibody fragment.
27. The method of claim 24, wherein the anti-FGFR2 antibody is a
chimeric or humanized antibody.
28. The method of claim 24, wherein the anti-FGFR2 antibody is a
human antibody.
29. The method of claim 23, wherein the FGFR2 antagonist is an
organic molecule that binds to FGFR2.
30. The method of claim 23, wherein the FGFR2 antagonist is an
oligopeptide that binds to FGFR2.
31. The method of claim 23, wherein the FGFR2 antagonist is a
soluble form of FGFR2.
32. The method of claim 23, wherein the FGFR2 antagonist is an
antisense nucleic acid of 10-30 nucleotides in length that binds to
and reduces expression of a nucleic acid encoding FGFR2.
33. The method of claim 23, wherein the pharmaceutical formulation
is administered in combination with at least one additional
therapeutic agent and/or adjuvant.
34. The method of claim 33, wherein the additional therapeutic
agent is an unconjugated cytotoxic agent, a chemotherapeutic agent,
or a growth inhibitory agent.
35. The method of claim 34, wherein the chemotherapeutic agent is
an agent or a combination of agents used in the treatment of
colorectal cancer.
36. The method of claim 35, wherein the agents include, but are not
limited to, fluorouracil (5FU) alone or in combination with
leucovorin or levamisole; edrocolomab; irinotecan; oxaliplatin;
raltitrexed; and fluoropyrimidines.
37. A method for determining whether an individual having a
colorectal cancer will respond to a therapeutic that targets FGFR2
or the FGFR2 gene, the method comprising a) obtaining a test
colorectal sample or a colorectal cancer sample from the mammal; b)
detecting the copy number of the FGFR2 gene in the test colorectal
sample or colorectal cancer sample; c) comparing said copy number
of the FGFR2 gene detected in step b) with the copy number of the
FGFR2 gene in a control normal sample; and d) identifying that the
individual having a colorectal cancer will respond to a therapeutic
that targets FGFR2 when a higher copy number of the FGFR2 gene is
detected relative to a control normal sample.
38. The method of claim 37, wherein the therapeutic is (a) an FGFR2
antagonist, (b) a cytotoxic anti-FGFR2 antibody, or (c) an
immunoconjugate comprising an anti-FGFR2 antibody and a cytotoxic
agent.
39. The method of claim 37, wherein determining whether the FGFR2
gene is amplified comprises detecting whether the copy number of
the FGFR2 gene is increased by at least 5-fold.
40. The method of claim 37, wherein the amplification of the FGFR2
gene is detected using an amplification assay or a hybridization
assay.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/799,772, filed May 12, 2006, the disclosure of
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and compositions
for the diagnosis and treatment of cancers associated with gene
amplification.
BACKGROUND
[0003] Cancer is characterized by an increase in the number of
abnormal, or neoplastic, cells derived from a normal tissue that
proliferate and, under certain circumstances, invade adjacent
tissues and eventually metastasize via the blood or lymphatic
system. Alteration of gene expression is intimately related to
uncontrolled cell growth and de-differentiation, which are common
features of cancer. Certain cancers are characterized by
overexpression of certain genes, e.g., oncogenes. A well known
mechanism of gene overexpression in cancer cells is gene
amplification. Gene amplification is a process in which multiple
copies of one or more genes are produced in the chromosome of a
cell. In certain instances, the process involves unscheduled
replication of the region of the chromosome comprising those genes,
followed by recombination of the replicated segments back into the
chromosome (Alitalo et al., Adv. Cancer Res., 47:235-281 [1986]).
In certain cases, overexpression of a gene is correlated with gene
amplification, i.e., is proportional to the number of copies
made.
[0004] Amplification and/or overexpression of certain
proto-oncogenes, e.g., those that encode growth factors and growth
factor receptors, play important roles in the pathogenesis of
various human malignancies. In certain instances, amplification
and/or overexpression are associated with more malignant forms of
cancer and thus may predict clinical outcome (Schwab et al., Genes
Chromosomes Cancer, 1:181-193 [1990]; Alitalo et al., supra). For
example, the human erbB2 gene (also known as her2 or c-erbB-2),
which encodes a 185-kd transmembrane glycoprotein receptor
(p185.sup.HER2 or HER2) related to the epidermal growth factor
receptor EGFR, is overexpressed in about 25% to 30% of human breast
cancers (Slamon et al., Science, 235:177-182 [1987]; Slamon et al.,
Science, 244:707-712 [1989]). Overexpression of erbB2 is considered
a predictor of a poor prognosis, especially in patients with
primary disease that involves axillary lymph nodes (Slamon et al.,
[1987] and [1989], supra; Ravdin and Chamness, Gene, 159:19-27
[1995]; and Hynes and Stern, Biochim. Biophys. Acta, 1198:165-184
[1994]). Overexpression of erbB2 has also been linked to
sensitivity and/or resistance to certain hormone therapy and
chemotherapeutic regimens, including CMF (cyclophosphamide,
methotrexate, and fluoruracil) and anthracyclines (Baselga et al.,
Oncology, 11 (3 Suppl 1):43-48 [1997]). However, patients that
overexpress erbB2 show greater response to treatment with taxanes.
Id.
[0005] Overexpression of erbB2 has provided the basis for targeted
breast cancer therapies. A recombinant humanized anti-ErbB2
(anti-HER2) monoclonal antibody (Herceptin.TM., Genentech, Inc.)
has been successfully used to treat patients with
ErbB2-overexpressing metastatic breast cancer. (Baselga et al., J.
Clin. Oncol., 14:737-744 [1996]).
[0006] A continuing need exists for compositions and methods that
target amplified genes and the products of those genes in the
diagnosis and treatment of cancer.
[0007] A continuing need also exists for compositions and methods
for the diagnosis and/or treatment of colorectal cancer. Over
56,000 people died of colorectal cancer in the year 2000. See Holen
and Kemeny (2002) "Colorectal Cancer: Epidemiology and Treatment,"
in Encyclopedia of Cancer, vol. 2 (Elsevier Sciences, USA), pages
1-8. There are approximately 110,000 new cases of colon cancer
diagnosed in the United States each year, accounting for
approximately 15% of all cancer cases. Id. There are approximately
45,000 new cases of rectal cancer diagnosed in the United States
each year, accounting for approximately 30% of all colorectal
cancers. Id.
[0008] The invention described herein meets the above-described
needs and provides other benefits.
SUMMARY
[0009] In one aspect, methods and compositions are provided for the
diagnosis and treatment of colorectal cancers associated with
amplification and/or overexpression of the FGFR2 gene.
[0010] In one aspect, a method of diagnosing the presence of a
colorectal cancer in a mammal is provided, the method comprising
detecting whether the FGFR2 gene is amplified in a test colorectal
sample from the mammal relative to a control sample, wherein
amplification of the FGFR2 gene indicates the presence of
colorectal cancer in the mammal. In one embodiment, detecting
whether the FGFR2 gene is amplified comprises detecting whether the
copy number of the FGFR2 gene is increased by at least 5-fold.
[0011] In another aspect, a method of diagnosing the presence of a
colorectal cancer in a mammal is provided, the method comprising
detecting expression of the FGFR2 gene in a test colorectal sample
from the mammal, wherein a higher level of FGFR2 gene expression in
the test colorectal sample relative to a control sample indicates
the presence of colorectal cancer in the mammal. In one embodiment,
detecting expression of the FGFR2 gene comprises determining the
level of mRNA transcription from the FGFR2 gene. In one embodiment,
a higher level of FGFR2 expression comprises at least a 5-fold
increase in mRNA transcription from the FGFR2 gene in the test
colorectal sample relative to the control sample. In one
embodiment, detecting expression of the FGFR2 gene comprises
determining the level of FGFR2. In one embodiment, detecting
expression of the FGFR2 gene comprises contacting the test
colorectal sample with an anti-FGFR2 antibody and determining the
level of expression of FGFR2 in the test colorectal sample by
detecting binding of the anti-FGFR2 antibody to FGFR2. In one
embodiment, a higher level of FGFR2 expression comprises at least a
5-fold increase in FGFR2 levels.
[0012] In another aspect, a method of inhibiting the proliferation
of a colorectal cancer cell is provided, the method comprising
exposing the cell to an FGFR2 antagonist. In one embodiment, the
FGFR2 antagonist is an anti-FGFR2 antibody. In one embodiment, the
anti-FGFR2 antibody binds to the extracellular domain of FGFR2. In
one embodiment, the anti-FGFR2 antibody is an antibody fragment. In
one embodiment, the anti-FGFR2 antibody is a chimeric or humanized
antibody. In one embodiment, the anti-FGFR2 antibody is a human
antibody. In one embodiment, the FGFR2 antagonist is an organic
molecule that binds to FGFR2. In one embodiment, the FGFR2
antagonist is an oligopeptide that binds to FGFR2. In one
embodiment, the FGFR2 antagonist is a soluble form of FGFR2. In one
embodiment, the FGFR2 antagonist is an antisense nucleic acid of
10-30 nucleotides in length that binds to and reduces expression of
a nucleic acid encoding FGFR2.
[0013] In another aspect, a method of inhibiting the proliferation
of a colorectal cancer cell is provided, the method comprising
exposing the cell to (a) a cytotoxic anti-FGFR2 antibody or (b) an
immunoconjugate comprising an anti-FGFR2 antibody and a cytotoxic
agent. In one embodiment, the method comprises exposing the cell to
a cytotoxic anti-FGFR2 antibody. In one embodiment, the method
comprises exposing the cell to an immunoconjugate comprising an
anti-FGFR2 antibody and a cytotoxic agent. In one embodiment, the
cytotoxic agent is a maytansinoid or an auristatin.
[0014] In another aspect, a method of treating a colorectal cancer
associated with amplification or overexpression of the FGFR2 gene
is provided, the method comprising administering to an individual
having the colorectal cancer an effective amount of a
pharmaceutical formulation comprising an antagonist of FGFR2. In
one embodiment, the FGFR2 antagonist is an anti-FGFR2 antibody. In
one embodiment, the anti-FGFR2 antibody binds to the extracellular
domain of FGFR2. In one embodiment, the anti-FGFR2 antibody is an
antibody fragment. In one embodiment, the anti-FGFR2 antibody is a
chimeric or humanized antibody. In one embodiment, the anti-FGFR2
antibody is a human antibody. In one embodiment, the FGFR2
antagonist is an organic molecule that binds to FGFR2. In one
embodiment, the FGFR2 antagonist is an oligopeptide that binds to
FGFR2. In one embodiment, the FGFR2 antagonist is a soluble form of
FGFR2. In one embodiment, the FGFR2 antagonist is an antisense
nucleic acid of 10-30 nucleotides in length that binds to and
reduces expression of a nucleic acid encoding FGFR2.
[0015] In another aspect, a method of treating a colorectal cancer
associated with amplification or overexpression of the FGFR2 gene
is provided, the method comprising administering to an individual
having the colorectal cancer an effective amount of a
pharmaceutical formulation comprising (a) a cytotoxic anti-FGFR2
antibody or (b) an immunoconjugate comprising an anti-FGFR2
antibody and a cytotoxic agent. In one embodiment, the method
comprises administering to an individual having the colorectal
cancer an effective amount of a pharmaceutical formulation
comprising a cytotoxic anti-FGFR2 antibody. In one embodiment, the
method comprises administering to an individual having the
colorectal cancer an effective amount of a pharmaceutical
formulation comprising an immunoconjugate comprising an anti-FGFR2
antibody and a cytotoxic agent. In one embodiment, the cytotoxic
agent is a maytansinoid or an auristatin.
[0016] In another aspect, a method for determining whether an
individual having a colorectal cancer will respond to a therapeutic
that targets FGFR2 or the FGFR2 gene is provided, the method
comprising determining whether the FGFR2 gene is amplified in the
colorectal cancer, wherein amplification of the FGFR2 gene
indicates that the individual will respond to the therapeutic. In
one embodiment, the therapeutic is selected from (a) an FGFR2
antagonist, (b) a cytotoxic anti-FGFR2 antibody, or (c) an
immunoconjugate comprising an anti-FGFR2 antibody and a cytotoxic
agent.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows the analysis of DNA copy number and mRNA
expression for the FGFR2 gene in a particular colorectal tumor
sample.
DETAILED DESCRIPTION OF EMBODIMENTS
[0018] Methods and compositions for the diagnosis and treatment of
cancers associated with gene amplification are provided. In certain
embodiments, the invention provides methods and compositions for
the treatment of colorectal cancer associated with amplification
and/or overexpression of the FGFR2 gene.
I. DEFINITIONS
[0019] The phrases "gene amplification" and "gene duplication" (and
variants such as "amplification of a gene" or "duplication of a
gene") are used interchangeably and refer to a process by which
multiple copies of a gene or gene fragment are formed in a
particular cell or cell line. The duplicated region (a stretch of
amplified DNA) is often referred to as an "amplicon." Usually, the
amount of the messenger RNA (mRNA) produced, i.e., the level of
gene expression, also increases in proportion to the number of
copies made of the particular gene.
[0020] The term "FGFR2," as used herein, refers to any native
fibroblast growth factor receptor 2 from any vertebrate source,
including mammals such as primates (e.g. humans and monkeys) and
rodents (e.g., mice and rats), unless otherwise indicated. The term
encompasses "full-length," unprocessed FGFR2 as well as any form of
FGFR2 that results from processing in the cell. The term also
encompasses naturally occurring variants of FGFR2, e.g., splice
variants, allelic variants, and other isoforms. The term also
encompasses fragments or variants of a native FGFR2 that maintain
at least one biological activity of FGFR2.
[0021] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0022] "Tumor," as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer,"
"cancerous," "cell proliferative disorder," "proliferative
disorder" and "tumor" are not mutually exclusive as referred to
herein.
[0023] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth/proliferation. Examples of cancer
include, but are not limited to, carcinoma, lymphoma (e.g.,
Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and
leukemia. More particular examples of such cancers include squamous
cell cancer, small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, squamous carcinoma of the lung, cancer
of the peritoneum, hepatocellular cancer, gastrointestinal cancer,
pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer, colon cancer,
rectal cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer, liver cancer, prostate
cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia
and other lymphoproliferative disorders, and various types of head
and neck cancer.
[0024] The term "colorectal cancer" refers to any cancer of the
large bowel, which includes the colon (the large intestine from the
cecum to the rectum) and the rectum.
[0025] The term "neoplasm" or "neoplastic cell" refers to an
abnormal tissue or cell that proliferates more rapidly than
corresponding normal tissues or cells and continues to grow after
removal of the stimulus that initiated the growth.
[0026] A "colorectal cancer cell" refers to a colon cancer cell or
a rectal cancer cell, either in vivo or in vitro, and encompasses
cell lines derived from colorectal cancer cells.
[0027] As used herein, "treatment" (and variations such as "treat"
or "treating") refers to clinical intervention in an attempt to
alter the natural course of the individual or cell being treated,
and can be performed either for prophylaxis or during the course of
clinical pathology. Desirable effects of treatment include
preventing occurrence or recurrence of disease, alleviation of
symptoms, diminishment of any direct or indirect pathological
consequences of the disease, preventing metastasis, decreasing the
rate of disease progression, amelioration or palliation of the
disease state, and remission or improved prognosis.
[0028] An "individual" is a vertebrate. In certain embodiments, the
vertebrate is a mammal. Mammals include, but are not limited to,
farm animals (such as cows), sport animals, pets (such as cats,
dogs, and horses), primates, mice and rats. In certain embodiments,
a mammal is a human.
[0029] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result.
[0030] A "therapeutically effective amount" of a substance/molecule
of the invention may vary according to factors such as the disease
state, age, sex, and weight of the individual, and the ability of
the substance/molecule, to elicit a desired response in the
individual. A therapeutically effective amount encompasses an
amount in which any toxic or detrimental effects of the
substance/molecule are outweighed by the therapeutically beneficial
effects. A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically, but not necessarily,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount
would be less than the therapeutically effective amount.
[0031] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. The term is intended to include
radioactive isotopes (e.g., Ar.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu), chemotherapeutic agents
(e.g., methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A "tumoricidal" agent causes destruction of tumor cells.
[0032] A "toxin" is any substance capable of having a detrimental
effect on the growth or proliferation of a cell.
[0033] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylolomelamine; acctogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e. g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omega1I (see, e.g., Agnew,
Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; an esperamicin; as well as ncocarzinostatin
chromophore and related chromoprotein enediyne antiobiotic
chromophores), aclacinomysins, actinomycin, authramycin, azaserine,
bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin,
chromomycins, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. docetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE0); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0034] Also included in this definition are anti-hormonal agents
that act to regulate, reduce, block, or inhibit the effects of
hormones that can promote the growth of cancer, and are often in
the form of systemic, or whole-body treatment. They may be hormones
themselves. Examples include anti-estrogens and selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen
(including NOLVADEX.RTM. tamoxifen), EVISTA raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. toremifene; anti-progesterones;
estrogen receptor down-regulators (ERDs); agents that function to
suppress or shut down the ovaries, for example, leutinizing
hormone-releasing hormone (LHRH) agonists such as LUPRON.RTM. and
ELIGARD.RTM. leuprolide acetate, goserelin acetate, buserelin
acetate and tripterelin; other anti-androgens such as flutamide,
nilutamide and bicalutamide; and aromatase inhibitors that inhibit
the enzyme aromatase, which regulates estrogen production in the
adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASIN.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole. In addition,
such definition of chemotherapeutic agents includes bisphosphonates
such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.),
DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic
acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM.
pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); antisense oligonucleotides, particularly those that
inhibit expression of genes in signaling pathways implicated in
abherant cell proliferation, such as, for example, PKC-alpha, Raf,
H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such
as THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0035] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell (such as a
cell expressing FGFR2) either in vitro or in vivo. Thus, the growth
inhibitory agent may be one which significantly reduces the
percentage of cells (such as a cell expressing FGFR2) in S phase.
Examples of growth inhibitory agents include agents that block cell
cycle progression (at a place other than S phase), such as agents
that induce G1 arrest and M-phase arrest. Classical M-phase
blockers include the vincas (vincristine and vinblastine), taxanes,
and topoisomerase II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. Those agents that arrest G1
also spill over into S-phase arrest, for example, DNA alkylating
agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in The Molecular Basis of Cancer,
Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic drugs" by Murakami et al.
(WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes
(paclitaxel and docetaxel) are anticancer drugs both derived from
the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer),
derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0036] As used herein, the term "EGFR inhibitor" refers to
compounds that bind to or otherwise interact directly with EGFR and
prevent or reduce its signaling activity, and is alternatively
referred to as an "EGFR antagonist." Examples of such agents
include antibodies and small molecules that bind to EGFR. Examples
of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB
8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528
(ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.)
and variants thereof, such as chimerized 225 (C225 or Cetuximab;
ERBUTIX.RTM.) and reshaped human 225 (H225) (see, WO 96/40210,
Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targeted
antibody (Imclone); antibodies that bind type II mutant EGFR (U.S.
Pat. No. 5,212,290); humanized and chimeric antibodies that bind
EGFR as described in U.S. Pat. No. 5,891,996; and human antibodies
that bind EGFR, such as ABX-EGF or Panitumumab (see WO98/50433,
Abgenix/Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer
32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody
directed against EGFR that competes with both EGF and TGF-alpha for
EGFR binding (EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab);
fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4,
E2.11, E6. 3 and E7.6. 3 and described in U.S. Pat. No. 6,235,883;
MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et
al., J. Biol. Chem. 279(29):30375-30384 (2004)). The anti-EGFR
antibody may be conjugated with a cytotoxic agent, thus generating
an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH).
EGFR antagonists include small molecules such as compounds
described in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001,
5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620,
6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602,
6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008,
and 5,747,498, as well as the following PCT publications:
WO98/14451, WO98/50038, WO99/09016, and WO99/24037. Particular
small molecule EGFR antagonists include OSI-774 (CP-358774,
erlotinib, TARCEVA.RTM. Genentech/OSI Pharmaceuticals); PD 183805
(CI 1033, 2-propenamide,
N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quin-
azolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib
(IRESSA.TM.)
4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoli-
ne, AstraZeneca); ZM 105180
((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382
(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4--
d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166
((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol)-
;
(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimi-
dine); CL-387785
(N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569
(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(-
dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU
5271; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors such as
lapatinib (TYKERB.RTM., GSK572016 or N-[3-chloro-4-[(3
fluorophenyl)methoxy]phenyl]6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-f-
uranyl]-4-quinazolinamine; Glaxo-SmithKline).
[0037] A "tyrosine kinase inhibitor" is a molecule which inhibits
tyrosine kinase activity of a tyrosine kinase such as a HER
receptor. Examples of such inhibitors include the EGFR-targeted
drugs noted in the preceding paragraph; small molecule HER2
tyrosine kinase inhibitor such as TAK165 available from Takeda;
CP-724,714, an oral selective inhibitor of the ErbB2 receptor
tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as
EKB-569 (available from Wyeth) which preferentially binds EGFR but
inhibits both HER2 and EGFR-overexpressing cells; lapatinib
(GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR
tyrosine kinase inhibitor; PKI-166 (available from Novartis);
pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1
inhibitors such as antisense agent ISIS-5132 available from ISIS
Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted TK
inhibitors such as imatinib mesylate (GLEEVEC.TM., available from
Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such
as sunitinib (SUTENT.RTM., available from Pfizer); VEGF receptor
tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584,
available from Novartis/Schering AG); MAPK extracellular regulated
kinase I inhibitor CI-1040 (available from Pharmacia);
quinazolines, such as PD 153035,4-(3-chloroanilino) quinazoline;
pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as
CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,
4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloyl
methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines
containing nitrothiophene moieties; PD-0183805 (Warner-Lamber);
antisense molecules (e.g. those that bind to HER-encoding nucleic
acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S.
Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787
(Novartis/Schering AG); pan-HER inhibitors such as CI-1033
(Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinib mesylate
(GLEEVEC.TM.); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);
CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474
(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone);
or as described in any of the following patent publications: U.S.
Pat. No. 5,804,396; WO 1999/09016 (American Cyanamid); WO
1998/43960 (American Cyanamid); WO 1997/38983 (Warner Lambert); WO
1999/06378 (Warner Lambert); WO 1999/06396 (Warner Lambert); WO
1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO 1996/3397
(Zeneca); and WO 1996/33980 (Zeneca).
[0038] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a polypeptide, such as FGFR2,
or the transcription or translation thereof. Suitable antagonist
molecules include, but are not limited to, antagonist antibodies,
polypeptide fragments, oligopeptides, organic molecules (including
small molecules), and anti-sense nucleic acids.
[0039] "Antibodies" (Abs) and "immunoglobulins" (Igs) refer to
glycoproteins having similar structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules which generally lack antigen specificity. Polypeptides of
the latter kind are, for example, produced at low levels by the
lymph system and at increased levels by myelomas.
[0040] The terms "antibody" and "immunoglobulin" are used
interchangeably in the broadest sense and include monoclonal
antibodies (e.g., full length or intact monoclonal antibodies),
polyclonal antibodies, monovalent antibodies, multivalent
antibodies, multispecific antibodies (e.g., bispecific antibodies
so long as they exhibit the desired biological activity) and may
also include certain antibody fragments (as described in greater
detail herein). An antibody can be chimeric, human, humanized
and/or affinity matured.
[0041] The term "anti-FGFR2 antibody" or "an antibody that binds to
FGFR2" refers to an antibody that is capable of binding FGFR2 with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting FGFR2. Preferably,
the extent of binding of an anti-FGFR2 antibody to an unrelated,
non-FGFR2 protein is less than about 10% of the binding of the
antibody to FGFR2 as measured, e.g., by a radioimmunoassay (RIA).
In certain embodiments, an antibody that binds to FGFR2 has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM. In certain
embodiments, an anti-FGFR2 antibody binds to an epitope of FGFR2
that is conserved among FGFR2 from different species.
[0042] The terms "full length antibody," "intact antibody" and
"whole antibody" are used herein interchangeably to refer to an
antibody in its substantially intact form, not antibody fragments
as defined below. The terms particularly refer to an antibody with
heavy chains that contain the Fc region.
[0043] "Antibody fragments" comprise only a portion of an intact
antibody, wherein the portion retains at least one, and as many as
most or all, of the functions normally associated with that portion
when present in an intact antibody. In one embodiment, an antibody
fragment comprises an antigen binding site of the intact antibody
and thus retains the ability to bind antigen. In another
embodiment, an antibody fragment, for example, one that comprises
the Fc region, retains at least one of the biological functions
normally associated with the Fc region when present in an intact
antibody, such as FcRn binding, antibody half life modulation, ADCC
function and complement binding. In one embodiment, an antibody
fragment is a monovalent antibody that has an in vivo half life
substantially similar to an intact antibody. For example, such an
antibody fragment may comprise an antigen binding arm linked to an
Fc sequence capable of conferring in vivo stability to the
fragment.
[0044] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0045] "Fv" is a minimum antibody fragment which contains a
complete antigen-binding site. In one embodiment, a two-chain Fv
species consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent association. In a
single-chain Fv (scFv) species, one heavy- and one light-chain
variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a
"dimeric" structure analogous to that in a two-chain Fv species. It
is in this configuration that the three CDRs of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six CDRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three CDRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0046] The Fab fragment contains the heavy- and light-chain
variable domains and also contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0047] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv see Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
[0048] 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
(VL) 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 may be bivalent or bispecific. Diabodies are described
more fully in, for example, EP 404,097; W093/1161; Hudson et al.
(2003) Nat. Med. 9:129-134; and Hollinger et al., Proc. Natl. Acad.
Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also
described in Hudson et al. (2003) Nat. Med. 9:129-134.
[0049] 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 mutations, e.g.,
naturally occurring mutations, that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies. In
certain embodiments, such a monoclonal antibody typically includes
an antibody comprising a polypeptide sequence that binds a target,
wherein the target-binding polypeptide sequence was obtained by a
process that includes the selection of a single target binding
polypeptide sequence from a plurality of polypeptide sequences. For
example, the selection process can be the selection of a unique
clone from a plurality of clones, such as a pool of hybridoma
clones, phage clones, or recombinant DNA clones. It should be
understood that a selected target binding sequence can be further
altered, for example, to improve affinity for the target, to
humanize the target binding sequence, to improve its production in
cell culture, to reduce its immunogenicity in vivo, to create a
multispecific antibody, etc., and that an antibody comprising the
altered target binding sequence is also a monoclonal antibody of
this invention. In contrast to polyclonal antibody preparations
which typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody of a
monoclonal antibody preparation is directed against a single
determinant on an antigen. In addition to their specificity,
monoclonal antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins.
[0050] 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. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler et al., Nature, 256:
495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold
Spring Harbor Laboratory Press, 2.sup.nd ed. 1988); Hammerling et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567), phage display technologies (see, e.g.,
Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.
Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):
299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004);
Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004);
and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004), and
technologies for producing human or human-like antibodies in
animals that have parts or all of the human immunoglobulin loci or
genes encoding human immunoglobulin sequences (see, e.g.,
WO98/24893; WO96/34096; WO96/33735; WO91/10741; Jakobovits et al.,
Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,
Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol.
7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016; Marks et al., Bio.Technology 10:
779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994);
Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature
Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14:
826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93
(1995).
[0051] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA 81:6851-6855 (1984)).
[0052] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. In one embodiment, a humanized antibody
is a human immunoglobulin (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit, or nonhuman primate having
the desired specificity, affinity, and/or capacity. In some
instances, framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
not found in the recipient antibody or in the donor antibody. These
modifications may be made to further refine antibody performance.
In general, a humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin, and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will 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); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the
following review articles and references cited therein: Vaswani and
Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);
Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and
Gross, Curr. Op. Biotech. 5:428-433 (1994).
[0053] A "human antibody" is one which comprises an amino acid
sequence corresponding to that of an antibody produced by a human
and/or has been made using any of the techniques for making human
antibodies as disclosed herein. Such techniques include screening
human-derived combinatorial libraries, such as phage display
libraries (see, e.g., Marks et al., J. Mol. Biol., 222: 581-597
(1991) and Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137
(1991)); using human myeloma and mouse-human heteromyeloma cell
lines for the production of human monoclonal antibodies (see, e.g.,
Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987); and Boemer et al., J. Immunol., 147:
86 (1991)); and generating monoclonal antibodies in transgenic
animals (e.g., mice) that are capable of producing a full
repertoire of human antibodies in the absence of endogenous
immunoglobulin production (see, e.g., Jakobovits et al., Proc.
Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature,
362: 255 (1993); Bruggermann et al., Year in Immunol., 7: 33
(1993)). This definition of a human antibody specifically excludes
a humanized antibody comprising antigen-binding residues from a
non-human animal.
[0054] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s). In
one embodiment, an affinity matured antibody has nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced by procedures known in the art. Marks et
al. Bio/Technology 10:779-783 (1992) describes affinity maturation
by VH and VL domain shuffling. Random mutagenesis of HVR and/or
framework residues is described by: Barbas et al. Proc Nat. Acad.
Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155
(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol.
Biol. 226:889-896 (1992).
[0055] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces a biological activity of the antigen it
binds. Certain blocking antibodies or antagonist antibodies
partially or completely inhibit the biological activity of the
antigen.
[0056] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: Clq binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g. B cell receptor); and B cell activation.
[0057] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. In some embodiments, an FcR is a
native human FcR. In some embodiments, an FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of those
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain (see Daeron, Annu. Rev.
Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and
Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,
Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.
Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR"
herein.
[0058] The term "Fc receptor" or "FcR" also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim
et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of
immunoglobulins. Methods of measuring binding to FcRn are known.
Binding to human FcRn in vivo and serum half life of human FcRn
high affinity binding polypeptides can be assayed, e.g., in
transgenic mice or transfected human cell lines expressing human
FcRn, or in primates administered with Fc variant polypeptides.
[0059] WO00/42072 (Presta) describes antibody variants with
improved or diminished binding to FcRs. The content of that patent
publication is specifically incorporated herein by reference. See,
also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001).
[0060] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. In certain embodiments,
the cells express at least Fc.gamma.RIII and perform ADCC effector
function(s). Examples of human leukocytes which mediate ADCC
include peripheral blood mononuclear cells (PBMC), natural killer
(NK) cells, monocytes, cytotoxic T cells and neutrophils. The
effector cells may be isolated from a native source, e.g., from
blood.
[0061] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which immunoglobulin bound to
Fc receptors (FcRs) present on certain cytotoxic effector cells
(e.g. Natural Killer (NK) cells, neutrophils, and macrophages)
enables those cytotoxic effector cells to bind specifically to an
antigen-bearing target cell and subsequently kill the target cell
with cytotoxins. The primary cells for mediating ADCC, NK cells,
express Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI,
Fc.gamma.RII and Fc.gamma.RIII. FcR expression on hematopoietic
cells is summarized in Table 3 on page 464 of Ravetch and Kinet,
Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a
molecule of interest, an in vitro ADCC assay, such as that
described in U.S. Pat. No. 5,500,362 or 5,821,337 or Presta U.S.
Pat. No. 6,737,056 may be performed. Useful effector cells for such
assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in an animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
[0062] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (Clq) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0063] Polypeptide variants with altered Fc region amino acid
sequences and increased or decreased Clq binding capability are
described in U.S. Pat. No. 6,194,551B1 and WO99/51642. The contents
of those patent publications are specifically incorporated herein
by reference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184
(2000).
[0064] The term "Fc region-comprising polypeptide" refers to a
polypeptide, such as an antibody or immunoadhesin, which comprises
an Fc region. The C-terminal lysine (residue 447 according to the
EU numbering system) of the Fc region may be removed, for example,
during purification of the polypeptide or by recombinant
engineering the nucleic acid encoding the polypeptide. Accordingly,
a composition comprising a polypeptide having an Fc region
according to this invention can comprise polypeptides with K447,
with all K447 removed, or a mixture of polypeptides with and
without the K447 residue.
[0065] A "cytotoxic antibody" is an antibody that is capable of an
effector function and/or inducing cell death upon binding to its
target antigen.
[0066] An "immunoconjugate" refers to an antibody conjugated to one
or more cytotoxic agents.
[0067] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[0068] A "small molecule" or "small organic molecule" is defined
herein as an organic molecule having a molecular weight below about
500 Daltons.
[0069] An "FGFR2-binding oligopeptide" or an "oligopeptide that
binds FGFR2" is an oligopeptide that is capable of binding FGFR2
with sufficient affinity such that the oligopeptide is useful as a
diagnostic and/or therapeutic agent in targeting FGFR2. In certain
embodiments, the extent of binding of an FGFR2-binding oligopeptide
to an unrelated, non-FGFR2 protein is less than about 10% of the
binding of the FGFR2-binding oligopeptide to FGFR2 as measured,
e.g., by a surface plasmon resonance assay. In certain embodiments,
an FGFR2-binding oligopeptide has a dissociation constant (Kd) of
.ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, or
.ltoreq.0.1 nM.
[0070] An "FGFR2-binding organic molecule" or "an organic molecule
that binds FGFR2" is an organic molecule other than an oligopeptide
or antibody as defined herein that is capable of binding FGFR2 with
sufficient affinity such that the organic molecule is useful as a
diagnostic and/or therapeutic agent in targeting FGFR2. In certain
embodiments, the extent of binding of an FGFR2-binding organic
molecule to an unrelated, non-FGFR2 protein is less than about 10%
of the binding of the FGFR2-binding organic molecule to FGFR2 as
measured, e.g., by a surface plasmon resonance assay. In certain
embodiments, an FGFR2-binding organic molecule has a dissociation
constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM,
.ltoreq.1 nM, or .ltoreq.0.1 nM.
[0071] The dissociation constant (Kd) of any molecule that binds a
target polypeptide may conveniently be measured using a surface
plasmon resonance assay. Such assays may employ a BIAcore.TM.-2000
or a BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, N.J.) at
25.degree. C. with immobilized target polypeptide CM5 chips at
.about.10 response units (RU). Briefly, carboxymethylated dextran
biosensor chips (CM5, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Target polypeptide is diluted with 10 mM sodium
acetate, pH 4.8, to 5 .mu.g/ml (-0.2 .mu.M) before injection at a
flow rate of 5 .mu.l/minute to achieve approximately 10 response
units (RU) of coupled protein. Following the injection of target
polypeptide, 1 M ethanolamine is injected to block unreacted
groups. For kinetics measurements, two-fold serial dilutions of the
binding molecule (0.78 nM to 500 nM) are injected in PBS with 0.05%
Tween 20 (PBST) at 25.degree. C. at a flow rate of approximately 25
.mu.l/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIAcore Evaluation Software version 3.2) by
simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (Kd) is
calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen, Y., et
al., (1999) J. Mol. Biol. 293:865-881. If the on-rate of an
antibody exceeds 10.sup.6 M.sup.-1 s.sup.-1 by the surface plasmon
resonance assay above, then the on-rate can be determined by using
a fluorescent quenching technique that measures the increase or
decrease in fluorescence emission intensity (excitation=295 nm;
emission=340 nm, 16 nm band-pass) at 25.degree. C. of a 20 nM
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophometer (Aviv Instruments) or a
8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a
stirred cuvette.
[0072] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of an agent, e.g., a drug, to a mammal. The components of
the liposome are commonly arranged in a bilayer formation, similar
to the lipid arrangement of biological membranes.
[0073] The word "label" when used herein refers to a detectable
compound or composition. The label may be detectable by itself
(e.g., radioisotope labels or fluorescent labels) or, in the case
of an enzymatic label, may catalyze chemical alteration of a
substrate compound or composition which results in a detectable
product. Radionuclides that can serve as detectable labels include,
for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211,
Cu-67, Bi-212, and Pd-109.
[0074] An "isolated" biological molecule, such as a nucleic acid,
polypeptide, or antibody, is one which has been identified and
separated and/or recovered from at least one component of its
natural environment.
II. EMBODIMENTS OF THE INVENTION
[0075] Methods and compositions for the diagnosis and treatment of
cancers associated with gene amplification are provided. In one
aspect, methods and compositions for the diagnosis and treatment of
colorectal cancer are provided. Those methods and compositions are
based, in part, on the discovery that a region of chromosome 10
comprising the FGFR2 gene is amplified in a particular colorectal
cancer, and this amplification is correlated with increased
expression of FGFR2 mRNA.
[0076] FGFR2 is a member of the fibroblast growth factor receptor
(FGFR) family of receptor protein tyrosine kinases, which also
includes FGFR1, FGFR3, and FGFR4. Like other members of the FGFR
family, FGFR2 contains an N-terminal extracellular ligand-binding
domain, a single transmembrane domain, and a C-terminal cytoplasmic
domain. The extracellular ligand-binding domain contains three
immunoglobulin (Ig)-like domains; the second and third Ig-like
domains are involved in ligand binding, as determined by X-ray
crystallography studies. The cytoplasmic domain contains the
catalytic protein tyrosine kinase core. For review, see, e.g.,
Eswarakumar et al. (2005) Cytokine & Growth Factor Rev.
16:139-149.
[0077] A full length, unprocessed form of human FGFR2 is shown in
SEQ ID NO:1. That sequence contains the following features:
TABLE-US-00001 Amino Acid Feature Residues Signal peptide 1-21
Predicted extracellular domain 22-377 First Ig-like domain 39-125
Second Ig-like domain 154-247 Third Ig-like domain 256-358
Predicted transmembrane domain 378-398 Predicted cytoplasmic domain
399-821 Protein tyrosine kinase domain 481-770
[0078] Alternative splicing of FGFR2 mRNA generates various
isoforms. Major isoforms include FGFR2b (also called KGFR in the
scientific literature; SEQ ID NO:2 is representative of the human
FGFR2b isoform); FGFR2c (also called BEK and FGFR2 in the
scientific literature; SEQ ID NO:1 is representative of the human
FGFR2c isoform); and an isoform called "K-SAM," which lacks the
first Ig-like domain. See, e.g., Miki et al. (1992) Proc. Natl
Acad. Sci. USA 89:246-250, and Dell et al. (1992) J. Biol. Chem.
267:21225-21229 (FGFR2b); Dionne et al. (1990) EMBO J. 9:2685-2692
(FGFR2c); and Hattori et al. (1990) Proc. Natl. Acad. Sci. USA
87:5983-5987 (K-SAM). The sequences of FGFR2b and FGFR2c are
identical, except for a divergent 49-amino acid stretch spanning
the second half of the third Ig-like domain. See Miki, supra.
Accordingly, the features defined above for SEQ ID NO:1 also apply
to SEQ ID NO:2. FGFR2b and FGFR2c show different ligand binding
specificities, although both bind to fibroblast growth factor 1
(FGF 1) with high affinity. See Ornitz et al. (1996) J. Biol. Chem.
271:15292-15297.
[0079] A. Methods of Diagnosis and Detection
[0080] In one aspect, methods of diagnosing colorectal cancer are
provided. As described below in the Examples, a colorectal tumor
was discovered in which a region of chromosome 10 was amplified.
The only gene present within that amplified region is the FGFR2
gene, as shown in FIG. 1. (The chromosomal location of the FGFR2
gene is 10q26.) Thus, FGFR2 or the FGFR2 gene is an attractive
target for colorectal cancer diagnostics and therapeutics.
[0081] Accordingly, in one aspect, a method of diagnosing the
presence of a colorectal cancer in a mammal is provided, the method
comprising detecting whether the FGFR2 gene is amplified in a test
colorectal sample from the mammal relative to a control sample,
wherein amplification of the FGFR2 gene indicates the presence of
colorectal cancer in the mammal. As used herein, the term
"detecting" encompasses quantitative or qualitative detection. A
"test colorectal sample" is a biological sample derived from
colorectal tissue that may or may not be cancerous, e.g., a sample
of colorectal cells suspected of being cancerous or a whole cell
extract or fractionated cell extract (such as a membrane
preparation) derived from colorectal cells. A "control sample" is a
biological sample derived from (a) normal tissue, e.g., normal
colorectal cells or a whole cell extract or fractionated cell
extract (such as a membrane preparation) derived from such cells,
or (b) colorectal cancer tissue in which the FGFR2 gene is known
not to be amplified or overexpressed, or a whole cell extract or
fractionated cell extract derived therefrom. The FGFR2 gene is said
to be "amplified" if the copy number of the FGFR2 gene is increased
by at least 3-, 5-, 7-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, or
50-fold in the test colorectal sample relative to the control
sample.
[0082] In certain embodiments, detecting amplification of the FGFR2
gene is achieved using certain techniques known to those skilled in
the art. For example, comparative genome hybridization may be used
to produce a map of DNA sequence copy number as a function of
chromosomal location. See, e.g., Kallioniemi et al. (1992) Science
258:818-821. Amplification of the FGFR2 gene may also be detected,
e.g., by Southern hybridization using a probe specific for the
FGFR2 gene or by real-time quantitative PCR.
[0083] In certain embodiments, detecting amplification of the FGFR2
gene is achieved by directly assessing the copy number of the FGFR2
gene, for example, by using a probe that hybridizes to the FGFR2
gene. In certain embodiments, detecting amplification of the FGFR2
gene is achieved by indirectly assessing the copy number of the
FGFR2 gene, for example, by assessing the copy number of a
chromosomal region that lies outside the FGFR2 gene but is
co-amplified with the FGFR2 gene. Guidance for selecting such a
region is provided, e.g., in FIG. 1, Panel C.
[0084] In another aspect, a method of diagnosing the presence of a
colorectal cancer in a mammal is provided, the method comprising
detecting expression of the FGFR2 gene in a test colorectal sample
from the mammal, wherein a higher level of FGFR2 gene expression in
the test colorectal sample relative to a control sample indicates
the presence of colorectal cancer in the mammal. In certain
embodiments, expression of the FGFR2 gene is detected by
determining the level of mRNA transcription from the FGFR2 gene.
Levels of mRNA transcription may be determined, either
quantitatively or qualitatively, by various methods known to those
skilled in the art. Levels of mRNA transcription may also be
determined directly or indirectly by detecting levels of cDNA
generated from the mRNA. Exemplary methods for determining levels
of mRNA transcription include, but are not limited to, real-time
quantitative RT-PCR and hybridization-based assays, including
microarray-based assays and filter-based assays such as Northern
blots. In certain embodiments, "a higher level of FGFR2 gene
expression" means at least a 3-, 5-, 7-, 10-, 15-, 20-, 25-, 30-,
35-, 40-, 45-, or 50-fold increase in mRNA transcription from the
FGFR2 gene.
[0085] In other embodiments, expression of the FGFR2 gene is
detected by determining the level of FGFR2. Levels of FGFR2 may be
determined, either quantitatively or quantitatively, by certain
methods known to those skilled in the art, including antibody-based
detection methods. In one embodiment, detecting expression of the
FGFR2 gene in a test colorectal sample comprises contacting the
test colorectal sample with an anti-FGFR2 antibody and determining
the level of expression (either quantitatively or qualitatively) of
FGFR2 in the test colorectal sample by detecting binding of the
anti-FGFR2 antibody to FGFR2. In certain embodiments, binding of an
anti-FGFR2 antibody to FGFR2 may be detected by various methods
known to those skilled in the art including, but not limited to,
fluorescence activated cell sorting, Western blot,
radioimmunoassay, ELISA, and the like. In certain embodiments, "a
higher level of FGFR2 gene expression" means at least a 3-, 5-, 7-,
10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, or 50-fold increase in
FGFR2 levels.
[0086] For any of the above methods, the stated purpose of
"diagnosing the presence of a colorectal cancer in a mammal" is
nonlimiting and encompasses classifying the type of colorectal
cancer present in a mammal by detecting whether the FGFR2 gene is
amplified and/or expressed at a higher level in a test sample of
colorectal cancer relative to a control sample. Classifying a
colorectal cancer based on whether or not the FGFR2 gene is
amplified and/or overexpressed is useful, e.g., for determining
whether an individual having the colorectal cancer will respond to
a therapeutic that targets FGFR2 or the FGFR2 gene, and thus, for
selecting the optimal regimen for treating the colorectal cancer,
as further described below. For example, a method is provided
herein for determining whether an individual having colorectal
cancer will respond to a therapeutic that targets FGFR2 or the
FGFR2 gene, the method comprising determining whether the FGFR2
gene is amplified and/or overexpressed in the colorectal cancer
(e.g., by using any of the methods described above), wherein
amplification and/or overexpression of the FGFR2 gene indicates
that the individual will respond to the therapeutic. A "therapeutic
that targets FGFR2 or the FGFR2 gene" means any agent that affects
the expression and/or an activity of FGFR2 or the FGFR2 gene
including, but not limited to, any of the FGFR2 antagonists,
cytotoxic antibodies, or immunoconjugates described below, Part B,
including such therapeutics that are already known in the art as
well as those that are later developed.
[0087] B. Compositions and Pharmaceutical Formulations
[0088] Pharmaceutical formulations for treating colorectal cancer
are provided. In certain embodiments, a pharmaceutical formulation
comprises at least one FGFR2 antagonist, a pharmaceutically
acceptable carrier, and optionally, at least one additional
therapeutic agent. In certain embodiments, an FGFR2 antagonist
comprises an anti-FGFR2 antibody, an oligopeptide, an organic
molecule, a soluble FGFR2 receptor, or an antisense nucleic acid.
In certain embodiments, a pharmaceutical formulation comprises at
least one cytotoxic anti-FGFR2 antibody, pharmaceutically
acceptable carrier, and optionally, at least one additional
therapeutic agent. In certain embodiments, a pharmaceutical
formulation comprises at least one immunoconjugate, wherein the
immunoconjugate comprises an antibody that binds FGFR2 and a
cytotoxic agent; a pharmaceutically acceptable carrier; and
optionally, at least one additional therapeutic agent.
[0089] I. FGFR2 Antagonists
[0090] In one aspect, an FGFR2 antagonist is an anti-FGFR2
antibody. In certain embodiments, an anti-FGFR2 antibody is a
"blocking antibody," e.g, an antibody that fully or partially
blocks the interaction of FGFR2 with its ligand. In certain
embodiments, an anti-FGFR2 antibody binds to the extracellular
domain of an FGFR2, e.g., a region within or overlapping amino
acids 22-377 of SEQ ID NO:1 or SEQ ID N0:2. In certain embodiments,
an anti-FGFR2 antibody binds to or otherwise occludes all or a
portion of the ligand binding domain of an FGFR2. The ligand
binding domain of FGFR2 has been examined by X-ray crystallography
and includes the second and third Ig-like domains from about amino
acid 154-247 and amino acid 256-358, respectively, of SEQ ID NO:1
or SEQ ID NO:2. See Part II, supra, and Plotnikov et al. (2000)
Cell 101:413-424. Accordingly, in certain embodiments, an
anti-FGFR2 antibody binds to or otherwise occludes all or a portion
of the second or third Ig-like domain of an FGFR2.
[0091] In various embodiments of the invention, an anti-FGFR2
antibody (including antagonist anti-FGFR2 antibodies and cytotoxic
anti-FGFR2 antibodies, discussed below, Part 2) is a monoclonal
antibody. In various embodiments, an anti-FGFR2 antibody is an
antibody fragment, e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab).sub.2
fragment, or a single domain antibody (Domantis, Inc., Waltham,
Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In certain
embodiments, an anti-FGFR2 antibody is a bispecific antibody (see,
e.g., WO94/04690 and Suresh et al. (1986) Methods in Enzymology
121:210). In certain embodiments, an anti-FGFR2 antibody is a
chimeric, humanized, or human antibody.
[0092] In another aspect, an FGFR2 antagonist is an oligopeptide
that binds to an FGFR2. In one embodiment, an oligopeptide binds to
the extracellular domain of an FGFR2. In one such embodiment, an
oligopeptide binds to or otherwise occludes a region of the ligand
binding domain, e.g., by binding to all or a portion of the second
and/or third Ig-like domain. In another embodiment, an oligopeptide
binds to the protein tyrosine kinase domain of an FGFR2 and/or
reduces the activity of the protein tyrosine kinase domain of an
FGFR2.
[0093] The above oligopeptides may be chemically synthesized using
known oligopeptide synthesis methodology or may be prepared and
purified using recombinant technology. Such oligopeptides are
usually at least about 5 amino acids in length, alternatively at
least about 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, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in
length. Such oligopeptides may be identified without undue
experimentation using well known techniques. In this regard, it is
noted that techniques for screening oligopeptide libraries for
oligopeptides that are capable of specifically binding to a
polypeptide target are well known in the art (see, e.g., U.S. Pat.
Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409,
5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506
and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. USA,
81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA,
82:178-182 (1985); Geysen et al., in Synthetic Peptides as
Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth.,
102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616
(1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA,
87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832;
Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al.
(1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc.
Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current
Opin. Biotechnol., 2:668). In certain embodiments, an oligopeptide
may be conjugated to a cytotoxic agent.
[0094] In yet another aspect, an FGFR2 antagonist is an organic
molecule that binds to FGFR2, other than an oligopeptide or
antibody as described herein. An organic molecule may be, for
example, a small molecule. In one embodiment, an organic molecule
binds to the extracellular domain of an FGFR2. In one such
embodiment, an organic molecule binds to or otherwise occludes a
region of the ligand binding domain, e.g., by binding to all or a
portion of the second and/or third Ig-like domain. In another
embodiment, an organic molecule binds to the protein tyrosine
kinase domain and/or reduces the activity of the protein tyrosine
kinase domain of an FGFR2.
[0095] An organic molecule that binds to FGFR2 may be identified
and chemically synthesized using known methodology (see, e.g., PCT
Publication Nos. WO00/00823 and WO00/39585). Such organic molecules
are usually less than about 2000 daltons in size, alternatively
less than about 1500, 750, 500, 250 or 200 daltons in size, wherein
such organic molecules that are capable of binding to FGFR2 may be
identified without undue experimentation using well known
techniques. In this regard, it is noted that techniques for
screening organic molecule libraries for molecules that are capable
of binding to a polypeptide target are well known in the art (see,
e.g., PCT Publication Nos. WO00/00823 and WO00/39585). In certain
embodiments, an organic molecule may be conjugated to a cytotoxic
agent.
[0096] Certain small molecule antagonists that bind to FGFR2 and
inhibit the protein tyrosine kinase activity of FGFR2 are known in
the art. Such molecules include, e.g.,
1-tert-butyl-3-[6-(3,5-dimethoxy-phenyl)-2-(4-diethylamino-butylamino)-py-
rido[2,3-d]pyrimidin-7-yl]-urea ("PD173074") (see, e.g., Moffa et
al. (2004) Mol. Cancer Res. 2:643-652); and
3-[3-(2-carboxyethyl)-4-methylpyrrol-2-methylidenyl]-2-indolinone
("SU5402," Calbiochem) (see, e.g., Bemard-Pierrot (2004) Oncogene
23:9201-9211. Indolinones are a class of small molecules known to
inhibit the receptor protein tyrosine kinase activity of FGFRs. See
Mohammadi et al. (1997) 276:9555-960. In certain embodiments, an
FGFR2 antagonist is a tyrosine kinase inhibitor, as defined
herein.
[0097] In yet another aspect, an FGFR2 antagonist is a soluble form
of FGFR2, i.e., a form of FGFR2 that is not anchored to the plasma
membrane. Such soluble forms of FGFR2 may compete with
membrane-bound FGFR2 for binding to an FGFR2 ligand. In certain
embodiments, a soluble form of FGFR2 may comprise all or a
ligand-binding portion of an extracellular domain of FGFR2, e.g.,
all or a ligand-binding portion of a polypeptide comprising amino
acids 22-377 of SEQ ID NO:1 or SEQ ID NO:2. In certain embodiments,
a soluble form of FGFR2 may comprise all or a ligand-binding
portion of one or more ligand binding domains of FGFR2, e.g., all
or a ligand-binding portion of a polypeptide comprising amino acids
154-247 and/or amino acids 256-368 of SEQ ID NO:1 or SEQ ID NO:2.
In any of the above embodiments, a soluble form of FGFR2 may or may
not further comprise a protein tyrosine kinase domain.
[0098] Naturally occurring, soluble forms of FGFR2 are reported in
Katoh et al. (1992) Proc. Natl Acad. Sci. USA 89:2960-2964. Such
forms include secreted forms of FGFR2 that either possess or lack a
protein tyrosine kinase domain. Id. Additionally, two oligopeptides
have been shown to be effective in competing with a membrane-bound
isoform of FGFR2 (FGFR2b) for ligand binding. Bottaro et al. (1993)
J. Biol. Chem. 268:9180-9183. Those peptides correspond to a 20-
and 25-amino acid stretch, respectively, that spans a portion of
one of the ligand binding domains (the third immunoglobulin-like
domain). Thus, soluble forms of FGFR2 are well within the skill in
the art.
[0099] In yet another aspect, an FGFR2 antagonist is an antisense
nucleic acid that decreases expression of the FGFR2 gene (i.e.,
that decreases transcription of the FGFR2 gene and/or translation
of FGFR2 mRNA). In certain embodiments, an antisense nucleic acid
binds to a nucleic acid (DNA or RNA) encoding FGFR2. In certain
embodiments, an antisense nucleic acid is an oligonucleotide of
about 10-30 nucleotides in length (including all points between
those endpoints). In certain embodiments, an antisense
oligonucleotide comprises a modified sugar-phosphodiester backbones
(or other sugar linkages, including phosphorothioate linkages and
linkages as described in WO 91/06629), wherein such modified
sugar-phosphodiester backbones are resistant to endogenous
nucleases. In one embodiment, an antisense nucleic acid is an
oligodeoxyribonucleotide, which results in the degradation and/or
reduced transcription or translation of FGFR2 mRNA. Certain
examples of FGFR2-specific antisense nucleic acids are known to
those skilled in the art and are described, e.g., in the following
publications: Post et al. (1996) Development 122:3107-3115
(describing a phosphorothioate oligodeoxyribonucleotide (15-mer)
spanning the translational start site and two isoform-specific
phosphorothioate oligodeoxyribonucleotides (16- and 19-mers));
Yamada et al. (1999) Glia 28:66-76 (describing a phosphorothioate
oligodeoxyribonucleotide complementary to the translational start
site); and WO03/024987 (describing phosphorothioate
oligodeoxyribonucleotide (20-mers) targeting various regions of
FGFR2 mRNA).
[0100] In certain embodiments, an antisense nucleic acid is an RNA
that reduces expression of a target nucleic acid by "RNA
interference" ("RNAi"). For review of RNAi, see, e.g., Novina et
al. (2004) Nature 430:161-164. Such RNAs are derived from, for
example, short interfering RNAs (siRNAs) and microRNAs. siRNAs,
e.g., may be synthesized as double stranded oligoribonucleotides of
about 18-26 nucleotides in length. Id. Thus, antisense nucleic
acids that decrease expression of FGFR2 are well within the skill
in the art.
[0101] 2. Cytotoxic Antibodies
[0102] In one aspect, cytotoxic antibodies are provided. In certain
embodiments, a cytotoxic antibody is an anti-FGFR2 antibody, such
as those provided above, which effects an effector function and/or
induces cell death. In certain embodiments, a cytotoxic anti-FGFR2
antibody binds to the extracellular domain of an FGFR2, e.g., a
region within amino acids 22-377 of SEQ ID NO:1 or SEQ ID NO:2.
[0103] 3. Immunoconjugates
[0104] Immunoconjugates, or "antibody-drug conjugates," are useful
for the local delivery of cytotoxic agents in the treatment of
cancer. See, e.g., Syrigos et al. (1999) Anticancer Research
19:605-614; Niculescu-Duvaz et al. (1997) Adv. Drug Deliv. Rev.
26:151-172; U.S. Pat. No. 4,975,278. Immunoconjugates allow for the
targeted delivery of a drug moiety to a tumor, whereas systemic
administration of unconjugated cytotoxic agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated. See Baldwin et al. (Mar. 15,
1986) Lancet pp. 603-05; Thorpe (1985) "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies '84: Biological and Clinical Applications (A. Pinchera
et al., eds.) pp. 475-506.
[0105] In one aspect, an immunoconjugate comprises an antibody that
binds FGFR2 (or an extracellular domain thereof), such as those
provided above, and a cytotoxic agent, such as a chemotherapeutic
agent, a growth inhibitory agent, a toxin (e.g., an enzymatically
active toxin of bacterial, fungal, plant, or animal origin, or
fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
[0106] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.122Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re.
[0107] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0108] Maytansine and Maytansinoids
[0109] In one embodiment, an immunoconjugate comprises an
anti-FGFR2 antibody conjugated to one or more maytansinoid
molecules. Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the
disclosures of which are hereby expressly incorporated by
reference.
[0110] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies that bind to
antigens on the surface of tumor cells. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 131, the disclosures of which are hereby expressly incorporated
by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) described immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansonid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
[0111] Anti-FGFR2 antibody-maytansinoid conjugates are prepared by
chemically linking an anti-FGFR2 antibody to a maytansinoid
molecule without significantly diminishing the biological activity
of either the antibody or the maytansinoid molecule. An average of
3-4 maytansinoid molecules conjugated per antibody molecule has
shown efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin per antibody would be expected
to enhance cytotoxicity over the use of naked antibody.
Maytansinoids are well known in the art and can be synthesized
using known techniques or isolated from natural sources. Suitable
maytansinoids are disclosed, for example, in U.S. Pat. No.
5,208,020 and in the other patents and nonpatent publications
referred to hereinabove. Preferred maytansinoids are maytansinol
and maytansinol analogues modified in the aromatic ring or at other
positions of the maytansinol molecule, such as various maytansinol
esters.
[0112] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al., Cancer Research 52:127-131 (1992). The linking groups
include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred.
[0113] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Certain coupling agents, including
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), provide for a
disulfide linkage.
[0114] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
[0115] Auristatins and Dolastatins
[0116] In some embodiments, an immunoconjugate comprises an
anti-FGFR2 antibody conjugated to a dolastatin or dolostatin
peptidic analog or derivative, e.g., an auristatin (U.S. Pat. Nos.
5,635,483; 5,780,588). Dolastatins and auristatins have been shown
to interfere with microtubule dynamics, GTP hydrolysis, and nuclear
and cellular division (Woyke et al (2001) Antimicrob. Agents and
Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No.
5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob.
Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug
moiety may be attached to the antibody through the N (amino)
terminus or the C (carboxyl) terminus of the peptidic drug moiety
(WO 02/088172).
[0117] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, disclosed in
"Monomethylvaline Compounds Capable of Conjugation to Ligands," US
Patent Application Publication No. US 2005-0238649 A1, the
disclosure of which is expressly incorporated by reference in its
entirety.
[0118] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;
Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al
(1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.
Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.
Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat.
Biotechnol. 21(7):778-784; US Patent Application Publication No.
2005-0238649 A1, hereby incorporated by reference in its entirety
(disclosing, e.g., linkers and methods of preparing
monomethylvaline compounds such as MMAE and MMAF conjugated to
linkers).
[0119] Calicheamicin
[0120] Another immunoconjugate of interest comprises an anti-FGFR2
antibody conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1.sup.1, .alpha..sub.2.sup.1,
.alpha..sub.3.sup.1, N-acetyl-.gamma..sub.1.sup.1, PSAG and
.theta..sup.1.sub.1 (Hinman et al., Cancer Research 53:3336-3342
(1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the
aforementioned U.S. patents to American Cyanamid). Another
anti-tumor drug to which the antibody can be conjugated is QFA
which is an antifolate. Both calicheamicin and QFA have
intracellular sites of action and do not readily cross the plasma
membrane. Therefore, cellular uptake of these agents through
antibody mediated internalization greatly enhances their cytotoxic
effects.
[0121] Other Cytotoxic Agents
[0122] Other antitumor agents that can be conjugated to an
anti-FGFR2 antibody include BCNU, streptozoicin, vincristine and
5-fluorouracil, the family of agents known collectively as
LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710,
as well as esperamicins (U.S. Pat. No. 5,877,296).
[0123] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0124] In another aspect, an immunoconjugate may comprise an
anti-FGFR2 antibody and a compound with nucleolytic activity (e.g.,
a ribonuclease or a DNA endonuclease such as a deoxyribonuclease;
DNase).
[0125] For selective destruction of a tumor, an immunoconjugate may
comprise an anti-FGFR2 antibody and a highly radioactive atom. A
variety of radioactive isotopes are available for the production of
radioconjugated anti-FGFR2 antibodies. Examples include At.sup.211,
I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, pb.sup.212 and radioactive isotopes of
Lu.
[0126] When the conjugate is used for diagnosis, it may comprise a
radioactive atom for scintigraphic studies, for example tc.sup.99m
or I.sup.123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic resonance imaging, mri), such as
iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0127] The radio- or other labels may be incorporated in the
immunoconjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun, 80: 49-57 can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
[0128] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
[0129] 4. Additional Therapeutic Agents
[0130] Pharmaceutical formulations may optionally comprise at least
one additional therapeutic agent (i.e., in addition to an FGFR2
antagonist, cytotoxic antibody, or immunoconjugate). Such
additional therapeutic agents are described in further detail
below, Part C.
[0131] 5. Preparation of Pharmaceutical Formulations
[0132] Pharmaceutical formulations comprising any of the above
agents are prepared for storage by mixing the antibody or
immunoconjugate having the desired degree of purity with optional
physiologically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)) in the form of aqueous solutions or lyophilized or other
dried formulations. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, histidine
and other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride); phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG). Pharmaceutical formulations to be used
for in vivo administration are generally sterile. This is readily
accomplished by filtration through sterile filtration
membranes.
[0133] An agent may also be entrapped in microcapsule prepared, for
example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0134] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the agent of
interest, which matrices are in the form of shaped articles, e.g.,
films, or microcapsule. Examples of sustained-release matrices
include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated agents remain in the
body for a long time, they may denature or aggregate as a result of
exposure to moisture at 37.degree. C., resulting in a loss of
biological activity and, for antibodies, possible changes in
immunogenicity. Rational strategies can be devised for
stabilization depending on the mechanism involved. For example, if
the aggregation mechanism is discovered to be intermolecular S--S
bond formation through thio-disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from
acidic solutions, controlling moisture content, using appropriate
additives, and developing specific polymer matrix compositions.
[0135] C. Methods of Treatment and Related Methods
[0136] Therapeutic methods using an FGFR2 antagonist, a cytotoxic
antibody, or an immunoconjugate are provided. Such methods include
in vitro, ex vivo, and/or in vivo therapeutic methods, unless
otherwise indicated.
[0137] In one aspect, the invention provides a method of inhibiting
the proliferation of a colorectal cancer cell, the method
comprising exposing the cell to 1) an FGFR2 antagonist, 2) a
cytotoxic anti-FGFR2 antibody, or 3) an immunoconjugate comprising
an anti-FGFR2 antibody and a cytotoxic agent. In certain
embodiments, the FGFR2 gene is amplified or overexpressed in the
colorectal cancer cell. In certain embodiments, the colorectal
cancer cell is derived from a colorectal tumor, e.g., a colorectal
tumor in which the FGFR2 gene is amplified or overexpressed. In
certain embodiments, the colorectal cancer cell may be of any of
the following cell lines: C70, HT29, LIM1863, SW1417, SW403, SW480,
SW620, SW837, VACO4A, DLD-1, GP2d, HCA7, HCT-15, HCT116, LoVo,
LS174T, LS411, VACO5, VACO400, or VAC0429. "Inhibiting the
proliferation" means decreasing a cell's proliferation by at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and
includes inducing cell death.
[0138] Inhibition of cell proliferation may be measured using
methods known to those skilled in the art. For example, a
convenient assay for measuring cell proliferation is the
CellTiter-Glo.TM. Luminescent Cell Viability Assay, which is
commercially available from Promega (Madison, Wis.). That assay
determines the number of viable cells in culture based on
quantitation of ATP present, which is an indication of
metabolically active cells. See Crouch et al (1993) J. Immunol.
Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may be
conducted in 96- or 384-well format, making it amenable to
automated high-throughput screening (HTS). See Cree et al (1995)
AntiCancer Drugs 6:398-404. The assay procedure involves adding a
single reagent (CellTiter-Glo.RTM. Reagent) directly to cultured
cells. This results in cell lysis and generation of a luminescent
signal produced by a luciferase reaction. The luminescent signal is
proportional to the amount of ATP present, which is directly
proportional to the number of viable cells present in culture. Data
can be recorded by luminometer or CCD camera imaging device. The
luminescence output is expressed as relative light units (RLU).
[0139] In another aspect, a method of treating a colorectal cancer
is provided, the method comprising administering to an individual
having the colorectal cancer an effective amount of a
pharmaceutical formulation comprising 1) an FGFR2 antagonist, 2) a
cytotoxic anti-FGFR2 antibody, or 3) an immunoconjugate comprising
an anti-FGFR2 antibody and a cytotoxic agent. In certain
embodiments, the colorectal cancer is associated with amplification
or overexpression of the FGFR2 gene. In certain embodiments, the
individual is a non-human animal model for colorectal cancer. Mouse
models of colorectal cancer are discussed in detail in Heijstek et
al. (2005) Dig. Surg. 22:16-25. In certain embodiments, the
individual is a human. In certain embodiments, an effective amount
of the pharmaceutical formulation results in any one of the
following: reduction in the number of cancer cells or elimination
of the cancer cells; reduction in the tumor size; full or partial
inhibition of cancer cell infiltration into peripheral organs,
including the spread of cancer into soft tissue and bone; full or
partial inhibition of tumor metastasis; full or partial inhibition
of tumor growth; and/or full or partial relief of one or more of
the symptoms associated with the cancer; and reduced morbidity and
mortality.
[0140] In certain embodiments, a pharmaceutical formulation
comprising 1) an FGFR2 antagonist, 2) a cytotoxic anti-FGFR2
antibody, or 3) an immunoconjugate comprising an anti-FGFR2
antibody and a cytotoxic agent is administered in combination with
at least one additional therapeutic agent and/or adjuvant. In
certain embodiments, an additional therapeutic agent is a cytotoxic
agent, a chemotherapeutic agent, or a growth inhibitory agent. In
one of such embodiments, a chemotherapeutic agent is an agent or a
combination of agents used in the treatment of colorectal cancer.
Such agents include, but are not limited to, fluorouracil (5FU)
alone or in combination with leucovorin or levamisole; edrocolomab;
irinotecan; oxaliplatin; raltitrexed; and fluoropyrimidines.
[0141] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of an FGFR2 antagonist, cytotoxic
antibody, or immunoconjugate can occur prior to, simultaneously,
and/or following, administration of the additional therapeutic
agent and/or adjuvant. An FGFR2 antagonist, cytotoxic antibody, or
immunoconjugate can also be used in combination with radiation
therapy.
[0142] An FGFR2 antagonist, cytotoxic antibody, or immunoconjugate
(and any additional therapeutic agent or adjuvant) can be
administered by any suitable means, including parenteral,
subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and,
if desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration. In
addition, the FGFR2 antagonist, cytotoxic antibody, or
immunoconjugate is suitably administered by pulse infusion,
particularly with declining doses of the FGFR2 antagonist,
cytotoxic antibody, or immunoconjugate. Dosing can be by any
suitable route, e.g. by injections, such as intravenous or
subcutaneous injections, depending in part on whether the
administration is brief or chronic.
[0143] Where the FGFR2 antagonist is an antisense nucleic acid,
guidance for dosage and in vivo administration of antisense nucleic
acids may be found in Khan et al. (2004) J. Drug Targeting
12:393-404.
[0144] Where the therapeutic agent is an anti-FGFR2 antibody or
immunoconjugate thereof, the appropriate dosage of the antibody or
immunoconjugate (when used alone or in combination with one or more
other additional therapeutic agents, such as chemotherapeutic
agents) will depend on the particular antibody or immunoconjugate,
the severity and course of the disease, whether the antibody or
immunoconjugate is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the antibody or immunoconjugate, and the discretion of
the attending physician. The antibody or immunoconjugate is
suitably administered to the patient at one time or over a series
of treatments. Depending on the type and severity of the disease,
about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody
or immunoconjugate can be an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. One typical
daily dosage might range from about 1 .mu.g/kg to 100 mg/kg or
more, depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of an antibody or immunoconjugate would be in the range from
about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of
about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any
combination thereof) may be administered to the patient. Such doses
may be administered intermittently, e.g. every week or every three
weeks (e.g. such that the patient receives from about two to about
twenty, or, e.g., about six doses of the antibody or
immunoconjugate). An initial higher loading dose, followed by one
or more lower doses may be administered. An exemplary dosing
regimen comprises administering an initial loading dose of about 4
mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of
the antibody or immunoconjugate. However, other dosage regimens may
be useful.
III. EXAMPLES
A. Samples
[0145] Thirty fresh frozen colorectal tumors, each from a different
patient sample, were selected for analysis. Each tumor sample had
greater than 75% neoplastic cell content, as estimated by a
pathologist. From each tumor both RNA and DNA were extracted and
purified by standard methods.
B. DNA Copy Number Analysis
[0146] The GeneChip.RTM. Human Mapping 500K Array Set (Affymetrix,
Santa Clara, Calif.) was used to measure DNA copy number changes in
the thirty colorectal tumors. The Gene Chip.RTM. Human Mapping 500K
Array Set consists of two arrays (the 250K "Sty I" array and the
250K "Nsp I" array), each containing probes specific for
approximately 250,000 SNPs, for a total of approximately 500,000
SNPs. The SNPs are distributed throughout the genome, thereby
permitting a genome-wide analysis of DNA copy number. Each array in
the array set includes more than 6.5 million features, with each
feature consisting of over 1 million copies of a 25-bp
oligonucleotide of defined sequence.
[0147] From each tumor sample, DNA was amplified, labeled, and
digested with either Sty 1 or Nsp 1 as per Affymetrix's standard
protocols, and the resulting preparation was allowed to hybridize
to both arrays of the GeneChip.RTM. Human Mapping 500K Array
Set.
[0148] Hybridization to the microarrays was detected according to
Affymetrix's standard protocols, and intensity values for each
feature were generated. Intensity values were normalized to a
reference set of normal genomic DNA. Features were then mapped to
the human genome. Thus, the normalized intensity values reflected
the DNA copy number at a particular genomic locus.
C. Expression Analysis
[0149] The GeneChip.RTM. Human Genome U133A 2.0 Array and the
GeneChip.RTM. Human Genome U133 Plus 2.0 Array (Affymetrix, Santa
Clara, Calif.) were used to measure relative mRNA expression in the
thirty colorectal tumors. Purified RNA samples were reverse
transcribed, amplified, labeled and otherwise treated as per
Affymetrix's standard protocols and allowed to hybridize to one or
the other of the arrays. Hybridization to the arrays was detected
according to Affymetrix's standard methods, and intensity values
for each feature were generated. The intensity value for each
feature was normalized to the median intensity of that feature
across all tumor samples. Features were then mapped to the
corresponding coding regions in the genome. Thus, the normalized
intensity values reflected mRNA expression levels for each feature,
and each feature was correlated with a particular position in the
genome.
D. Analysis and Results
[0150] One of the thirty colorectal tumor samples (designated
glgcX05362) displayed a gene amplification and expression profile
as shown in FIG. 1. In Panel A of that figure, the normalized
intensity value from the DNA copy number analysis (Part B, above)
for each feature is represented as a vertical line. The vertical
lines are plotted along the horizontal axis in Panel A, which
represents the length of chromosome 10. The height of each vertical
line reflects the normalized intensity value, which is a measure of
the DNA copy number at that point on the chromosome. A spike of
signal intensity was observed near the right end of the
chromosome.
[0151] Panel B shows an enlargement of the right end of chromosome
10 from 121,000,000 nucleotides to 126,000,000 nucleotides. As for
Panel A, normalized intensity values from the DNA copy number
analysis are shown as vertical lines. A cluster of normalized
intensity values within that region of chromosome 10 showed about a
10-fold increase in copy number.
[0152] In Panel C, normalized intensity values from the expression
analysis (Part C, above) are shown as vertical lines. The
horizontal axis represents the same chromosomal region as in Panel
B. Thus, the vertical lines in Panel C show the relative levels of
mRNA expression from the coding regions within that chromosomal
region. The height of each vertical line reflects the relative mRNA
expression level for each feature.
[0153] Panel D shows the coding regions of genes known to map to
the region of chromosome 10 depicted in Panels B and C.
[0154] Comparison of Panels B, C, and D, shows that only one gene,
the FGFR2 gene, is present within the region of increased copy
number observed in Panel B. The increase in DNA copy number of the
FGFR2 gene is correlated with marked overexpression (at least about
10-40 fold overexpression) of the FGFR2 transcript, as shown in
Panel C.
[0155] The high level amplification of the FGFR2 gene suggests that
an increase in copy number of that gene causes overexpression of
the encoded growth factor receptor, thereby promoting the growth
and proliferation of colorectal tumor cells. The observed
overexpression of FGFR2 mRNA is consistent with that
conclusion.
[0156] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literatures cited herein are expressly
incorporated in their entirety by reference.
Sequence CWU 1
1
21821PRTHomo sapiens 1Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val
Val Val Thr Met1 5 10 15Ala Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser
Leu Val Glu Asp 20 25 30Thr Thr Leu Glu Pro Glu Glu Pro Pro Thr Lys
Tyr Gln Ile Ser 35 40 45Gln Pro Glu Val Tyr Val Ala Ala Pro Gly Glu
Ser Leu Glu Val 50 55 60Arg Cys Leu Leu Lys Asp Ala Ala Val Ile Ser
Trp Thr Lys Asp 65 70 75Gly Val His Leu Gly Pro Asn Asn Arg Thr Val
Leu Ile Gly Glu 80 85 90Tyr Leu Gln Ile Lys Gly Ala Thr Pro Arg Asp
Ser Gly Leu Tyr 95 100 105Ala Cys Thr Ala Ser Arg Thr Val Asp Ser
Glu Thr Trp Tyr Phe 110 115 120Met Val Asn Val Thr Asp Ala Ile Ser
Ser Gly Asp Asp Glu Asp 125 130 135Asp Thr Asp Gly Ala Glu Asp Phe
Val Ser Glu Asn Ser Asn Asn 140 145 150Lys Arg Ala Pro Tyr Trp Thr
Asn Thr Glu Lys Met Glu Lys Arg 155 160 165Leu His Ala Val Pro Ala
Ala Asn Thr Val Lys Phe Arg Cys Pro 170 175 180Ala Gly Gly Asn Pro
Met Pro Thr Met Arg Trp Leu Lys Asn Gly 185 190 195Lys Glu Phe Lys
Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg 200 205 210Asn Gln His
Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp 215 220 225Lys Gly
Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile 230 235 240Asn
His Thr Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg 245 250
255Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val Val 260
265 270Gly Gly Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp Ala Gln
275 280 285Pro His Ile Gln Trp Ile Lys His Val Glu Lys Asn Gly Ser
Lys 290 295 300Tyr Gly Pro Asp Gly Leu Pro Tyr Leu Lys Val Leu Lys
Ala Ala 305 310 315Gly Val Asn Thr Thr Asp Lys Glu Ile Glu Val Leu
Tyr Ile Arg 320 325 330Asn Val Thr Phe Glu Asp Ala Gly Glu Tyr Thr
Cys Leu Ala Gly 335 340 345Asn Ser Ile Gly Ile Ser Phe His Ser Ala
Trp Leu Thr Val Leu 350 355 360Pro Ala Pro Gly Arg Glu Lys Glu Ile
Thr Ala Ser Pro Asp Tyr 365 370 375Leu Glu Ile Ala Ile Tyr Cys Ile
Gly Val Phe Leu Ile Ala Cys 380 385 390Met Val Val Thr Val Ile Leu
Cys Arg Met Lys Asn Thr Thr Lys 395 400 405Lys Pro Asp Phe Ser Ser
Gln Pro Ala Val His Lys Leu Thr Lys 410 415 420Arg Ile Pro Leu Arg
Arg Gln Val Thr Val Ser Ala Glu Ser Ser 425 430 435Ser Ser Met Asn
Ser Asn Thr Pro Leu Val Arg Ile Thr Thr Arg 440 445 450Leu Ser Ser
Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser Glu 455 460 465Tyr Glu
Leu Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp Lys 470 475 480Leu
Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln Val 485 490
495Val Met Ala Glu Ala Val Gly Ile Asp Lys Asp Lys Pro Lys Glu 500
505 510Ala Val Thr Val Ala Val Lys Met Leu Lys Asp Asp Ala Thr Glu
515 520 525Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu Met Met Lys
Met 530 535 540Ile Gly Lys His Lys Asn Ile Ile Asn Leu Leu Gly Ala
Cys Thr 545 550 555Gln Asp Gly Pro Leu Tyr Val Ile Val Glu Tyr Ala
Ser Lys Gly 560 565 570Asn Leu Arg Glu Tyr Leu Arg Ala Arg Arg Pro
Pro Gly Met Glu 575 580 585Tyr Ser Tyr Asp Ile Asn Arg Val Pro Glu
Glu Gln Met Thr Phe 590 595 600Lys Asp Leu Val Ser Cys Thr Tyr Gln
Leu Ala Arg Gly Met Glu 605 610 615Tyr Leu Ala Ser Gln Lys Cys Ile
His Arg Asp Leu Ala Ala Arg 620 625 630Asn Val Leu Val Thr Glu Asn
Asn Val Met Lys Ile Ala Asp Phe 635 640 645Gly Leu Ala Arg Asp Ile
Asn Asn Ile Asp Tyr Tyr Lys Lys Thr 650 655 660Thr Asn Gly Arg Leu
Pro Val Lys Trp Met Ala Pro Glu Ala Leu 665 670 675Phe Asp Arg Val
Tyr Thr His Gln Ser Asp Val Trp Ser Phe Gly 680 685 690Val Leu Met
Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr Pro 695 700 705Gly Ile
Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly His 710 715 720Arg
Met Asp Lys Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met Met 725 730
735Met Arg Asp Cys Trp His Ala Val Pro Ser Gln Arg Pro Thr Phe 740
745 750Lys Gln Leu Val Glu Asp Leu Asp Arg Ile Leu Thr Leu Thr Thr
755 760 765Asn Glu Glu Tyr Leu Asp Leu Ser Gln Pro Leu Glu Gln Tyr
Ser 770 775 780Pro Ser Tyr Pro Asp Thr Arg Ser Ser Cys Ser Ser Gly
Asp Asp 785 790 795Ser Val Phe Ser Pro Asp Pro Met Pro Tyr Glu Pro
Cys Leu Pro 800 805 810Gln Tyr Pro His Ile Asn Gly Ser Val Lys Thr
815 820 2822PRTHomo sapiens 2Met Val Ser Trp Gly Arg Phe Ile Cys
Leu Val Val Val Thr Met1 5 10 15Ala Thr Leu Ser Leu Ala Arg Pro Ser
Phe Ser Leu Val Glu Asp 20 25 30Thr Thr Leu Glu Pro Glu Glu Pro Pro
Thr Lys Tyr Gln Ile Ser 35 40 45Gln Pro Glu Val Tyr Val Ala Ala Pro
Gly Glu Ser Leu Glu Val 50 55 60Arg Cys Leu Leu Lys Asp Ala Ala Val
Ile Ser Trp Thr Lys Asp 65 70 75Gly Val His Leu Gly Pro Asn Asn Arg
Thr Val Leu Ile Gly Glu 80 85 90Tyr Leu Gln Ile Lys Gly Ala Thr Pro
Arg Asp Ser Gly Leu Tyr 95 100 105Ala Cys Thr Ala Ser Arg Thr Val
Asp Ser Glu Thr Trp Tyr Phe 110 115 120Met Val Asn Val Thr Asp Ala
Ile Ser Ser Gly Asp Asp Glu Asp 125 130 135Asp Thr Asp Gly Ala Glu
Asp Phe Val Ser Glu Asn Ser Asn Asn 140 145 150Lys Arg Ala Pro Tyr
Trp Thr Asn Thr Glu Lys Met Glu Lys Arg 155 160 165Leu His Ala Val
Pro Ala Ala Asn Thr Val Lys Phe Arg Cys Pro 170 175 180Ala Gly Gly
Asn Pro Met Pro Thr Met Arg Trp Leu Lys Asn Gly 185 190 195Lys Glu
Phe Lys Gln Glu His Arg Ile Gly Gly Tyr Lys Val Arg 200 205 210Asn
Gln His Trp Ser Leu Ile Met Glu Ser Val Val Pro Ser Asp 215 220
225Lys Gly Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly Ser Ile 230
235 240Asn His Thr Tyr His Leu Asp Val Val Glu Arg Ser Pro His Arg
245 250 255Pro Ile Leu Gln Ala Gly Leu Pro Ala Asn Ala Ser Thr Val
Val 260 265 270Gly Gly Asp Val Glu Phe Val Cys Lys Val Tyr Ser Asp
Ala Gln 275 280 285Pro His Ile Gln Trp Ile Lys His Val Glu Lys Asn
Gly Ser Lys 290 295 300Tyr Gly Pro Asp Gly Leu Pro Tyr Leu Lys Val
Leu Lys His Ser 305 310 315Gly Ile Asn Ser Ser Asn Ala Glu Val Leu
Ala Leu Phe Asn Val 320 325 330Thr Glu Ala Asp Ala Gly Glu Tyr Ile
Cys Lys Val Ser Asn Tyr 335 340 345Ile Gly Gln Ala Asn Gln Ser Ala
Trp Leu Thr Val Leu Pro Lys 350 355 360Gln Gln Ala Pro Gly Arg Glu
Lys Glu Ile Thr Ala Ser Pro Asp 365 370 375Tyr Leu Glu Ile Ala Ile
Tyr Cys Ile Gly Val Phe Leu Ile Ala 380 385 390Cys Met Val Val Thr
Val Ile Leu Cys Arg Met Lys Asn Thr Thr 395 400 405Lys Lys Pro Asp
Phe Ser Ser Gln Pro Ala Val His Lys Leu Thr 410 415 420Lys Arg Ile
Pro Leu Arg Arg Gln Val Thr Val Ser Ala Glu Ser 425 430 435Ser Ser
Ser Met Asn Ser Asn Thr Pro Leu Val Arg Ile Thr Thr 440 445 450Arg
Leu Ser Ser Thr Ala Asp Thr Pro Met Leu Ala Gly Val Ser 455 460
465Glu Tyr Glu Leu Pro Glu Asp Pro Lys Trp Glu Phe Pro Arg Asp 470
475 480Lys Leu Thr Leu Gly Lys Pro Leu Gly Glu Gly Cys Phe Gly Gln
485 490 495Val Val Met Ala Glu Ala Val Gly Ile Asp Lys Asp Lys Pro
Lys 500 505 510Glu Ala Val Thr Val Ala Val Lys Met Leu Lys Asp Asp
Ala Thr 515 520 525Glu Lys Asp Leu Ser Asp Leu Val Ser Glu Met Glu
Met Met Lys 530 535 540Met Ile Gly Lys His Lys Asn Ile Ile Asn Leu
Leu Gly Ala Cys 545 550 555Thr Gln Asp Gly Pro Leu Tyr Val Ile Val
Glu Tyr Ala Ser Lys 560 565 570Gly Asn Leu Arg Glu Tyr Leu Arg Ala
Arg Arg Pro Pro Gly Met 575 580 585Glu Tyr Ser Tyr Asp Ile Asn Arg
Val Pro Glu Glu Gln Met Thr 590 595 600Phe Lys Asp Leu Val Ser Cys
Thr Tyr Gln Leu Ala Arg Arg Met 605 610 615Glu Tyr Leu Ala Ser Gln
Lys Cys Ile His Arg Asp Leu Ala Ala 620 625 630Arg Asn Val Leu Val
Thr Glu Asn Asn Val Met Lys Ile Ala Asp 635 640 645Phe Gly Leu Ala
Arg Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys 650 655 660Thr Thr Asn
Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala 665 670 675Leu Phe
Asp Arg Val Tyr Thr His Gln Ser Asp Val Trp Ser Phe 680 685 690Gly
Val Leu Met Trp Glu Ile Phe Thr Leu Gly Gly Ser Pro Tyr 695 700
705Pro Gly Ile Pro Val Glu Glu Leu Phe Lys Leu Leu Lys Glu Gly 710
715 720His Arg Met Asp Lys Pro Ala Asn Cys Thr Asn Glu Leu Tyr Met
725 730 735Met Met Arg Asp Cys Trp His Ala Val Pro Ser Gln Arg Pro
Thr 740 745 750Phe Lys Gln Leu Val Glu Asp Leu Asp Arg Ile Leu Thr
Leu Thr 755 760 765Thr Asn Glu Glu Tyr Leu Asp Leu Ser Gln Pro Leu
Glu Gln Tyr 770 775 780Ser Pro Ser Tyr Pro Asp Thr Arg Ser Ser Cys
Ser Ser Gly Asp 785 790 795Asp Ser Val Phe Ser Pro Asp Pro Met Pro
Tyr Glu Pro Cys Leu 800 805 810Pro Gln Tyr Pro His Ile Asn Gly Ser
Val Lys Thr 815 820
* * * * *