U.S. patent application number 13/891815 was filed with the patent office on 2013-11-28 for compositions and methods for diagnosing and treating cancer.
This patent application is currently assigned to OncoMed Pahrmaceuticals, Inc.. The applicant listed for this patent is OncoMed Pharmaceuticals, Inc.. Invention is credited to Aaron Ken SATO, Edward Thein Htun VAN DER HORST.
Application Number | 20130316450 13/891815 |
Document ID | / |
Family ID | 41340727 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130316450 |
Kind Code |
A1 |
VAN DER HORST; Edward Thein Htun ;
et al. |
November 28, 2013 |
Compositions and Methods for Diagnosing and Treating Cancer
Abstract
Isolated antibodies that specifically bind the human MET
Receptor and inhibit MET signaling are described. Also described
are methods of treating cancer, the methods comprising
administering a therapeutically effective amount of the provided
MET antibodies and combinations thereof.
Inventors: |
VAN DER HORST; Edward Thein
Htun; (Palo Alto, CA) ; SATO; Aaron Ken;
(Burlingame, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OncoMed Pharmaceuticals, Inc. |
Redwood City |
CA |
US |
|
|
Assignee: |
OncoMed Pahrmaceuticals,
Inc.
Redwood City
CA
|
Family ID: |
41340727 |
Appl. No.: |
13/891815 |
Filed: |
May 10, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12993656 |
Feb 22, 2011 |
8455623 |
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PCT/US09/03136 |
May 21, 2009 |
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13891815 |
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61055088 |
May 21, 2008 |
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61091265 |
Aug 22, 2008 |
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61161275 |
Mar 18, 2009 |
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Current U.S.
Class: |
435/334 ;
435/252.33; 435/320.1; 536/23.53 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 2317/732 20130101; A61K 2039/505 20130101; A61K 2039/507
20130101; A61P 35/00 20180101; C07K 2317/21 20130101; C07K 16/2863
20130101; C07K 2317/73 20130101; C07K 16/30 20130101; C07K 2317/565
20130101 |
Class at
Publication: |
435/334 ;
536/23.53; 435/320.1; 435/252.33 |
International
Class: |
C07K 16/30 20060101
C07K016/30 |
Claims
1-135. (canceled)
136. An isolated polynucleotide encoding an antibody that
specifically binds human MET, wherein the antibody is selected from
the group consisting of: (a) an antibody comprising (i) a heavy
chain CDR1 comprising GFSLSTSGMVVS (SEQ ID NO:3), a heavy chain
CDR2 comprising FISWDDDKYYSTSLKT (SEQ ID NO:4), and a heavy chain
CDR3 comprising EPGRYGGYYFDY (SEQ ID NO:5); and (ii) a light chain
CDR1 comprising RASQTISHYLA (SEQ ID NO:8), a light chain CDR2
comprising AASILQS (SEQ ID NO:9), and a light chain CDR3 comprising
QQYSGFPV (SEQ ID NO:10); (b) an antibody-comprising (i) a heavy
chain CDR1 comprising GGSISGYYWS (SEQ ID NO: 13), a heavy chain
CDR2 comprising EIYYAGSTLYNPSLKG (SEQ ID NO:14), and a heavy chain
CDR3 comprising HYGLDWFGDTGMDV (SEQ ID NO:15); and (ii) a light
chain CDR1 comprising SGDNLGDKYVH (SEQ ID NO:18), a light chain
CDR2 comprising DDNERPSG (SEQ ID NO:19), and a light chain CDR3
comprising SAYGSHSGT (SEQ ID NO:20); and (c) an antibody comprising
(i) a heavy chain CDR1 comprising GGSISGYYWS (SEQ ID NO:13), a
heavy Chain CDR2 comprising EIYYAGSTLYNPSLKG (SEQ ID NO:14), and a
heavy chain CDR3 comprising HYGLDWFGDTGMDV (SEQ ID NO:15); and (ii)
a light chain. CDR1 comprising SGDNLGEQYVH (SEQ ID NO:23), a light
chain CDR2 comprising DDSERPSG (SEQ ID NO:24), and a light chain
CDR3 comprising QSYTFYPNSR (SEQ ID NO:25).
137. The polynucleotide of claim 136, wherein the antibody
comprises: (a) a heavy chain variable region having at least 95%
identity to SEQ ID NO:2 and a light chain variable region having at
least 95% identity to SEQ ID NO:7; (b) a heavy chain variable
region having at least 95% identity to SEQ ID NO:12 and a light
chain variable region having at least 95% identity to SEQ ID NO:17;
or (c) a heavy chain variable region having at least 95% identity
to SEQ ID NO:12 and a light chain variable region having at least
95% identity to SEQ ID NO:22.
138. The isolated polynucleotide of claim 136, wherein the antibody
comprises: (a) a polypeptide having the amino acid sequence of SEQ
ID NO:2 and a polypeptide having the amino acid sequence of SEQ ID
NO:7; (b) a polypeptide having the amino acid sequence of SEQ ID
NO:12 and a polypeptide having the amino acid sequence of SEQ ID
NO:17; or (c) a polypeptide having the amino acid sequence of SEQ
ID NO:12 and a polypeptide having the amino acid sequence of SEQ ID
NO:22.
139. The polynucleotide of claim 136, wherein the antibody is a
monovalent antibody.
140. The polynucleotide of claim 136, wherein the antibody is an
antigen-binding antibody fragment.
145. The polynucleotide of claim 140, wherein the antibody fragment
is monovalent.
146. A vector comprising the polynucleotide of claim 136.
147. An isolated cell comprising the polynucleotide of claim
136.
148. An isolated cell comprising the vector of claim 147.
149. An isolated polynucleotide comprising at least one
polynucleotide sequence selected from the group consisting of SEQ
ID NO:1, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:16, and SEQ
NO:21.
150. The polynucleotide of claim 149 comprising SEQ ID NO:1 and SEQ
ID NO:6.
151. The polynucleotide of claim 149 comprising SEQ ID NO:11 and
SEQ ID NO:16.
152. The polynucleotide of claim 149 comprising SEQ ID NO:11 and
SEQ ID NO:21.
153. An isolated polynucleotide comprising a polynucleotide
sequence which hybridizes to a polynucleotide sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID
NO:11, SEQ ID NO:16, and SEQ ID NO:21, or its complement.
154. The polynucleotide of claim 153, wherein the hybridization is
under stringent hybridization conditions.
155. An isolated polynucleotide encoding a first arm of a
bispecific antibody, wherein the first arm of the bispecific
antibody specifically binds human MET and is selected from the
group consisting of: (a) an antibody comprising (i) a heavy chain
CDR1 comprising GFSLSTSGMVVS (SEQ ID NO:3), a heavy chain CDR2
comprising FISWDDDKYYSTSLKT (SEQ ID NO:4), and a heavy chain CDR3
comprising EPGRYGGYYFDY (SEQ ID NO:5); and (ii) a light chain CDR1
comprising RASQTISHYLA (SEQ ID NO:8), a light chain CDR2 comprising
AASILQS (SEQ ID NO:9), and a light chain CDR3 comprising, QQYSGFPV
(SEQ ID NO:10); (b) an antibody comprising (i) a heavy chain CDR1
comprising GGSISGYYWS (SEQ ID NO:13), a heavy chain CDR2 comprising
EIYYAGSTLYNPSLKG (SEQ ID NO:14), and a heavy chain CDR3 comprising
HYGLDWFGDTGMDV (SEQ ID NO:15); and (ii) a light chain CDR1
comprising SGDNLGDKYVH (SEQ ID NO:18), a light chain CDR2
comprising DDNERPSG (SEQ ID NO:19), and a light chain CDR3
comprising SAYGSHSGT (SEQ ID NO:20); and (c) an antibody comprising
(i) a heavy chain CDR1 comprising GGSISGYYWS (SEQ ID NO:13), a
heavy chain CDR2 comprising EIYYAGSTLYNPSLKG (SEQ ID NO:14), and a
heavy chain CDR3 comprising HYGLDWFGDTGMDV: (SEQ ID NO:15); and
(ii) a light chain CDR1 comprising SGDNLGEQYVH (SEQ ID NO:23), a
light chain CDR2 comprising DDSERPSG (SEQ ID NO:24), and a light
chain CDR3 comprising QSYTFYPNSR (SEQ ID NO 25).
156. The polynucleotide of claim 155, Wherein the first arm of the
bispecific antibody comprises: (a) a heavy chain variable region
having at least 95% identity to SEQ ID NO:2 and a light chain
variable region having at least 95% identity to SEQ ID NO:7; (b) a
heavy chain variable region having at least 95% identity to SEQ ID
NO:12 and a light chain variable region having at least 95%
identity to SEQ ID NO:17; or (c) a heavy chain variable region
having at least 95% identity to SEQ ID NO:12 and a light chain
variable region having at least 95% identity to SEQ ID NO:22.
157. The isolated polynucleotide of claim 155, wherein the first
arm of the bispecific antibody comprises: (a) a polypeptide having
the amino acid sequence of SEQ ID NO:2 and a polypeptide having the
amino acid sequence of SEQ ID NO:7; (b) a polypeptide having the
amino acid sequence of SEQ ID NO:12 and a polypeptide having the
amino acid sequence of SEQ ID NO: 17; or (c) a polypeptide having
the amino acid sequence of SEQ ID NO:12 and a polypeptide having
the amino acid sequence of SEQ ID NO:22.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the field of oncology and
provides novel compositions and methods for diagnosing and treating
cancer.
[0003] 2. Background Art
[0004] Cancer is one of the leading causes of death in the
developed world, with over one million people diagnosed with cancer
and 500,000 deaths per year in the United States alone. Overall it
is estimated that more than 1 in 3 people will develop some form of
cancer during their lifetime. There are more than 200 different
types of cancer, four of which--breast, lung, colorectal, and
prostate--account for over half of all new cases (Jemal et al.,
2003, Cancer J. Clin. 53:5-26).
[0005] Breast cancer is the most common cancer in women, with an
estimated 12% of women at risk of developing the disease during
their lifetime. Although mortality rates have decreased due to
earlier detection and improved treatments, breast cancer remains a
leading cause of death in middle-aged women, and metastatic breast
cancer is still an incurable disease. On presentation, most
patients with metastatic breast cancer have only one or two organ
systems affected, but as the disease progresses, multiple sites
usually become involved. The most common sites of metastatic
involvement are locoregional recurrences in the skin and soft
tissues of the chest wall, as well as in axilla and supraclavicular
areas. The most common site for distant metastasis is the bone
(30-40% of distant metastasis), followed by the lungs and liver.
And although only approximately 1-5% of women with newly diagnosed
breast cancer have distant metastasis at the time of diagnosis,
approximately 50% of patients with local disease eventually relapse
with metastasis within five years. At present the median survival
from the manifestation of distant metastases is about three
years.
[0006] Current methods of diagnosing and staging breast cancer
include the tumor-node-metastasis (TNM) system that relies on tumor
size, tumor presence in lymph nodes, and the presence of distant
metastases (American Joint Committee on Cancer: AJCC Cancer Staging
Manual. Philadelphia, Pa.: Lippincott-Raven Publishers, 5th ed.,
1997, pp 171-180; Harris, J R: "Staging of breast carcinoma" in
Harris, J. R., Hellman, S., Henderson, I. C., Kinne D. W. (eds.):
Breast Diseases. Philadelphia, Lippincott, 1991). These parameters
are used to provide a prognosis and select an appropriate therapy.
The morphologic appearance of the tumor can also be assessed but
because tumors with similar histopathologic appearance can exhibit
significant clinical variability, this approach has serious
limitations. Finally assays for cell surface markers can be used to
divide certain tumors types into subclasses. For example, one
factor considered in the prognosis and treatment of breast cancer
is the presence of the estrogen receptor (ER) as ER-positive breast
cancers typically respond more readily to hormonal therapies such
as tamoxifen or aromatase inhibitors than ER-negative tumors. Yet
these analyses, though useful, are only partially predictive of the
clinical behavior of breast tumors, and there is much phenotypic
diversity present in breast cancers that current diagnostic tools
fail to detect and current therapies fail to treat.
[0007] Prostate cancer is the most common cancer in men in the
developed world, representing an estimated 33% of all new cancer
cases in the U.S., and is the second most frequent cause of death
(Jemal et al., 2003, CA Cancer J. Clin. 53:5-26). Since the
introduction of the prostate specific antigen (PSA) blood test,
early detection of prostate cancer has dramatically improved
survival rates; the five year survival rate for patients with local
and regional stage prostate cancers at the time of diagnosis is
nearing 100%. Yet more than 50% of patients will eventually develop
locally advanced or metastatic disease (Muthuramalingam et al.,
2004, Clin. Oncol. 16:505-16).
[0008] Currently radical prostatectomy and radiation therapy
provide curative treatment for the majority of localized prostate
tumors. However, therapeutic options are very limited for advanced
cases. For metastatic disease, androgen ablation with luteinising
hormone-releasing hormone (LHRH) agonist alone or in combination
with anti-androgens is the standard treatment. Yet despite maximal
androgen blockage, the disease nearly always progresses with the
majority developing androgen-independent disease. At present there
is no uniformly accepted treatment for hormone refractory prostate
cancer, and chemotherapeutic regimes are commonly used
(Muthuramalingam et al., 2004, Clin. Oncol. 16:505-16; Trojan et
al., 2005, Anticancer Res. 25:551-61).
[0009] Colorectal cancer is the third most common cancer and the
fourth most frequent cause of cancer deaths worldwide (Weitz et
al., 2005, Lancet 365:153-65). Approximately 5-10% of all
colorectal cancers are hereditary with one of the main forms being
familial adenomatous polyposis (FAP), an autosomal dominant disease
in which about 80% of affected individuals contain a germline
mutation in the adenomatous polyposis coli (APC) gene. Colorectal
carcinomas invade locally by circumferential growth and elsewhere
by lymphatic, hematogenous, transperitoneal, and perineural spread.
The most common site of extralymphatic involvement is the liver,
with the lungs the most frequently affected extra-abdominal organ.
Other sites of hematogenous spread include the bones, kidneys,
adrenal glands, and brain.
[0010] The current staging system for colorectal cancer is based on
the degree of tumor penetration through the bowel wall and the
presence or absence of nodal involvement. This staging system is
defined by three major Duke's classifications: Duke's A disease is
confined to submucosa layers of colon or rectum; Duke's B disease
has tumors that invade through the muscularis propria and may
penetrate the wall of the colon or rectum; and Duke's C disease
includes any degree of bowel wall invasion with regional lymph node
metastasis. While surgical resection is highly effective for early
stage colorectal cancers, providing cure rates of 95% in Duke's A
patients, the rate is reduced to 75% in Duke's B patients and the
presence of positive lymph node in Duke's C disease predicts a 60%
likelihood of recurrence within five years. Treatment of Duke's C
patients with a post surgical course of chemotherapy reduces the
recurrence rate to 40%-50% and is now the standard of care for
these patients.
[0011] Lung cancer is the most common cancer worldwide, the third
most commonly diagnosed cancer in the United States, and by far the
most frequent cause of cancer deaths (Spiro et al., 2002, Am. J.
Respir. Crit. Care Med. 166:1166-96; Jemal et al., 2003, CA Cancer
J. Clin. 53:5-26). Cigarette smoking is believed responsible for an
estimated 87% of all lung cancers making it the most deadly
preventable disease. Lung cancer is divided into two major types
that account for over 90% of all lung cancers: small cell lung
cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC accounts
for 15-20% of cases and is characterized by its origin in large
central airways and histological composition of sheets of small
cells with little cytoplasm. SCLC is more aggressive than NSCLC,
growing rapidly and metastasizing early. NSCLC accounts for 80-85%
of all cases and is further divided into three major subtypes based
on histology: adenocarcinoma, squamous cell carcinoma (epidermoid
carcinoma), and large cell undifferentiated carcinoma.
[0012] Lung cancer typically presents late in its course, and thus
has a median survival of only 6-12 months after diagnosis and an
overall 5 year survival rate of only 5-10%. Although surgery offers
the best chance of a cure, only a small fraction of lung cancer
patients are eligible with the majority relying on chemotherapy and
radiotherapy. Despite attempts to manipulate the timing and dose
intensity of these therapies, survival rates have increased little
over the last 15 years (Spiro et al., 2002, Am. J. Respir. Crit.
Care Med. 166:1166-96).
[0013] These four cancers, as well as many others, present as solid
tumors that are composed of heterogeneous cell populations. For
example, breast cancers are a mixture of cancer cells and normal
cells, including mesenchymal (stromal) cells, inflammatory cells,
and endothelial cells. Several models of cancer provide different
explanations for the presence of this heterogeneity. One model, the
classic model of cancer, holds that phenotypically distinct cancer
cell populations all have the capacity to proliferate and give rise
to a new tumor. In the classical model, tumor cell heterogeneity
results from environmental factors as well as ongoing mutations
within cancer cells resulting in a diverse population of
tumorigenic cells. This model rests on the idea that all
populations of tumor cells have some degree of tumorigenic
potential. (Pandis et al., 1998, Genes, Chromosomes & Cancer
12:122-129; Kuukasjrvi et al., 1997, Cancer Res. 57:1597-1604;
Bonsing et al., 1993, Cancer 71:382-391; Bonsing et al., 2000,
Genes Chromosomes & Cancer 82: 173-183; Beerman H et al., 1991,
Cytometry 12:147-54; Aubele M & Werner M, 1999, Analyt. Cell.
Path. 19:53; Shen L et al., 2000, Cancer Res. 60:3884).
[0014] An alternative model for the observed solid tumor cell
heterogeneity derives from the impact of stem cells on tumor
development. According to this model, cancer arises from
dysregulation of the mechanisms that control normal tissue
development and maintenance. (Beachy et al., 2004, Nature 432:324).
During normal animal development, cells of most or all tissues are
derived from normal precursors, called stem cells (Morrison et al.,
1997, Cell 88:287-98; Morrison et al., 1997, Curr. Opin. Immunol.
9:216-21; Morrison et al., 1995, Annu. Rev. Cell. Dev. Biol.
11:35-71). Stem cells are cells that: (1) have extensive
proliferative capacity; 2) are capable of asymmetric cell division
to generate one or more kinds of progeny with reduced proliferative
and/or developmental potential; and (3) are capable of symmetric
cell divisions for self-renewal or self-maintenance. The
best-studied example of adult cell renewal by the differentiation
of stem cells is the hematopoietic system where developmentally
immature precursors (hematopoietic stem and progenitor cells)
respond to molecular signals to form the varied blood and lymphoid
cell types. Other cells, including cells of the gut, breast ductal
system, and skin are constantly replenished from a small population
of stem cells in each tissue, and recent studies suggest that most
other adult tissues also harbor stem cells, including the brain.
Tumors derived from a "solid tumor stem cell" (or "cancer stem
cell" from a solid tumor) subsequently undergoes chaotic
development through both symmetric and asymmetric rounds of cell
divisions. In this stem cell model, solid tumors contain a distinct
and limited (possibly even rare) subset of cells that share the
properties of normal "stem cells", in that they extensively
proliferate and efficiently give rise both to additional solid
tumor stem cells (self-renewal) and to the majority of tumor cells
of a solid tumor that lack tumorigenic potential. Indeed, mutations
within a long-lived stem cell population may initiate the formation
of cancer stem cells that underlie the growth and maintenance of
tumors and whose presence contributes to the failure of current
therapeutic approaches.
[0015] The stem cell nature of cancer was first revealed in the
blood cancer, acute myeloid leukemia (AML) (Lapidot et al., 1994,
Nature 17:645-8). More recently it has been demonstrated that
malignant human breast tumors similarly harbor a small, distinct
population of cancer stern cells enriched for the ability to form
tumors in immunodeficient mice. An ESA+, CD44+, CD24-/low, Lin-cell
population was found to be 50-fold enriched for tumorigenic cells
compared to unfractionated tumor cells (Al-Hajj et al., 2003, Proc.
Nat'l Acad. Sci. 100:3983-8). The ability to prospectively isolate
the tumorigenic cancer cells has permitted investigation of
critical biological pathways that underlie tumorigenicity in these
cells, and thus promises the development of better diagnostic
assays and therapeutics for cancer patients. It is toward this
purpose that this invention is directed.
BRIEF SUMMARY OF THE INVENTION
[0016] Provided are antibodies that specifically bind to receptors
such as the human MET Receptor. In certain embodiments, the
antibodies are humanized antibodies or human antibodies. In certain
embodiments, these antibodies inhibit MET Receptor interactions
with HGF ligand binding and downstream MET Receptor signaling. Also
provided are pharmaceutical compositions comprising the antibodies
of the present disclosure and a pharmaceutically acceptable
vehicle. Further provided are methods of treating cancer comprising
administering the antibodies of the present disclosure in a
therapeutically effective amount.
[0017] In another aspect, the invention provides a method of
inhibiting the functioning or signaling by a receptor (e.g., a
human Met receptor or other human receptor tyrosine kinase) on a
cell comprising contacting the cell with an effective amount of
either (a) an antibody that specifically binds both a first epitope
and a second epitope on the extracellular domain of the receptor,
or (b) a combination of antibodies comprising (i) a first antibody
that binds a first epitope on the extracellular domain of the
receptor and (ii) a second antibody that binds a second epitope on
the extracellular domain of the same receptor. In certain
embodiments, the first epitope does not overlap with the second
epitope. In certain embodiments, the binding of the antibody to the
first epitope inhibits binding of a ligand to the receptor by
direct competition and/or binds to the ligand binding site on the
receptor. In some embodiments, the second epitope is a
conformational epitope and/or binding to the second epitope does
not compete directly with ligand binding, but nonetheless inhibits
binding of the ligand to the receptor. In some embodiments, binding
of the antibody or antibodies to the combination of both the first
and second epitopes synergistically inhibits ligand binding.
[0018] In still another aspect, the invention provides a method of
inhibiting tumor growth and/or treating cancer in a patient
comprising administering to the patient a therapeutically effective
amount of a multispecific antibody that specifically binds both a
first epitope and a second epitope on the extracellular domain of a
human Met receptor. In certain embodiments, the first epitope does
not overlap with the second epitope. In certain embodiments,
binding of the antibody to the first epitope inhibits HGF binding
to the Met receptor and/or binding of the antibody to the second
epitope inhibits HGF binding to the Met receptor. In certain
embodiments, binding both the first and second epitope
synergistically inhibits HGF binding. In certain embodiments,
binding of the antibody to the first epitope directly competes with
binding of HGF to the Met receptor and/or the first epitope is in
the SEMA domain of the Met receptor. In certain embodiments,
binding of the antibody to the second epitope does not directly
compete with binding of HGF to the Met receptor and/or the second
epitope is a conformational epitope. In certain embodiments,
binding of the antibody to the first epitope increases the avidity
of the antibody to the second epitope.
[0019] In yet another aspect, the invention provides a method of
inhibiting tumor growth and/or treating cancer in a patient
comprising administering to the patient a therapeutically effective
amount of a combination of antibodies comprising (i) a first
antibody that binds a first epitope on the extracellular domain of
a human Met receptor and (ii) a second antibody that binds a second
epitope on the extracellular domain of the same receptor. In
certain embodiments, the first epitope does not overlap with the
second epitope. In certain embodiments, binding of each of the
antibodies alone inhibits HGF binding to the Met receptor. In some
embodiments, the binding of the two antibodies together
synergistically inhibits HGF binding. In certain embodiments,
binding of the first antibody directly competes with binding of HGF
to the Met receptor and/or the first antibody binds to the SEMA
domain. In certain embodiments, the second antibody, although it
inhibits HGF binding to the Met receptor does not directly compete
with HGF for binding and/or the second epitope is a conformational
epitope. In certain embodiments, binding of the first antibody to
the first epitope increases the avidity of the second antibody for
the second epitope.
[0020] In another aspect, the invention provides the antibodies
13-MET, 21-MET, and 28-MET, each of which specifically binds to the
human Met receptor and inhibits HGF binding to the receptor.
[0021] In another aspect, the invention provides an antibody that
specifically binds to the extracellular domain of the human MET
receptor and comprises at least one (i.e., one, two, three, four,
five, or six) CDRs of 13-MET, 21-MET, or 28-MET.
[0022] In another aspect, the invention provides an antibody that
specifically binds to the extracellular domain of the human MET
receptor and comprises a heavy chain variable region having at
least about 90% identity to the heavy chain variable region of
13-MET and/or at least about 90% identity to the light chain
variable region of 13-MET.
[0023] In another aspect, the invention provides an antibody that
specifically binds to the extracellular domain of the human MET
receptor and comprises a heavy chain variable region having at
least about 90% identity to the heavy chain variable region of
28-MET and/or at least about 90% identity to the light chain
variable region of 28-MET or 21-MET.
[0024] In another aspect, the invention provides an antibody that
competes for specific binding to a human Met receptor with the
13-MET, 21-MET, and/or 28-MET antibody.
[0025] In some embodiments of each of the aforementioned aspects,
as well as other aspects described herein, the antibodies inhibit
HGF binding to the human Met receptor.
[0026] In some embodiments of each of the aforementioned aspects,
as well as other aspects described herein, the antibodies are
monoclonal antibodies. In some embodiments, the antibodies are
human or humanized antibodies. In some embodiments, the antibodies
are multispecific (e.g., bispecific) antibodies. In some
embodiments, the multispecific or bispecific antibodies
specifically bind to more than one epitope on the extracellular
domain of the Met receptor. In certain embodiments, the different
epitopes on the Met receptor to which the multispecific or
bispecific antibody binds are non-overlapping. In some embodiments,
the antibodies are isolated. Methods of inhibiting the functioning
of or signaling by a Met receptor on a cell comprising contacting
the cell with an effective amount of one or more of the antibodies
described herein is also provided. Methods of inhibiting tumor
growth and methods of treating cancer in a patient comprising
administering to the patient a therapeutically effective amount of
the antibody of one or more of the antibodies described herein are
also provided.
[0027] Polypeptide comprising fragments of the antibodies, as well
as polynucleotides encoding the polypeptides or antibodies are also
provided.
[0028] Compositions, such as pharmaceutical compositions comprising
the antibodies are also provided.
[0029] Additional objects and advantages of the invention will be
set forth in part in the description which follows, and in part
will be obvious from the description, or may be learned by practice
of the invention. The objects and advantages of the invention will
be realized and attained by means of the elements and combinations
particularly pointed out in the appended claims. It is to be
understood that both the foregoing general description and the
following detailed description are exemplary and explanatory only
and are not restrictive of the invention, as claimed. The
accompanying drawings, which are incorporated in and constitute a
part of this specification, illustrate several embodiments of the
invention and, together with the description, serve to explain the
principles of the invention. In the specification and the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0030] FIG. 1: Anti-MET Antibodies Block Binding Between HGF and
the MET Receptor Extracellular Domain. (A) A schematic of the
different Met Receptor antibodies generated. Each monovalent Fab
(Fab 13 or 28 monomeric Fab) was linked to itself via a helix loop
helix motif to produce divalent dimer Fabs (dimeric Fab-dHLX) in
which two 13-MET Fabs were linked to produce the dimer 9-MET or two
28-MET Fabs were linked to produce the dimer 19-MET. Similarly,
each monovalent Fab was used to generate a human IgG1 antibody (IgG
13 or IgG 28). (B, C) The effect (y-axis) of monomeric anti-MET
antibodies 13-MET (B) and 28-MET (C) on blocking MET binding to HGF
is shown for increasing doses of each antibody (x-axis; nM). The
effect at each dose is marked with an "X", and a dose-effect curve
graphed for each antibody. Dm=IC50; m=slope of the curve,
r=curvefit value.
[0031] FIG. 2: Anti-MET Antibodies Block Binding Between HGF and
the MET Receptor Extracellular Domain. The effect (y-axis) of
dimeric anti-MET antibodies 9-MET (A) and 19-MET (B) of blocking
MET binding to HGF is shown for increasing doses of each antibody
(x-axis; nM). The effect at each dose is marked with an "X", and a
dose-effect curve graphed for each antibody. Dm=IC50; m=slope of
the curve, r=curvefit value.
[0032] FIG. 3: A Combination of Anti-MET Antibodies 9-MET and
19-MET Act Synergistically to Block Binding Between HGF and the MET
Receptor Extracellular Domain. (A) The effect (y-axis) of anti-MET
antibodies 19-MET (circles), 9-MET (crosses), and a combination of
9-MET/19-MET (1:5) (x's) of blocking MET binding to HGF is shown
for increasing doses of each antibody or antibody combination
(x-axis; nM). The effect at each dose is marked with its
corresponding symbol, and a dose-effect curve graphed for each
antibody. (B) A conservative isobologram demonstrates that the
9-MET/19-MET antibody combination acts synergistically to block HGF
binding to MET. The effective dose (ED) 50 (X), ED75 (crosses) and
ED90 (circles) are graphed. Dm=IC50; m=slope of the curve,
r=curvefit value. (C) A Combination of Anti-MET Antibodies 9-MET
and 19-MET Act Synergistically to induce antibody-dependent
cellular cytotoxicity in GTL-16 cancer cells (ADCC). The effect
(y-axis) of anti-MET antibodies 13-MET (crosses), 28-MET (circles),
and a combination of 13-MET/28-MET (1:5) (x's) inducing ADCC on
GTL-16 cells is shown for increasing doses of each antibody or
antibody combination (x-axis; nM). The effect at each dose is
marked with its corresponding symbol, and a dose-effect curve
graphed for each antibody. (D) A conservative isobologram
demonstrates that the 13-28-MET antibody combination acts
synergistically to induce ADCC on GTL-16 cells. The effective dose
(ED) 50 (X), ED75 (crosses) and ED90 (circles) are graphed.
Dm=IC50; m=slope of the curve, r=curvefit value.
[0033] FIG. 4: A Combination of Anti-MET Antibodies Eliminates
Detectable Phosphorylation of MET and Downstream Signaling Proteins
in Lung Tumor Cells. Cells incubated without (NS) or with HGF
(+HGF) in the presence or absence (-) of 13-MET (13m), 28-MET
(28m), and a combination of 13-MET/28-MET (13m 28m) antibodies (A)
or 9-MET (9d), 19-MET (19d), and a combination of 9-MET/19-MET (9d
19d) antibodies (B-D) were analyzed by immunblotting either as
whole cell lysates (WCL) or by immunoprecipitation (IP) with
antibodies that recognize the phosphorylated form (left blots) or
total protein (right blots) of the MET receptor (B, D); the
downstream signaling molecules SHC (A, C), AKT (D), and ERK1/2 (D);
or Actin as a loading control (D). While each antibody alone
decreases phosphorylation of MET and/or phosphorylation of
downstream signaling molecules, the antibody combinations eliminate
or nearly eliminate detectable phosphorylation.
[0034] FIG. 5: A Combination of Anti-MET Antibodies Disrupts MET
Signaling as Effectively as SU11274 in Lung Tumor Cells. Cells
incubated without (NS) or with HGF (+HGF) in the presence or
absence (-) of 13-MET (13), 28-MET (28), a combination of
13-MET/28-MET (13 28) antibodies (C, D), a combination of
9-MET/19-MET (9d 19d) antibodies (A, B), or SU11274 (SU) were
analyzed by immunblotting either as whole cell lysates (WCL) or by
immunoprecipitation (IP) with antibodies that recognize the
phosphorylated form (left blots) or total protein (right blots) of
the MET receptor (B, C); the downstream signaling molecules SHC
(A), AKT (B), and ERK1/2 (B, D); or Actin as a loading control (B,
D). The antibody combinations eliminate detectable phosphorylation
of MET and downstream signaling molecules as effectively as
SU11274. (E-I) The dose-effect of the 9-MET/19-MET combination
(1:5) on phosphorylation of MET at Y1230/Y1234/Y1235 (E) or at
Y1349 (F); AKT1 at S473 (G), and ERK1/2 at T185/Y187 (H) are
graphed and the Dm=IC50; m=slope of the curve, r=curvefit value are
calculated for each (I).
[0035] FIG. 6: A Combination of Anti-MET Antibodies Disrupts MET
Signaling and HGF-Mediated Proliferation of HUVEC Cells. Cells
incubated without (NS) or with HGF (+HGF) in the presence or
absence (-) of a combination of 9-MET/19-MET (9d 19d) antibodies or
SU11274 were analyzed by immunoblotting either as whole cell
lysates (WCL) or by immunoprecipitation (IP) with antibodies that
recognize the phosphorylated form (left blots) or total protein
(right blots) of the MET receptor (A); the downstream signaling
molecules SHC (B), AKT (C), and ERK1/2 (C); or Actin as a loading
control (C). The 9-MET/19-MET antibody combination eliminated
detectable phosphorylation of MET and downstream signaling
molecules as effectively as SU11274 in HUVEC cells. (D) Cell
proliferation was measured in the absence (upper graph) or the
presence (lower graph) of HGF. Cells were incubated with 9-19-MET
antibodies (squares) or control medium (diamonds) over 7 days.
Treatment of HUVEC cells with 9-19-MET antibodies disrupted
HGF-mediated cell proliferation.
[0036] FIG. 7: A Combination of Anti-MET Antibodies Disrupts
HGF-Mediated Cell Migration. (A) H441 cells in which a scrape has
been made through the monolayer were treated with control media
(top row) or HGF (bottom row) either alone (left), in the presence
of SU11274 (middle), or in the presence of a combination of
13-MET/28-MET antibodies at 30 ug/ml (right). Photographs show
results 16 hours after exposure to HGF. (B) HUVEC cells in which a
scrape has been made through the monolayer were treated with
control media (top, left) or HGF either alone (top, right) or in
the presence of SU11274 (bottom, left) or 9-MET/19-MET Fab
combination of antibodies (bottom, right). Photographs show results
17 hours after exposure to HGF.
[0037] FIG. 8: Anti-MET Antibody Combinations Synergistically Block
MET Receptor function. AF647-conjugated R28 (black bars) or R13
(white bars) were used as FACS-reagents to detect MET-receptor on
GTL-16 cells. AF647-labeled antibodies (AF647-R28 or AF647-R13)
were used at fixed concentrations (360 nM, AF647-R28; nM,
AF647-R13) and unlabeled R13 (0.5 nM, 20 nM) or R28 (90 nM, 360 nM)
was titered in. Note that 20 nM of R13 increased MFI-values
(.DELTA.MFI) for AF647-R28 by 2.6 fold, whereas adding R28 to
AF647-R13 did not show any effect. .DELTA.MFI values were
determined by substracting background MFI. NS indicates not
stimulated. Arrows indicate the detected proteins. bars, SD. Kd
values for the antibodies remained unchanged (data not shown).
[0038] FIG. 9: Anti-MET Antibodies Reduce In Vivo Growth of Colon
Tumors and Met-Expressing Gastric Carcinoma Cell Line GTL-16. (A)
Immunodeficient mice were injected with GLT-16 cells and
established tumors treated with either control antibody 1B7.11
(squares) or anti-MET antibodies 13-MET and 28-MET at a ratio of
1:8 (inverted triangles). Tumor volume (x-axis) is plotted over
time (y-axis). Administration of a 1:8 ratio of 13-MET to 28-MET
antibodies resulted in a statistically significant decrease in
tumor volume compared to control antibody treated animals at day 15
(p<0.01) and at day 19 to 22 (p<0.001) post-injection. (B)
Immunodeficient mice were injected with OMP-C12 colon tumor cells
and established tumors treated with anti-MET antibodies 13-MET and
28-MET at a ratio of 1:8 (inverted triangles). Tumor volume
.alpha.-axis) is plotted over time (y-axis). Administration of a
1:8 ratio of 13-MET to 28-MET antibodies resulted in a
statistically significant decrease in tumor volume compared to
control antibody treated animals at day 77 (p<0.01) and at day
81 to day 105 (p<0.001) post-injection. (C) Immunodeficient mice
were injected with OMP-C17 colon tumor cells and established tumors
treated with anti-MET antibodies 13-MET and 28-MET at a ratio of
1:8 (inverted triangles). Tumor volume (x-axis) is plotted over
time (y-axis). Administration of a 1:8 ratio of 13-MET to 28-MET
antibodies resulted in a statistically significant decrease in
tumor volume compared to control antibody treated animals at day 58
(p<0.01) and day 62 (p<0.001) post-injection. (D)
Immunodeficient mice were injected with OMP-C27 colon tumor cells
and established tumors treated with anti-MET antibodies 13-MET and
28-MET at a ratio of 1:8 (inverted triangles). Tumor volume
.alpha.-axis) is plotted over time (y-axis). Administration of a
1:8 ratio of 13-MET to 28-MET antibodies resulted in a
statistically significant decrease in tumor volume compared to
control antibody treated animals at day 41 (p<0.05) and day 44
to 48 (p<0.001) post-injection. (E) Immunodeficient mice were
injected with OMP-C28 colon tumor cells and treated with anti-MET
antibodies 13-MET and 28-MET at a ratio of 1:8 (inverted
triangles). Tumor volume (x-axis) is plotted over time (y-axis).
Administration of a 1:8 ratio of 13-MET to 28-MET antibodies
resulted in a statistically significant decrease in tumor volume
compared to control antibody treated animals at day 40 to 48
(p<0.001) post-injection. (F) Immunodeficient mice were injected
with GLT-16 cells and established tumors treated with either
control antibody 1B7.11 (squares) or an anti-MET bispecific
antibody 73R21.13 (triangles). Tumor volume (x-axis) is plotted
over time (y-axis). Administration of the anti-MET bispecific
antibody 73R21.13 resulted in a statistically significant decrease
in tumor volume compared to control antibody treated animals.
[0039] FIG. 10: Anti-MET Antibodies Increase Survival by Decreasing
Lung Metastases. Mice were injected with GTL-16 cells stably
expressing the luciferase (luc)-gene and treated weekly with
13-MET/28-MET antibodies at a ratio of 1:8 or control antibody
1B711. Treatment was stopped after three weeks and the disease
recurrence was measured by non-invasive imaging every week. At day
170, only one mouse in the 13-MET/28-MET-treatment group had died,
whereas in the control group six out of seven mice had died.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The term "antibody" is used to mean an immunoglobulin
molecule that recognizes and specifically binds to a target, such
as a protein, polypeptide, peptide, carbohydrate, polynucleotide,
lipid, or combinations of the foregoing through at least one
antigen recognition site within the variable region of the
immunoglobulin molecule. In certain embodiments, antibodies of the
present invention include antagonist antibodies that specifically
bind to a cancer stem cell marker protein and interfere with, for
example, ligand binding, receptor dimerization, expression of a
cancer stem cell marker protein, and/or downstream signaling of a
cancer stem cell marker protein. In certain embodiments, disclosed
antibodies include agonist antibodies that specifically bind to a
cancer stem cell marker protein and promote, for example, ligand
binding, receptor dimerization, and/or signaling by a cancer stem
cell marker protein. In certain embodiments, disclosed antibodies
do not interfere with or promote the biological activity of a
cancer stem cell marker protein but inhibit tumor growth by, for
example, antibody internalization and/or recognized by the immune
system. As used herein, the term "antibody" encompasses intact
polyclonal antibodies, intact monoclonal antibodies, antibody
fragments (such as Fab, Fab', F(ab').sub.2, and Fv fragments),
single chain Fv (scFv) mutants, multispecific antibodies such as
bispecific antibodies generated from at least two intact
antibodies, chimeric antibodies, humanized antibodies, human
antibodies, fusion proteins comprising an antigen determination
portion of an antibody, and any other modified immunoglobulin
molecule comprising an antigen recognition site so long as the
antibodies exhibit the desired biological activity. An antibody can
be of any the five major classes of immunoglobulins: IgA, IgD, IgE,
IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2,
IgG3, IgG4, IgA1 and IgA2), based on the identity of their
heavy-chain constant domains referred to as alpha, delta, epsilon,
gamma, and mu, respectively. The different classes of
immunoglobulins have different and well known subunit structures
and three-dimensional configurations. Antibodies can be naked or
conjugated to other molecules such as toxins, radioisotopes,
etc.
[0041] As used herein, the term "antibody fragment" refers to a
portion of an intact antibody and refers to the antigenic
determining variable regions of an intact antibody. Examples of
antibody fragments include, but are not limited to Fab, Fab',
F(ab')2, and Fv fragments, linear antibodies, single chain
antibodies, and multispecific antibodies formed from antibody
fragments.
[0042] An "Fv antibody" refers to the minimal antibody fragment
that contains a complete antigen-recognition and -binding site
either as two-chains, in which one heavy and one light chain
variable domain form a non-covalent dimer, or as a single-chain
(scFv), in which one heavy and one light chain variable domain are
covalently linked by a flexible peptide linker so that the two
chains associate in a similar dimeric structure. In this
configuration the complementary determining regions (CDRs) of each
variable domain interact to define the antigen-binding specificity
of the Fv dimer. Alternatively a single variable domain (or half of
an Fv) can be used to recognize and bind antigen, although
generally with lower affinity.
[0043] A "monoclonal antibody" as used herein refers to homogenous
antibody population involved in the highly specific recognition and
binding of a single antigenic determinant, or epitope. This is in
contrast to polyclonal antibodies that typically include different
antibodies directed against different antigenic determinants. The
term "monoclonal antibody" encompasses both intact and full-length
monoclonal antibodies as well as antibody fragments (such as Fab,
Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins
comprising an antibody portion, and any other modified
immunoglobulin molecule comprising an antigen recognition site.
Furthermore, "monoclonal antibody" refers to such antibodies made
in any number of manners including but not limited to by hybridoma,
phage selection, recombinant expression, and transgenic
animals.
[0044] As used herein, the term "humanized antibody" refers to
forms of non-human (e.g. rodent) antibodies that are specific
immunoglobulin chains, chimeric immunoglobulins, or fragments
thereof that contain minimal non-human sequences. Typically,
humanized antibodies are human immunoglobulins in which residues
from the complementary determining regions (CDRs) within the
antigen determination region (or hypervariable region) of the
variable region of an antibody chain or chains are replaced by
residues from the CDR of a non-human species (e.g. mouse, rat,
rabbit, hamster) that have the desired specificity, affinity, and
capability. In some instances, residues from the variable chain
framework region (FR) of a human immunoglobulin are replaced with
the corresponding residues in an antibody from a non-human species
that has the desired specificity, affinity, and capability. The
humanized antibody can be further modified by the substitution of
additional residue either in the variable framework region and/or
within the replaced non-human residues to refine and optimize
antibody specificity, affinity, and/or capability. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two or three or four, variable domains containing all
or substantially all of the CDR regions that correspond to the
non-human immunoglobulin whereas all or substantially all of the FR
regions are those of a human immunoglobulin consensus sequence. The
humanized antibody can also comprise at least a portion of an
immunoglobulin constant region or domain (Fc), typically that of a
human immunoglobulin. Examples of methods used to generate
humanized antibodies are described in U.S. Pat. No. 5,225,539.
[0045] The term "human antibody" as used herein means an antibody
produced by a human or an antibody having an amino acid sequence
corresponding to an antibody produced by a human made using any
technique known in the art. This definition of a human antibody
includes intact or full-length antibodies, fragments thereof,
and/or antibodies comprising at least one human heavy and/or light
chain polypeptide such as, for example, an antibody comprising
murine light chain and human heavy chain polypeptides.
[0046] "Hybrid antibodies" are immunoglobulin molecules in which
pairs of heavy and light chains from antibodies with different
antigenic determinant regions are assembled together so that two
different epitopes or two different antigens can be recognized and
bound by the resulting tetramer.
[0047] The term "chimeric antibodies" refers to antibodies wherein
the amino acid sequence of the immunoglobulin molecule is derived
from two or more species. Typically, the variable region of both
light and heavy chains corresponds to the variable region of
antibodies derived from one species of mammals (e.g. mouse, rat,
rabbit, etc) with the desired specificity, affinity, and capability
while the constant regions are homologous to the sequences in
antibodies derived from another (usually human) to avoid eliciting
an immune response in that species.
[0048] 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).
Preferred affinity matured antibodies will have 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 CDR and/or
framework residues is described by: Barbas et al. Proc. Nat. Acad.
Sci, USA 91:3809-3813 (1994); Schieret 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.
226389-896 (1992).
[0049] The term "epitope" or "antigenic determinant" are used
interchangeably herein and refer to that portion of an antigen
capable of being recognized and specifically bound by a particular
antibody. When the antigen is a polypeptide, epitopes can be formed
both from contiguous amino acids and noncontiguous amino acids
juxtaposed by tertiary folding of a protein. Epitopes formed from
contiguous amino acids are typically retained upon protein
denaturing, whereas epitopes formed by tertiary folding are
typically lost upon protein denaturing. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation.
[0050] Competition between antibodies is determined by an assay in
which the immunoglobulin under test inhibits specific binding of a
reference antibody to a common antigen. Numerous types of
competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay (ETA), sandwich competition assay (see
Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid phase
direct biotin-avidin EIA (see Kirkland et al., J. Immunol.
137:3614-3619 (1986)); solid phase direct labeled assay, solid
phase direct labeled sandwich assay (see Harlow and Lane,
"Antibodies, A Laboratory Manual," Cold Spring Harbor Press
(1988)); solid phase direct label RIA using 1-125 label (see Morel
et al., Molec. Immunol. 25(1):7-15 (1988)); solid phase direct
biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and
direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82
(1990)). Typically, such an assay involves the use of purified
antigen bound to a solid surface or cells bearing either of these,
an unlabeled test immunoglobulin and a labeled reference
immunoglobulin. Competitive inhibition is measured by determining
the amount of label bound to the solid surface or cells in the
presence of the test immunoglobulin. Usually the test
immunoglobulin is present in excess. Antibodies identified by
competition assay (competing antibodies) include antibodies binding
to the same epitope as the reference antibody and antibodies
binding to an adjacent epitope sufficiently proximal to the epitope
bound by the reference antibody for steric hindrance to occur.
Usually, when a competing antibody is present in excess, it will
inhibit specific binding of a reference antibody to a common
antigen by at least 50 or 75%.
[0051] That an antibody "selectively binds" or "specifically binds"
means that the antibody reacts or associates more frequently, more
rapidly, with greater duration, with greater affinity, or with some
combination of the above to an epitope than with alternative
substances, including unrelated proteins. "Selectively binds" or
"specifically binds" means, for instance, that an antibody binds to
a protein with a K.sub.D of at least about 0.1 mM, but more usually
at least about 1 .mu.M. "Selectively binds" or "specifically binds"
means at times that an antibody binds to a protein at times with a
K.sub.D of at least about 0.1 .mu.M or better, and at other times
at least about 0.01 .mu.M or better. Because of the sequence
identity between homologous proteins in different species, specific
binding can include an antibody that recognizes a cancer stem cell
marker protein in more than one species.
[0052] As used herein, the terms "non-specific binding" and
"background binding" when used in reference to the interaction of
an antibody and a protein or peptide refer to an interaction that
is not dependent on the presence of a particular structure (i.e.,
the antibody is binding to proteins in general rather that a
particular structure such as an epitope).
[0053] The terms "isolated" or "purified" refer to material that is
substantially or essentially free from components that normally
accompany it in its native state. Purity and homogeneity are
typically determined using analytical chemistry techniques such as
polyacrylamide gel electrophoresis or high performance liquid
chromatography. A protein (e.g. an antibody) or nucleic acid of the
present disclosure that is the predominant species present in a
preparation is substantially purified. In particular, an isolated
nucleic acid is separated from open reading frames that naturally
flank the gene and encode proteins other than protein encoded by
the gene. An isolated antibody is separated from other
non-immunoglobulin proteins and from other immunoglobulin proteins
with different antigen binding specificity. It can also mean that
the nucleic acid or protein is in some embodiments at least 80%
pure, in some embodiments at least 85% pure, in some embodiments at
least 90% pure, in some embodiments at least 95% pure, and in some
embodiments at least 99% pure.
[0054] As used herein, the terms "cancer" and "cancerous" refer to
or describe the physiological condition in mammals in which a
population of cells are characterized by unregulated cell growth.
Examples of cancer include, but are not limited to, carcinoma,
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,
glioblastoma, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, hepatoma, breast cancer, colon cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney cancer, liver cancer, prostate cancer, vulval cancer,
thyroid cancer, hepatic carcinoma and various types of head and
neck cancers.
[0055] The terms "proliferative disorder" and "proliferative
disease" refer to disorders associated with abnormal cell
proliferation such as cancer.
[0056] "Tumor" and "neoplasm" as used herein refer to any mass of
tissue that result from excessive cell growth or proliferation,
either benign (noncancerous) or malignant (cancerous) including
pre-cancerous lesions.
[0057] "Metastasis" as used herein refers to the process by which a
cancer spreads or transfers from the site of origin to other
regions of the body with the development of a similar cancerous
lesion at the new location. A "metastatic" or "metastasizing" cell
is one that loses adhesive contacts with neighboring cells and
migrates via the bloodstream or lymph from the primary site of
disease to invade neighboring body structures.
[0058] The terms "cancer stem cell", "tumor stem cell", or "solid
tumor stem cell" are used interchangeably herein and refer to a
population of cells from a solid tumor that: (1) have extensive
proliferative capacity; 2) are capable of asymmetric cell division
to generate one or more kinds of differentiated progeny with
reduced proliferative or developmental potential; and (3) are
capable of symmetric cell divisions for self-renewal or
self-maintenance. These properties of "cancer stem cells", "tumor
stem cells" or "solid tumor stem cells" confer on those cancer stem
cells the ability to form palpable tumors upon serial
transplantation into an immunocompromised mouse compared to the
majority of tumor cells that fail to form tumors. Cancer stem cells
undergo self-renewal versus differentiation in a chaotic manner to
form tumors with abnormal cell types that can change over time as
mutations occur. Solid tumor stem cells differ from the "cancer
stem line" provided by U.S. Pat. No. 6,004,528. In that patent, the
"cancer stem line" is defined as a slow growing progenitor cell
type that itself has few mutations but which undergoes symmetric
rather than asymmetric cell divisions as a result of tumorigenic
changes that occur in the cell's environment. This "cancer stem
line" hypothesis thus proposes that highly mutated, rapidly
proliferating tumor cells arise largely as a result of an abnormal
environment, which causes relatively normal stem cells to
accumulate and then undergo mutations that cause them to become
tumor cells. U.S. Pat. No. 6,004,528 proposes that such a model can
be used to enhance the diagnosis of cancer. The solid tumor stem
cell model is fundamentally different from the "cancer stem line"
model and as a result exhibits utilities not offered by the "cancer
stem line" model. First, solid tumor stem cells are not
"mutationally spared". The "mutationally spared cancer stem line"
described by U.S. Pat. No. 6,004,528 can be considered a
pre-cancerous lesion, while solid tumor stem cells are cancer cells
that may themselves contain the mutations that are responsible for
tumorigenesis starting at the pre-cancerous stage through later
stage cancer. That is, solid tumor stem cells ("cancer stem cells")
would be included among the highly mutated cells that are
distinguished from the "cancer stem line" in U.S. Pat. No.
6,004,528. Second, the genetic mutations that lead to cancer can be
largely intrinsic within the solid tumor stem cells as well as
being environmental. The solid tumor stem cell model predicts that
isolated solid tumor stem cells can give rise to additional tumors
upon transplantation (thus explaining metastasis) while the "cancer
stem line" model would predict that transplanted "cancer stem line"
cells would not be able to give rise to a new tumor, since it was
their abnormal environment that was tumorigenic. Indeed, the
ability to transplant dissociated, and phenotypically isolated
human solid tumor stem cells to mice (into an environment that is
very different from the normal tumor environment) where they still
form new tumors distinguishes the present invention from the
"cancer stem line" model. Third, solid tumor stem cells likely
divide both symmetrically and asymmetrically, such that symmetric
cell division is not an obligate property. Fourth, solid tumor stem
cells can divide rapidly or slowly, depending on many variables,
such that a slow proliferation rate is not a defining
characteristic.
[0059] The terms "cancer cell", "tumor cell" and grammatical
equivalents refer to the total population of cells derived from a
tumor or a pre-cancerous lesion including both non-tumorigenic
cells, which comprise the bulk of the tumor cell population, and
tumorigenic stem cells (cancer stem cells).
[0060] As used herein "tumorigenic" refers to the functional
features of a solid tumor stem cell including the properties of
self-renewal (giving rise to additional tumorigenic cancer stem
cells) and proliferation to generate all other tumor cells (giving
rise to differentiated and thus non-tumorigenic tumor cells) that
allow solid tumor stem cells to form a tumor.
[0061] As used herein, the terms "stem cell cancer marker(s)",
"cancer stem cell marker(s)", "tumor stem cell marker(s)", or
"solid tumor stem cell marker(s)" refer to a gene or genes or a
protein, polypeptide, or peptide expressed by the gene or genes
whose expression level, alone or in combination with other genes,
is correlated with the presence of tumorigenic cancer cells
compared to non-tumorigenic cells. The correlation can relate to
either an increased or decreased expression of the gene (e.g.
increased or decreased levels of mRNA or the peptide encoded by the
gene).
[0062] As used herein, the terms "biopsy" or "biopsy tissue" refer
to a sample of tissue or fluid that is removed from a subject for
the purpose of determining if the sample contains cancerous tissue.
In some embodiments, biopsy tissue or fluid is obtained because a
subject is suspected of having cancer, and the biopsy tissue or
fluid is then examined for the presence or absence of cancer.
[0063] As used herein, the term "subject" refers to any animal
(e.g., a mammal), including, but not limited to humans, non-human
primates, rodents, and the like, which is to be the recipient of a
particular treatment. Typically, the terms "subject" and "patient"
are used interchangeably herein in reference to a human
subject.
[0064] "Pharmaceutically acceptable" refers to approved or
approvable by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, including humans.
[0065] "Pharmaceutically acceptable vehicle" refers to a diluent,
adjuvant, excipient, or carrier with which at least one antibody of
the present disclosure is administered.
[0066] The term "therapeutically effective amount" refers to an
amount of an antibody, polypeptide, polynucleotide, small organic
molecule, or other drug effective to "treat" a disease or disorder
in a subject or mammal. In the case of cancer, the therapeutically
effective amount of the drug can reduce the number of cancer cells;
reduce the tumor size; inhibit or stop cancer cell infiltration
into peripheral organs including, for example, the spread of cancer
into soft tissue and bone; inhibit and stop tumor metastasis;
inhibit and stop tumor growth; relieve to some extent one or more
of the symptoms associated with the cancer, reduce morbidity and
mortality; improve quality of life; or a combination of such
effects. To the extent the drug prevents growth and/or kills
existing cancer cells, it can be referred to as cytostatic and/or
cytotoxic.
[0067] As used herein, "providing a diagnosis" or "diagnostic
information" refers to any information, including for example the
presence of cancer stern cells, that is useful in determining
whether a patient has a disease or condition and/or in classifying
the disease or condition into a phenotypic category or any category
having significance with regards to the prognosis of or likely
response to treatment (either treatment in general or any
particular treatment) of the disease or condition. Similarly,
diagnosis refers to providing any type of diagnostic information,
including, but not limited to, whether a subject is likely to have
a condition (such as a tumor), whether a subject's tumor comprises
cancer stem cells, information related to the nature or
classification of a tumor as for example a high risk tumor or a low
risk tumor, information related to prognosis and/or information
useful in selecting an appropriate treatment. Selection of
treatment can include the choice of a particular chemotherapeutic
agent or other treatment modality such as surgery or radiation or a
choice about whether to withhold or deliver therapy.
[0068] As used herein, the terms "providing a prognosis",
"prognostic information", or "predictive information" refer to
providing information, including for example the presence of cancer
stem cells in a subject's tumor, regarding the impact of the
presence of cancer (e.g., as determined by the diagnostic methods
of the present invention) on a subject's future health (e.g.,
expected morbidity or mortality, the likelihood of getting cancer,
and the risk of metastasis).
[0069] Terms such as "treating" or "treatment" or "to treat" or
"alleviating" or "to alleviate" refer to both 1) therapeutic
measures that cure, slow down, lessen symptoms of, and/or halt
progression of a diagnosed pathologic condition or disorder and 2)
prophylactic or preventative measures that prevent and/or slow the
development of a targeted pathologic condition or disorder. Thus
those in need of treatment include those already with the disorder;
those prone to have the disorder; and those in whom the disorder is
to be prevented. A subject is successfully "treated" according to
the methods of the present invention if the patient shows one or
more of the following: a reduction in the number of or complete
absence of cancer cells; a reduction in the tumor size; inhibition
of or an absence of cancer cell infiltration into peripheral organs
including, for example, the spread of cancer into soft tissue and
bone; inhibition of or an absence of tumor metastasis; inhibition
or an absence of tumor growth; relief of one or more symptoms
associated with the specific cancer; reduced morbidity and
mortality; improvement in quality of life; or some combination of
effects.
[0070] As used herein, the terms "polynucleotide" or "nucleic acid"
refer to a polymer composed of a multiplicity of nucleotide units
(ribonucleotide or deoxyribonucleotide or related structural
variants) linked via phosphodiester bonds, including but not
limited to, DNA or RNA. The term encompasses sequences that include
any of the known base analogs of DNA and RNA. including, but not
limited to, 4 acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl
2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil,
inosine, N6-isopentenyladenine, 1-methyladenine,
1-methylpseudouracil,1-methylguanine, 1-methylinosine,
2,2-dimethylgaanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N6-methyladenine,
7-methylguanine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,
queosine, 2-thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil,
4-thiouracil, 5-methyluracil, N-uracil 5-oxyacetic acid
methylester, uracil 5-oxyacetic acid, pseudouracil, queosine,
2-thiocytosine, and 2,6-diaminopurine.
[0071] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (Tm) for the specific sequence at a defined
ionic strength pH. The Tm is the temperature (under defined ionic
strength, pH, and nucleic concentration) at which 50% of the probes
complementary to the target hybridize to the target sequence at
equilibrium (as the target sequences are present in excess, at Tm,
50% of the probes are occupied at equilibrium). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. For selective or specific hybridization,
a positive signal is at least two times background, preferably 10
times background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or, 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C.
[0072] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA).
The polypeptide can be encoded by a full length coding sequence or
by any portion of the coding sequence so long as the desired
activity or functional properties (e.g., enzymatic activity, ligand
binding, signal transduction, immunogenicity, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends for a
distance of about 1 kb or more on either end such that the gene
corresponds to the length of the full-length mRNA. Sequences
located 5' of the coding region and present on the mRNA are
referred to as 5' non-translated sequences. Sequences located 3' or
downstream of the coding region and present on the mRNA are
referred to as 3.degree. non-translated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene. A genomic form
or done of a gene contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene that are
transcribed into nuclear RNA (hnRNA); introns can contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide. In addition to containing
introns, genomic forms of a gene can also include sequences located
on both the 5' and 3.degree. end of the sequences that are present
on the RNA transcript. These sequences are referred to as
"flanking" sequences or regions (these flanking sequences are
located 5' or 3' to the non-translated sequences present on the
mRNA transcript). The 5' flanking region can contain regulatory
sequences such as promoters and enhancers that control or influence
the transcription of the gene. The 3' flanking region can contain
sequences that direct the termination of transcription, post
transcriptional cleavage and polyadenylation.
[0073] The term "recombinant" when used with reference to a cell,
nucleic acid, protein or vector indicates that the cell, nucleic
acid, protein or vector has been modified by the introduction of a
heterologous nucleic acid or protein, the alteration of a native
nucleic acid or protein, or that the cell is derived from a cell so
modified. Thus, e.g., recombinant cells express genes that are not
found within the native (non-recombinant) form of the cell or
express native genes that are overexpressed or otherwise abnormally
expressed such as, for example, expressed as non-naturally
occurring fragments or splice variants. By the term "recombinant
nucleic acid" herein is meant nucleic acid, originally formed in
vitro, in general, by the manipulation of nucleic acid, e.g., using
polymerases and endonucleases, in a form not normally found in
nature. In this manner, operably linkage of different sequences is
achieved. Thus an isolated nucleic acid, in a linear form, or an
expression vector formed in vitro by ligating DNA molecules that
are not normally joined, are both considered recombinant for the
purposes of this invention. It is understood that once a
recombinant nucleic acid is made and introduced into a host cell or
organism, it will replicate non-recombinantly, i.e., using the in
vivo cellular machinery of the host cell rather than in vitro
manipulations; however, such nucleic acids, once produced
recombinantly, although subsequently replicated non-recombinantly,
are still considered recombinant for the purposes of the invention.
Similarly, a "recombinant protein" is a protein made using
recombinant techniques, i.e., through the expression of a
recombinant nucleic acid as depicted above.
[0074] As used herein, the term "heterologous gene" refers to a
gene that is not in its natural environment. For example, a
heterologous gene includes a gene from one species introduced into
another species. A heterologous gene also includes a gene native to
an organism that has been altered in some way (e.g., mutated, added
in multiple copies, linked to non-native regulatory sequences,
etc). Heterologous genes are distinguished from endogenous genes in
that the heterologous gene sequences are typically joined to DNA
sequences that are not found naturally associated with the gene
sequences in the chromosome or are associated with portions of the
chromosome not found in nature (e.g., genes expressed in loci where
the gene is not normally expressed).
[0075] As used herein, the term "vector" is used in reference to
nucleic acid molecules that transfer DNA segment(s) from one cell
to another. The term "vehicle" is sometimes used interchangeably
with "vector." Vectors are often derived from plasmids,
bacteriophages, or plant or animal viruses.
[0076] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments. Unless
otherwise provided, ligation can be accomplished using known
buffers and conditions with 10 units to T4 DNA ligase ("ligase")
per 0.5 ug of approximately equimolar amounts of the DNA fragments
to be ligated. Ligation of nucleic acid can serve to link two
proteins together in-frame to produce a single protein, or fusion
protein.
[0077] As used herein, the term "gene expression" refers to the
process of converting genetic information encoded in a gene into
RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of
the gene (e.g., via the enzymatic action of an RNA polymerase), and
for protein encoding genes, into protein through "translation" of
mRNA. Gene expression can be regulated at many stages in the
process. "Up regulation" or "activation" refers to regulation that
increases the production of gene expression products (e.g., RNA or
protein), while "down-regulation" or "repression" refers to
regulation that decrease production. Molecules (e.g., transcription
factors) that are involved in up-regulation or down-regulation are
often called "activators" and "repressors," respectively.
[0078] The terms "polypeptide," "peptide," "protein," and "protein
fragment" are used interchangeably herein to refer to a polymer of
amino acid residues. The terms apply to amino acid polymers in
which one or more amino acid residue is an artificial chemical
mimetic of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers and non-naturally
occurring amino acid polymers.
[0079] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
Amino acid analogs refers to compounds that have the same basic
chemical structure as a naturally occurring amino acid, e.g., an
alpha carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide, methionine methyl sulfonium. Such analogs can
have modified R groups (e.g., norleucine) or modified peptide
backbones, but retain the same basic chemical structure as a
naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions
similarly to a naturally occurring amino acid.
[0080] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. "Amino acid variants" refers to
amino acid sequences. With respect to particular nucleic acid
sequences, conservatively modified variants refers to those nucleic
acids which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical or associated (e.g., naturally
contiguous) sequences. Because of the degeneracy of the genetic
code, a large number of functionally identical nucleic acids encode
most proteins. For instance, the codons GCA, GCC, GCG and GCU all
encode the amino acid alanine. Thus, at every position where an
alanine is specified by a codon, the codon can be altered to
another of the corresponding codons described without altering the
encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified
variations. Every nucleic acid sequence herein which encodes a
polypeptide also describes silent variations of the nucleic acid.
One of skill will recognize that in certain contexts each codon in
a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, silent variations of a nucleic acid which
encodes a polypeptide is implicit in a described sequence with
respect to the expression product, but not with respect to actual
probe sequences. As to amino acid sequences, one of skill will
recognize that individual substitutions, deletions or additions to
a nucleic acid, peptide, polypeptide, or protein sequence which
alters, adds or deletes a single amino acid or a small percentage
of amino acids in the encoded sequence is a "conservatively
modified variant" including where the alteration results in the
substitution of an amino acid with a chemically similar amino acid.
Conservative substitution tables providing functionally similar
amino acids are well known in the art. Such conservatively modified
variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles of the invention.
Typically conservative substitutions include: 1) Alanine (A),
Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine
(N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0081] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptides, refer to two or more
sequences or subsequences that are the same or have a specified
percentage of nucleotides or amino acid residues that are the same,
when compared and aligned for maximum correspondence, as measured
using a sequence comparison algorithm such as those described below
for example, or by visual inspection.
[0082] In some embodiments, two nucleic acids or polypeptides of
the invention are substantially identical, meaning they have at
least 70%, at least 75%, preferably at least 80%, more preferably
at least 85%, more preferably at least 90%, and in some embodiments
at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue
identity, when compared and aligned for maximum correspondence, as
measured using a sequence comparison algorithm or by visual
inspection. Preferably, identity exists over a region of the
sequences that is at least about 10, preferably about 20, more
preferable about 40-60 residues in length or any integral value
therebetween, preferably over a longer region than 60-80 residues,
more preferably at least about 90-100 residues, and most preferably
the sequences are substantially identical over the full length of
the sequences being compared, such as the coding region of a
nucleotide sequence for example.
[0083] The term "epitope tagged" as used herein refers to a
chimeric polypeptide comprising a cancer stem cell marker protein,
or a domain sequence or portion thereof, fused to an "epitope tag".
The epitope tag polypeptide comprises enough amino acid residues to
provide an epitope for recognition by an antibody, yet is short
enough such that it does not interfere with the activity of the
cancer stem cell marker protein. Suitable epitope tags generally
have at least six amino acid residues, usually between about 8 to
about 50 amino acid residues, and at times between about 10 to
about 20 residues. Commonly used epitope tags include Fc, HA, H is,
and FLAG tags.
[0084] The present invention provides compositions and methods for
studying, diagnosing, characterizing, and treating cancer. In
particular, the present invention provides antibodies against solid
tumor stem cell markers and methods of using these antibodies to
inhibit tumor growth and treat cancer in human patients. In certain
embodiments, antibodies of the present invention include antagonist
antibodies that specifically bind to a cancer stem cell marker
protein and interfere with, for example, ligand binding, receptor
dimerization, expression of a cancer stem cell marker protein,
and/or signaling of a cancer stem cell marker protein. In certain
embodiments, disclosed antibodies include agonist antibodies that
specifically bind to a cancer stem cell marker protein and promote,
for example, ligand binding, receptor dimerization, and/or
signaling by a cancer stem cell marker protein. In certain
embodiments, disclosed antibodies do not interfere with or promote
the biological activity of a cancer stem cell marker protein but
inhibit tumor growth by, for example, internalization and/or
recognized by the immune system. In certain embodiments, the
antibodies specifically recognize more than one solid tumor tem
cells marker protein.
[0085] Like the tissues in which they originate, solid tumors
consist of a heterogeneous population of cells. That the majority
of these cells lack tumorigenicity suggested that the development
and maintenance of solid tumors also relies on a small population
of stem cells (i.e., tumorigenic cancer cells) with the capacity to
proliferate and efficiently give rise both to additional tumor stem
cells (self-renewal) and to the majority of more differentiated
tumor cells that lack tumorigenic potential (i.e., non-tumorigenic
cancer cells). The concept of cancer stem cells was first
introduced soon after the discovery of hematopoietic stem cells
(HSC) and was established experimentally in acute myelogenous
leukemia (AML) (Park et al., 1971, J. Natl. Cancer Inst. 46:411-22;
Lapidot et al., 1994, Nature 367:645-8; Bonnet & Dick, 1997,
Nat. Med. 3:730-7; Hope et al., 2004, Nat. Immunol. 5:738-43). Stem
cells from solid tumors have more recently been isolated based on
their expression of a unique pattern of cell-surface receptors and
on the assessment of their properties of self-renewal and
proliferation in culture and in xenograft animal models. An ESA+
CD44+ CD24-/low Lineage-population greater than 50-fold enriched
for the ability to form tumors relative to unfractionated tumor
cells was discovered (Al-Hajj et al., 2003, Proc. Nat'l. Acad. Sci.
100:3983-8). The ability to isolate tumorigenic cancer stem cells
from the bulk of non-tumorigenic tumor cells has led to the
identification of cancer stem cell markers, genes with differential
expression in cancer stem cells compared to non-tumorigenic tumor
cells or normal breast epithelium, using microarray analysis. The
present invention employs the knowledge of these identified cancer
stem cell markers to diagnosis and treat cancer.
[0086] The cancer stem cell marker of the present invention relates
to the human MET Receptor (SEQ ID NOs: 26 and 27). MET is a
receptor tyrosine kinase which has mitogenic and morphogenic
activities that are activated by the mesenchyme-derived
pleiotrophic factor, hepatocyte growth factor (HGF) (SEQ ID NOs 28
and 29). Aberrant HGF and MET expression are frequently observed in
a variety of tumors, see, e.g., Maulik et al., Cytokine &
Growth Factor Reviews (2002), 13:41-59; Danilkov'tch-Miagkova &
Zbar, J. Clin. Invest. (2002), 109(7):863-867, and regulation of
the HGF/c-Met signaling pathway is implicated in tumor progression
and metastasis. See Trusolino & Comoglio, Nature Rev. (2002),
2:289-300.
[0087] HGF/MET signaling regulates a diverse array of biological
processes, including cell scattering, proliferation, and survival.
Signaling is essential for normal embryonic development especially
in migration of muscle progenitor cells and development of the
liver and nervous system (Bladt et al., 1995; Hamanoue et al.,
1996; Maina et al., 1996; Schmidt et al., 1995; Uehara et al.,
1995). Developmental phenotypes of Met and HGF knockout mice are
very similar suggesting that HGF is the cognate ligand for the MET
receptor (Schmidt et al., 1995; Uehara et al., 1995). HGF-Met also
plays a role in liver regeneration, angiogenesis, and wound healing
(Bussolino et al., 1992; Matsumoto & Nakamura, 1993; Nusrat et
al., 1994). Upon HGF binding, activation of MET leads to tyrosine
phosphorylation and downstream signaling through Gab1 and Grb2/Sos
mediated P13-kinase and Ras/MAPK activation respectively, which
drives cell motility and proliferation (Furge et al., 2000;
Hartmann et al., 1994; Ponzetto et al., 1996; Royal and Park,
1995).
[0088] Met was shown to be transforming in a carcinogen-treated
osteosarcoma cell line (Cooper et al., 1984; Park et al., 1986).
MET overexpression or gene-amplification has been observed in a
variety of human cancers. For example, MET protein is overexpressed
at least 5-fold in colorectal cancers and reported to be
gene-amplified in liver metastasis (Di Renzo et al., 1995; Liu et
al., 1992). MET protein is also reported to be overexpressed in
oral squamous cell carcinoma, hepatocellular carcinoma, renal cell
carcinoma, breast carcinoma, and lung carcinoma (Jin et al., 1997;
Morello et al., 2001; Natali et al., 1996; Olivero et al., 1996;
Suzuki et al., 1994). In addition, overexpression of Met mRNA has
been observed in hepatocellular carcinoma, gastric carcinoma, and
colorectal carcinoma (Boix et al., 1994; Kuniyasu et al., 1993; Liu
et al., 1992).
[0089] A number of mutations in the kinase domain of MET have been
found in renal papillary, carcinoma which leads to constitutive
receptor activation (Olivero et al., 1999; Schmidt et al., 1997;
Schmidt et al., 1999). These activating mutations confer
constitutive MET tyrosine phosphorylation and result in MAPK
activation, focus formation, and tumorigenesis (Jeffers et al.,
1997), In addition, these mutations enhance cell motility and
invasion (Giordano et al., 2000; Lorenzato et al., 2002).
HGF-dependent MET activation in transformed cells mediates
increased motility, scattering, and migration which eventually
leads to invasive tumor growth and metastasis (Jeffers et al.,
1996; Meiners et al., 1998).
[0090] MET has been shown to interact with other proteins that
drive receptor activation, transformation, and invasion. In
neoplastic cells, MET is reported to interact with alpha6beta4
integrin, a receptor for extracellular matrix (ECM) components such
as laminins, to promote HGF-dependent invasive growth (Trusolino et
al., 2001). In addition, the extracellular domain of MET has been
shown to interact with a member of the semaphorin family, plexin
B1, and to enhance invasive growth (Giordano et al., 2002).
Furthermore, CD44v6, which has been implicated in tumorigenesis and
metastasis, is also reported to form a complex with MET and HGF and
result in MET receptor activation (Orian-Rousseau et al.,
2002).
[0091] The extracellular domain structure of MET suggests it shares
homology with the semaphorins and plexins: the N-terminus of MET
contains a Sema domain of approximately 500 amino acids that is
conserved in all semaphorins and plexins. The MET Sema domain is
sufficient for HGF and heparin binding, (Gherardi et al., 2003),
and is necessary for receptor dimerization and activation. (Cancer
Cell (2004), 6:61-73).
[0092] Numerous molecules targeted at the HGF/MET pathway have been
reported. These molecules include antibodies such as those
described in U.S. Pat. Nos. 5,686,292; 6,214,344; 6,468,529;
5,233,960; and 6,134,104. A divalent antibody 5D5, which inhibits
HGF binding to MET, is reported to be a potent MET activator, while
a monovalent 5D5 antibody is a MET antagonist. (Schwall et al.,
AACR Meeting Abstract, 2004). The identification of MET antagonists
suitable for development as therapeutic agents remains a continual
challenge.
[0093] As described herein, the identification of MET expression in
cancer stem cells suggested targeting MET to eliminate not only the
majority of non-tumorigenic cancer cells, but also the tumorigenic
cells responsible for the formation and reoccurrence of solid
tumors. Thus, the present invention provides a cancer stem cell
marker, the expression of which can be analyzed to diagnosis or
monitor a disease associated with cancer and to provide
therapeutics for the treatment of cancer.
[0094] In certain embodiments, antibodies against the cancer stem
cell marker MET are provided. In the context of the present
invention, a suitable antibody is an agent that can have one or
more of the following effects, for example: interfere with the
expression of a cancer stem cell marker; interfere with activation
of a cancer stem cell signal transduction pathway by, for example,
sterically inhibiting interactions between a cancer stem cell
marker and its ligand, receptor or co-receptors; activate a cancer
stem cell signal transduction pathway by, for example, acting as a
ligand or promoting the binding of an endogenous ligand; or bind to
a cancer stem cell marker and inhibit tumor cell proliferation.
[0095] In certain embodiments, a combination of at least two
different antibodies are administered to interfere with MET
receptor signaling. Combinations of antibodies that bind to
different epitopes synergistically block MET receptor functioning.
The synergistic activity results from one antibody facilitating the
binding of the second antibody to the MET receptor. In certain
embodiments, the first antibody causes a change in the conformation
of the antigen thereby giving the second antibody greater access to
bind its correlate epitope.
[0096] In certain embodiments, antibodies against a cancer stem
cell marker act extracellularly to modulate the function of a
cancer stem cell marker protein. In some embodiments, extracellular
binding of an antibody against a cancer stem cell marker can
inhibit the signaling of a cancer stem cell marker protein by, for
example, inhibiting intrinsic activation (e.g. kinase activity) of
a cancer stem cell marker and/or by sterically inhibiting the
interaction, for example, of a cancer stem cell marker with its
ligand, with its receptor, with a co-receptor, or with the
extracellular matrix. In some embodiments, extracellular binding of
an antibody against a cancer stem cell marker can down-regulate
cell-surface expression of a cancer stem cell marker such as, for
example, by internalization of a cancer stem cell marker protein or
decreasing cell surface trafficking of a cancer stem cell marker.
In some embodiments, extracellular binding of an antibody against a
cancer stem cell marker can promote the signaling of a cancer stem
cell marker protein by, for example, acting as a decoy ligand or
increasing ligand binding.
[0097] In certain embodiments, antibodies against a cancer stem
cell marker bind to a cancer stem cell marker protein and have one
or more of the following effects: inhibit proliferation of tumor
cells, trigger cell death of tumor cells, or prevent metastasis of
tumor cells. In certain embodiments, antibodies against a cancer
stem cell marker trigger cell death via a conjugated toxin,
chemotherapeutic agent, radioisotope, or other such agent. For
example, an antibody against a cancer stem cell marker is
conjugated to a toxin that is activated in tumor cells expressing
the cancer stem cell marker by protein internalization.
[0098] In certain embodiments, antibodies against a cancer stem
cell marker mediate cell death of a cell expressing the cancer stem
cell marker protein via antibody-dependent cellular cytotoxicity
(ADCC). ADCC involves cell lysis by effector cells that recognize
the Fc portion of an antibody. Many lymphocytes, monocytes, tissue
macrophages, granulocytes and eosinophiles, for example, have Fc
receptors and can mediate cytolysis (Dillman, 1994, J. Clin. Oncol.
12:1497).
[0099] In certain embodiments, antibodies against a cancer stem
cell marker trigger cell death of a cell expressing a cancer stern
cell marker protein by activating complement-dependent cytotoxicity
(CDC). CPC involves binding of serum complement to the Fc portion
of an antibody and subsequent activation of the complement protein
cascade, resulting in cell membrane damage and eventual cell death.
Biological activity of antibodies is known to be determined, to a
large extent, by the constant domains or Fc region of the antibody
molecule (Uananue and Benacerraf, Textbook of Immunology, 2nd
Edition, Williams & Wilkins, p. 218 (1984)). Antibodies of
different classes and subclasses differ in this respect, as do
antibodies of the same subclass but from different species. Of
human antibodies, IgM is the most efficient class of antibodies to
bind complement, followed by IgG1, IgG3, and IgG2 whereas IgG4
appears quite deficient in activating the complement cascade
(Dillman, 1994, J. Clin, Oncol. 12:1497; Jefferis et al., 1998.
Immunol. Rev. 163:59-76). According to the present invention,
antibodies of those classes having the desired biological activity
are prepared.
[0100] The ability of any particular antibody against a cancer stem
cell to mediate lysis of the target cell by complement activation
and/or ADCC can be assayed. The cells of interest are grown and
labeled in vitro; the antibody is added to the cell culture in
combination with either serum complement or immune cells which can
be activated by the antigen antibody complexes. Cytolysis of the
target cells is detected, for example, by the release of label from
the lysed cells. In fact, antibodies can be screened using the
patient's own serum as a source of complement and/or immune cells.
The antibody that is capable of activating complement or mediating
ADCC in the in vitro test can then be used therapeutically in that
particular patient.
[0101] In certain embodiments, antibodies against a cancer stem
cell marker can trigger cell death inhibiting angiogenesis.
Angiogenesis is the process by which new blood vessels form from
pre-existing vessels and is a fundamental process required for
normal growth, for example, during embryonic development, wound
healing, and in response to ovulation. Solid tumor growth larger
than 1-2 mm.sup.2 also requires angiogenesis to supply nutrients
and oxygen without which tumor cells die. In certain embodiments,
an antibody against a cancer stem cell marker targets vascular
cells that express the cancer stem cell marker including, for
example, endothelial cells, smooth muscle cells, or components of
the extracellular matrix required for vascular assembly. In certain
embodiments, an antibody against a cancer stem cell marker inhibits
growth factor signaling required by vascular cell recruitment,
assembly, maintenance, or survival.
[0102] The antibodies against a cancer stem cell marker find use in
the diagnostic and therapeutic methods described herein. In certain
embodiments, the antibodies of the present invention are used to
detect the expression of a cancer stem cell marker protein in
biological samples such as, for example, a patient tissue biopsy,
pleural effusion, or blood sample. Tissue biopsies can be sectioned
and protein detected using, for example, immunofluorescence or
immunohistochemistry. In addition, individual cells from a sample
can be isolated, and protein expression detected on fixed or live
cells by FACS analysis. In certain embodiments, antibodies can be
used on protein arrays to detect expression of a cancer stem cell
marker, for example, on tumor cells, in cell lysates, or in other
protein samples. In certain embodiments, the antibodies of the
present invention are used to inhibit the growth of tumor cells by
contacting the antibodies with tumor cells in in vitro cell based
assays, in vivo animal models, etc. In certain embodiments, the
antibodies are used to treat cancer in a patient by administering a
therapeutically effective amount of an antibody against a cancer
stem cell marker.
[0103] The antibodies of the invention can be prepared by any
conventional means known in the art. For example, polyclonal
antibodies can be prepared by immunizing an animal (e.g. a rabbit,
rat, mouse, donkey, etc) by multiple subcutaneous or
intraperitoneal injections of the relevant antigen (a purified
peptide fragment, full-length recombinant protein, fusion protein,
etc) optionally conjugated to keyhole limpet hemocyanin (KLH),
serum albumin, etc. diluted in sterile saline and combined with an
adjuvant (e.g. Complete or Incomplete Freund's Adjuvant) to form a
stable emulsion. The polyclonal antibody is then recovered from
blood, ascites and the like, of an animal so immunized. Collected
blood is clotted, and the serum decanted, clarified by
centrifugation, and assayed for antibody titer. The polyclonal
antibodies can be purified from serum or ascites according to
standard methods in the art including affinity chromatography,
ion-exchange chromatography, gel electrophoresis, dialysis,
etc.
[0104] Monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein (1975)
Nature 256:495. Using the hybridoma method, a mouse, hamster, or
other appropriate host animal, is immunized as described above to
elicit the production by lymphocytes of antibodies that will
specifically bind to an immunizing antigen. Lymphocytes can also be
immunized in vitro. Following immunization, the lymphocytes are
isolated and fused with a suitable myeloma cell line using, for
example, polyethylene glycol, to form hybridoma cells that can then
be selected away from unfused lymphocytes and myeloma cells.
Hybridomas that produce monoclonal antibodies directed specifically
against a chosen antigen as determined by immunoprecipitation,
immunoblotting, or by an in vitro binding assay (e.g.
radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA))
can then be propagated either in vitro culture using standard
methods (Goding, Monoclonal Antibodies: Principles and Practice,
Academic Press, 1986) or in vivo as ascites tumors in an animal.
The monoclonal antibodies can then be purified from the culture
medium or ascites fluid as described for polyclonal antibodies
above.
[0105] Alternatively monoclonal antibodies can also be made using
recombinant DNA methods as described in U.S. Pat. No. 4,816,567.
The polynucleotides encoding a monoclonal antibody are isolated
from mature B-cells or hybridoma cell, such as by RT-PCR using
oligonucleotide primes that specifically amplify the genes encoding
the heavy and light chains of the antibody, and their sequence is
determined using conventional procedures. The isolated
polynucleotides encoding the heavy and light chains are then cloned
into suitable expression vectors, which when transfected into host
cells such as E. coli cells, simian COS cells, Chinese hamster
ovary (CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, monoclonal antibodies are generated by the
host cells. Also, recombinant monoclonal antibodies or fragments
thereof of the desired species can be isolated from phage display
libraries expressing CDRs of the desired species as described
(McCafferty et al., 1990, Nature, 348:552-554; Clackson et al.,
1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,
222:581-597).
[0106] The polynucleotide(s) encoding a monoclonal antibody can
further be modified in a number of different manners using
recombinant DNA technology to generate alternative antibodies. In
some embodiments, the constant domains of the light and heavy
chains of, for example, a mouse monoclonal antibody can be
substituted 1) for those regions of, for example, a human antibody
to generate a chimeric antibody or 2) for a non-immunoglobulin
polypeptide to generate a fusion antibody. In some embodiments, the
constant regions are truncated or removed to generate the desired
antibody fragment of a monoclonal antibody. Site-directed or
high-density mutagenesis of the variable region can be used to
optimize specificity, affinity, etc. of a monoclonal antibody.
[0107] In some embodiments of the present invention, the monoclonal
antibody against the cancer stem cell marker is a humanized
antibody. Humanized antibodies are antibodies that contain minimal
sequences from non-human (e.g rodent) antibodies within the antigen
determination or hypervariable region that comprise the three
complementary determination regions (CDRs) within each antibody
chain. Such antibodies are used therapeutically to reduce
antigenicity and HAMA (human anti-mouse antibody) responses when
administered to a human subject. In practice, humanized antibodies
are typically human antibodies with minimum to virtually no
non-human sequences. A human antibody is an antibody produced by a
human or an antibody having an amino acid sequence corresponding to
an antibody produced by a human.
[0108] Humanized antibodies can be produced using various
techniques known in the art. An antibody can be humanized by
substituting the CDRs of a human antibody with those of a non-human
antibody (e.g. mouse, rat, rabbit, hamster, etc.) having the
desired specificity, affinity, and capability following the methods
of Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988,
Nature, 332:323-327; Verhoeyen et al., 1988, Science,
239:1534-1536. The humanized antibody can be further modified by
the substitution of additional residue either in the variable human
framework region and/or within the replaced non-human residues to
refine and optimize antibody specificity, affinity, and/or
capability.
[0109] The choice of human heavy and/or light chain variable
domains to be used in making humanized antibodies can be important
for reducing antigenicity. According to the "best-fit" method, the
sequence of the variable domain of a rodent antibody is screened
against the entire library of known human variable-domain amino
acid sequences. Thus in certain embodiments, the human amino acid
sequence which is most homologous to that of the rodent antibody
from which the CDRs are taken is used as the human framework region
(FR) for the humanized antibody (Sims et al., 1993, J. Immunol.,
151: 2296; Chothia et al., 1987, J. Mol. Biol., 196: 901). Another
method uses a particular FR derived from the consensus sequence of
all human antibodies of a particular subgroup of light or heavy
chains and can be used for several difference humanized antibodies
(Carter et al., 1992, PNAS, 89; 4285; Presta et al., 1993, J.
Immunol., 151: 2623). In certain embodiments, a combination of
methods is used to pick the human variable FR to use in generation
of humanized antibodies.
[0110] It is further understood that antibodies (e.g. rodent) to be
humanized must retain high affinity for the antigen as well as
other favorable biological properties. To achieve this goal,
humanized antibodies can be prepared by a process of analysis of
the parental sequence from the rodent antibody to be humanized and
the various candidate humanizing sequences. Three-dimensional
immunoglobulin models are available and familiar to those skilled
in the art. Computer programs can be used to illustrate and display
probable three-dimensional conformational structures of selected
candidate antibody sequences. Use of such models permits analysis
of the likely role of the residues in the function of the antibody
to be humanized, i.e., the analysis of residues that influence the
ability of the candidate antibody to bind its antigen. In this way,
FR residues can be selected and combined from the parental antibody
to the recipient humanized antibody so that the desired antibody
characteristics are achieved. In general, the residues in the CDRs
of the antigen determination region (or hypervariable region) are
retained from the parental antibody (e.g. the rodent antibody with
the desired antigen binding properties) in the humanized antibody
for antigen binding. In certain embodiments, at least one
additional residue within the variable FR is retained from the
parental antibody in the humanized antibody. In certain
embodiments, up to six additional residues within the variable FR
are retained from the parental antibody in the humanized
antibody.
[0111] Amino acids from the variable regions of the mature heavy
and light chains of immunoglobulins are designated Hx and Lx
respectively, where x is a number designating the position of an
amino acid according to the scheme of Kabat, Sequences of Proteins
of Immunological Interest, U.S. Department of Health and Human
Services, 1987, 1991. Kabat lists many amino acid sequences for
antibodies for each subgroup, and lists the most commonly occurring
amino acid for each residue position in that subgroup to generate a
consensus sequence. Kabat uses a method for assigning a residue
number to each amino acid in a listed sequence, and this method for
assigning residue numbers has become standard in the field. Kabat's
scheme is extendible to other antibodies not included in his
compendium by aligning the antibody in question with one of the
consensus sequences in Kabat by reference to conserved amino acids.
The use of the Kabat numbering system readily identifies amino
acids at equivalent positions in different antibodies. For example,
an amino acid at the L50 position of a human antibody occupies the
equivalent position to an amino acid position L50 of a mouse
antibody. Moreover, any two antibody sequences can be uniquely
aligned, for example to determine percent identity, by using the
Kabat numbering system so that each amino acid in one antibody
sequence is aligned with the amino acid in the other sequence that
has the same Kabat number. In some embodiments, after alignment, if
a subject antibody region (e.g., the entire mature variable region
of a heavy or light chain) is being compared with the same region
of a reference antibody, the percentage sequence identity between
the subject and reference antibody regions is the number of
positions occupied by the same amino acid in both the subject and
reference antibody region divided by the total number of aligned
positions of the two regions, with gaps not counted, multiplied by
100 to convert to percentage.
[0112] In addition to humanized antibodies, fully human antibodies
can be directly prepared using various techniques known in the art.
Immortalized human B lymphocytes immunized in vitro or isolated
from an immunized individual that produce an antibody directed
against a target antigen can be generated (See, e.g., Cole et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77
(1985); Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S.
Pat. No. 5,750,373). Also, the human antibody can be selected from
a phage library, where that phage library expresses human
antibodies (Vaughan et al., 1996, Nat. Biotech., 14:309-314; Sheets
et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162; Hoogenboom and
Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol.
Biol., 222:581). Human antibodies can also be made in transgenic
mice containing human immunoglobulin loci that are capable upon
immunization of producing the full repertoire of human antibodies
in the absence of endogenous immunoglobulin production. This
approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016.
[0113] This invention also encompasses bispecific antibodies that
specifically recognize a cancer stem cell marker. Bispecific
antibodies are antibodies that are capable of specifically
recognizing and binding at least two different epitopes (See, e.g.,
Wu et al., Simultaneous Targeting of Multiple Disease Mediators by
a Dual-Variable-Domain Immunoglobulin, Nature Biotech.,
25(11):1290-97). The different epitopes can either be within the
same molecule (e.g. the same cancer stem cell marker polypeptide)
or on different molecules such that both, for example, can
specifically recognize and bind a cancer stem cell marker as well
as, for example, 1) an effector molecule on a leukocyte such as a
T-cell receptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or
CD16) or 2) a cytotoxic agent as described in detail below.
Bispecific antibodies can be intact antibodies or antibody
fragments.
[0114] Exemplary bispecific antibodies can bind to two different
epitopes, at least one of which originates in a polypeptide of the
invention. Alternatively, an anti-antigenic arm of an
immunoglobulin molecule can be combined with an arm which binds to
a triggering molecule on a leukocyte such as a T cell receptor
molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG so
as to focus cellular defense mechanisms to the cell expressing the
particular antigen. Bispecific antibodies can also be used to
direct cytotoxic agents to cells which express a particular
antigen. These antibodies possess an antigen-binding arm and an arm
which binds a cytotoxic agent or a radionuclide chelator, such as
EOTUBE, DPTA, DOTA, or TETA. Techniques for making bispecific
antibodies are common in the art (Millstein et al., 1983, Nature
305:537-539; Brennan et al., 1985, Science 229:81; Suresh et al,
1986, Methods in Enzymol. 121:120; Traunecker et al., 1991, EMBO J.
10:3655-3659; Shalaby et al., 1992, J. Exp. Med. 175:217-225;
Kostelny et al., 1992, J. Immunol. 148:1547-1553; Gruber et al.,
1994, J. Immunol. 152:5368; and U.S. Pat. No. 5,731,168).
Antibodies with more than two valencies are also contemplated. For
example, trispecific antibodies can be prepared (Tutt et al., J.
Immunol. 147:60 (1991))
[0115] In certain embodiments are provided an antibody fragment to,
for example, increase tumor penetration. Various techniques are
known for the production of antibody fragments: Traditionally,
these fragments are derived via proteolytic digestion of intact
antibodies (for example Morimoto et al., 1993, Journal of
Biochemical and Biophysical Methods 24:107-117; Brennan et al.,
1985, Science, 229:81). In certain embodiments, antibody fragments
are produced recombinantly. Fab, Fv, and scFv antibody fragments
can all be expressed in and secreted from E. coli or other host
cells, thus allowing the production of large amounts of these
fragments. Such antibody fragments can also be isolated from the
antibody phage libraries discussed above. The antibody fragment can
also be linear antibodies as described in U.S. Pat. No. 5,641,870,
for example, and can be monospecific or bispecific. Other
techniques for the production of antibody fragments will be
apparent to the skilled practitioner.
[0116] According to the present invention, techniques can be
adapted for the production of single-chain antibodies specific to a
polypeptide of the invention (see U.S. Pat. No. 4,946,778). In
addition, methods can be adapted for the construction of Fab
expression libraries (Huse, et al., Science 246:1275-1281 (1989))
to allow rapid and effective identification of monoclonal Fab
fragments with the desired specificity for the MET receptor, or
derivatives, fragments, or homologs thereof. Antibody fragments
that contain the idiotypes to a polypeptide of the invention may be
produced by techniques in the art including, but not limited to:
(a) an F(ab')2 fragment produced by pepsin digestion of an antibody
molecule; (b) an Fab fragment generated by reducing the disulfide
bridges of an F(ab')2 fragment, (c) an Fab fragment generated by
the treatment of the antibody molecule with papain and a reducing
agent, and (d) Fv fragments.
[0117] It can further be desirable, especially in the case of
antibody fragments, to modify an antibody in order to increase its
serum half-life. This can be achieved, for example, by
incorporation of a salvage receptor binding epitope into the
antibody fragment by mutation of the appropriate region in the
antibody fragment or by incorporating the epitope into a peptide
tag that is then fused to the antibody fragment at either end or in
the middle (e.g., by DNA or peptide synthesis).
[0118] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune cells to unwanted cells (U.S. Pat.
No. 4,676,980). It is contemplated that the antibodies can be
prepared in vitro using known methods in synthetic protein
chemistry, including those involving crosslinking agents. For
example, immunotoxins can be constructed using a disulfide exchange
reaction or by forming a thioether bond. Examples of suitable
reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate.
[0119] For the purposes of the present invention, it should be
appreciated that modified antibodies can comprise any type of
variable region that provides for the association of the antibody
with the polypeptides of human MET. In this regard, the variable
region may comprise or be derived from any type of mammal that can
be induced to mount a humoral response and generate immunoglobulins
against the desired tumor associated antigen. As such, the variable
region of the modified antibodies can be, for example, of human,
murine, non-human primate (e.g. cynomolgus monkeys, macaques, etc.)
or lupine origin. In some embodiments both the variable and
constant regions of the modified immunoglobulins are human. In
other embodiments the variable regions of compatible antibodies
(usually derived from a non-human source) can be engineered or
specifically tailored to improve the binding properties or reduce
the immunogenicity of the molecule. In this respect, variable
regions useful in the present invention can be humanized or
otherwise altered through the inclusion of imported amino acid
sequences.
[0120] The variable domains in both the heavy and light chains are
altered by at least partial replacement of one or more CDRs and, if
necessary, by partial framework region replacement and sequence
changing. Although the CDRs may be derived from an antibody of the
same class or even subclass as the antibody from which the
framework regions are derived, it is envisaged that the CDRs will
be derived from an antibody of different class and preferably from
an antibody from a different species. It may not be necessary to
replace all of the CDRs with the complete CDRs from the donor
variable region to transfer the antigen binding capacity of one
variable domain to another. Rather, it may only be necessary to
transfer those residues that are necessary to maintain the activity
of the antigen binding site. Given the explanations set forth in
U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well
within the competence of those skilled in the art, either by
carrying out routine experimentation or by trial and error testing
to obtain a functional antibody with reduced immunogenicity.
[0121] Alterations to the variable region notwithstanding, those
skilled in the art will appreciate that the modified antibodies of
this invention will comprise antibodies, or immunoreactive
fragments thereof, in which at least a fraction of me or more of
the constant region domains has been deleted or otherwise altered
so as to provide desired biochemical characteristics such as
increased tumor localization or reduced serum half-life when
compared with an antibody of approximately the same immunogenicity
comprising a native or unaltered constant region. In some
embodiments, the constant region of the modified antibodies will
comprise a human constant region. Modifications to the constant
region compatible with this invention comprise additions, deletions
or substitutions of one or more amino acids in one or more domains.
That is, the modified antibodies disclosed herein may comprise
alterations or modifications to one or more of the three heavy
chain constant domains (CH 1, CH2 or CH3) and/or to the light chain
constant domain (CL). In some embodiments of the invention modified
constant regions wherein one or more domains are partially or
entirely deleted are contemplated. In some embodiments the modified
antibodies will comprise domain deleted constructs or variants
wherein the entire CH2 domain has been removed (.DELTA.CH2
constructs). In some embodiments the omitted constant region domain
will be replaced by a short amino acid spacer (e.g. 10 residues)
that provides some of the molecular flexibility typically imparted
by the absent constant region.
[0122] Besides their configuration, it is known in the art that the
constant region mediates several effector functions. For example,
binding of the C1 component of complement to antibodies activates
the complement system. Activation of complement is important in the
opsonisation and lysis of cell pathogens. The activation of
complement also stimulates the inflammatory response and can also
be involved in autoimmune hypersensitivity. Further, antibodies
bind to cells via the Fc region, with a Fc receptor site on the
antibody Fc region binding to a Fc receptor (FcR) on a cell, There
are a number of Fc receptors which are specific for different
classes of antibody, including IgG (gamma receptors), IgE (eta
receptors), IgA (alpha receptors) and IgM (mu receptors). Binding
of antibody to Fc receptors on cell surfaces triggers a number of
important and diverse biological responses including engulfment and
destruction of antibody-coated particles, clearance of immune
complexes, lysis of antibody-coated target cells by killer cells
(called antibody-dependent cell-mediated cytotoxicity, or ADCC),
release of inflammatory mediators, placental transfer and control
of immunoglobulin production. Although various Fc receptors and
receptor sites have been studied to a certain extent, there is
still much which is unknown about their location, structure and
functioning.
[0123] While not limiting the scope of the present invention, it is
believed that antibodies comprising constant regions modified as
described herein provide for altered effector functions that, in
turn, affect the biological profile of the administered antibody.
For example, the deletion or inactivation (through point mutations
or other means) of a constant region domain may reduce Fc receptor
binding of the circulating modified antibody thereby increasing
tumor localization. In other cases it may be that constant region
modifications, consistent with this invention, moderate complement
binding and thus reduce the serum half life and nonspecific
association of a conjugated cytotoxin. Yet other modifications of
the constant region may be used to eliminate disulfide linkages or
oligosaccharide moieties that allow for enhanced localization due
to increased antigen specificity or antibody flexibility.
Similarly, modifications to the constant region in accordance with
this invention may easily be made using well known biochemical or
molecular engineering techniques well within the purview of the
skilled artisan.
[0124] It will be noted that the modified antibodies may be
engineered to fuse the CH3 domain directly to the hinge region of
the respective modified antibodies. In other constructs it may be
desirable to provide a peptide spacer between the hinge region and
the modified CH2 and/or CH3 domains. For example, compatible
constructs could be expressed wherein the CH2 domain has been
deleted and the remaining CH3 domain (modified or unmodified) is
joined to the hinge region with a 5-20 amino acid spacer. Such a
spacer may be added, for instance, to ensure that the regulatory
elements of the constant domain remain free and accessible or that
the hinge region remains flexible. However, it should be noted that
amino acid spacers can, in some cases, prove to be immunogenic and
elicit an unwanted immune response against the construct.
Accordingly, any spacer added to the construct be relatively
non-immunogenic or, even omitted altogether if the desired
biochemical qualities of the modified antibodies may be
maintained.
[0125] Besides the deletion of whole constant region domains, it
will be appreciated that the antibodies of the present invention
may be provided by the partial deletion or substitution of a few or
even a single amino acid. For example, the mutation of a single
amino acid in selected areas of the CH2 domain may be enough to
substantially reduce Fc binding and thereby increase tumor
localization. Similarly, it may be desirable to simply delete that
part of one or more constant region domains that control the
effector function (e.g. complement CLQ binding) to be modulated.
Such partial deletions of the constant regions may improve selected
characteristics of the antibody (serum half-life) while leaving
other desirable functions associated with the subject constant
region domain intact. Moreover, as alluded to above, the constant
regions of the disclosed antibodies may be modified through the
mutation or substitution of one or more amino acids that enhances
the profile of the resulting construct. In this respect it may be
possible to disrupt the activity provided by a conserved binding
site (e.g. Fc binding) while substantially maintaining the
configuration and immunogenic profile of the modified antibody.
Certain embodiments can comprise the addition of one or more amino
acids to the constant region to enhance desirable characteristics
such as effector function or provide for more cytotoxin or
carbohydrate attachment. In such embodiments it can be desirable to
insert or replicate specific sequences derived from selected
constant region domains.
[0126] The present invention further embraces variants and
equivalents which are substantially homologous to the chimeric,
humanized and human antibodies, or antibody fragments thereof, set
forth herein. These can contain, for example, conservative
substitution mutations, i.e. the substitution of one or more amino
acids by similar amino acids. For example, conservative
substitution refers to the substitution of an amino acid with
another within the same general class such as, for example, one
acidic amino acid with another acidic amino acid, one basic amino
acid with another basic amino acid or one neutral amino acid by
another neutral amino acid. What is intended by a conservative
amino acid substitution is well known in the art.
[0127] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent. Cytotoxic agents
include chemotherapeutic agents, growth inhibitory agents, toxins
(e.g., an enzymatically active toxin of bacterial, fungal, plant,
or animal origin, or fragments thereof), radioactive isotopes
(i.e., a radioconjugate), etc. Chemotherapeutic agents useful in
the generation of such immunoconjugates include, for example,
methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents.
Enzymatically active toxins and fragments thereof that can be used
include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAN, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. In some embodiments,
the antibodies can be conjugated to radioisotopes, such as
.sup.90Y, .sup.125I, .sup.131I, .sup.123I, .sup.111In, .sup.105Rh,
.sup.153Sm, .sup.67Cu, .sup.67Ga, .sup.166Ho, .sup.177Lu,
.sup.186Re and .sup.188Re using anyone of a number of well known
chelators or direct labeling. In other embodiments, the disclosed
compositions can comprise antibodies coupled to drugs, prodrugs or
lymphokines such as interferon. Conjugates of the antibody and
cytotoxic agent are 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). 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, can also be used. In some
embodiments, the modified antibodies can be complexed with other
immunologically active ligands (e.g. antibodies or fragments
thereof) wherein the resulting molecule binds to both the
neoplastic cell and an effector cell such as a T cell.
[0128] Regardless of how useful quantities are obtained, the
antibodies of the present invention can be used in any one of a
number of conjugated (i.e. an immunoconjugate) or unconjugated
forms. Alternatively, the antibodies of this invention can be used
in a nonconjugated or "naked" form to harness the subject's natural
defense mechanisms including complement-dependent cytotoxicity
(CDC) and antibody dependent cellular toxicity (ADCC) to eliminate
the malignant cells. The selection of which conjugated or
unconjugated modified antibody to use will depend of the type and
stage of cancer, use of adjunct treatment (e.g., chemotherapy or
external radiation) and patient condition. It will be appreciated
that one skilled in the art could readily make such a selection in
view of the teachings herein.
[0129] The antibodies of the present invention can be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include, but are not limited to,
competitive and non-competitive assay systems using techniques such
as BIAcore analysis, FACS analysis, immunofluorescence,
immunocytochemistry, Western blots, radioimmunoassays, ELISA,
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, and protein A
immunoassays. Such assays are routine and well known in the art
(see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York,
which is incorporated by reference herein in its entirety).
[0130] In some embodiments, the immunospecificity of an antibody
against a cancer stem cell marker is determined using ELISA. An
ELISA assay comprises preparing antigen, coating wells of a 96 well
microtiter plate with antigen, adding the antibody against a cancer
stem cell marker conjugated to a detectable compound such as an
enzymatic substrate (e.g. horseradish peroxidase or alkaline
phosphatase) to the well, incubating for a period of time and
detecting the presence of the antigen. In some embodiments, the
antibody against a cancer stem cell marker is not conjugated to a
detectable compound, but instead a second conjugated antibody that
recognizes the antibody against a cancer stem cell marker is added
to the well. In some embodiments, instead of coating the well with
the antigen, the antibody against a cancer stem cell marker can be
coated to the well and a second antibody conjugated to a detectable
compound can be added following the addition of the antigen to the
coated well. One of skill in the art would be knowledgeable as to
the parameters that can be modified to increase the signal detected
as well as other variations of ELISAs known in the art (see e.g.
Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,
Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).
[0131] The binding affinity of an antibody to a cancer stem cell
marker antigen and the off-rate of an antibody-antigen interaction
can be determined by competitive binding assays. One example of a
competitive binding assay is a radioimmunoassay comprising the
incubation of labeled antigen (e.g. .sup.3H or .sup.125I), or
fragment or variant thereof, with the antibody of interest in the
presence of increasing amounts of unlabeled antigen followed by the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody against a cancer stem cell marker and the
binding off-rates can be determined from the data by scatchard plot
analysis. In some embodiments, BIAcore kinetic analysis is used to
determine the binding on and off rates of antibodies against a
cancer stem cell marker. BIAcore kinetic analysis comprises
analyzing the binding and dissociation of antibodies from chips
with immobilized cancer stem cell marker antigens on their
surface.
[0132] In certain embodiments, the present invention provides
antibodies that are substantially identical to the antibody
sequences of the invention, meaning they have at least 70%, at
least 75%, preferably at least 80%, more preferably at least 85%,
more preferably at least 90%, and in some embodiments at least 95%,
96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when
compared and aligned for maximum correspondence, as measured using
a sequence comparison algorithm or by visual inspection. In certain
embodiments, the invention provides an antibody that specifically
binds a human MET receptor, comprising (a) a heavy chain variable
region comprising an amino acid sequence at least about 90%
identical to SEQ ID NO:2 and a light chain variable region
comprising an amino acid sequence that is at least about 90%
identical to SEQ ID NO:7; and/or (b) a heavy chain variable region
comprising an amino acid sequence at least about 90% identical to
SEQ ID NO:12 and a light chain variable region comprising an amino
acid sequence that is at least about 90% identical to SEQ ID NO:17
or to SEQ ID NO:22.
[0133] Example 1 below describes the production of exemplary human
anti-MET antibodies which specifically bind to the cancer stem cell
marker MET and inhibit HGF binding and downstream MET signaling. In
certain embodiments, the invention provides an isolated antibody
that specifically binds to a human MET Receptor, wherein the
antibody comprises a heavy chain variable region comprising CDR
sequences SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In certain
embodiments, the isolated MET Receptor antibody further comprising
a light chain variable region comprising CDR sequences SEQ ID NO:
8, SEQ ID NO: 9, and SEQ ID NO: 10. In certain embodiments, the
isolated MET Receptor antibody comprises a heavy chain variable
region that comprises an amino acid sequence at least 95% identical
to SEQ ID NO: 2. In certain embodiments, the isolated MET Receptor
antibody comprises a heavy chain variable region that comprises an
amino acid sequence at least 99% identical to SEQ ID NO: 2. In
certain embodiments, the isolated MET Receptor antibody comprises a
light chain variable region that comprises an amino acid sequence
at least 95% identical to SEQ ID NO: 7. In certain embodiments, the
isolated MET Receptor antibody comprises a light chain variable
region that comprises an amino acid sequence at least 99% identical
to SEQ ID NO: 7. In certain embodiments, the isolated MET Receptor
antibody comprises heavy chain SEQ ID NO: 2 and light chain SEQ ID
NO: 7. In certain embodiments, the isolated MET Receptor antibody
is a human antibody. In certain embodiments, the human MET Receptor
antibody is a human IgG antibody. In certain embodiments, the human
IgG antibody is 13-MET IgG, the IgG encoded by the plasmid DNA
deposited with the American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, Va., USA, on Apr. 10, 2008, under
the provisions of the Budapest Treaty, and having ATCC deposit no.
PTA-9148.
[0134] In certain embodiments, the invention provides an isolated
antibody that competes with antibody 13-MET for specific binding to
a human MET Receptor, wherein the 13-MET antibody comprises: (i) a
heavy chain variable region comprising SEQ ID NO: 2; and (ii) a
light chain variable region comprising SEQ ID NO: 7. In certain
embodiments, the invention provides an isolated antibody that
competes with antibody 13-MET for specific binding to a human MET
Receptor, wherein the 13-MET antibody comprise the antibody encoded
by the plasmid DNA deposited with ATCC on Apr. 10, 2008 and having
ATCC deposit no. PTA-9148.
[0135] In certain embodiments, the present invention provides an
isolated antibody that specifically binds to a human MET Receptor
wherein the antibody comprises a heavy chain variable region
comprising CDR sequences SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID
NO: 15. In certain embodiments, the isolated MET Receptor antibody
further comprises a light chain variable region comprising CDR
sequences SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In
certain embodiments, the isolated MET Receptor antibody further
comprises a light chain variable region comprising CDR sequences
SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25. In certain
embodiments, the isolated MET Receptor antibody comprises a heavy
chain variable region comprising an amino acid sequence at least
95% identical to SEQ ID NO: 12. In certain embodiments, the
isolated MET Receptor antibody comprises a heavy chain variable
region comprising an amino acid sequence at least 99% identical to
SEQ ID NO: 12. In certain embodiments, the isolated MET Receptor
antibody comprises a light chain variable region comprising an
amino acid sequence at least 95% identical to SEQ ID NO: 17. In
certain embodiments, the isolated MET Receptor antibody comprises a
light chain variable region comprising an amino acid sequence at
least 99% identical to SEQ ID NO: 17. In certain embodiments, the
isolated MET Receptor antibody comprises a light chain variable
region comprising an amino acid sequence at least 95% identical to
SEQ ID NO: 22. In certain embodiments, the isolated MET Receptor
antibody comprises a light chain variable region comprising an
amino acid sequence at least 99% identical to SEQ ID NO: 22. In
certain embodiments, the isolated MET Receptor antibody comprises
heavy chain SEQ ID NO: 12 and light chain SEQ ID NO: 17. In certain
embodiments, the isolated MET Receptor antibody is a human
antibody. In certain embodiments, the human MET Receptor antibody
is a human IgG antibody. In certain embodiments, human IgG antibody
is 28-MET IgG, the antibody encoded by the plasmid DNA deposited
with ATCC on Apr. 10, 2008 under the provisions of the Budapest
Treaty, and having ATCC deposit no. PTA-9149.
[0136] In certain embodiments, the present invention provides an
isolated antibody that competes with antibody 28-MET for specific
binding to a human MET Receptor, wherein the 28-MET antibody
comprises: (i) a heavy chain variable region comprising SEQ ID NO:
12; and (ii) a light chain variable region comprising SEQ ID NO:
17. In certain embodiments, the present invention provides an
isolated antibody that competes with antibody 21-MET for specific
binding to a human MET Receptor, wherein the 21-MET antibody
comprises: (i) a heavy chain variable region comprising SEQ ID NO:
12; and (ii) a light chain variable region comprising SEQ ID NO:
22. In certain embodiments, the present invention provides an
isolated antibody that competes with antibody 28-MET for specific
binding to a human MET Receptor, wherein the 28-MET antibody
comprises the antibody encoded by the plasmid DNA deposited with
ATCC on Apr. 10, 2008 and having ATCC deposit no. PTA-9149.
[0137] In certain embodiments, the present invention provides a
method of treating cancer in a patient comprising administering to
the patient a therapeutically effective amount of an antibody which
specifically binds to a human MET Receptor, wherein the antibody
comprises a heavy chain variable region comprising CDR sequences
SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In certain
embodiments, the antibody further comprises a light chain variable
region comprising CDR sequences SEQ ID NO: 8, SEQ ID NO: 9, and SEQ
ID NO: 10. In certain embodiments, the antibody comprises SEQ ID
NO: 2 and SEQ ID NO: 7. In certain embodiments, the isolated MET
Receptor antibody is a human antibody. In certain embodiments, the
human MET Receptor antibody is a human IgG antibody. In certain
embodiments, human IgG antibody is 13-MET IgG deposited with ATCC'
on Apr. 10, 2008 and having ATCC deposit no. PTA-9148. In certain
embodiments, the present invention provides a method of treating
cancer in a patient comprising administering to the patient a
therapeutically effective amount of an antibody that competes with
antibody 13-MET for specific binding to a human MET Receptor,
wherein the 13-MET antibody comprises: (i) a heavy chain variable
region comprising SEQ ID NO: 2; and (ii) a light chain variable
region comprising SEQ ID NO: 7. In certain embodiments, the present
invention provides a method of treating cancer in a patient
comprising administering to the patient a therapeutically effective
amount of an antibody that competes with antibody 13-MET for
specific binding to a human MET Receptor, wherein the 13-MET
antibody comprises the antibody encoded by the plasmid DNA
deposited with ATCC on Apr. 10, 2008 and having ATCC deposit no.
PTA-9148.
[0138] In certain embodiments, the present invention provides a
method of treating cancer in a patient comprising administering to
the patient a therapeutically effective amount of an antibody which
specifically binds to a human MET Receptor, wherein the antibody
comprises a heavy chain variable region comprising CDR sequences
SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In certain
embodiments, the antibody, further comprises a light chain variable
region comprising CDR sequences selected from the group consisting
of: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: .delta. 20, SEQ ID
NO:23, SEQ ID NO: 24, and SEQ ID NO: 25. In certain embodiments,
the antibody comprises SEQ ID NO: 12 and SEQ ID NO: 17. In certain
embodiments, the antibody comprises SEQ ID NO: 12 and SEQ ID NO:
22. In certain embodiments, the isolated MET Receptor antibody is a
human antibody. In certain embodiments, the human MET Receptor
antibody is a human IgG antibody. In certain embodiments, human IgG
antibody is 28-MET IgG, the antibody encoded by the plasmid DNA
deposited with ATCC on Apr. 10, 2008 and having ATCC deposit no.
PTA-9149.
[0139] In certain embodiments, the present invention provides a
method of treating cancer in a patient comprising administering to
the patient a therapeutically effective amount of an antibody that
competes with antibody 13-MET for specific binding to a human MET
Receptor, wherein the 13-MET antibody comprises: (i) a heavy chain
variable region comprising SEQ ID NO: 2; and (ii) a light chain
variable region comprising SEQ ID NO: 7. In certain embodiments,
the present invention provides a method of treating cancer in a
patient comprising administering to the patient a therapeutically
effective amount of an antibody that competes with antibody 13-MET
for specific binding to a human MET Receptor, wherein the 28-MET
antibody comprises the antibody encoded by the plasmid DNA
deposited with ATCC on Apr. 10, 2008 and having ATCC deposit no.
PTA-9149.
[0140] In certain embodiments, the present invention provides a
method of treating cancer in a patient comprising administering to
the patient a therapeutically effective amount of an antibody that
competes with antibody 21-MET for specific binding to a human MET
Receptor, wherein the 21-MET antibody comprises: (i) a heavy chain
variable region comprising SEQ ID NO: 12; and (ii) a light chain
variable region comprising SEQ ID NO: 22.
[0141] In certain embodiments, the present invention provides a
method of treating cancer in a patient comprising administering to
the patient a therapeutically effective amount of an antibody that
specifically binds a human MET receptor, comprising (a) a heavy
chain variable region comprising an amino acid sequence at least
about 90% identical to SEQ ID NO:2 and a light chain variable
region comprising an amino acid sequence that is at least about 90%
identical to SEQ ID NO:7; and/or (b) a heavy chain variable region
comprising an amino acid sequence at least about 90% identical to
SEQ ID NO:12 and a light chain variable region comprising an amino
acid sequence that is at least about 90% identical to SEQ ID NO:17
or to SEQ ID NO:22.
[0142] In certain additional embodiments, the invention provides a
bispecific antibody that specifically binds a first and second
epitope on the extracellular domain of a human Met receptor and
inhibits binding of HGF to the receptor, wherein the first epitope
is in the SEMA domain and the second epitope is a conformational
epitope that does not overlap with the first epitope. In certain
embodiments, binding to the SEMA domain directly blocks HGF binding
to the MET receptor. In certain embodiments, binding to the SEMA
domain increases the avidity of the bispecific antibody for binding
to the other epitope. Methods of using the antibody to inhibit
signaling by a human MET receptor on a cell, comprising contacting
the cell with the bispecific antibody are further provided. Methods
of inhibiting growth of a tumor in a patient and methods of
treating cancer, comprising administering to the patient a
therapeutically effective amount of the antibody is also
provided.
[0143] The invention further provides, in some embodiments, methods
of treating cancer comprising administering (a) a first antibody
that binds to a first epitope in the SEMA domain of a human Met
receptor, and (b) a second antibody that binds to a second,
conformational epitope on the extracellular domain of the human Met
receptor. In certain embodiments, the combination of the first and
second antibody synergistically inhibits HGF binding to the Met
receptor. In certain embodiments, binding of the first antibody to
the SEMA domain directly blocks HGF binding to the MET receptor. In
certain embodiments, binding of the first antibody to the SEMA
domain increases the avidity of the second antibody for the MET
receptor.
[0144] In certain embodiments, the present invention provides a
method of treating cancer in a patient comprising administering to
the patient a therapeutically effective amount of a combination of
antibodies that specifically bind different epitopes on the same
antigen. In certain embodiments, the present invention provides a
method of inhibiting receptor functioning comprising administering
to the patient a therapeutically effective amount of a combination
of antibodies that specifically bind different epitopes on the same
antigen. In certain embodiments, the different epitopes are on the
extracellular domain of the receptor. In some embodiments, the
epitopes are non-overlapping. In certain embodiments, the receptor
is a receptor tyrosine kinase. In certain embodiments the receptor
is a human MET receptor. As demonstrated herein, combinations of
antibodies act synergistically by increasing the availability of
epitopes for antibody binding. The proper ratio of the antibody
combination can readily be determined by one of ordinary skill in
the art. In certain embodiments, the first antibody and the second
antibody are administered at a ratio of 1:X, wherein X is any
integer. In certain embodiments, X is at least about 1, at least
about 2, at least about 3, at least about 4, at least about 5, at
least about 6, at least about 7, at least about 8, at least about
9, at least about 10, at least about 15, at least about 20, at
least about 25, or at least about 50.
[0145] In certain embodiments, the invention provides a bispecific
antibody that specifically binds to two different epitopes on the
extracellular domain of a receptor, such as a receptor tyrosine
kinase. In certain embodiments, the receptor is a human MET
receptor. In certain embodiments, the two different epitopes are
non-overlapping. In certain embodiments, the bispecific antibody
inhibits the signaling or other activity of the receptor.
[0146] In certain embodiments, the present invention provides a
method of treating cancer in a patient comprising administering to
the patient a therapeutically effective amount of a combination of
antibodies which specifically binds to a human MET Receptor,
wherein the combination of antibodies comprises: (i) a first
antibody comprising: (a) a heavy chain variable region comprising
CDR sequences SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; and (b)
a light chain variable region comprising CDR sequences SEQ ID NO:
8, SEQ ID NO: 9, and SEQ ID NO: 10; and (ii) a second antibody
comprising (a) a heavy chain variable region comprising CDR
sequences SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; and (b)
a light chain variable region comprising CDR sequences selected
from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID
NO: 20, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25. In certain
embodiments, the first antibody comprises SEQ ID NO: 2 and SEQ ID
NO: 7 and the second antibody comprises SEQ ID NO: 12 and SEQ ID
NO: 17. In another embodiment, the first antibody comprises SEQ ID
NO: 2 and SEQ ID NO: 7 and the second antibody comprises SEQ ID
NO:12 and SEQ ID NO:22. In certain embodiments, the first antibody
and the second antibody are human antibodies. In certain
embodiment, the first human antibody and the second human antibody
are human IgG antibodies. In certain embodiments, the first
antibody and the second antibody are administered at a ratio of
1:1. In certain embodiments, the first antibody and the second
antibody are administered at a ratio of 1:5. In certain
embodiments, the first human antibody is the 13-MET IgG antibody
encoded by the plasmid deposited with ATCC on Apr. 10, 2008 and
having ATCC deposit no. PTA-9148 and the second human antibody is
the 28-MET IgG antibody encoded by the plasmid DNA deposited with
ATCC on Apr. 10, 2008 and having ATCC deposit no. PTA-9149. In
certain embodiments, the first antibody and the second antibody are
administered at a ratio of 1:1. In certain embodiments, the 13-MET
and 28-MET antibodies are administered at a ratio of 1:8, In
certain embodiments, the 9-MET and 19-MET antibodies are
administered at a ratio of 1:8. In certain embodiments, the first
antibody and the second antibody are administered to the patient
sequentially. In certain embodiments, the first antibody and the
second antibody are administered to the patient in separate
compositions.
[0147] In certain embodiments, the present invention provides a
bispecific antibody that specifically binds to a human MET
Receptor, the antibody comprising: (i) a first heavy chain variable
region comprising CDR sequences SEQ ID NO: 3, SEQ ID NO: 4, and SEQ
ID NO: 5; and/or (ii) a first light chain variable region
comprising CDR sequences SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:
10. In certain embodiments, the bispecific antibody further
comprises: (iii) a second heavy chain variable region comprising
CDR sequences SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15;
and/or (iv) a second light chain variable region comprising CDR
sequences selected from the group consisting of SEQ ID NO: 18, SEQ
ID NO: 19, SEQ ID NO: 20, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID
NO:25. In certain embodiments, the second light chain variable
region comprises CDR sequences SEQ ID NO: 18, SEQ ID NO: 19, and
SEQ ID NO: 20. In certain alternative embodiments, the second light
chain variable region comprises CDR sequences SEQ ID NO:23, SEQ ID
NO:24, and SEQ ID NO:25.
[0148] In certain embodiments, the present invention provides a
bispecific antibody that specifically binds to a human MET
Receptor, the antibody comprising: (i) a first heavy chain variable
region comprising CDR sequences SEQ ID NO: 13, SEQ ID NO: 14, and
SEQ ID NO: 15; and/or (ii) a first light chain variable region
comprising CDR sequences selected from the group consisting of SEQ
ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:23, SEQ ID
NO:24, and SEQ ID NO:25. In certain embodiments, the first light
chain variable region comprises CDR sequences SEQ ID NO: 18, SEQ ID
NO: 19, and SEQ ID NO: 20. In certain alternative embodiments, the
first light chain variable region comprises CDR sequences SEQ ID
NO:23, SEQ ID NO:24, and SEQ ID NO:25. In certain embodiments, the
bispecific antibody further comprises: (iii) a second heavy chain
variable region comprising CDR sequences SEQ ID NO: 3, SEQ ID NO:
4, and SEQ ID NO: 5; and (iv) a second light chain variable region
comprising CDR sequences SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:
10.
[0149] In certain embodiments, the invention encompasses isolated
polynucleotides that encode a polypeptide comprising a human
antibody, or fragment thereof, against human MET. Thus, the term
"polynucleotide encoding a polypeptide" encompasses a
polynucleotide which includes only coding sequences for the
polypeptide as well as a polynucleotide which includes additional
coding and/or non-coding sequences. The polynucleotides of the
invention can be in the form of RNA or in the form of DNA. DNA
includes cDNA, genomic DNA, and synthetic DNA; and can be
double-stranded or single-stranded, and if single stranded can be
the coding stand or non-coding (anti-sense) strand.
[0150] The present invention further relates to variants of the
hereinabove described polynucleotides encoding, for example,
fragments, analogs, and derivatives. The variant of the
polynucleotide can be a naturally occurring allelic variant of the
polynucleotide or a non-naturally occurring variant of the
polynucleotide. In certain embodiments, the polynucleotide can have
a coding sequence which is a naturally occurring allelic variant of
the coding sequence of the disclosed polypeptides. As known in the
art, an allelic variant is an alternate form of a polynucleotide
sequence that have, for example, a substitution, deletion, or
addition of one or more nucleotides, which does not substantially
alter the function of the encoded polypeptide.
[0151] In certain embodiments the polynucleotides comprise the
coding sequence for the mature polypeptide fused in the same
reading frame to a polynucleotide which aids, for example, in
expression and secretion of a polypeptide from a host cell (e.g. a
leader sequence which functions as a secretory sequence for
controlling transport of a polypeptide from the cell). The
polypeptide having a leader sequence is a preprotein and can have
the leader sequence cleaved by the host cell to form the mature
form of the polypeptide. The polynucleotides can also encode for a
proprotein which is the mature protein plus additional 5' amino
acid residues. A mature protein having a prosequence is a
proprotein and is an inactive form of the protein. Once the
prosequence is cleaved an active mature protein remains.
[0152] In certain embodiments the polynucleotides comprise the
coding sequence for the mature polypeptide fused in the same
reading frame to a marker sequence that allows, for example, for
purification of the encoded polypeptide. For example, the marker
sequence can be a hexa-histidine tag supplied by a pQE-9 vector to
provide for purification of the mature polypeptide fused to the
marker in the case of a bacterial host, or the marker sequence can
be a hemagglutinin (HA) tag derived from the influenza
hemagglutinin protein when a mammalian host (e.g. COS-7 cells) is
used.
[0153] In certain embodiments, the present invention provides
isolated nucleic acid molecules having a nucleotide sequence at
least 80% identical, at least 85% identical, at least 90%
identical, at least 95% identical, and in some embodiments, at
least 96%, 97%, 98% or 99% identical to a polynucleotide encoding a
polypeptide comprising a humanized antibody, or fragment thereof,
against human MET.
[0154] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence is
intended that the nucleotide sequence of the polynucleotide is
identical to the reference sequence except that the polynucleotide
sequence can include up to five point mutations per each 100
nucleotides of the reference nucleotide sequence. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence can be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence can be inserted into
the reference sequence. These mutations of the reference sequence
can occur at the amino- or carboxy-terminal positions of the
reference nucleotide sequence or anywhere between those terminal
positions, interspersed either individually among nucleotides in
the reference sequence or in one or more contiguous groups within
the reference sequence.
[0155] As a practical matter, whether any particular nucleic acid
molecule is at least 80% identical, at least 85% identical, at
least 90% identical, and in some embodiments, at least 95%, 96%,
97%, 98%, or 99% identical to a reference sequence can be
determined conventionally using known computer programs such as the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). Bestfit uses the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2: 482 489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5%
of the total number of nucleotides in the reference sequence are
allowed.
[0156] The polynucleotide variants can contain alterations in the
coding regions, non-coding regions, or both. In some embodiments
the polynucleotide variants contain alterations which produce
silent substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide. In some
embodiments, nucleotide variants are produced by silent
substitutions due to the degeneracy of the genetic code.
Polynucleotide variants can be produced for a variety of reasons,
e.g., to optimize codon expression for a particular host (change
codons in the human mRNA to those preferred by a bacterial host
such as E. coli).
[0157] The polypeptides of the present invention can be recombinant
polypeptides, natural polypeptides, or synthetic polypeptides
comprising an antibody, or fragment thereof, against human MET. It
will be recognized in the art that some amino acid sequences of the
invention can be varied without significant effect of the structure
or function of the protein. Thus, the invention further includes
variations of the polypeptides which show substantial activity or
which include regions of a humanized antibody, or fragment thereof,
against human MET protein. Such mutants include deletions,
insertions, inversions, repeats, and type substitutions.
[0158] The polypeptides and analogs can be further modified to
contain additional chemical moieties not normally part of the
protein. Those derivatized moieties can improve the solubility, the
biological half life or absorption of the protein. The moieties can
also reduce or eliminate any desirable side effects of the proteins
and the like. An overview for those moieties can be found in
REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Co.,
Easton, Pa. (2000).
[0159] The isolated polypeptides described herein can be produced
by any suitable method known in the art. Such methods range from
direct protein synthetic methods to constructing a DNA sequence
encoding isolated polypeptide sequences and expressing those
sequences in a suitable transformed host. In some embodiments, a
DNA sequence is constructed using recombinant technology by
isolating or synthesizing a DNA sequence encoding a wild-type
protein of interest. Optionally, the sequence can be mutagenized by
site-specific mutagenesis to provide functional analogs thereof.
See, e.g. Zoeller et al., Proc. Nat'l. Acad, Sci. USA 81:5662-5066
(1984) and U.S. Pat. No. 4,588,585.
[0160] In some embodiments a DNA sequence encoding a polypeptide of
interest would be constructed by chemical synthesis using an
oligonucleotide synthesizer. Such oligonucleotides can be designed
based on the amino acid sequence of the desired polypeptide and
selecting those codons that are favored in the host cell in which
the recombinant polypeptide of interest will be produced. Standard
methods can be applied to synthesize an isolated polynucleotide
sequence encoding an isolated polypeptide of interest. For example,
a complete amino acid sequence can be used to construct a
back-translated gene. Further, a DNA oligomer containing a
nucleotide sequence coding for the particular isolated polypeptide
can be synthesized. For example, several small oligonucleotides
coding for portions of the desired polypeptide can be synthesized
and then ligated. The individual oligonucleotides typically contain
5' or 3' overhangs for complementary assembly.
[0161] Once assembled (by synthesis, site-directed mutagenesis or
another method), the polynucleotide sequences encoding a particular
isolated polypeptide of interest will be inserted into an
expression vector and operatively linked to an expression control
sequence appropriate for expression of the protein in a desired
host. Proper assembly can be confirmed by nucleotide sequencing,
restriction mapping, and expression of a biologically active
polypeptide in a suitable host. As is well known in the art, in
order to obtain high expression levels of a transfected gene in a
host, the gene must be operatively linked to transcriptional and
translational expression control sequences that are functional in
the chosen expression host.
[0162] Recombinant expression vectors are used to amplify and
express DNA encoding cancer stem cell marker polypeptide fusions.
Recombinant expression vectors are replicable DNA constructs which
have synthetic or cDNA-derived DNA fragments encoding a cancer stem
cell marker polypeptide fusion or a bioequivalent analog
operatively linked to suitable transcriptional or translational
regulatory elements derived from mammalian, microbial, viral or
insect genes. A transcriptional unit generally comprises an
assembly of (1) a genetic element or elements having a regulatory
role in gene expression, for example, transcriptional promoters or
enhancers, (2) a structural or coding sequence which is transcribed
into mRNA and translated into protein, and (3) appropriate
transcription and translation initiation and termination sequences,
as described in detail below. Such regulatory elements can include
an operator sequence to control transcription. The ability to
replicate in a host, usually conferred by an origin of replication,
and a selection gene to facilitate recognition of transformants can
additionally be incorporated. DNA regions are operatively linked
when they are functionally related to each other. For example, DNA
for a signal peptide (secretory leader) is operatively linked to
DNA for a polypeptide if it is expressed as a precursor which
participates in the secretion of the polypeptide; a promoter is
operatively linked to a coding sequence if it controls the
transcription of the sequence; or a ribosome binding site is
operatively linked to a coding sequence if it is positioned so as
to permit translation. Generally, operatively linked means
contiguous and, in the case of secretory leaders, means contiguous
and in reading frame. Structural elements intended for use in yeast
expression systems include a leader sequence enabling extracellular
secretion of translated protein by a host cell. Alternatively,
where recombinant protein is expressed without a leader or
transport sequence, it can include an N-terminal methionine
residue. This residue can optionally be subsequently cleaved from
the expressed recombinant protein to provide a final product.
[0163] The choice of expression control sequence and expression
vector will depend upon the choice of host. A wide variety of
expression host/vector combinations can be employed. Useful
expression vectors for eukaryotic hosts, include, for example,
vectors comprising expression control sequences from SV40, bovine
papilloma virus, adenovims and cytomegalovirus. Useful expression
vectors for bacterial hosts include known bacterial plasmids, such
as plasmids from Esherichia coli, including pCR 1, pBR322, pMB9 and
their derivatives, wider host range plasmids, such as M13 and
filamentous single-stranded DNA phages.
[0164] Suitable host cells for expression of a cancer stem cell
marker protein include prokaryotes, yeast, insect or higher
eukaryotic cells under the control of appropriate promoters.
Prokaryotes include gram negative or gram positive organisms, for
example E. coli or bacilli. Higher eukaryotic cells include
established cell lines of mammalian origin as described below.
Cell-free translation systems could also be employed. Appropriate
cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described by Pouwels et al.
(Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), the
relevant disclosure of which is hereby incorporated by
reference.
[0165] Various mammalian or insect cell culture systems are also
advantageously employed to express recombinant protein. Expression
of recombinant proteins in mammalian cells can be performed because
such proteins are generally correctly folded, appropriately
modified and completely functional. Examples of suitable mammalian
host cell lines include the COS-7 lines of monkey kidney cells,
described by Gluzman (Cell 23:175, 1981), and other cell lines
capable of expressing an appropriate vector including, for example,
L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell
lines. Mammalian expression vectors can comprise nontranscribed
elements such as an origin of replication, a suitable promoter and
enhancer linked to the gene to be expressed, and other 5' or 3'
flanking nontranscribed sequences, and 5' or 3' nontranslated
sequences, such as necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites, and
transcriptional termination sequences. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow and Summers, Bio; Technology 6:47 (1988).
[0166] The proteins produced by a transformed host can be purified
according to any suitable method. Such standard methods include
chromatography (e.g., ion exchange, affinity and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for protein purification. Affinity tags
such as hexahistidine, maltose binding domain, influenza coat
sequence and glutathione-S-transferase can be attached to the
protein to allow easy purification by passage over an appropriate
affinity column. Isolated proteins can also be physically
characterized using such techniques as proteolysis, nuclear
magnetic resonance and x-ray crystallography.
[0167] For example, supernatants from systems which secrete
recombinant protein into culture media can be first concentrated
using a commercially available protein concentration filter, for
example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the concentration step, the concentrate can be applied to
a suitable purification matrix. Alternatively, an anion exchange
resin can be employed, for example, a matrix or substrate having
pendant diethylaminoethyl (DEAE) groups. The matrices can be
acrylamide, agarose, dextran, cellulose or other types commonly
employed in protein purification. Alternatively, a cation exchange
step can be employed. Suitable cation exchangers include various
insoluble matrices comprising sulfopropyl or carboxymethyl groups.
Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify a cancer stem cell protein-Fc
composition. Some or all of the foregoing purification steps, in
various combinations, can also be employed to provide a homogeneous
recombinant protein.
[0168] Recombinant protein produced in bacterial culture can be
isolated, for example, by initial extraction from cell pellets,
followed by one or more concentration, salting-out, aqueous ion
exchange or size exclusion chromatography steps. High performance
liquid chromatography (HPLC) can be employed for final purification
steps. Microbial cells employed in expression of a recombinant
protein can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
[0169] The present invention provides methods for inhibiting the
growth of tumorigenic cells expressing a cancer stem cell marker
using the antibodies against a cancer stem cell marker described
herein. In certain embodiments, the method of inhibiting the growth
of tumorigenic cells expressing a cancer stem cell marker comprises
contacting the cell with an antibody against a cancer stem cell
marker in vitro. For example, an immortalized cell line or a cancer
cell line that expresses a cancer stem cell marker is cultured in
medium to which is added an antibody against the expressed cancer
stem cell marker to inhibit cell growth. In some embodiments, tumor
cells comprising tumor stem cells are isolated from a patient
sample such as, for example, a tissue biopsy, pleural effusion, or
blood sample and cultured in medium to which is added an antibody
against a cancer stem cell marker to inhibit cell growth.
[0170] In some embodiments, the method of inhibiting the growth of
tumorigenic cells expressing a cancer stem cell marker comprises
contacting the cell with an antibody against a cancer stem cell
marker in vivo. In certain embodiments, contacting a tumorigenic
cell with an antibody against a cancer stem cell marker is
undertaken in an animal model. For example, xenografts expressing a
cancer stem cell marker are grown in immunocompromised mice (e.g.
NOD/SCID mice) that are administered an antibody against a cancer
stem cell marker to inhibit tumor growth. In some embodiments,
cancer stem cells that express a cancer stem cell marker are
isolated from a patient sample such as, for example, a tissue
biopsy, pleural effusion, or blood sample and injected into
immunocompromised mice that are then administered an antibody
against the cancer stem cell marker to inhibit tumor cell growth.
In some embodiments, the antibody against a cancer stem cell marker
is administered at the same time or shortly after introduction of
tumorigenic cells into the animal to prevent tumor growth. In some
embodiments, the antibody against a cancer stem cell marker is
administered as a therapeutic after the tumorigenic cells have
grown to a specified size.
[0171] In certain embodiments, antibodies administered in
combination act synergistically to inhibit any receptor
functioning. In certain embodiments, inhibiting receptor
functioning is performed in vitro. In certain embodiments,
inhibiting receptor functioning is performed in vivo. In some
embodiments, the antibodies are administered as a composition
comprising at least two antibodies that bind to different epitopes
within the same antigen. In certain embodiments, each of the
antibodies binds to a separate epitope on the receptor. In certain
embodiments, each of the two epitopes are on the extracellular
domain of the antigen. In some other embodiments, one or more of
the antibodies is a bispecific antibody that binds to two different
epitopes on the same antigen. When administered in combination, the
first antibody causes a change in the receptor such that the second
antibody is able to have a greater effect on blocking receptor
functioning. In certain embodiments, the first antibody causes a
conformational change in the receptor so that the second antibody
has greater access to the epitope on the receptor to which it
binds. In certain embodiments, one of the antibodies stabilizes a
particular conformation of the receptor upon binding to the
receptor. In certain embodiments, a second antibody binds or
preferentially binds to that stabilized conformation of the
receptor. In certain embodiments, the avidity of the second
antibody for the receptor is increased by the binding of the first
antibody to the receptor.
[0172] In certain embodiments, receptor tyrosine kinase activity is
blocked by administration of a combination of at least two
antibodies that bind to different epitopes of the receptor tyrosine
kinase (e.g., two epitopes on the extracellular domain of the
receptor tyrosine kinase). In certain embodiments, the receptor
tyrosine kinase is a receptor that is stimulated by dimerization.
In another embodiment, human MET receptor activity is blocked by
administration of a combination of at least two antibodies that
bind different epitopes of the human MET receptor (e.g., different
epitopes on the extracellular domain of the human MET receptor). In
certain embodiments, the receptor activity that is inhibited is
signaling by the receptor. In certain embodiments, one antibody
competes for specific binding to a human MET receptor with (1) a
first antibody comprising (a) a heavy chain variable region
comprising SEQ ID NO: 2; and (b) a light chain variable region
comprising SEQ ID NO: 7, and the other antibody competes for
specific binding to a human MET receptor with (2) a second antibody
comprising (a) a heavy chain variable region comprising SEQ ID NO:
12; and (b) a light chain variable region comprising SEQ ID NO: 17
or SEQ ID NO:22.
[0173] The present invention further provides pharmaceutical
compositions comprising antibodies that target a cancer stem cell
marker and a pharmaceutically acceptable excipient. In certain
embodiments, the antibodies are bispecific antibodies. These
bispecific antibodies may bind to two different epitopes on the
extracellular domain of the same receptor. In certain embodiments
the pharmaceutical composition comprises at least two antibodies
and a pharmaceutically acceptable excipient. In some embodiments,
each of the two antibodies specifically binds a different epitope
on the extracellular domain of the same receptor tyrosine kinase.
In certain embodiments, the receptor tyrosine kinase is human MET
receptor. In some embodiments, the first antibody competes for
specific binding to a human MET receptor with an antibody
comprising (a) a heavy chain variable region comprising SEQ ID NO:
2; and (b) a light chain variable region comprising SEQ ID NO: 7,
and (2) the second antibody competes for specific binding to a
human MET receptor with an antibody comprising (a) a heavy chain
variable region comprising SEQ ID NO: 12; and (b) a light chain
variable region comprising SEQ ID NO: 17 or SEQ ID NO:22. These
pharmaceutical compositions find use in inhibiting tumor cell
growth and treating cancer in human patients.
[0174] Formulations are prepared for storage and use by combining a
purified antibody of the present invention with a pharmaceutically
acceptable vehicle (e.g. carrier, excipient) (Remington, The
Science and Practice of Pharmacy 20th Edition Mack Publishing,
2000). Suitable pharmaceutically acceptable vehicles include, but
are not limited to, nontoxic buffers such as phosphate, citrate,
and other organic acids; salts such as sodium chloride;
antioxidants including ascorbic acid and methionine; preservatives
(e.g. 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 polypeptides (e.g. less than about
10 amino acid residues); proteins such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; carbohydrates such as
monosacchandes, disaccharides, 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 non-ionic
surfactants such as TWEEN or polyethylene glycol (PEG).
[0175] The pharmaceutical composition of the present invention can
be administered in any number of ways for either local or systemic
treatment. Administration can be topical (such as to mucous
membranes including vaginal and rectal delivery) such as
transdermal patches, ointments, lotions, creams, gels, drops,
suppositories, sprays, liquids and powders; pulmonary (e.g., by
inhalation or insufflation of powders or aerosols, including by
nebulizer; intratracheal, intranasal, epidermal and transdermal);
oral; or parenteral including intravenous, intraarterial,
subcutaneous, intraperitoneal or intramuscular injection or
infusion; or intracranial (e.g., intrathecal or intraventricular)
administration.
[0176] The therapeutic formulation can be in unit dosage form. Such
formulations include tablets, pills, capsules, powders, granules,
solutions or suspensions in water or non-aqueous media, or
suppositories for oral, parenteral, or rectal administration or for
administration by inhalation. In solid compositions such as tablets
the principal active ingredient is mixed with a pharmaceutical
carrier. Conventional tableting ingredients include corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other diluents (e.g. water) to
form a solid preformulation composition containing a homogeneous
mixture of a compound of the present invention, or a non-toxic
pharmaceutically acceptable salt thereof. The solid preformulation
composition is then subdivided into unit dosage forms of the type
described above. The tablets, pills, etc of the novel composition
can be coated or otherwise compounded to provide a dosage form
affording the advantage of prolonged action. For example, the
tablet or pill can comprise an inner composition covered by an
outer component. Furthermore, the two components can be separated
by an enteric layer that serves to resist disintegration and
permits the inner component to pass intact through the stomach or
to be delayed in release. A variety of materials can be used for
such enteric layers or coatings, such materials including a number
of polymeric acids and mixtures of polymeric acids with such
materials as shellac, cetyl alcohol and cellulose acetate.
[0177] Pharmaceutical formulations include antibodies of the
present invention complexed with liposomes (Epstein, et al., 1985,
Proc. Natl. Acad. Sci. USA 82:3688; Hwang, et al., 1980, Proc.
Natl. Acad. Sci. USA 77:4030; and U.S. Pat. Nos. 4,485,045 and
4,544,545). Liposomes with enhanced circulation time are disclosed
in U.S. Pat. No. 5,013,556. Some liposomes can be generated by the
reverse phase evaporation with a lipid composition comprising
phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
[0178] The antibodies can also be entrapped in microcapsules. Such
microcapsules are prepared, for example, by coacervation techniques
or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions as described in Remington, The Science and
Practice of Pharmacy 20th Ed. Mack Publishing (2000).
[0179] In addition sustained-release preparations can be prepared.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
antibody, which matrices are in the form of shaped articles (e.g.
films, or microcapsules). Examples of sustained-release matrices
include polyesters, hydrogels such as
poly(2-hydroxyethyl-methacrylate) or poly(v nylalcohol),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and 7 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), sucrose
acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0180] In some embodiments, the treatment involves the combined
administration of an antibody of the present invention and a
chemotherapeutic agent or cocktail of multiple different
chemotherapeutic agents. Treatment with an antibody can occur prior
to, concurrently with, or subsequent to administration of
chemotherapies. Chemotherapies contemplated by the invention
include chemical substances or drugs which are known in the art and
are commercially available, such as Doxorubicin, 5-Fluorouracil,
Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa,
Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan,
Vinblastine and Carboplatin. Combined administration can include
co-administration, either in a single pharmaceutical formulation or
using separate formulations, or consecutive administration in
either order but generally within a time period such that all
active agents can exert their biological activities simultaneously.
Preparation and dosing schedules for such chemotherapeutic agents
can be used according to manufacturers' instructions or as
determined empirically by the skilled practitioner. Preparation and
dosing schedules for such chemotherapy are also described in
Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,
Baltimore, Md. (1992).
[0181] In other embodiments, the treatment involves the combined
administration of an antibody of the present invention and
radiation therapy. Treatment with the antibody can occur prior to,
concurrently with, or subsequent to administration of radiation
therapy. Any dosing schedules for such radiation therapy can be
used as determined by the skilled practitioner.
[0182] In other embodiments, the treatment can involve the combined
administration of antibodies of the present invention with other
antibodies against additional tumor associated antigens including,
but not limited to, antibodies that bind to the EGF receptor (EGFR)
(Erbitux.RTM.), the erbB2 receptor (HER2) (Herceptin.RTM.), and
vascular endothelial growth factor (VEGF) (Avastin.RTM.).
Furthermore, treatment can include administration of one or more
cytokines, can be accompanied by surgical removal of cancer cells
or any other therapy deemed necessary by a treating physician.
[0183] For the treatment of the disease, the appropriate dosage of
an antibody of the present invention depends on the type of disease
to be treated, the severity and course of the disease, the
responsiveness of the disease, whether the antibody is administered
for therapeutic or preventative purposes, previous therapy,
patient's clinical history, and so on all at the discretion of the
treating physician. The antibody can be administered one time or
over a series of treatments lasting from several days to several
months, or until a cure is effected or a diminution of the disease
state is achieved (e.g. reduction in tumor size). Optimal dosing
schedules can be calculated from measurements of drug accumulation
in the body of the patient and will vary depending on the relative
potency of an individual antibody. The administering physician can
easily determine optimum dosages, dosing methodologies and
repetition rates. In general, dosage is from 0.01 .mu.g to 100 mg
per kg of body weight, and can be given once or more daily, weekly,
monthly or yearly. The treating physician can estimate repetition
rates for dosing based on measured residence times and
concentrations of the drug in bodily fluids or tissues.
[0184] The present invention provides kits comprising the
antibodies described herein and that can be used to perform the
methods described herein. In certain embodiments, a kit comprises
at least one purified antibody against a cancer stem cell marker in
one or more containers. In certain embodiments, a kit comprises at
least two antibodies, wherein each of the two antibodies
specifically binds a different epitope on the same receptor
tyrosine kinase. In certain embodiments, each of the two antibodies
specifically binds to the extracellular domain of the receptor
tyrosine kinase. In certain embodiments, the receptor tyrosine
kinase is human MET receptor. In certain embodiments, the first
antibody competes for specific binding to a human MET receptor with
an antibody comprising (a) a heavy chain variable region comprising
SEQ ID NO: 2; and (b) a light chain variable region comprising SEQ
ID NO: 7, and (2) the second antibody competes for specific binding
to a human MET receptor with an antibody comprising (a) a heavy
chain variable region comprising SEQ ID NO: 12; and (b) a light
chain variable region comprising SEQ ID NO: 17 or SEQ ID NO:22. In
some embodiments, the kits contain all of the components necessary
and/or sufficient to perform a detection assay, including all
controls, directions for performing assays, and any necessary
software for analysis and presentation of results. One skilled in
the art will readily recognize that the disclosed antibodies of the
present invention can be readily incorporated into one of the
established kit formats which are well known in the art.
[0185] Embodiments of the present disclosure can be further defined
by reference to the following examples, which describe in detail
preparation of antibodies of the present disclosure and methods for
using antibodies of the present disclosure. It will be apparent to
those skilled in the art that many modifications, both to materials
and methods, may be practiced without departing from the scope of
the present disclosure. Wherever possible, the same reference
numbers will be used throughout the drawings to refer to the same
or like parts. As used herein and it the appended claims, the
singular forms "a," "or," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "an antibody" includes a plurality of such antibodies
or one or more antibodies and equivalents thereof known to those
skilled in the art. Furthermore, all numbers expressing quantities
of ingredients, reaction conditions, purity, polypeptide and
polynucleotide lengths, and so forth, used in the specification,
are modified by the term "about," unless otherwise indicated.
Accordingly, the numerical parameters set forth in the
specification and claims are approximations that may vary depending
upon the desired properties of the present invention.
[0186] All of the various embodiments or options described herein
can be combined in any and all variations.
EXAMPLES
Example 1
Production of Functional Anti-MET Antibodies
[0187] Using the Morphosys HuCAL GOLD Fab library, functional
anti-Met antibodies were discovered using a series of novel
selections against human cancer cell lines (GTL-16 & SNU-5)
which over-express Met, and recombinant Met extracellular domain
(ECD) (and isolated domains thereof). Specifically,
2.times.10.sup.13 Fab displaying phage particles were incubated
with GTL-16 (>10.sup.6 cells) in round one, non-specific phage
were washed off, and then specific phage were eluted with low pH.
The eluted output was used to infect TG1 F+ bacteria, rescued with
helper phage, and then Fab display induced with IPTG (0.25 mM).
[0188] This rescued round one output was used as the starting point
for wide array of selections in rounds two, three, and four as
indicated below:
TABLE-US-00001 R1 R2 R3 R4 GTL-16 .fwdarw. Met Fc .fwdarw. GTL-16
GTL-16 .fwdarw. Met Fc .fwdarw. SEMA GTL-16 .fwdarw. SNU-5 .fwdarw.
Met Fc .fwdarw. Met Fc GTL-16 .fwdarw. Met His .fwdarw. Met His
GTL-16 .fwdarw. SNU-5 .fwdarw. Met His .fwdarw. Met His GTL-16
.fwdarw. Met His .fwdarw. GTL-16(E) .fwdarw. Met His
[0189] For selections on recombinant protein, protein was passively
immobilized on polystyrene plates and blocked with milk. After
washing with PBS-Tween (PBS/T), specific phage were eluted with
dithiothreitol (DTT). Fab-13 was isolated from the selections 1 and
2; whereas Fab-28 was isolated from selections 3-6. In all cases,
the functional Fabs were only discovered from selections initiated
on cancer cell lines and were not found in selections conducted
only on recombinant protein.
[0190] Two monovalent Fabs were isolated for further analysis:
Fab-13 (13-MET) (R13) and Fab-28 (28-MET) (R28). The 13-MET heavy
chain variable nucleotide and amino acid sequences are provided in
SEQ ID NO: 1 and 2, respectively, along with the amino acid
sequences of the heavy chain CDR1 (SEQ ID NO: 3); CDR2 (SEQ ID NO:
4); and CDR3 (SEQ ID NO 5). The 13-MET light chain variable
nucleotide and amino acid sequences are provided in SEQ ID NO: 6
and 7, respectively, along with the amino acid sequences of the
light chain CDR1 (SEQ ID NO: 8); CDR2 (SEQ ID NO: 9); and CDR3 (SEQ
ID NO 10). The 28-MET heavy chain variable nucleotide and amino
acid sequences are provided in SEQ ID NO: 11 and 12, respectively,
along with the amino acid sequences of the heavy chain CDR1 (SEQ ID
NO: 13); CDR2 (SEQ ID NO: 14); and CDR3 (SEQ ID NO 15). The 28-MET
light chain variable nucleotide and amino acid sequences are
provided in SEQ ID NO: 16 and 17, respectively, along with the
amino acid sequences of the light chain CDR1 (SEQ ID NO: 18); CDR2
(SEQ ID NO: 19); and CDR3 (SEQ ID NO 20). Each monovalent Fab was
combined with human constant regions to generated full human
IgG.sub.1 13-MET and 28-MET antibodies.
[0191] Each monovalent Fab was also linked to itself via a
helix-loop-helix motif to produce divalent dimer Fabs (FIG. 1A).
Specifically, two 13-MET Fabs were linked to produce the dimer
9-MET, and two 28-MET Fabs were linked to produce the dimer 19-MET.
Similarly, each monovalent Fab was combined to generate a human
IgG1 molecule (FIG. 1A). 13-MET and 28-MET monovalent Fabs are
similarly linked to produce bispecific antibodies.
[0192] The 28-MET antibody was further subjected to affinity
maturation. The 21-MET antibody (R21) is an affinity matured 28-MET
antibody comprising an altered light chain variable region. The
21-MET light chain variable nucleotide and amino acid sequences are
provided in SEQ ID NO: 21 and 22, respectively, along with the
amino acid sequences of the light chain CDR1 (SEQ ID NO: 23); CDR2
(SEQ ID NO: 24); and CDR3 (SEQ ID NO 25).
[0193] BIAcore was used to determine binding affinities, shown in
Table 1 below for 13-MET and 28-MET. Fab and IgG affinities were
determined using a BIAcore 2000 instrument. Briefly, MET ECD was
immobilized on a CM5 chip using standard amine based chemistry
(NHS/EDC). For each Fab and IgG, different concentrations (100-1
nM) were injected over the MET ECD surface and kinetic data were
collected over time. The data was fit using the simultaneous global
fit equation to yield affinity constants (KD) for each Fab and
IgG.
TABLE-US-00002 TABLE 1 Affinity of 13-MET and 28-MET Antibodies nM
Monomer Dimer IgG Clone K.sub.D K.sub.D K.sub.D 13 0.56 0.90 28
49.00 8.70 47.9
[0194] In addition, a bispecific antibody, 73R21.13, was produced
using the method described in Wu et al., which is incorporated
herein by reference (Wu, C., et al. Simultaneous targeting of
multiple disease mediators by a dual-variable-domain
immunoglobulin. Nat Biotechnol, 25: 1290-1297, 2007). The
bispecific antibody comprised the light and heavy chain variable
regions of both the 13-MET antibody and a 21-MET antibody (an
affinity matured 28-MET antibody). The sequences of the light chain
and heavy chain variable regions of the 13-MET antibody that were
included in the bispecific antibody are shown in SEQ ID NO:7 and
SEQ ID NO:2, respectively. The sequences of the light chain and
heavy chain variable regions of the 21-MET antibody are shown in
SEQ ID NO:22 and SEQ ID NO:12, respectively.
Example 2
[0195] Anti-MET Antibodies Block HGF Ligand Binding to the MET
Receptor Extracellular Domain and Bind to Different Epitopes
[0196] MET antibodies block hHGF binding to the MET Receptor in
vitro. In certain embodiments, a HGF/MET blocking assay was used to
assess HGF ligand binding to the MET Receptor. Maxi-sorp 384-well
microtiter plates (Nunc, Rochester, N.Y.) were coated with
recombinant human HGF (1 mg/mL.times.25 pt; R&D Systems,
Minneapolis, Minn.) at room temperature (30.degree. C.) for 2
hours. After washing the wells once with 0.2% PBS/T, they were
blocked with 5% PBS/milk for one hour. Monomeric anti-MET
antibodies (Fab), 13-MET and 28-MET, or dimeric anti-MET
antibodies, 9-MET and 19-MET, were preincubated with 25 ng/well
recombinant human MET/Fc chimeric protein (R&D Systems,
Minneapolis, Minn.) at room temperature for 1 hour. The Fab/MET-Fc
mixtures with increasing concentrations of anti-MET antibodies were
then added to the HGF-coated wells and allowed to incubate for one
hour at room temperature on a rocker and washed three times with
0.2% PBS/T. The secondary antibody (goat anti-human Fc,
HRP-conjugated) was added at 1:5,000 dilution and allowed to
incubate for one hour at room temperature. Washing was repeated as
described above. Fifty microliters of substrate were added per well
until a yellow color developed. The reaction was then stopped with
50 .mu.L of 1 M H.sub.2SO.sub.4 and the absorbance at 450 nm
determined with a standard plate reader.
[0197] Both 13-MET and 28-MET Fab monomeric antibodies effectively
blocked binding between the MET extracellular domain and HGF (FIG.
1B, C) with an IC.sub.50 of 12.44 and 36.08, the slope of the curve
(m) of 0.81443 and 0.77698, and curvefit value (r) of 0.96229 and
0.98873, respectively. Similarly, 9-MET and 19-MET dimeric Fab
antibodies effectively blocked binding between MET and its ligand
HGF (FIG. 2A, B) with a Dm of 5.08 and 44.34, respectively.
[0198] Epitope mapping of the MET-13 and MET-28 IgG antibodies was
performed using 384-well microtiter plates coated with the human
recombinant proteins MET-ECD, SEMA, and PSI-IPT. Binding of MET-13
and MET-28 IgG antibodies to individual domains of MET was
determined with a standard plate reader.
[0199] The epitope analysis revealed that the 13-MET IgG antibody
binds to the SEMA domain of MET-ECD and not to PSI-IPT, whereas the
28-MET IgG antibody exclusively binds to the full-length MET-ECD
molecule and not to either the SEMA or PSI-IST domains in
isolation. Since the SEMA domain of MET-ECD is known to be critical
for HGF binding, the binding of the 13-MET antibody to the SEMA
domain suggests that R13 is a direct HGF-competitor. The 28-MET
antibody, on the other hand, competes with HGF but does not bind to
the critical SEMA domain of MET-ECD, indicating that the 28-MET
antibody recognizes an epitope in its tertiary conformation.
Example 3
Combinations of Anti-MET Antibodies Act Synergistically to Block
HGF Binding to the MET Receptor Extracellular Domain and Induce
ADCC 6 cells
[0200] Combinations of MET antibodies act synergistically to block
hHGF binding to the MET Receptor in vitro. In certain embodiments,
the HGF/MET blocking assay described in detail above was used to
assess HGF ligand binding to the MET Receptor. A combination of
9-MET and 19-MET antibodies (1:5) demonstrated increased efficacy
in blocking binding between HGF and MET compared to either antibody
alone (FIG. 3A), with an IC50 of 4.44 compared to 8.88 for 9-MET
and 105.9 for 19-MET.
[0201] MET antibodies induce Antibody-dependent Cellular
Cytotoxicity (ADCC) in vitro. In certain embodiments, an ADCC assay
was used to assess ADCC activity of the MET antibodies. Blood was
collected from normal volunteers and mixed with 33% (v/v) volume of
PBS without Ca.sup.2+ or Mg.sup.2+. The mixture was layered onto a
Ficoll gradient and centrifuged at 400 relative centrifugal force x
g for 40 min. Peripheral blood mononuclear cells were collected at
the interface and washed three times in PBS. The cells were
pelleted and then resuspended in RPMI 1640 medium with 10% fetal
calf serum. GTL-16 cells were europium-labeled according to the
manufacturer's protocol (Perkin Elmer) and plated at a density of
10,000 cells/well in 50 .mu.l in a 96-well U-bottomed plate, and
incubated with the indicated anti-MET antibodies (50 .mu.l) at
37.degree. C. for 30 minutes. Peripheral blood mononuclear cells
were added to triplicate wells in a volume of 100 .mu.l at an
effector/tumor cell ratios of 100/1 and incubated at 37.degree. C.
for 4 hours. After centrifugation at 1500 rpm for 10 minutes, 10
.mu.l of supernatant was transferred to a 96-well flat-bottom
plate, followed by the addition of 100 .mu.l/well
europium-releasing reagent, an incubation of 15 minutes under
rocking conditions and a reading of the fluorescence at 615 nm.
Target maximum fluorescence was determined by lysing the cells with
10 .mu.l of Lysis buffer, whereas target spontaneous fluorescence
was determined in the absence of antibody and effector cells. The
percentage of specific cell lysis mediated by the antibodies was
calculated as: the percentage of cell lysis in the antibody-treated
group: (experimental EM615 nm-target spontaneous)/(target
maximum-target spontaneous).times.100. Similarly, a combination of
13-MET and 28-MET IgG (1:5) demonstrated increased efficacy in
inducing antibody-dependent cellular cytotoxicity (ADCC) on GTL-16
cells to either antibody alone (FIG. 3C), with an EC50 of 58.9
compared to 479.1 for 13-MET IgG and 3,771 for 28-MET IgG.
[0202] Antibody interactions were then analyzed using CalcuSyn
(Chou and Hayball, 1996). This software calculates the IC50 of the
antibody combinations using the median effect equation.
Determination of synergy or antagonism was based on the multiple
drug effect equation of Chou and Talalay (1977, 1983) and was
quantified by the combination index (CI). CI=1 indicates an
additive effect; <1, synergy, >1, antagonism. Results are
shown for the mutually exclusive assumption of modes of activity of
the drugs, however, applying the alternative assumption showed the
same pattern of results (FIG. 3B, D). The CI of the 9-MET/19-MET
(1:5) was 0.71 at ED50; 0.52 at ED75, and 0.40 at ED90 (FIG. 3B).
The CI of 13-MET/28-MET (1:5) was 0.20 at ED50; 0.044 at ED7S; and
0.018 at EC90 (FIG. 3D).
[0203] R13 was also shown to enhance R28 avidity to MET-receptor on
GTL-16 cells (FIG. 3E). AF647-conjugated R28 (black bars) or R13
(white bars) were used as FACS-reagents to detect MET-receptor on
GTL-16 cells. AF647-labeled antibodies (AF647-R28 or AF647-R13)
were used at fixed concentrations (360 nM, AF647-R28; 20 nM,
AF647-R13) and unlabeled R13 (0.5 mM, 20 nM) or R28 (90 nM, 360 nM)
was titered in. Note that 20 nM of R13 increased MFI-values
(.DELTA.MFI) for AF647-R28 by 2.6 fold, whereas adding R28 to
AF647-R13 did not show any effect. .DELTA.MFI values were
determined by substracting background MFI. NS indicates not
stimulated. Arrows indicate the detected proteins. bars, SD.
Example 4
Combinations of Anti-MET Antibodies Eliminate Detectable
HGF-Mediated MET Signaling
[0204] Combinations of MET antibodies eliminated hHGF-mediated MET
Receptor signaling in vivo. A549 lung tumor cells were plated onto
culture plates and serum-starved (0.1% FBS in DMEM) for 24 hours.
The cells were then either left untreated or were pretreated with
the indicated Fab-monomers, -dimers or IgGs (30 .mu.g/ml) for one
to two hours following stimulation with 80 ng/ml recombinant human
HGF for 10 min at 37.degree. C. After HGF stimulation, the cells
were lysed on ice in lysis buffer (50 mM HEPES pH 7.5, containing
150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100,
1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 2 mM
sodium orthovanadate, 5 mM .beta.-glycerolphosphate, 10 mg/ml
aprotinin). Crude lysates were centrifuged at 13,000 g for 20
minutes at 4.degree. C. before protein concentrations were
determined.
[0205] For immunoprecipitations, the appropriate antibody and
protein A/G Sepharose (Pharmacia) were added to the cleared lysate
and incubated for 3 hours at 4.degree. C. Immunoprecipitates were
washed with a washing buffer (20 mM HEPES pH 7.5, containing 150 mM
NaCl, 1 mM EDTA, 1 mM sodium fluoride, 10% (v/v) glycerol, 0.1%
(v/v) Triton X-100). Sample buffer containing SDS and DTT was added
and the samples were denaturated by heating at 75.degree. C. for 10
minutes. Proteins were fractionated by SDS-PAGE and
electrophoretically transferred to nitrocellulose filters.
[0206] For immunoblot analysis, nitrocellulose filters were first
incubated with mouse monoclonal or rabbit polyclonal primary
antibodies for 3 hours at 4.degree. C. Next, a HRP-coupled goat
anti-mouse or goat anti-rabbit secondary antibody was added,
followed by an enhanced chemiluminescence (ECL) substrate reaction
(Amersham). The substrate reaction was detected on Kodak X-Omat
film. Filters that were used more than once with different
antibodies were stripped according to the manufacturer's protocol,
blocked and reprobed. Antibodies raised against following proteins
were used: MET (monoclonal mouse antibody (mmAb) DO-24, UBI and
polyclonal rabbit antibody (prAb) C-12, Santa Cruz), phospho-MET
(monoclonal rabbit antibody (mrAb) 3D7, Cell Signaling), SHC (prAb
UBI), phospho-AKT (mrAb 193H12, Cell Signaling), phospho-MAPK (mrAb
197G2, Cell Signaling) and phosphotyrosine (mAb 4G10, UBI).
[0207] The combination of 13-28-MET monomeric antibodies eliminated
detectable phosphorylation of the downstream MET signaling protein,
SHC (FIG. 4A). Similarly, the combination of 9-19-MET dimeric
antibodies eliminated or nearly eliminated detectable
phosphorylation of the MET Receptor as well as phosphorylation of
the downstream signaling molecules SHC, AKT, and ERK1/2 (FIG.
4B-D). The elimination of downstream signaling activation by
anti-MET antibody combinations in lung tumor cells mimicked the
block of HGF-mediated MET signaling by SU11248 (FIG. 5). Similar
results were obtained using HUVEC cells (FIG. 6A-C).
Example 5
Combinations of Anti-MET Antibodies Prevent HGF-Mediated Cell
Proliferation
[0208] Combinations of MET antibodies prevented hHGF-mediated cell
proliferation in vitro. In certain embodiments, HUVEC cells were
plated at 8.times.10.sup.4 per ml onto Collagen I coated 96-well
culture plates in complete media (serum and growth factors; EGM-2),
incubated for 24 hours and subsequently serum-starved for 24 hours
in EBM-2 supplemented with 5% fetal bovine serum (FBS). HUVEC cells
were either left untreated or pretreated with the indicated
Fab-monomers, -dimers or IgGs (30 .mu.g/ml) for 1-2 hours and
subsequently challenged with 50 ng/ml recombinant human HGF. Cells
were incubated for seven days and cell number was quantified using
Cell-Titer-Glo Reagent (Promega) according to the manufacturer's
protocol every 48 hours.
[0209] The 9-19-MET antibody combination had no effect on cell
proliferation in the absence of HGF (FIG. 6D; upper graph). In
contrast, the antibody combination eliminated cell proliferation in
the presence of HGF compared to control treated cells (FIG.
6D).
Example 6
Combinations of Anti-MET Antibodies Prevent HGF-Mediated Cell
Migration
[0210] Combinations of MET antibodies prevented HGF-mediated cell
migration in vitro. H441 cells were seeded at a density of
3.times.10.sup.5 cells/well in a 24-well plate. HUVECs were seeded
at a density of 2.5.times.10.sup.5 cells/well in a 24-well
pre-coated with Collagen I. The next day, cells were serum-starved
for 24 hours with media containing 0.5% FCS. Then a single scrape
was made in the confluent monolayer in each well as described
previously (Lorenzato et al., 2002). For HGF-dependent studies,
cells were pre-incubated with indicated amounts of Fab dimers for 1
hour prior to HGF addition. Photographs were taken when the gap in
HGF-treated cells had closed completely 24 hours later. For
HGF-independent studies, cells were treated as above without
addition of HGF. The scrape was monitored and photographed.
[0211] H441 cells fail to proliferate into the scrape in the
absence of HGF, but nearly completely fill in the scrape in the
presence of HGF after 16 hours. Incubation with the 13-28-MET IgG
combination disrupts this cell migration to a similar extent as
SU11274 (FIG. 7A).
Example 7
[0212] Anti-MET Antibody Combinations Alter the Conformation of the
MET Receptor
[0213] This example describes the conformational changes induced in
the MET receptor by the anti-MET antibodies. Since the
above-described inhibitory effects of 13-MET and 28-MET antibodies
are synergistic and the antibodies do not compete for the same
epitope, the ability of one antibody to decrease the flexibility of
MET, thereby enabling the other antibody to block the HGF binding
site was tested. The belief was that one antibody would increase
the binding of the other antibody to cells expressing MET in its
active form. Therefore, one unlabeled antibody (13-MET IgG ("R13")
or 28-MET ("R28")) was titered to the other AF647-conjugated
antibody (28-MET ('R28'') or 13-MET ("R13")) and then the mean
fluorescence intensity (MFI) on GTL-16 cells was measured.
[0214] More specifically, GTL-16 cells were non-enzymatically
detached from cell culture plates, washed and blocked with PBS/2%
FCS for 30 min prior to incubation with the antibodies. 100 .mu.g
of 13-MET IgG antibody and 28-MET IgG antibody were chemically
conjugated with fluorochrome AF-647 following the supplier's
protocol (Invitrogen (Carlsbad, Calif.)), Cells were then incubated
with unlabeled and/or labeled 13-MET/28-MET at the concentrations
indicated in FIG. 8 for 30 min at RT. After extensive washing cells
were resuspended in PBS/2% FCS and analyzed by FACS.
[0215] The results are shown in FIG. 8. The .DELTA.MFI values of
AF647-conjugated 28-MET increased by increasing amounts of
unlabeled 13-MET. Since the Kd values remained unchanged (data not
shown), this suggests that 13-MET increases the antigen
accessibility of 28-MET for MET on GTL-16 cells, which leads to an
increase in .DELTA.MFI values. Conversely, unlabeled 28-MET did not
increase 13-MET-induced .DELTA.MFI on GTL-16 cells. Taken together,
this experiment indicates that 13-MET facilitates the binding of
28-MET on GTL-16 cells, thereby potentiating the binding of 28-MET
to the MET extracellular domain and "locking" it into a
non-functional receptor.
Example 8
In Vivo Treatment of Established Tumors Using Anti-MET
Antibodies
[0216] This example describes the use of a combination of human
anti-MET antibodies to treat cancer in a xenograft model. In
certain embodiments, tumor cells from a patient sample (solid tumor
biopsy or pleural effusion) that have been passaged as a xenograft
in mice were prepared for repassaging into experimental animals.
Tumor tissue was removed, cut up into small pieces, minced
completely using sterile blades, and single cell suspensions
obtained by enzymatic digestion and mechanical disruption.
Dissociated tumor cells are then injected subcutaneously either
into the mammary fat pads, for breast tumors, or were injects at
50,000 per animal into the right flank, for OMP-C12, C17, C27 and
C28 colon tumors, of NOD/SCID mice to elicit tumor growth.
Alternatively, ESA+, CD44+, CD24-/low, Lin-tumorigenic tumor cells
are isolated as described in detail above and injected.
[0217] Following tumor cell injection, animals were monitored for
tumor growth. Once the tumors have reached an average size of
approximately 65 to 200 mm.sup.3 mice were randomized and antibody
treatment began. Each animal received i.p. either 45 mg/kg (C12,
C27 and C28) or 30 mg/kg (C17) of 13-MET and 28-MET antibodies at a
ratio of 1:8 once a week. Tumor size was assessed twice a week.
Humanized MET antibodies reduced tumor growth compared to control
antibody 1B7.11 (murine IgG from ATCC) in C12, C17, C27 and C28
tumors (FIG. 9B-E). Administration of a 1:8 ratio of 13-MET to
28-MET antibodies resulted in a statistically significant decrease
in C12 tumor volume compared to control antibody treated animals at
day 77 post-injection (p<0.01) and day 81 to 105 (p<0.001)
post-injection (FIG. 9B). Administration of a 1:8 ratio of 13-MET
to 28-MET antibodies resulted in a statistically significant
decrease in C17 tumor volume compared to control antibody treated
animals at day 58 (p<0.01) and day 62 (p<0.001)
post-injection (FIG. 9C). Administration of a 1:8 ratio of 13-MET
to 28-MET antibodies resulted in a statistically significant
decrease in C27 tumor volume compared to control antibody treated
animals at day 41 (p<0.05) and day 44 to 48 (p<0.001)
post-injection (FIG. 9D). Administration of a 1:8 ratio of 13-MET
to 28-MET antibodies resulted in a statistically significant
decrease in C28 tumor volume compared to control antibody treated
animals at day 40 to 48 post-injection (p<0.01) (FIG. 9E).
[0218] Histologic assessment showed extensive hypoxic areas in
13-MET/28-MET(R13/28)-treatment groups when compared to control
C-27-tumors. Staining for hypoxic regions was performed as reported
previously (Raleigh, J. A., et al, Int J Radiat Oncol Biol Phys,
42:727-730 (1998)). Briefly, to measure hypoxia,
pimonidazole-hydrochloride (HypoxyProbe, NPI, Burlington, Mass.),
which forms long-lived protein adducts at partial pressure of
oxygen less than approximately 10 mmHg, was injected
intraperitoneally at 60 mg/kg 1 hr prior to sacrifice. Tumors were
then processed for histological analysis, and tumor sections (5
.mu.m thick) were stained using anti-pimonidazole antibody.
Photographs were taken using a BX51 microscope (Olympus, Center
Valley, Pa.).
[0219] Even more pronounced effects were observed the experiment
was repeated with GTL-16 cells: combination of
13-MET/28-METstrongly inhibited GTL-16 tumor-growth and increased
hypoxic regions almost over the entire tumor surface area,
especially in the center of the tumor. Healthy cells were only
detectable at the rim of the GTL-16 tumors. The data indicate that
inhibition of MET signaling abrogated pro-angiogenic signals, which
results in diminished tumor growth. This mechanism resembles the
treatment of tumors with anti-HER2 antibody trastuzumab, which also
indirectly lead to a decrease in pro-angiogenic factors, but to a
dramatic increase in thrombospondin, a negative-regulator of
angiogenesis (Izumi, Y., et al., Nature, 416: 279-280 (2002)).
[0220] The gene-expression profile of 13-MET/28-MET-treated C27
tumors was analyzed by microarray. Global gene expression profiling
analysis was performed on Affymetrix HG-U133 plus 2.0 microarray
(Affymetrix, Santa Clara, Calif.). Three independent RNA samples of
xenograft whole tumors from the control and treatment groups were
isolated and hybridized to the microarrays according to the
manufacturer's instructions. Scanned array background adjustment
and signal intensity normalization were performed with GCRMA
algorithm in the open-source biocoriductor software
(www.bioconductor.org). Genes differentially expressed (P<0.05
and fold change>2.0) between the two groups were identified with
Bayesian t-test (Baldi, P. and Long, A.D. Bioinformatics, 17:
509-519 (2001)).
TABLE-US-00003 TABLE 2 Gene-expression profile of
13-MET/28-MET-treated tumors Gene Fold PVal LOC441453 -2.25 3.10
.times. 10.sup.-4 KITLG -2.43 1.46 .times. 10.sup.-4 DOK7 -2.56
6.91 .times. 10.sup.-5 DCLK1 -7.05 6.79 .times. 10.sup.-8 BCL11A
-3.53 3.07 .times. 10.sup.-5 ACTA1 -2.08 2.32 .times. 10.sup.-4
PRKACB -2.74 1.20 .times. 10.sup.-4 DHRS3 -3.58 1.42 .times.
10.sup.-5 CRYAB -3.17 2.58 .times. 10.sup.-4 METTL7B -2.05 8.53
.times. 10.sup.-4 MYLPF -3.82 1.61 .times. 10.sup.-4 C11orf31 -2.80
7.30 .times. 10.sup.-4 CST6 -6.56 1.86 .times. 10.sup.-4 SERPINE2
-4.20 5.03 .times. 10.sup.-4 PCDH10 -6.45 8.97 .times. 10.sup.-4
RAB3B 3.53 8.37 .times. 10.sup.-4 LMO7 2.01 2.86 .times. 10.sup.-4
EDN1 2.36 9.81 .times. 10.sup.-4 KLHL24 2.91 4.75 .times. 10.sup.-4
LCE3D 2.86 1.01 .times. 10.sup.-4 KLF6 2.29 5.27 .times. 10.sup.-4
SLC12A6 2.49 6.14 .times. 10.sup.-4 NOS3 2.42 3.99 .times.
10.sup.-4 PIK3IP1 2.32 2.69 .times. 10.sup.-4 HAS3 2.92 2.20
.times. 10.sup.-4 HAPLN3 2.15 3.65 .times. 10.sup.-4 SPTAN1 2.43
5.43 .times. 10.sup.-4 ATG9B 3.75 3.02 .times. 10.sup.-5 HLA-F 2.97
7.33 .times. 10.sup.-5 KRT17 3.17 7.01 .times. 10.sup.-6 AHNAK2
2.03 1.47 .times. 10.sup.-5 MGC3260 2.36 5.15 .times. 10.sup.-4
ARHGAP29 2.21 1.61 .times. 10.sup.-4 MAP3K8 3.40 4.19 .times.
10.sup.-5 TncRNA 3.74 1.32 .times. 10.sup.-5 IL6R 3.03 8.30 .times.
10.sup.-5 MEF2A 2.30 3.60 .times. 10.sup.-4 MAST4 2.77 1.96 .times.
10.sup.-4 CEACAM1 2.14 3.20 .times. 10.sup.-4 TGFA 2.02 2.90
.times. 10.sup.-4 ECM1 2.49 1.06 .times. 10.sup.-4 BMP2 2.76 2.40
.times. 10.sup.-4 DUSP5 3.86 8.29 .times. 10.sup.-7 PRDM1 2.47 8.60
.times. 10.sup.-7 FRMD4A 2.42 5.51 .times. 10.sup.-5 LOC162993 3.66
5.02 .times. 10.sup.-6 HSD11B1 48.85 .sup. 8.88 .times.
10.sup.-16
[0221] Interestingly, inhibition of HGF/MET pathway significantly
up-regulated the known tumor-suppressors KLF6, CEACAM1 and BMP2
(2.3-fold, 2.1-fold and 2.8-fold, P<0.001) and the
negative-regulator of Phosphatidyl-inositol-3-OH-kinase (PI3K)
PIK3IP1 (2.3-fold, P<0.001). Concurrently, SCF and SERPINE2,
both enhancers of proliferation and invasiveness, were
significantly suppressed (SCF, 2.4-fold, SERPIINE2, 4.2-fold,
P<0.001). It has previously been shown, that the tumor
suppressor functions of KLF6, CEACAM1 and BMP2 are inactivated in
CRC (Miyaki, M., et al., Oncology 71:131-135 (2006); Mukai, S. et
al., World J Gastroenterol 13:3932-3938 (2007); Shively, J. E.
Oncogene 23:9303-9305 (2004)). Moreover, SCF/KIT-receptor signaling
has been implicated in proliferation and invasiveness of CRC
through the PI3K/AKT pathway (Yasuda, A. et al., Dig Dis Sci,
52:2292-2300 (2007)). A recent report by Zhu et al. suggests that
PIK31P1 is a novel p110 interacting protein, which directly down
modulates PI3K-activity (Biochem Biophys Res Commun, 358:66-72
(2007)). On the other hand, SERPINE2 has been involved in enhancing
invasive potential of pancreas cancer cells in nude mice (Buchholz,
M. et al., Cancer Res, 63:4945-4951 (2003)). One mode of action of
13-MET/28-MET could be the restoration of the tumor suppressor
function of KLF6, CEACAM1 and BMP2, which ultimately would inhibit
tumor progression. Additionally, 13-MET/28-MET-induced PIK31P1
could amplify the inhibition of AKT1 phosphorylation and also
potentiate 13-MET/28-MET-induced down-regulation of SCF via
abrogated PI3K-activity, resulting in diminished
anti-apoptotic/migratory signaling.
[0222] In certain embodiments, combinations of anti-MET antibodies
are administered. In certain embodiments, a combination of 13-MET
and 28-MET human IgG antibodies are administered. In certain
embodiments, a combination of 9-MET and 19-MET dimeric Fab
antibodies are administered. In certain embodiments, the 13-MET and
28-MET antibodies are administered at a ratio of 1:5. In certain
embodiments, the 9-MET and 19-MET antibodies are administered at a
ratio of 1:5.
[0223] In certain embodiments, 1.times.10.sup.7 GTL-16 tumor cells
were injected subcutaneously into the right posterior flanks of 6-8
week-old immunodeficient female mice on a Swiss CD-1 background
(Harlan). The injected total volume per mouse was 200 .mu.l with
50% being Matrigel (BD Biosciences). Once the tumors have reached
an average size of approximately 65 to 200 mm.sup.3 mice were
randomized and antibody treatment began with either 13-MET and
28-MET antibodies administered weekly at a ratio of 1:8 at 45 mg/kg
(FIG. 9A), or at 15 mg/kg of the 13-21 MET bispecific antibody
73R21.13 (FIG. 9F). Tumor volume was measured twice weekly as
described in Michieli et al., Cancer Cell 2004 July; 6(1):61-73.
All the experiments were performed on groups of at least ten
animals per experimental point. Administration of a 1:8 ratio of
13-MET to 28-MET antibodies resulted in a statistically significant
decrease in tumor volume compared to control antibody treated
animals at day 15 post-injection (p<0.01) and at day 19
(p<0.001) (FIG. 9A). Administration of the bispecific 13-21 MET
antibody 73R21.13 also resulted in a statistically significant
decrease in tumor volume compared to control antibody treated
animals (FIG. 9F).
[0224] At the end point of antibody treatment, tumors are harvested
for further analysis. In some embodiments, a portion of the tumor
is analyzed by immunofluorescence to assess antibody penetration
into the tumor and tumor response. A portion of each harvested
tumor from anti-MET treated and control antibody treated mice is
fresh-frozen in liquid nitrogen, embedded in O.C.T., and cut on a
cryostat as 10 .mu.n3 sections onto glass slides. In some
embodiments, a portion of each tumor is formalin-fixed,
paraffin-embedded, and cut on a microtome as 10 .mu.m section onto
glass slides. Sections are post-fixed and incubated with
chromophore labeled antibodies that specifically recognize injected
antibodies to detect anti-MET receptor or control antibodies
present in the tumor biopsy. Furthermore antibodies that detect
different tumor and tumor-recruited cell types such as, for
example, anti-VE cadherin (CD144) or anti-PECAM-1 (CD31) antibodies
to detect vascular endothelial cells, anti-smooth muscle
alpha-actin antibodies to detect vascular smooth muscle cells,
anti-Ki67 antibodies to detect proliferating cells, TUNEL assays to
detect dying cells, anti-intracellular domain (ICD) Notch fragment
antibodies to detect Notch signaling can be used to assess the
effects of antibody treatment on, for example, angiogenesis, tumor
growth and tumor morphology.
[0225] In certain embodiments, the effect of humanized anti-MET
antibody treatment on tumor cell gene expression is also assessed.
Total RNA is extracted from a portion of each harvested tumor from
MET antibody treated and control antibody treated mice and used for
quantitative RT-PCR. Expression levels of the MET receptor, MET
ligands, components of the MET signaling pathway, as well as
addition cancer stem cell markers previously identified (e.g. CD44)
are analyzed relative to the house-keeping gene GAPDH as an
internal control. Changes in tumor cell gene expression upon MET
antibody treatment are thus determined.
[0226] In addition, the effect of humanized anti-MET receptor
antibody treatment on the presence of cancer stem cells in a tumor
is assessed, Tumor samples from MET versus control antibody treated
mice are cut up into small pieces, minced completely using sterile
blades, and single cell suspensions obtained by enzymatic digestion
and mechanical disruption. Dissociated tumor cells are then
analyzed by FACS analysis for the presence of tumorigenic cancer
stem cells based on ESA+, CD44+, CD24-/low, Lin-surface cell marker
expression as described in detail above.
[0227] The tumorigenicity of cells isolated based on ESA+, CD44+,
CD24-/low, Lin-expression following humanized anti-MET antibody
treatment can then assessed. ESA+, CD44+, CD24-/low, Lin-cancer
stem cells isolated from MET antibody treated versus control
antibody treated mice are re-injected subcutaneously into the
mammary fat pads of NOD/SCID mice. The tumorigenicity of cancer
stem cells based on the number of injected cells required for
consistent tumor formation is then determined.
Example 9
Decrease in Lung Metastases Using Anti-MET Antibodies
[0228] This example describes the use of a combination of human
anti-MET antibodies to increase survival by decreasing lung
metastases in vivo. It has previously been reported that inhibition
of MET in GTL-16 strongly decreased lung metastases in vivo (Corso,
S. et al. Oncogene, 27: 684-693, 2008). Therefore, the ability of
13-MET and 28-MET to increase survival by diminishing lung
metastases in an experimental metastasis model with GTL-16 cells
was tested. Experimental metastasis-assays were performed as
described previously (Corso, S. et al.) with the exception that
GTL-16-luc were used, and tumor burden in mice was visualized by
non-invasive imaging with an IVIS200 instrument (Caliper, Mountain
View, Calif.), as previously published (Zhang, G. J. et al.,
Neoplasia 9:652-661 (2007)). Antibodies were administered weekly or
as indicated and tumors measured twice weekly. Tumor volume was
calculated as described in Al-Hajj et al. (Proc Natl Acad Sci USA
100:3983-3988 (2003)). All the experiments were performed on groups
of at least ten animals per experimental point.
[0229] Mice were injected with GTL-16 cells stably expressing the
luciferase (luc)-gene and treated weekly with 13-MET/28-MET
(R13/28) antibodies or control antibody 1B7.11. Treatment was
stopped after three weeks and the disease recurrence was measured
by non-invasive imaging every week. After 70 days five out of seven
mice were alive, and 4 out of five showed strong Inc-activity in
the lungs, whereas 13-MET/28-MET -treated animals were all alive
and did not show any luc-activity (data not shown). At day 170,
only one mouse in the 13-MET/28-MET -treatment group had died,
whereas in the control group six out of seven mice had died (FIG.
10). This suggests that the antibody-treatment with 13-MET/28-MET
antibodies inhibited either the extravasation of the tumor cells
and/or diminished the survival capabilities of GTL-16 in the lungs,
by preventing the binding of stromal-derived HGF to MET. Another
explanation is that 13-MET or 28-MET act as inverse agonists,
thereby shifting the equilibrium from the activated state towards
the inactive form of MET. This shift could then lead to decrease of
proliferation and increase of the apoptotic rate of GTL-16
cells.
Example 10
Treatment of Human Cancer Using Anti-MET Antibodies
[0230] This example describes methods for treating cancer using
human antibodies against MET to target tumors comprising cancer
stem cells. The presence of cancer stem cell marker expression can
first be determined from a tumor biopsy. Tumor cells from a biopsy
from a patient diagnosed with cancer are removed under sterile
conditions. In some embodiments the tissue biopsy is fresh-frozen
in liquid nitrogen, embedded in O.C.T., and cut on a cryostat as 10
.mu.m sections onto glass slides. In some embodiments, the tissue
biopsy is formalin-fixed, paraffin-embedded, and cut on a microtome
as 10 .mu.m section onto glass slides. In some embodiments,
sections are incubated with antibodies against MET to detect
protein expression.
[0231] The presence of cancer stem cells can also be determined.
Tissue biopsy samples are cut up into small pieces, minced
completely using sterile blades, and cells subject to enzymatic
digestion and mechanical disruption to obtain a single cell
suspension. Dissociated tumor cells are then incubated with
anti-ESA, --CD44, --CD24, and -Lin antibodies to detect cancer stem
cells, and the presence of ESA+, CD44+, CD24-/low, and Lin-tumor
stem cells is determined by flow cytometry as described in detail
above.
[0232] Cancer patients are treated with human anti-MET antibodies.
In certain embodiments, anti-MET antibodies generated as described
above are purified and formulated with a suitable pharmaceutical
vehicle for injection. In certain embodiments, combinations of
anti-MET antibodies are administered. In certain embodiments, a
combination of 13-MET and 28-MET human IgG antibodies are
administered. In certain embodiments, a combination of 9-MET and
19-MET dimeric Fab antibodies are administered. In certain
embodiments, the 13-MET and 28-MET antibodies are administered at a
ratio of 1:5. In certain embodiments, the 9-MET and 19-MET
antibodies are administered at a ratio of 1:5. In certain
embodiments bispecific antibodies comprising the antigen
determination region of 13-MET and 28-MET are administered.
[0233] In certain embodiments, patients are treated with the
antibodies at least once a month for at least ten weeks. In certain
embodiments, patients are treated with the antibodies at least once
a week for at least about fourteen weeks. Each administration of
the antibody should be a pharmaceutically effective dose. In some
embodiments, between about 2 to about 100 mg/ml of an antibody is
administered. In some embodiments, between about 5 to about 40
mg/ml of a humanized antibody is administered. The antibody can be
administered prior to, concurrently with, or after standard
radiotherapy regimens or chemotherapy regimens using one or more
chemotherapeutic agent, such as oxaliplatin, fluorouracil,
leucovorin, or streptozocin. Patients are monitored to determine
whether such treatment has resulted in an anti-tumor response based
on, for example, tumor regression, reduction in the incidences of
new tumors, lower tumor antigen expression, decreased numbers of
cancer stem cells, or other means of evaluating disease
prognosis.
[0234] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims. All publications and patents mentioned in the
above specification are herein incorporated by reference in their
entirety.
Sequence CWU 1
1
291366DNAArtificial SequenceSynthetic Fab-13 VH nucleotide sequence
1caggtgcaat tgaaagaaag cggcccggcc ctggtgaaac cgacccaaac cctgaccctg
60acctgtacct tttccggatt tagcctgtct acttctggta tggttgtgtc ttggattcgc
120cagccgcctg ggaaagccct cgagtggctg gcttttatct cttgggatga
tgataagtat 180tatagcacca gcctgaaaac gcgtctgacc attagcaaag
atacttcgaa aaatcaggtg 240gtgctgacta tgaccaacat ggacccggtg
gatacggcca cctattattg cgcgcgtgag 300cctggtcgtt atggtggtta
ttattttgat tattggggcc aaggcaccct ggtgacggtt 360agctca
3662122PRTArtificial SequenceSynthetic Fab-13 VH protein sequence
2Gln Val Gln Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1
5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr
Ser 20 25 30 Gly Met Val Val Ser Trp Ile Arg Gln Pro Pro Gly Lys
Ala Leu Glu 35 40 45 Trp Leu Ala Phe Ile Ser Trp Asp Asp Asp Lys
Tyr Tyr Ser Thr Ser 50 55 60 Leu Lys Thr Arg Leu Thr Ile Ser Lys
Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met
Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Glu Pro
Gly Arg Tyr Gly Gly Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 312PRTArtificial
SequenceSynthetic Fab-13 VH CDR1 3Gly Phe Ser Leu Ser Thr Ser Gly
Met Val Val Ser 1 5 10 416PRTArtificial SequenceSynthetic Fab-13 VH
CDR2 4Phe Ile Ser Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser Leu Lys
Thr 1 5 10 15 512PRTArtificial SequenceSynthetic Fab-13 VH CDR3
5Glu Pro Gly Arg Tyr Gly Gly Tyr Tyr Phe Asp Tyr 1 5 10
6327DNAArtificial SequenceSynthetic Fab-13 VL nucleotide sequence
6gatatccaga tgacccagag cccgtctagc ctgagcgcga gcgtgggtga tcgtgtgacc
60attacctgca gagcgagcca gactatttct cattatctgg cttggtacca gcagaaacca
120ggtaaagcac cgaaactatt aatttatgct gcttctattt tgcaaagcgg
ggtcccgtcc 180cgttttagcg gctctggatc cggcactgat tttaccctga
ccattagcag cctgcaacct 240gaagactttg cggtttatta ttgccagcag
tattctggtt ttcctgttac ctttggccag 300ggtacgaaag ttgaaattaa acgtacg
3277109PRTArtificial SequenceSynthetic Fab-13 VL protein sequence
7Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser His
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ile Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Ser Gly Phe Pro Val 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr 100 105 811PRTArtificial
SequenceSynthetic Fab-13 VL CDR1 8Arg Ala Ser Gln Thr Ile Ser His
Tyr Leu Ala 1 5 10 97PRTArtificial SequenceSynthetic Fab-13 VL CDR2
9Ala Ala Ser Ile Leu Gln Ser 1 5 108PRTArtificial SequenceSynthetic
Fab-13 VL CDR3 10Gln Gln Tyr Ser Gly Phe Pro Val 1 5
11366DNAArtificial SequenceSynthetic Fab-28 VH nucleotide sequence
11caggtgcaat tgcaagaaag tggtccgggc ctggtgaaac cgggcgaaac cctgagcctg
60acctgcaccg tttccggagg cagcatttct ggttattatt ggtcttggat tcgccaggcc
120cctgggaagg gtctcgagtg gattggcgag atctattatg ctggctctac
cctttataat 180ccgagcctga aaggccgggt gaccattagc gttgatactt
cgaaaaacca gtttagcctg 240aaactgagca gcgtgacggc ggaagatacg
gccgtgtatt attgcgcgcg tcattatggt 300cttgattggt ttggtgatac
tggtatggat gtttggggcc aaggcaccct ggtgacggtt 360agctca
36612122PRTArtificial SequenceSynthetic Fab-28 VH protein sequence
12Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Gly Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Gly
Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Tyr Ala Gly Ser Thr Leu Tyr
Asn Pro Ser Leu Lys 50 55 60 Gly Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg His Tyr Gly Leu
Asp Trp Phe Gly Asp Thr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 1310PRTArtificial
SequenceSynthetic Fab-28 VH CDR1 13Gly Gly Ser Ile Ser Gly Tyr Tyr
Trp Ser 1 5 10 1416PRTArtificial SequenceSynthetic Fab-28 VH CDR2
14Glu Ile Tyr Tyr Ala Gly Ser Thr Leu Tyr Asn Pro Ser Leu Lys Gly 1
5 10 15 1514PRTArtificial SequenceSynthetic Fab-28 VH CDR3 15His
Tyr Gly Leu Asp Trp Phe Gly Asp Thr Gly Met Asp Val 1 5 10
16324DNAArtificial SequenceSynthetic Fab-28 VL nucleotide sequence
16gatatcgaac tgacccagcc gccttcagtg agcgttgcac caggtcagag cattaccatc
60tcgtgtagcg gcgataatct tggtgataag tatgttcatt ggtaccagca gaaacccggg
120caggcgccag ttcttgtgat ttatgatgat aatgagcgtc cctcaggcat
cccggaacgc 180tttagcggat ccaacagcgg caacaccgcg accctgacca
ttagcggcac tcaggcggaa 240gacgaagcgg attattattg ctctgcttat
ggttctcatt ctggtactgt gtttggcggc 300ggcacgaagt taaccgttct tggc
32417108PRTArtificial SequenceSynthetic Fab-28 VL protein sequence
17Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1
5 10 15 Ser Ile Thr Ile Ser Cys Ser Gly Asp Asn Leu Gly Asp Lys Tyr
Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu
Val Ile Tyr 35 40 45 Asp Asp Asn Glu Arg Pro Ser Gly Ile Pro Glu
Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr
Ile Ser Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys
Ser Ala Tyr Gly Ser His Ser Gly Thr 85 90 95 Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly 100 105 1811PRTArtificial
SequenceSynthetic Fab-28 VL CDR1 18Ser Gly Asp Asn Leu Gly Asp Lys
Tyr Val His 1 5 10 198PRTArtificial SequenceSynthetic Fab-28 VL
CDR2 19Asp Asp Asn Glu Arg Pro Ser Gly 1 5 209PRTArtificial
SequenceSynthetic Fab-28 VL CDR3 20Ser Ala Tyr Gly Ser His Ser Gly
Thr 1 5 21327DNAArtificial SequenceSynthetic Fab-21 VL nucleotide
sequence 21gatatcgaac tgacccagcc gccttcagtg agcgttgcac caggtcagac
cgcgcgtatc 60tcgtgtagcg gcgataatct tggtgagcag tatgttcatt ggtaccagca
gaaacccggg 120caggcgccag ttcttgtgat ttatgatgat tctgagcgtc
cctcaggcat cccggaacgc 180tttagcggat ccaacagcgg caacaccgcg
accctgacca ttagcggcac tcaggcggaa 240gacgaagcgg attattattg
ccagtcttat actttttatc ctaattctcg tgtgtttggc 300ggcggcacga
agttaaccgt tcttggc 32722109PRTArtificial SequenceSynthetic Fab-21
VL protein sequence 22Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser
Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Ser Cys Ser Gly Asp
Asn Leu Gly Glu Gln Tyr Val 20 25 30 His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Asp Asp Ser Glu Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly
Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu 65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Thr Phe Tyr Pro Asn Ser 85 90
95 Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105
2311PRTArtificial SequenceSynthetic Fab-21 VL CDR1 23Ser Gly Asp
Asn Leu Gly Glu Gln Tyr Val His 1 5 10 248PRTArtificial
SequenceSynthetic Fab-21 VL CDR2 24Asp Asp Ser Glu Arg Pro Ser Gly
1 5 2510PRTArtificial SequenceSynthetic Fab-21 VL CDR3 25Gln Ser
Tyr Thr Phe Tyr Pro Asn Ser Arg 1 5 10 264173DNAHomo sapiens
26atgaaggccc ccgctgtgct tgcacctggc atcctcgtgc tcctgtttac cttggtgcag
60aggagcaatg gggagtgtaa agaggcacta gcaaagtccg agatgaatgt gaatatgaag
120tatcagcttc ccaacttcac cgcggaaaca cccatccaga atgtcattct
acatgagcat 180cacattttcc ttggtgccac taactacatt tatgttttaa
atgaggaaga ccttcagaag 240gttgctgagt acaagactgg gcctgtgctg
gaacacccag attgtttccc atgtcaggac 300tgcagcagca aagccaattt
atcaggaggt gtttggaaag ataacatcaa catggctcta 360gttgtcgaca
cctactatga tgatcaactc attagctgtg gcagcgtcaa cagagggacc
420tgccagcgac atgtctttcc ccacaatcat actgctgaca tacagtcgga
ggttcactgc 480atattctccc cacagataga agagcccagc cagtgtcctg
actgtgtggt gagcgccctg 540ggagccaaag tcctttcatc tgtaaaggac
cggttcatca acttctttgt aggcaatacc 600ataaattctt cttatttccc
agatcatcca ttgcattcga tatcagtgag aaggctaaag 660gaaacgaaag
atggttttat gtttttgacg gaccagtcct acattgatgt tttacctgag
720ttcagagatt cttaccccat taagtatgtc catgcctttg aaagcaacaa
ttttatttac 780ttcttgacgg tccaaaggga aactctagat gctcagactt
ttcacacaag aataatcagg 840ttctgttcca taaactctgg attgcattcc
tacatggaaa tgcctctgga gtgtattctc 900acagaaaaga gaaaaaagag
atccacaaag aaggaagtgt ttaatatact tcaggctgcg 960tatgtcagca
agcctggggc ccagcttgct agacaaatag gagccagcct gaatgatgac
1020attcttttcg gggtgttcgc acaaagcaag ccagattctg ccgaaccaat
ggatcgatct 1080gccatgtgtg cattccctat caaatatgtc aacgacttct
tcaacaagat cgtcaacaaa 1140aacaatgtga gatgtctcca gcatttttac
ggacccaatc atgagcactg ctttaatagg 1200acacttctga gaaattcatc
aggctgtgaa gcgcgccgtg atgaatatcg aacagagttt 1260accacagctt
tgcagcgcgt tgacttattc atgggtcaat tcagcgaagt cctcttaaca
1320tctatatcca ccttcattaa aggagacctc accatagcta atcttgggac
atcagagggt 1380cgcttcatgc aggttgtggt ttctcgatca ggaccatcaa
cccctcatgt gaattttctc 1440ctggactccc atccagtgtc tccagaagtg
attgtggagc atacattaaa ccaaaatggc 1500tacacactgg ttatcactgg
gaagaagatc acgaagatcc cattgaatgg cttgggctgc 1560agacatttcc
agtcctgcag tcaatgcctc tctgccccac cctttgttca gtgtggctgg
1620tgccacgaca aatgtgtgcg atcggaggaa tgcctgagcg ggacatggac
tcaacagatc 1680tgtctgcctg caatctacaa ggttttccca aatagtgcac
cccttgaagg agggacaagg 1740ctgaccatat gtggctggga ctttggattt
cggaggaata ataaatttga tttaaagaaa 1800actagagttc tccttggaaa
tgagagctgc accttgactt taagtgagag cacgatgaat 1860acattgaaat
gcacagttgg tcctgccatg aataagcatt tcaatatgtc cataattatt
1920tcaaatggcc acgggacaac acaatacagt acattctcct atgtggatcc
tgtaataaca 1980agtatttcgc cgaaatacgg tcctatggct ggtggcactt
tacttacttt aactggaaat 2040tacctaaaca gtgggaattc tagacacatt
tcaattggtg gaaaaacatg tactttaaaa 2100agtgtgtcaa acagtattct
tgaatgttat accccagccc aaaccatttc aactgagttt 2160gctgttaaat
tgaaaattga cttagccaac cgagagacaa gcatcttcag ttaccgtgaa
2220gatcccattg tctatgaaat tcatccaacc aaatctttta ttagtggtgg
gagcacaata 2280acaggtgttg ggaaaaacct gaattcagtt agtgtcccga
gaatggtcat aaatgtgcat 2340gaagcaggaa ggaactttac agtggcatgt
caacatcgct ctaattcaga gataatctgt 2400tgtaccactc cttccctgca
acagctgaat ctgcaactcc ccctgaaaac caaagccttt 2460ttcatgttag
atgggatcct ttccaaatac tttgatctca tttatgtaca taatcctgtg
2520tttaagcctt ttgaaaagcc agtgatgatc tcaatgggca atgaaaatgt
actggaaatt 2580aagggaaatg atattgaccc tgaagcagtt aaaggtgaag
tgttaaaagt tggaaataag 2640agctgtgaga atatacactt acattctgaa
gccgttttat gcacggtccc caatgacctg 2700ctgaaattga acagcgagct
aaatatagag tggaagcaag caatttcttc aaccgtcctt 2760ggaaaagtaa
tagttcaacc agatcagaat ttcacaggat tgattgctgg tgttgtctca
2820atatcaacag cactgttatt actacttggg tttttcctgt ggctgaaaaa
gagaaagcaa 2880attaaagatc tgggcagtga attagttcgc tacgatgcaa
gagtacacac tcctcatttg 2940gataggcttg taagtgcccg aagtgtaagc
ccaactacag aaatggtttc aaatgaatct 3000gtagactacc gagctacttt
tccagaagat cagtttccta attcatctca gaacggttca 3060tgccgacaag
tgcagtatcc tctgacagac atgtccccca tcctaactag tggggactct
3120gatatatcca gtccattact gcaaaatact gtccacattg acctcagtgc
tctaaatcca 3180gagctggtcc aggcagtgca gcatgtagtg attgggccca
gtagcctgat tgtgcatttc 3240aatgaagtca taggaagagg gcattttggt
tgtgtatatc atgggacttt gttggacaat 3300gatggcaaga aaattcactg
tgctgtgaaa tccttgaaca gaatcactga cataggagaa 3360gtttcccaat
ttctgaccga gggaatcatc atgaaagatt ttagtcatcc caatgtcctc
3420tcgctcctgg gaatctgcct gcgaagtgaa gggtctccgc tggtggtcct
accatacatg 3480aaacatggag atcttcgaaa tttcattcga aatgagactc
ataatccaac tgtaaaagat 3540cttattggct ttggtcttca agtagccaaa
ggcatgaaat atcttgcaag caaaaagttt 3600gtccacagag acttggctgc
aagaaactgt atgctggatg aaaaattcac agtcaaggtt 3660gctgattttg
gtcttgccag agacatgtat gataaagaat actatagtgt acacaacaaa
3720acaggtgcaa agctgccagt gaagtggatg gctttggaaa gtctgcaaac
tcaaaagttt 3780accaccaagt cagatgtgtg gtcctttggc gtgctcctct
gggagctgat gacaagagga 3840gccccacctt atcctgacgt aaacaccttt
gatataactg tttacttgtt gcaagggaga 3900agactcctac aacccgaata
ctgcccagac cccttatatg aagtaatgct aaaatgctgg 3960caccctaaag
ccgaaatgcg cccatccttt tctgaactgg tgtcccggat atcagcgatc
4020ttctctactt tcattgggga gcactatgtc catgtgaacg ctacttatgt
gaacgtaaaa 4080tgtgtcgctc cgtatccttc tctgttgtca tcagaagata
acgctgatga tgaggtggac 4140acacgaccag cctccttctg ggagacatca tag
4173271390PRTHomo sapiens 27Met Lys Ala Pro Ala Val Leu Ala Pro Gly
Ile Leu Val Leu Leu Phe 1 5 10 15 Thr Leu Val Gln Arg Ser Asn Gly
Glu Cys Lys Glu Ala Leu Ala Lys 20 25 30 Ser Glu Met Asn Val Asn
Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala 35 40 45 Glu Thr Pro Ile
Gln Asn Val Ile Leu His Glu His His Ile Phe Leu 50 55 60 Gly Ala
Thr Asn Tyr Ile Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys 65 70 75 80
Val Ala Glu Tyr Lys Thr Gly Pro Val Leu Glu His Pro Asp Cys Phe 85
90 95 Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser Gly Gly Val
Trp 100 105 110 Lys Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr Tyr
Tyr Asp Asp 115 120 125 Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly
Thr Cys Gln Arg His 130 135 140 Val Phe Pro His Asn His Thr Ala Asp
Ile Gln Ser Glu Val His Cys 145 150 155 160 Ile Phe Ser Pro Gln Ile
Glu Glu Pro Ser Gln Cys Pro Asp Cys Val 165 170 175 Val Ser Ala Leu
Gly Ala Lys Val Leu Ser Ser Val Lys Asp Arg Phe 180 185 190 Ile Asn
Phe Phe Val Gly Asn Thr Ile Asn Ser Ser Tyr Phe Pro Asp 195 200 205
His Pro Leu His Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Lys Asp 210
215 220 Gly Phe Met Phe Leu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro
Glu 225 230 235 240 Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala
Phe Glu Ser Asn 245 250 255 Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg
Glu Thr Leu Asp Ala Gln 260 265 270 Thr Phe His Thr Arg Ile Ile Arg
Phe Cys Ser Ile Asn Ser Gly Leu 275 280 285 His Ser Tyr Met Glu Met
Pro Leu Glu Cys Ile Leu Thr Glu Lys Arg 290 295 300 Lys Lys Arg Ser
Thr Lys Lys Glu Val Phe Asn Ile Leu Gln Ala Ala 305 310 315 320 Tyr
Val Ser Lys Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser 325 330
335 Leu Asn Asp Asp Ile Leu Phe Gly Val Phe Ala Gln Ser Lys Pro Asp
340 345 350 Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys Ala Phe Pro
Ile Lys 355 360 365 Tyr Val Asn Asp Phe Phe Asn Lys Ile Val Asn Lys
Asn Asn Val Arg 370 375 380 Cys Leu Gln His Phe Tyr Gly Pro Asn His
Glu His Cys Phe Asn Arg 385 390 395 400 Thr Leu Leu Arg Asn Ser Ser
Gly Cys Glu Ala Arg Arg Asp Glu Tyr 405 410 415 Arg Thr Glu Phe Thr
Thr Ala Leu Gln Arg Val Asp Leu Phe Met Gly 420 425
430 Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys Gly
435 440 445 Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe
Met Gln 450 455 460 Val Val Val Ser Arg Ser Gly Pro Ser Thr Pro His
Val Asn Phe Leu 465 470 475 480 Leu Asp Ser His Pro Val Ser Pro Glu
Val Ile Val Glu His Thr Leu 485 490 495 Asn Gln Asn Gly Tyr Thr Leu
Val Ile Thr Gly Lys Lys Ile Thr Lys 500 505 510 Ile Pro Leu Asn Gly
Leu Gly Cys Arg His Phe Gln Ser Cys Ser Gln 515 520 525 Cys Leu Ser
Ala Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp Lys 530 535 540 Cys
Val Arg Ser Glu Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln Ile 545 550
555 560 Cys Leu Pro Ala Ile Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu
Glu 565 570 575 Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp Asp Phe Gly
Phe Arg Arg 580 585 590 Asn Asn Lys Phe Asp Leu Lys Lys Thr Arg Val
Leu Leu Gly Asn Glu 595 600 605 Ser Cys Thr Leu Thr Leu Ser Glu Ser
Thr Met Asn Thr Leu Lys Cys 610 615 620 Thr Val Gly Pro Ala Met Asn
Lys His Phe Asn Met Ser Ile Ile Ile 625 630 635 640 Ser Asn Gly His
Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp 645 650 655 Pro Val
Ile Thr Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly 660 665 670
Thr Leu Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg 675
680 685 His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser
Asn 690 695 700 Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser
Thr Glu Phe 705 710 715 720 Ala Val Lys Leu Lys Ile Asp Leu Ala Asn
Arg Glu Thr Ser Ile Phe 725 730 735 Ser Tyr Arg Glu Asp Pro Ile Val
Tyr Glu Ile His Pro Thr Lys Ser 740 745 750 Phe Ile Ser Gly Gly Ser
Thr Ile Thr Gly Val Gly Lys Asn Leu Asn 755 760 765 Ser Val Ser Val
Pro Arg Met Val Ile Asn Val His Glu Ala Gly Arg 770 775 780 Asn Phe
Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile Cys 785 790 795
800 Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu Lys
805 810 815 Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr
Phe Asp 820 825 830 Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe
Glu Lys Pro Val 835 840 845 Met Ile Ser Met Gly Asn Glu Asn Val Leu
Glu Ile Lys Gly Asn Asp 850 855 860 Ile Asp Pro Glu Ala Val Lys Gly
Glu Val Leu Lys Val Gly Asn Lys 865 870 875 880 Ser Cys Glu Asn Ile
His Leu His Ser Glu Ala Val Leu Cys Thr Val 885 890 895 Pro Asn Asp
Leu Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys 900 905 910 Gln
Ala Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro Asp 915 920
925 Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile Ser Thr Ala
930 935 940 Leu Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu Lys Lys Arg
Lys Gln 945 950 955 960 Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr
Asp Ala Arg Val His 965 970 975 Thr Pro His Leu Asp Arg Leu Val Ser
Ala Arg Ser Val Ser Pro Thr 980 985 990 Thr Glu Met Val Ser Asn Glu
Ser Val Asp Tyr Arg Ala Thr Phe Pro 995 1000 1005 Glu Asp Gln Phe
Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg Gln 1010 1015 1020 Val Gln
Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr Ser Gly 1025 1030 1035
Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val His Ile 1040
1045 1050 Asp Leu Ser Ala Leu Asn Pro Glu Leu Val Gln Ala Val Gln
His 1055 1060 1065 Val Val Ile Gly Pro Ser Ser Leu Ile Val His Phe
Asn Glu Val 1070 1075 1080 Ile Gly Arg Gly His Phe Gly Cys Val Tyr
His Gly Thr Leu Leu 1085 1090 1095 Asp Asn Asp Gly Lys Lys Ile His
Cys Ala Val Lys Ser Leu Asn 1100 1105 1110 Arg Ile Thr Asp Ile Gly
Glu Val Ser Gln Phe Leu Thr Glu Gly 1115 1120 1125 Ile Ile Met Lys
Asp Phe Ser His Pro Asn Val Leu Ser Leu Leu 1130 1135 1140 Gly Ile
Cys Leu Arg Ser Glu Gly Ser Pro Leu Val Val Leu Pro 1145 1150 1155
Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile Arg Asn Glu Thr 1160
1165 1170 His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe Gly Leu Gln
Val 1175 1180 1185 Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe
Val His Arg 1190 1195 1200 Asp Leu Ala Ala Arg Asn Cys Met Leu Asp
Glu Lys Phe Thr Val 1205 1210 1215 Lys Val Ala Asp Phe Gly Leu Ala
Arg Asp Met Tyr Asp Lys Glu 1220 1225 1230 Tyr Tyr Ser Val His Asn
Lys Thr Gly Ala Lys Leu Pro Val Lys 1235 1240 1245 Trp Met Ala Leu
Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr Lys 1250 1255 1260 Ser Asp
Val Trp Ser Phe Gly Val Leu Leu Trp Glu Leu Met Thr 1265 1270 1275
Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe Asp Ile Thr 1280
1285 1290 Val Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln Pro Glu Tyr
Cys 1295 1300 1305 Pro Asp Pro Leu Tyr Glu Val Met Leu Lys Cys Trp
His Pro Lys 1310 1315 1320 Ala Glu Met Arg Pro Ser Phe Ser Glu Leu
Val Ser Arg Ile Ser 1325 1330 1335 Ala Ile Phe Ser Thr Phe Ile Gly
Glu His Tyr Val His Val Asn 1340 1345 1350 Ala Thr Tyr Val Asn Val
Lys Cys Val Ala Pro Tyr Pro Ser Leu 1355 1360 1365 Leu Ser Ser Glu
Asp Asn Ala Asp Asp Glu Val Asp Thr Arg Pro 1370 1375 1380 Ala Ser
Phe Trp Glu Thr Ser 1385 1390 281390PRTHomo sapiens 28Met Lys Ala
Pro Ala Val Leu Ala Pro Gly Ile Leu Val Leu Leu Phe 1 5 10 15 Thr
Leu Val Gln Arg Ser Asn Gly Glu Cys Lys Glu Ala Leu Ala Lys 20 25
30 Ser Glu Met Asn Val Asn Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala
35 40 45 Glu Thr Pro Ile Gln Asn Val Ile Leu His Glu His His Ile
Phe Leu 50 55 60 Gly Ala Thr Asn Tyr Ile Tyr Val Leu Asn Glu Glu
Asp Leu Gln Lys 65 70 75 80 Val Ala Glu Tyr Lys Thr Gly Pro Val Leu
Glu His Pro Asp Cys Phe 85 90 95 Pro Cys Gln Asp Cys Ser Ser Lys
Ala Asn Leu Ser Gly Gly Val Trp 100 105 110 Lys Asp Asn Ile Asn Met
Ala Leu Val Val Asp Thr Tyr Tyr Asp Asp 115 120 125 Gln Leu Ile Ser
Cys Gly Ser Val Asn Arg Gly Thr Cys Gln Arg His 130 135 140 Val Phe
Pro His Asn His Thr Ala Asp Ile Gln Ser Glu Val His Cys 145 150 155
160 Ile Phe Ser Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro Asp Cys Val
165 170 175 Val Ser Ala Leu Gly Ala Lys Val Leu Ser Ser Val Lys Asp
Arg Phe 180 185 190 Ile Asn Phe Phe Val Gly Asn Thr Ile Asn Ser Ser
Tyr Phe Pro Asp 195 200 205 His Pro Leu His Ser Ile Ser Val Arg Arg
Leu Lys Glu Thr Lys Asp 210 215 220 Gly Phe Met Phe Leu Thr Asp Gln
Ser Tyr Ile Asp Val Leu Pro Glu 225 230 235 240 Phe Arg Asp Ser Tyr
Pro Ile Lys Tyr Val His Ala Phe Glu Ser Asn 245 250 255 Asn Phe Ile
Tyr Phe Leu Thr Val Gln Arg Glu Thr Leu Asp Ala Gln 260 265 270 Thr
Phe His Thr Arg Ile Ile Arg Phe Cys Ser Ile Asn Ser Gly Leu 275 280
285 His Ser Tyr Met Glu Met Pro Leu Glu Cys Ile Leu Thr Glu Lys Arg
290 295 300 Lys Lys Arg Ser Thr Lys Lys Glu Val Phe Asn Ile Leu Gln
Ala Ala 305 310 315 320 Tyr Val Ser Lys Pro Gly Ala Gln Leu Ala Arg
Gln Ile Gly Ala Ser 325 330 335 Leu Asn Asp Asp Ile Leu Phe Gly Val
Phe Ala Gln Ser Lys Pro Asp 340 345 350 Ser Ala Glu Pro Met Asp Arg
Ser Ala Met Cys Ala Phe Pro Ile Lys 355 360 365 Tyr Val Asn Asp Phe
Phe Asn Lys Ile Val Asn Lys Asn Asn Val Arg 370 375 380 Cys Leu Gln
His Phe Tyr Gly Pro Asn His Glu His Cys Phe Asn Arg 385 390 395 400
Thr Leu Leu Arg Asn Ser Ser Gly Cys Glu Ala Arg Arg Asp Glu Tyr 405
410 415 Arg Thr Glu Phe Thr Thr Ala Leu Gln Arg Val Asp Leu Phe Met
Gly 420 425 430 Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser Thr Phe
Ile Lys Gly 435 440 445 Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu
Gly Arg Phe Met Gln 450 455 460 Val Val Val Ser Arg Ser Gly Pro Ser
Thr Pro His Val Asn Phe Leu 465 470 475 480 Leu Asp Ser His Pro Val
Ser Pro Glu Val Ile Val Glu His Thr Leu 485 490 495 Asn Gln Asn Gly
Tyr Thr Leu Val Ile Thr Gly Lys Lys Ile Thr Lys 500 505 510 Ile Pro
Leu Asn Gly Leu Gly Cys Arg His Phe Gln Ser Cys Ser Gln 515 520 525
Cys Leu Ser Ala Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp Lys 530
535 540 Cys Val Arg Ser Glu Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln
Ile 545 550 555 560 Cys Leu Pro Ala Ile Tyr Lys Val Phe Pro Asn Ser
Ala Pro Leu Glu 565 570 575 Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp
Asp Phe Gly Phe Arg Arg 580 585 590 Asn Asn Lys Phe Asp Leu Lys Lys
Thr Arg Val Leu Leu Gly Asn Glu 595 600 605 Ser Cys Thr Leu Thr Leu
Ser Glu Ser Thr Met Asn Thr Leu Lys Cys 610 615 620 Thr Val Gly Pro
Ala Met Asn Lys His Phe Asn Met Ser Ile Ile Ile 625 630 635 640 Ser
Asn Gly His Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp 645 650
655 Pro Val Ile Thr Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly
660 665 670 Thr Leu Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn
Ser Arg 675 680 685 His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys
Ser Val Ser Asn 690 695 700 Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln
Thr Ile Ser Thr Glu Phe 705 710 715 720 Ala Val Lys Leu Lys Ile Asp
Leu Ala Asn Arg Glu Thr Ser Ile Phe 725 730 735 Ser Tyr Arg Glu Asp
Pro Ile Val Tyr Glu Ile His Pro Thr Lys Ser 740 745 750 Phe Ile Ser
Gly Gly Ser Thr Ile Thr Gly Val Gly Lys Asn Leu Asn 755 760 765 Ser
Val Ser Val Pro Arg Met Val Ile Asn Val His Glu Ala Gly Arg 770 775
780 Asn Phe Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile Cys
785 790 795 800 Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu
Pro Leu Lys 805 810 815 Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu
Ser Lys Tyr Phe Asp 820 825 830 Leu Ile Tyr Val His Asn Pro Val Phe
Lys Pro Phe Glu Lys Pro Val 835 840 845 Met Ile Ser Met Gly Asn Glu
Asn Val Leu Glu Ile Lys Gly Asn Asp 850 855 860 Ile Asp Pro Glu Ala
Val Lys Gly Glu Val Leu Lys Val Gly Asn Lys 865 870 875 880 Ser Cys
Glu Asn Ile His Leu His Ser Glu Ala Val Leu Cys Thr Val 885 890 895
Pro Asn Asp Leu Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys 900
905 910 Gln Ala Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro
Asp 915 920 925 Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile
Ser Thr Ala 930 935 940 Leu Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu
Lys Lys Arg Lys Gln 945 950 955 960 Ile Lys Asp Leu Gly Ser Glu Leu
Val Arg Tyr Asp Ala Arg Val His 965 970 975 Thr Pro His Leu Asp Arg
Leu Val Ser Ala Arg Ser Val Ser Pro Thr 980 985 990 Thr Glu Met Val
Ser Asn Glu Ser Val Asp Tyr Arg Ala Thr Phe Pro 995 1000 1005 Glu
Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg Gln 1010 1015
1020 Val Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr Ser Gly
1025 1030 1035 Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val
His Ile 1040 1045 1050 Asp Leu Ser Ala Leu Asn Pro Glu Leu Val Gln
Ala Val Gln His 1055 1060 1065 Val Val Ile Gly Pro Ser Ser Leu Ile
Val His Phe Asn Glu Val 1070 1075 1080 Ile Gly Arg Gly His Phe Gly
Cys Val Tyr His Gly Thr Leu Leu 1085 1090 1095 Asp Asn Asp Gly Lys
Lys Ile His Cys Ala Val Lys Ser Leu Asn 1100 1105 1110 Arg Ile Thr
Asp Ile Gly Glu Val Ser Gln Phe Leu Thr Glu Gly 1115 1120 1125 Ile
Ile Met Lys Asp Phe Ser His Pro Asn Val Leu Ser Leu Leu 1130 1135
1140 Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu Val Val Leu Pro
1145 1150 1155 Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile Arg Asn
Glu Thr 1160 1165 1170 His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe
Gly Leu Gln Val 1175 1180 1185 Ala Lys Gly Met Lys Tyr Leu Ala Ser
Lys Lys Phe Val His Arg 1190 1195 1200 Asp Leu Ala Ala Arg Asn Cys
Met Leu Asp Glu Lys Phe Thr Val 1205 1210 1215 Lys Val Ala Asp Phe
Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu 1220 1225 1230 Tyr Tyr Ser
Val His Asn Lys Thr Gly Ala Lys Leu Pro Val Lys 1235 1240 1245 Trp
Met Ala Leu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr Lys 1250 1255
1260 Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Leu Met Thr
1265 1270 1275 Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe Asp
Ile Thr 1280 1285 1290 Val Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln
Pro Glu Tyr Cys 1295 1300
1305 Pro Asp Pro Leu Tyr Glu Val Met Leu Lys Cys Trp His Pro Lys
1310 1315 1320 Ala Glu Met Arg Pro Ser Phe Ser Glu Leu Val Ser Arg
Ile Ser 1325 1330 1335 Ala Ile Phe Ser Thr Phe Ile Gly Glu His Tyr
Val His Val Asn 1340 1345 1350 Ala Thr Tyr Val Asn Val Lys Cys Val
Ala Pro Tyr Pro Ser Leu 1355 1360 1365 Leu Ser Ser Glu Asp Asn Ala
Asp Asp Glu Val Asp Thr Arg Pro 1370 1375 1380 Ala Ser Phe Trp Glu
Thr Ser 1385 1390 29908PRTHomo sapiens 29Glu Cys Lys Glu Ala Leu
Ala Lys Ser Glu Met Asn Val Asn Met Lys 1 5 10 15 Tyr Gln Leu Pro
Asn Phe Thr Ala Glu Thr Pro Ile Gln Asn Val Ile 20 25 30 Leu His
Glu His His Ile Phe Leu Gly Ala Thr Asn Tyr Ile Tyr Val 35 40 45
Leu Asn Glu Glu Asp Leu Gln Lys Val Ala Glu Tyr Lys Thr Gly Pro 50
55 60 Val Leu Glu His Pro Asp Cys Phe Pro Cys Gln Asp Cys Ser Ser
Lys 65 70 75 80 Ala Asn Leu Ser Gly Gly Val Trp Lys Asp Asn Ile Asn
Met Ala Leu 85 90 95 Val Val Asp Thr Tyr Tyr Asp Asp Gln Leu Ile
Ser Cys Gly Ser Val 100 105 110 Asn Arg Gly Thr Cys Gln Arg His Val
Phe Pro His Asn His Thr Ala 115 120 125 Asp Ile Gln Ser Glu Val His
Cys Ile Phe Ser Pro Gln Ile Glu Glu 130 135 140 Pro Ser Gln Cys Pro
Asp Cys Val Val Ser Ala Leu Gly Ala Lys Val 145 150 155 160 Leu Ser
Ser Val Lys Asp Arg Phe Ile Asn Phe Phe Val Gly Asn Thr 165 170 175
Ile Asn Ser Ser Tyr Phe Pro Asp His Pro Leu His Ser Ile Ser Val 180
185 190 Arg Arg Leu Lys Glu Thr Lys Asp Gly Phe Met Phe Leu Thr Asp
Gln 195 200 205 Ser Tyr Ile Asp Val Leu Pro Glu Phe Arg Asp Ser Tyr
Pro Ile Lys 210 215 220 Tyr Val His Ala Phe Glu Ser Asn Asn Phe Ile
Tyr Phe Leu Thr Val 225 230 235 240 Gln Arg Glu Thr Leu Asp Ala Gln
Thr Phe His Thr Arg Ile Ile Arg 245 250 255 Phe Cys Ser Ile Asn Ser
Gly Leu His Ser Tyr Met Glu Met Pro Leu 260 265 270 Glu Cys Ile Leu
Thr Glu Lys Arg Lys Lys Arg Ser Thr Lys Lys Glu 275 280 285 Val Phe
Asn Ile Leu Gln Ala Ala Tyr Val Ser Lys Pro Gly Ala Gln 290 295 300
Leu Ala Arg Gln Ile Gly Ala Ser Leu Asn Asp Asp Ile Leu Phe Gly 305
310 315 320 Val Phe Ala Gln Ser Lys Pro Asp Ser Ala Glu Pro Met Asp
Arg Ser 325 330 335 Ala Met Cys Ala Phe Pro Ile Lys Tyr Val Asn Asp
Phe Phe Asn Lys 340 345 350 Ile Val Asn Lys Asn Asn Val Arg Cys Leu
Gln His Phe Tyr Gly Pro 355 360 365 Asn His Glu His Cys Phe Asn Arg
Thr Leu Leu Arg Asn Ser Ser Gly 370 375 380 Cys Glu Ala Arg Arg Asp
Glu Tyr Arg Thr Glu Phe Thr Thr Ala Leu 385 390 395 400 Gln Arg Val
Asp Leu Phe Met Gly Gln Phe Ser Glu Val Leu Leu Thr 405 410 415 Ser
Ile Ser Thr Phe Ile Lys Gly Asp Leu Thr Ile Ala Asn Leu Gly 420 425
430 Thr Ser Glu Gly Arg Phe Met Gln Val Val Val Ser Arg Ser Gly Pro
435 440 445 Ser Thr Pro His Val Asn Phe Leu Leu Asp Ser His Pro Val
Ser Pro 450 455 460 Glu Val Ile Val Glu His Thr Leu Asn Gln Asn Gly
Tyr Thr Leu Val 465 470 475 480 Ile Thr Gly Lys Lys Ile Thr Lys Ile
Pro Leu Asn Gly Leu Gly Cys 485 490 495 Arg His Phe Gln Ser Cys Ser
Gln Cys Leu Ser Ala Pro Pro Phe Val 500 505 510 Gln Cys Gly Trp Cys
His Asp Lys Cys Val Arg Ser Glu Glu Cys Leu 515 520 525 Ser Gly Thr
Trp Thr Gln Gln Ile Cys Leu Pro Ala Ile Tyr Lys Val 530 535 540 Phe
Pro Asn Ser Ala Pro Leu Glu Gly Gly Thr Arg Leu Thr Ile Cys 545 550
555 560 Gly Trp Asp Phe Gly Phe Arg Arg Asn Asn Lys Phe Asp Leu Lys
Lys 565 570 575 Thr Arg Val Leu Leu Gly Asn Glu Ser Cys Thr Leu Thr
Leu Ser Glu 580 585 590 Ser Thr Met Asn Thr Leu Lys Cys Thr Val Gly
Pro Ala Met Asn Lys 595 600 605 His Phe Asn Met Ser Ile Ile Ile Ser
Asn Gly His Gly Thr Thr Gln 610 615 620 Tyr Ser Thr Phe Ser Tyr Val
Asp Pro Val Ile Thr Ser Ile Ser Pro 625 630 635 640 Lys Tyr Gly Pro
Met Ala Gly Gly Thr Leu Leu Thr Leu Thr Gly Asn 645 650 655 Tyr Leu
Asn Ser Gly Asn Ser Arg His Ile Ser Ile Gly Gly Lys Thr 660 665 670
Cys Thr Leu Lys Ser Val Ser Asn Ser Ile Leu Glu Cys Tyr Thr Pro 675
680 685 Ala Gln Thr Ile Ser Thr Glu Phe Ala Val Lys Leu Lys Ile Asp
Leu 690 695 700 Ala Asn Arg Glu Thr Ser Ile Phe Ser Tyr Arg Glu Asp
Pro Ile Val 705 710 715 720 Tyr Glu Ile His Pro Thr Lys Ser Phe Ile
Ser Gly Gly Ser Thr Ile 725 730 735 Thr Gly Val Gly Lys Asn Leu Asn
Ser Val Ser Val Pro Arg Met Val 740 745 750 Ile Asn Val His Glu Ala
Gly Arg Asn Phe Thr Val Ala Cys Gln His 755 760 765 Arg Ser Asn Ser
Glu Ile Ile Cys Cys Thr Thr Pro Ser Leu Gln Gln 770 775 780 Leu Asn
Leu Gln Leu Pro Leu Lys Thr Lys Ala Phe Phe Met Leu Asp 785 790 795
800 Gly Ile Leu Ser Lys Tyr Phe Asp Leu Ile Tyr Val His Asn Pro Val
805 810 815 Phe Lys Pro Phe Glu Lys Pro Val Met Ile Ser Met Gly Asn
Glu Asn 820 825 830 Val Leu Glu Ile Lys Gly Asn Asp Ile Asp Pro Glu
Ala Val Lys Gly 835 840 845 Glu Val Leu Lys Val Gly Asn Lys Ser Cys
Glu Asn Ile His Leu His 850 855 860 Ser Glu Ala Val Leu Cys Thr Val
Pro Asn Asp Leu Leu Lys Leu Asn 865 870 875 880 Ser Glu Leu Asn Ile
Glu Trp Lys Gln Ala Ile Ser Ser Thr Val Leu 885 890 895 Gly Lys Val
Ile Val Gln Pro Asp Gln Asn Phe Thr 900 905
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