U.S. patent application number 15/817998 was filed with the patent office on 2018-08-09 for compositions and methods for diagnosing and treating cancer.
This patent application is currently assigned to OncoMed Pharmaceuticals, Inc.. The applicant listed for this patent is OncoMed Pharmaceuticals, Inc.. Invention is credited to Austin L. Gurney, Timothy Charles Hoey, John Lewicki, Sanjeev H. Satyal.
Application Number | 20180222997 15/817998 |
Document ID | / |
Family ID | 38006447 |
Filed Date | 2018-08-09 |
United States Patent
Application |
20180222997 |
Kind Code |
A1 |
Gurney; Austin L. ; et
al. |
August 9, 2018 |
COMPOSITIONS AND METHODS FOR DIAGNOSING AND TREATING CANCER
Abstract
An isolated antibody that specifically binds to an extracellular
domain of two or more human FZD receptors and inhibits growth of
tumor cells is described. Also described is a method of treating
cancer comprising administering an antibody of the present
disclosure in an amount effective to inhibit tumor cell growth.
Inventors: |
Gurney; Austin L.; (San
Francisco, CA) ; Lewicki; John; (Los Gatos, CA)
; Satyal; Sanjeev H.; (San Carlos, CA) ; Hoey;
Timothy Charles; (Hillsborough, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OncoMed Pharmaceuticals, Inc. |
Redwood City |
CA |
US |
|
|
Assignee: |
OncoMed Pharmaceuticals,
Inc.
|
Family ID: |
38006447 |
Appl. No.: |
15/817998 |
Filed: |
November 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11589993 |
Oct 31, 2006 |
9850311 |
|
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15817998 |
|
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60812966 |
Jun 13, 2006 |
|
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60731468 |
Oct 31, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 25/00 20180101; A61P 15/00 20180101; C07K 16/2896 20130101;
A61P 35/00 20180101; A61P 1/18 20180101; C07K 16/30 20130101; A61P
13/08 20180101; C07K 16/468 20130101; A61P 35/02 20180101; A61P
11/00 20180101; C07K 2317/732 20130101; A61K 2039/505 20130101;
C07K 2317/73 20130101; C07K 2317/734 20130101; A61P 1/04 20180101;
A61P 43/00 20180101; C07K 2317/76 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 45/06 20060101 A61K045/06; C07K 16/28 20060101
C07K016/28; C07K 16/46 20060101 C07K016/46 |
Claims
1. An isolated polynucleotide encoding an antibody that
specifically binds to an extracellular domain of two or more human
FZD receptors and inhibits growth of tumor cells.
2-14. (canceled)
15. A method of treating cancer, the method comprising
administering to a subject in need thereof a monoclonal antibody in
an amount effective to inhibit tumor cell growth, wherein the
monoclonal antibody comprises an antigen binding site that
specifically binds to the extracellular domains of human FZD5 and
FZD8, wherein the extracellular domain of human FZD5 comprises
approximately amino acid 27 to 233 of SEQ ID NO:3, and the
extracellular domain of human FZD8 comprises approximately amino
acid 28 to 158 of SEQ ID NO:6.
16. The method of claim 15 wherein the antibody is conjugated to a
cytotoxic moiety.
17. The method of claim 15 further comprising administering at
least one additional therapeutic agent appropriate for effecting
combination therapy.
18. The method of claim 15 wherein the tumor cells are chosen from
a breast tumor, colorectal tumor, lung tumor, pancreatic tumor,
prostate tumor, and a head and neck tumor.
19. The method of claim 17, wherein the at least one additional
therapeutic agent is a chemotherapeutic agent.
20. The method of claim 15, wherein the antibody is a chimeric
antibody.
21. The method of claim 15, wherein the antibody is a humanized
antibody.
22. The method of claim 15, wherein the antibody is a human
antibody.
23. The method of claim 15, wherein the antibody is an intact
antibody.
24. The method of claim 15, wherein the antigen binding site binds
to the extracellular domains of human FZD5 and FZD8 at a site
comprising amino acid residues that are identical between the
extracellular domains of human FZD5 and FZD8.
25. The method of claim 24, wherein the antigen binding site binds
to the extracellular domains of human FZD5 and FZD8 at a site
comprising at least 3 amino acid residues that are identical
between the extracellular domains of human FZD5 and FZD8.
26. The method of claim 24, wherein the amino acid residues that
are identical between the extracellular domains of human FZD5 and
FZD8 are contiguous.
27. The method of claim 25, wherein the at least 3 amino acid
residues that are identical between the extracellular domains of
human FZD5 and FZD8 are contiguous.
27. A method of inhibiting the growth of tumor cells comprising
contacting the tumor cells with an effective amount of a monoclonal
antibody that comprises an antigen binding site that specifically
binds to the extracellular domains of human FZD5 and FZD8, wherein
the extracellular domain of human FZD5 comprises approximately
amino acid 27 to 233 of SEQ ID NO:3, and the extracellular domain
of human FZD8 comprises approximately amino acid 28 to 158 of SEQ
ID NO:6.
28. The method of claim 27 wherein the tumor cells are chosen from
a breast tumor, colorectal tumor, lung tumor, pancreatic tumor,
prostate tumor, and a head and neck tumor.
29. The method of claim 27, wherein the antibody is a chimeric
antibody.
30. The method of claim 27, wherein the antibody is a humanized
antibody.
31. The method of claim 27, wherein the antibody is a human
antibody.
32. The method of claim 27, wherein the antibody is an intact
antibody.
33. The method of claim 27, wherein the antigen binding site binds
to the extracellular domains of human FZD5 and FZD8 at a site
comprising amino acid residues that are identical between the
extracellular domains of human FZD5 and FZD8.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/589,993, filed Oct. 31, 2006, which claims the benefit of
U.S. Prov. Appl. No. 60/731,468, filed Oct. 31, 2005 and U.S. Prov.
Appl. No. 60/812,966, filed Jun. 13, 2006, each of which is herein
incorporated by reference.
REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA
EFS-WEB
[0002] The content of the electronically submitted sequence listing
(Name: 2293_016000A_SL.txt; Size: 12,388 bytes; and Date of
Creation: Nov. 14, 2017) is herein incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates to the field of oncology and
provides novel compositions and methods for diagnosing and treating
cancer. The present invention provides antibodies against a cancer
stem cell marker for the diagnosis and treatment of solid
tumors.
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
estimate 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 stem 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 is an isolated monoclonal antibody that
specifically binds to an extracellular domain of a human FZD8
receptor and inhibits growth of tumor cells. Also provided is an
isolated antibody that specifically binds to an extracellular
domain of two or more human FZD receptors and inhibits growth of
tumor cells. A pharmaceutical composition comprising an antibody of
the present disclosure and a pharmaceutically acceptable vehicle is
provided. Further provided is a method of treating cancer
comprising administering an antibody of the present disclosure in
an amount effective to inhibit tumor cell growth.
[0017] 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
[0018] FIGS. 1A-F: Analysis of Specific Binding of anti-FZD10,
anti-FZD7, anti-FZD5, anti-FZD6, anti-FZD4, and anti-FZD8
Antibodies to their Corresponding Membrane-Associated Receptors.
HEK293 cells expressing full-length FZD10, FZD7, FZD5, FZD6, FZD4,
and FZD8 without (FIG. 1A) or with (FIG. 1B, FIG. 1C, FIG. 1D, FIG.
1E, and FIG. 1F) co-transfection of GFP were incubated with
anti-FZD antibodies or control IgG and sorted by FACS. (FIG. 1A)
FACs analysis of antibodies against FZD10 are shown compared with
an IgG isotype negative control for each antibody. A FLAG-tagged
construct matched with anti-FLAG antibodies is shown as a positive
control (bottom). (FIG. 1B) FACs analysis of antibodies against
FZD7 in cells expressing FZD7 and GFP compared to a control IgG. A
FLAG-tagged construct matched with anti-FLAG antibodies is shown as
a positive control (bottom, far right). (FIG. 1C) FACs analysis of
antibodies against FZD5 in cells expressing FZD5 and GFP compared
to a control IgG. Serum from an animal immunized with FZD5 antigen
is shown on the bottom, right. (FIG. 1D) FACs analysis of
antibodies against FZD6 in cells expressing FZD6 and GFP compared
to a control IgG. A FLAG-tagged construct matched with anti-FLAG
antibodies is shown as a positive control (bottom, right). (FIG.
1E) FACs analysis of antibodies against FZD4 in cells expressing
FZD4 and GFP compared to a control IgG. A FLAG-tagged construct
matched with anti-FLAG antibodies is shown as a positive control
(bottom, right). (FIG. 1F) FACs analysis of antibodies against FZD8
in cells expressing FZD8 and GFP.
[0019] FIG. 2: FZD Fc Soluble Receptors Inhibit Wnt Signaling. HEK
293 cells stably transfected with 8.times.TCF-luciferase reporter
were incubated with increasing concentrations of FZD Fc soluble
receptors in the presence of different Wnt ligands including Wnt1,
Wnt2, Wnt3, Wnt3a, and Wnt7b. FZD4 Fc, FZD5 Fc, and FZD8 Fc fusion
proteins inhibited Wnt signaling mediated by all five Wnt ligands
as shown by loss of luciferase activity.
[0020] FIG. 3: Identification of anti-FZD5 Antibodies that
Interfere with Wnt3a Ligand Binding. Wnt signaling in HEK 293 cells
transfected with the Wnt 8.times.TCF-luciferase reporter vector was
measured by luciferase activity in the presence of Wnt3a and
thirty-two different antibodies against FZD5, either alone (left
bar) or in the presence of soluble FZD5 Fc (right bar). Antibodies
that interfere with binding between FZD5 Fc and Wnt3a result in
significant activation of Wnt signaling (right bar).
[0021] FIG. 4: Reduction of Tumor Growth by anti-FZD6 and anti-FZD5
Antibodies. Tumor growth in NOD/SCID mice injected with UM-C4 colon
tumor cells and treated with either anti-FZD6 or anti-FZD5
antibodies is plotted on the x-axis in mm3 over 8 weeks. Treatment
with anti-FZD6 antibody 23M2 (open bars) and anti-FZD5 antibody
44M13 (dashed bars) significantly reduced tumor growth as compared
to PBS injected controls (filled bars).
DETAILED DESCRIPTION OF THE INVENTION
[0022] 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')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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] As used herein, the term "humanized antibody" refers to
forms of non-human (e.g. murine) 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 region (CDR) 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, the Fv framework region (FR)
residues 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 Fv 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, 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.
[0027] 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.
[0028] "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.
[0029] 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.
[0030] 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.
[0031] 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 KD 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
KD 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.
[0032] 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).
[0033] 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.
[0034] 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.
[0035] The terms "proliferative disorder" and "proliferative
disease" refer to disorders associated with abnormal cell
proliferation such as cancer.
[0036] "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.
[0037] "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.
[0038] 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.
[0039] 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).
[0040] 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. These properties of
self-renewal and proliferation to generate all other tumor cells
confer on the cancer stem cells of this invention the ability to
form palpable tumors upon serial transplantation into an
immunocompromised mouse compared to the majority of tumor cells
that are unable to form tumors upon the serial transplantation.
Tumor cells, i.e. non-tumorigenic tumor cells, may form a tumor
upon transplantation into an immunocompromised mouse a limited
number of times (for example one or two times) after obtaining the
tumor cells from a solid tumor.
[0041] 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).
[0042] As used herein, the terms "unfractionated tumor cells",
"presorted tumor cells", "bulk tumor cells", and their grammatical
equivalents are used interchangeably to refer to a tumor cell
population isolated from a patient sample (e.g. a tumor biopsy or
pleural effusion) that has not been segregated, or fractionated,
based on cell surface marker expression.
[0043] As used herein, the terms "non-ESA+CD44+ tumor cells",
"non-ESA+44+", "sorted non-tumorigenic tumor cells",
"non-tumorigenic tumor cells," "non-stem cells," "tumor cells" and
their grammatical equivalents are used interchangeably to refer to
a tumor population from which the cancer stem cells of this
invention have been segregated, or removed, based on cell surface
marker expression.
[0044] 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.
[0045] 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.
[0046] "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.
[0047] "Pharmaceutically acceptable salt" refers to a salt of a
compound that is pharmaceutically acceptable and that possesses the
desired pharmacological activity of the parent compound.
[0048] "Pharmaceutically acceptable excipient, carrier or adjuvant"
refers to an excipient, carrier or adjuvant that can be
administered to a subject, together with at least one antibody of
the present disclosure, and which does not destroy the
pharmacological activity thereof and is nontoxic when administered
in doses sufficient to deliver a therapeutic amount of the
compound.
[0049] "Pharmaceutically acceptable vehicle" refers to a diluent,
adjuvant, excipient, or carrier with which at least one antibody of
the present disclosure is administered.
[0050] "Prodrug" refers to a derivative of a therapeutically
effective compound that requires a transformation within the body
to produce the therapeutically effective compound. Prodrugs can be
pharmacologically inactive until converted to the therapeutically
effective parent compound.
[0051] 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.
[0052] As used herein, "providing a diagnosis" or "diagnostic
information" refers to any information 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), 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.
[0053] As used herein, the terms "providing a prognosis",
"prognostic information", or "predictive information" refer to
providing information 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).
[0054] 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.
[0055] 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. 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' non-translated sequences. The term
"gene" encompasses both cDNA and genomic forms of a gene. A genomic
form or clone 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' 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.
[0056] 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.
[0057] 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).
[0058] 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.
[0059] "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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] "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)).
[0064] 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, His,
and FLAG tags.
[0065] Provided is an isolated antibody that specifically binds to
an extracellular domain of a human FZD8 receptor and inhibits
growth of tumor cells. In certain embodiments the antibody is a
monoclonal antibody. In certain embodiments the antibody is a
chimeric antibody. In certain embodiments the antibody is a
humanized antibody. In certain embodiments the antibody is a human
antibody.
[0066] Also provided is an isolated antibody that specifically
binds to an extracellular domain of two or more human FZD receptors
and inhibits growth of tumor cells. In certain embodiments the
antibody specifically binds to the extracellular domain of human
FZD2 and FZD6. In certain embodiments the antibody specifically
binds to the extracellular domain of human FZD7 and FZD10. In
certain embodiments the antibody specifically binds to the
extracellular domain of human FZD4 and FZD5. In certain embodiments
the antibody specifically binds to the extracellular domain of
human FZD4 and FZD8. In certain embodiments the antibody
specifically binds to the extracellular domain of human FZD5 and
FZD8. In some embodiments the antibody is a monoclonal antibody. In
some embodiments the antibody is a chimeric antibody. In some
embodiments the antibody is a humanized antibody. In some
embodiments the antibody is a human antibody.
[0067] Further provided is an isolated antibody that specifically
binds to the extracellular domain of three or more human FZD
receptors. In certain embodiments the antibody specifically binds
to the extracellular domain of human FZD4, FZD5, and FZD8. In some
embodiments the antibody is a monoclonal antibody. In some
embodiments the antibody is a chimeric antibody. In some
embodiments the antibody is a humanized antibody. In some
embodiments the antibody is a human antibody.
[0068] Also provided is a pharmaceutical composition comprising an
antibody of the present disclosure and a pharmaceutically
acceptable vehicle.
[0069] Also provided is a hybridoma that produces an antibody of
the present disclosure.
[0070] Further provided is a method of treating cancer comprising
administering a antibody or a pharmaceutical composition of the
present disclosure in an amount effective to inhibit tumor cell
growth. In certain embodiments the antibody is conjugated to a
cytotoxic moiety. In certain embodiments the method further
comprises administering at least one additional therapeutic agent
appropriate for effecting combination therapy. In certain
embodiments the tumor cells are chosen from a breast tumor,
colorectal tumor, lung tumor, prostate tumor, pancreatic tumor, and
a head and neck tumor.
[0071] 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 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.
[0072] The cancer stem cell markers of the present invention relate
to a human FZD receptor, including for example, human FZD4, FZD5,
and FZD8 as markers of cancer stem cells, implicating the Wnt
signaling pathway in the maintenance of cancer stem cells and as a
target for treating cancer via the elimination of these tumorigenic
cells. The Wnt signaling pathway is one of several critical
regulators of embryonic pattern formation, post-embryonic tissue
maintenance, and stem cell biology. More specifically, Wnt
signaling plays an important role in the generation of cell
polarity and cell fate specification including self-renewal by stem
cell populations. Unregulated activation of the Wnt pathway is
associated with numerous human cancers where it can alter the
developmental fate of tumor cells to maintain them in an
undifferentiated and proliferative state. Thus carcinogenesis can
proceed by usurping homeostatic mechanisms controlling normal
development and tissue repair by stem cells (reviewed in Reya &
Clevers, 2005, Nature 434:843; Beachy et al., 2004, Nature
432:324).
[0073] The Wnt signaling pathway was first elucidated in the
Drosophila developmental mutant wingless (wg) and from the murine
proto-oncogene int-1, now Wnt1 (Nusse & Varmus, 1982, Cell
31:99-109; Van Ooyen & Nusse, 1984, Cell 39:233-40; Cabrera et
al., 1987, Cell 50:659-63; Rijsewijk et al., 1987, Cell 50:649-57).
Wnt genes encode secreted lipid-modified glycoproteins of which 19
have been identified in mammals. These secreted ligands activate a
receptor complex consisting of a Frizzled (Fzd) receptor family
member and low-density lipoprotein (LDL) receptor-related protein 5
or 6 (LPRS/6). The Fzd receptors are seven transmembrane domain
proteins of the G-protein coupled receptor (GPCR) superfamily and
contain a large extracellular N-terminal ligand binding domain with
10 conserved cysteines, known as a cysteine-rich domain (CRD) or
Fri domain. There are ten human FZD receptors: FZD1-10. Different
Fzd CRDs have different binding affinities for specific Wnts (Wu
& Nusse, 2002, J. Biol. Chem. 277:41762-9), and Fzd receptors
have been grouped into those that activate the canonical
.beta.-catenin pathway and those that activate non-canonical
pathways described below (Miller et al., 1999, Oncogene
18:7860-72). To form the receptor complex that binds the FZD
ligands, FZD receptors interact with LRPS/6, single pass
transmembrane proteins with four extracellular EGF-like domains
separated by six YWTD amino acid repeats (Johnson et al., 2004, J.
Bone Mineral Res. 19:1749).
[0074] The canonical Wnt signaling pathway activated upon receptor
binding is mediated by the cytoplasmic protein Dishevelled (Dsh)
interacting directly with the Fzd receptor and results in the
cytoplasmic stabilization and accumulation of .beta.-catenin. In
the absence of a Wnt signal, .beta.-catenin is localized to a
cytoplasmic destruction complex that includes the tumor suppressor
proteins adenomatous polyposis coli (APC) and auxin. These proteins
function as critical scaffolds to allow glycogen synthase kinase
(GSK)-3.beta. to bind and phosphorylate .beta.-catenin, marking it
for degradation via the ubiquitin/proteasome pathway. Activation of
Dsh results in phophorylation of GSK3.beta. and the dissociation of
the destruction complex. Accumulated cytoplasmic .beta.-catenin is
then transported into the nucleus where it interacts with the
DNA-binding proteins of the Tcf/Lef family to activate
transcription.
[0075] In addition to the canonical signaling pathway, Wnt ligands
also active .beta.-catenin-independent pathways (Veeman et al.,
2003, Dev. Cell 5:367-77). Non-canonical Wnt signaling has been
implicated in numerous processes but most convincingly in
gastrulation movements via a mechanism similar to the Drosophila
planar cell polarity (PCP) pathway. Other potential mechanisms of
non-canonical Wnt signaling include calcium flux, JNK, and both
small and heterotrimeric G-proteins. Antagonism is often observed
between the canonical and non-canonical pathways, and some evidence
indicates that non-canonical signaling can suppress cancer
formation (Olson & Gibo, 1998, Exp. Cell Res. 241:134; Topol et
al., 2003, J. Cell Biol. 162:899-908). Thus in certain contexts,
Fzd receptors act as negative regulators of the canonical Wnt
signaling pathway. For example, FZD6 represses Wnt-3a-induced
canonical signaling when co-expressed with FZD1 via the TAK1-NLK
pathway (Golan et al., 2004, JBC 279:14879-88). Similarly, Fzd2
antagonized canonical Wnt signaling in the presence of Wnt-5a via
the TAK1-NLK MAPK cascade (Ishitani et al., 2003, Mol. Cell. Biol.
23:131-9).
[0076] Hematopoietic stem cells (HSCs) are the best understood stem
cells in the body, and Wnt signaling is implicated both in their
normal maintenance as well as in leukemic transformation (Reya
& Clevers, 2005, Nature 434:843). HSCs are a rare population of
cells that reside in a stomal niche within the adult bone marrow.
These cells are characterized both by a unique gene expression
profile as well as an ability to continuously give rise to more
differentiated progenitor cells to reconstitute the entire
hematopoietic system. Both HSCs and the cells of their stromal
microenvironment express Wnt ligands, and Wnt reporter activation
is present in HSCs in vivo. Furthermore, both .beta.-catenin and
purified Wnt3A promote self-renewal of murine HSCs in vitro and
enhance their ability to reconstitute the hematopoietic system in
vivo while Wnt5A promotes expansion of human hematopoietic
progenitors in vitro and re-population in a NOD-SCID xenotransplant
model (Reya et al., 2003, Nature 423:409-14; Willert et al., 2003,
Nature 423:448-52; Van Den Berg et al., 1998, Blood 92:3189-202;
Murdoch et al., 2003, Proc. Nat'l Acad. Sci. 100:3422-7).
[0077] More recently Wnt signaling has been found to play a role in
the oncogenic growth of both myeloid and lymphoid lineages. For
example, granulocyte-macrophage progenitors (GMPs) from chronic
myelogenous leukemias display activated Wnt signaling on which they
are depended for growth and renewal (Jamieson et al., 2004, N.
Engl. J. Med. 351:657-67) And while leukemias do not appear to
harbor mutations within the Wnt pathway, autocrine and/or paracrine
Wnt signaling can sustain cancerous self-renewal (Reya &
Clevers 2005, Nature 434:843).
[0078] The canonical Wnt signaling pathway also plays a central
role in the maintenance of stem cell populations in the small
intestine and colon, and the inappropriate activation of this
pathway plays a prominent role in colorectal cancers (Reya &
Clevers, 2005, Nature 434:843). The absorptive epithelium of the
intestines is arranged into villi and crypts. Stem cells reside in
the crypts and slowly divide to produce rapidly proliferating cells
which give rise to all the differentiated cell populations that
move up out of the crypts to occupy the intestinal villi. The Wnt
signaling cascade plays a dominant role in controlling cell fates
along the crypt-villi axis and is essential for the maintenance of
the stem cell population. Disruption of Wnt signaling either by
genetic loss of Tcf7/2 by homologous recombination (Korinek et al.,
1998, Nat. Genet. 19:379) or overexpression of Dickkopf-1 (Dkk1), a
potent secreted Wnt antagonist (Pinto et al., 2003, Genes Dev.
17:1709-13; Kuhnert et al., 2004, Proc. Nat'l. Acad. Sci.
101:266-71), results in depletion of intestinal stem cell
populations.
[0079] Colorectal cancer is most commonly initiated by activating
mutations in the Wnt signaling cascade. 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. Mutations
have also been identified in other Wnt pathway components including
auxin and .beta.-catenin. Individual adenomas are clonal outgrowths
of epithelial cell containing a second inactivated allele, and the
large number of FAP adenomas inevitably results in the development
of adenocarcinomas through addition mutations in oncogenes and/or
tumor suppressor genes. Furthermore, activation of the Wnt
signaling pathway, including gain-of-function mutations in APC and
.beta.-catenin, can induce hyperplastic development and tumor
growth in mouse models (Oshima et al., 1997, Cancer Res. 57:1644-9;
Harada et al., 1999, EMBO J. 18:5931-42).
[0080] A role for Wnt signaling in cancer was first uncovered with
the identification of Wnt1 (originally int1) as an oncogene in
mammary tumors transformed by the nearby insertion of a murine
virus (Nusse & Varmus, 1982, Cell 31:99-109). Additional
evidence for the role of Wnt signaling in breast cancer has since
accumulated. For instance, transgenic overexpression of
.beta.-catenin in the mammary glands results in hyperplasias and
adenocarcinomas (Imbert et al., 2001, J. Cell Biol. 153:555-68;
Michaelson & Leder, 2001, Oncogene 20:5093-9) whereas loss of
Wnt signaling disrupts normal mammary gland development (Tepera et
al., 2003, J. Cell Sc. 116:1137-49; Hatsell et al., 2003, J.
Mammary Gland Biol. Neoplasia 8:145-58). More recently mammary stem
cells have been shown to be activated by Wnt signaling (Liu et al.,
2004, Proc. Nat'l Acad. Sci. 101:4158). In human breast cancer,
.beta.-catenin accumulation implicates activated Wnt signaling in
over 50% of carcinomas, and though specific mutations have not been
identified, upregulation of Frizzled receptor expression has been
observed (Brennan & Brown, 2004, J. Mammary Gland Neoplasia
9:119-31; Malovanovic et al., 2004, Int. J. Oncol. 25:1337-42).
[0081] FZD10, FZD8, FZD7, FZD4, and FZD5 are five of ten identified
human Wnt receptors. In the mouse embryo Fzd10 is expressed with
Wnt7a in the neural tube, limb buds, and Mullerian duct (Nunnally
& Parr, 2004, Dev. Genes Evol. 214:144-8) and can act as a
receptor for Wnt-7a during limb bud development (Kawakami et al.,
2000, Dev. Growth Differ. 42:561-9). Fzd10 is co-expressed with
Wnt7b in the lungs, and cell transfection studies have demonstrated
that the Fzd10/LRPS co-receptor activates the canonical Wnt
signaling pathway in response to Wnt7b (Wang et al., 2005, Mol.
Cell Biol. 25:5022-30). FZD10 mRNA is upregulated in numerous
cancer cell lines, including cervical, gastric, and glioblastoma
cell lines, and in primary cancers including approximately 40% of
primary gastric cancers, colon cancers, and synovial sarcomas
(Saitoh et al., 2002, Int. J. Oncol. 20:117-20; Terasaki et al.,
2002, Int. J. Mol. Med. 9:107-12; Nagayama et al., 2005, Oncogene
1-12). FZD8 is upregulated in several human cancer cell lines,
primary gastric cancers, and renal carcinomas (Saitoh et al., 2001,
Int. J. Oncol. 18:991-96; Kirikoshi et al., 2001, Int. J. Oncol.
19:111-5; Janssens et al., 2004, Tumor Biol. 25:161-71). FZD7 is
expressed throughout the gastrointestinal tract and is up-regulated
in one out of six cases of human primary gastric cancer (Kirikoshi
et al., 2001, Int. J. Oncol. 19:111-5). Expression of the FZD7
ectodomain by a colon cancer cell line induced morphological
changes and decreased tumor growth in a xenograft model (Vincan et
al., 2005, Differentiation 73:142-53). FZD5 plays an essential role
in yolk sac and placental angiogenesis (Ishikawa et al., 2001, Dev.
128:25-33) and is upregulated in renal carcinomas in association
with activation of Wnt/.beta.-catenin signaling (Janssens et al.,
2004, Tumor Biology 25:161-71). FZD4 is highly expressed in
intestinal crypt epithelial cells and is one of several factors
that display differential expression in normal versus neoplastic
tissue (Gregorieff et al., 2005, Gastroenterology 129:626-38). The
identification of FZD receptors as markers of cancer stem cells
thus makes these proteins ideal targets for cancer
therapeutics.
[0082] The present invention provides a cancer stem cell marker the
expression of which can be analyzed to diagnosis or monitor a
disease associated with expression of a cancer stem cell marker. In
some embodiments, expression of a cancer stem cell marker is
determined by polynucleotide expression such as, for example, mRNA
encoding the cancer stem cell marker. The polynucleotide can be
detected and quantified by any of a number of means well known to
those of skill in the art. In some embodiments, mRNA encoding a
cancer stem cell marker is detected by in situ hybridization of
tissue sections from, from example, a patient biopsy. In some
embodiments, RNA is isolated from a tissue and detected by, for
example, Northern blot, quantitative RT-PCR, or microarrays. For
example, total RNA can be extracted from a tissue sample and
primers that specifically hybridize and amplify a cancer stem cell
marker can be used to detect expression of a cancer stem cell
marker polynucleotide using RT-PCR.
[0083] In certain embodiments, expression of a cancer stem cell
marker can be determined by detection of the corresponding
polypeptide. The polypeptide can be detected and quantified by any
of a number of means well known to those of skill in the art. In
some embodiments, a cancer stem cell marker polypeptide is detected
using analytic biochemical methods such as, for example,
electrophoresis, capillary electrophoresis, high performance liquid
chromatography (HPLC) or thin layer chromatography (TLC). The
isolated polypeptide can also be sequenced according to standard
techniques. In some embodiments, a cancer stem cell marker protein
is detected with antibodies raised against the protein using, for
example, immunofluorescence or immunohistochemistry on tissue
sections. Alternatively antibodies against a cancer stem cell
marker can detect expression using, for example, ELISA, FACS,
Western blot, immunoprecipitation or protein microarrays. For
example, cancer stem cells can be isolated from a patient biopsy
and expression of a cancer stem cell marker protein detected with
fluorescently labeled antibodies using FACS. In another method, the
cells expressing a cancer stem cell marker can be detected in vivo
using labeled antibodies in typical imaging system. For example,
antibodies labeled with paramagnetic isotopes can be used for
magnetic resonance imaging (MRI).
[0084] In some embodiments of the present invention, a diagnostic
assay comprises determining the expression or not of a cancer stem
cell marker in tumor cells using, for example,
immunohistochemistry, in situ hybridization, or RT-PCR. In other
embodiments, a diagnostic assay comprises determining expression
levels of a cancer stem cell marker using, for example,
quantitative RT-PCR. In some embodiments, a diagnostic assay
further comprises determining expression levels of a cancer stem
cell marker compared to a control tissue such as, for example,
normal epithelium.
[0085] Detection of a cancer stem cell marker expression can then
be used to provide a prognosis and select a therapy. A prognosis
can be based on any known risk expression of a cancer stem cell
marker indicates. Furthermore, detection of a cancer stem cell
marker can be used to select an appropriate therapy including, for
example, treatment with antibodies against the detected cancer stem
cell marker protein. In certain embodiments, the antibody
specifically binds to the extracellular domain of a cancer stem
cell marker protein such as a human FZD receptor.
[0086] 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.
[0087] 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 downregulate
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.
[0088] 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. 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).
[0089] In certain embodiments, antibodies against a cancer stem
cell marker trigger cell death of a cell expressing a cancer stem
cell marker protein by activating complement-dependent cytotoxicity
(CDC). CDC 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] Polyclonal antibodies can be prepared by any known method.
Polyclonal antibodies are raised 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.
[0094] 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.
[0095] 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 primers 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).
[0096] 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.
[0097] In some embodiments, of the present invention the monoclonal
antibody against a cancer stem cell marker is a humanized antibody.
Humanized antibodies are antibodies that contain minimal sequences
from non-human (e.g murine) antibodies within the variable regions.
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 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.
[0098] Humanized antibodies can be produced using various
techniques known in the art. An antibody can be humanized by
substituting the CDR of a human antibody with that of a non-human
antibody (e.g. mouse, rat, rabbit, hamster, etc.) having the
desired specificity, affinity, and capability (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 Fv framework region and/or within the
replaced non-human residues to refine and optimize antibody
specificity, affinity, and/or capability.
[0099] 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); Boemer 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). Humanized
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.
[0100] 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. 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, the antibodies 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.
[0101] 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))
[0102] 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.
[0103] 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 a FZD receptor, or
derivatives, fragments, analogs 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.
[0104] 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).
[0105] 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.
[0106] 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 a human FZD receptor. 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.
[0107] 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.
[0108] 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 one 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 (CH1, 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.
[0109] Besides their configuration, it is known in the art that the
constant region mediates several effector functions. For example,
binding of the Cl 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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 (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies including .sup.212Bi, .sup.131I, .sup.131In, .sup.90Y,
and .sup.186Re. Conjugates of the antibody and cytotoxic agent are
made using a variety of bifunctional protein-coupling agents such
as N-succinimidyl-3-(2-pyridyidithiol) 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.
[0115] Conjugate 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.
[0116] 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. 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 biological
response modifiers such as methotrexate, adriamycin, and
lymphokines such as interferon. Still other embodiments of the
present invention comprise the use of antibodies conjugated to
specific biotoxins such as ricin or diptheria toxin. In yet other
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. 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.
[0117] 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).
[0118] 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).
[0119] 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. 3H or 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.
[0120] In certain embodiments, the invention encompasses isolated
polynucleotides that encode a polypeptide comprising an antibody,
or fragment thereof, against a human FZD receptor. 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 strand or non-coding (anti-sense) strand.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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 an antibody, or fragment thereof, against a
human FZD receptor.
[0125] 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.
[0126] 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.
[0127] 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).
[0128] The polypeptides of the present invention can be recombinant
polypeptides, natural polypeptides, or synthetic polypeptides
comprising an antibody, or fragment thereof, against a human FZD
receptor. 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 an antibody, or
fragment thereof, against a human FZD receptor protein. Such
mutants include deletions, insertions, inversions, repeats, and
type substitutions.
[0129] 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).
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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).
[0137] 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.
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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.
[0142] The present invention further provides pharmaceutical
compositions comprising antibodies that target a cancer stem cell
marker. These pharmaceutical compositions find use in inhibiting
tumor cell growth and treating cancer in human patients.
[0143] 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).
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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).
[0148] 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.
[0149] 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).
[0150] 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.
[0151] 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 EGFR, HER2, and VEGF.
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.
[0152] 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.
[0153] 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 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.
[0154] 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.
EXAMPLES
Example 1
Production of FZD Antibodies
Antigen Production
[0155] Recombinant polypeptide fragments of the extracellular
domain of human FZD receptors were generated as antigens for
antibody production. Standard recombinant DNA technology was used
to isolate polynucleotides encoding amino acids 1-227 of FZD10 (SEQ
ID NO: 1), amino acids 1-255 of FZD7 (SEQ ID NO: 2), amino acids
1-233 of FZD5 (SEQ ID NO. 3), amino acids 1-207 of FZD6 (SEQ ID NO:
4), amino acids 1-224 of FZD4 (SEQ ID NO: 5); and amino acids 1-158
of FZD8 (SEQ ID NO: 6). These polynucleotides were ligated in-frame
N-terminal to either a human Fc-tag or histidine-tag and cloned
into a transfer plasmid vector for baculovirus mediated expression
in insect cells. Standard transfection, infection, and cell culture
protocols were used to produce recombinant insect cells expressing
the corresponding FZD polypeptides (O'Reilley et al., Baculovirus
expression vectors: A Laboratory Manual, Oxford: Oxford University
Press (1994)).
[0156] Cleavage of the endogenous signal sequence of human FZD
receptors was approximated using cleavage prediction software
SignalP 3.0, however the actual in vivo cleavage point can differ
by a couple of amino acids either direction. The predicated
cleavage of FZD10 is between amino acids 20 and 21, thus FZD10
antigen protein comprises approximately amino acid 21 through amino
acid 227. The predicated cleavage of FZD7 is between amino acids 31
and 32, thus FZD7 antigen protein comprises approximately amino
acids 32 to 255. The predicated cleavage of FZD5 is between amino
acids 26 and 27, thus FZD5 antigen protein comprises approximately
amino acid 27 through amino acid 233. The predicated cleavage of
FZD6 is between amino acids 17 and 18, thus FZD6 antigen protein
comprises approximately amino acid 18 through amino acid 207. The
predicated cleavage of FZD4 is between amino acids 39 and 40, thus
FZD4 antigen protein comprises approximately amino acid 40 through
amino acid 224. The predicated cleavage of FZD8 is between amino
acids 27 and 28, thus FZD8 antigen protein comprises approximately
amino acid 28 through amino acid 158.
[0157] Antigen protein was purified from insect cell conditioned
medium using Protein A and Ni++-chelate affinity chromatography.
Purified antigen protein was dialyzed against PBS (pH=7),
concentrated to approximately 1 mg/ml, and sterile filtered in
preparation for immunization.
Immunization
[0158] Mice (n=3) were immunized with purified FZD10, FZD7, FZD5,
FZD6, FZD4, and FZD8 antigen protein (Antibody Solutions; Mountain
View, Calif.) using standard techniques. Blood from individual mice
was screened approximately 70 days after initial immunization for
antigen recognition using ELISA and FACS analysis (described in
detail below). The two animals with the highest antibody titers
were selected for final antigen boost after which spleen cells were
isolated for hybridoma production. Hybridoma cells were plated at 1
cell per well in 96 well plates, and the supernatant from each well
screened by ELISA and FACS analysis against antigen protein.
Several hybridomas with high antibody titer were selected and
scaled up in static flask culture. Antibodies were purified from
the hybridoma supernatant using protein A or protein G agarose
chromatography. Purified monoclonal antibodies were again tested by
FACS and are isotyped to select for IgG and IgM antibodies.
Epitope Mapping
[0159] To identify antibodies that recognize specific regions of
the FZD extracellular domain including the cysteine-rich domain,
epitope mapping is performed. Mammalian expression plasmid vectors
comprising a CMV promoter upstream of polynucleotides that encode
fragments of the extracellular FZD domain are generated using
standard recombinant DNA technology. Recombinant proteins are then
expressed in cultured mammalian cells by transient transfection.
Twenty-four to 48 hours following transfection, cells are harvested
and cell lysate protein separated on SDS-PAGE acrylamide gels for
Western blotting using antibodies from mice immunized with FZD
antigen. Antibodies that recognize the ligand binding domain of FZD
can be further analyzed for competitive binding with Wnt proteins
by ELISA.
[0160] To identify specific epitopes within the extracellular
domains recognized by an antibody against FZD the SPOTs system is
used (Sigma Genosys, The Woodlands, Tex.). A series of 10-residue
linear peptides overlapping by one amino acid and covering the
entire FZD extracellular domain are synthesized and covalently
bound to a cellulose membrane by the SPOT synthesis technique. The
membrane is preincubated for 8 hours at room temperature with
blocking buffer and hybridized with antibody overnight at 4.degree.
C. The membrane is then washed, incubated with a secondary antibody
conjugated to horseradish peroxidase (HRP) (Amersham Bioscience,
Piscataway, N.J.), re-washed, and visualized with signal
development solution containing 3-amino-9-ethylcarbazole. Specific
epitopes recognized by an antibody are thus determined.
FACS Analysis
[0161] To select monoclonal antibodies produced by hybridomas
clones that recognize native cell-surface FZD protein, FACs
analysis was used. HEK293 cells were transfected with an expression
vector encoding a full-length cDNA clone of the corresponding FZD
either alone (FZD10) or co-transfected with a vector expressing GFP
(FZD7, FZD5, FZD6, FZD4, and FZD8). In the case of FZD10, FZD7,
FZD6, and FZD4 expression vectors, the Flag epitope tag was
introduced at the amino-terminus, which allowed verification of
expression of the tagged FZD receptors on the cell surface.
Twenty-four to 48-hours post-transfection, cells were collected in
suspension and incubated on ice with anti-FZD antibodies, FLAG
antibodies, immune serum (for FZD5 expressing cells), or control
IgG to detect background antibody binding. The cells were washed
and primary antibodies detected with anti-mouse secondary
antibodies conjugated to a fluorescent chromophore. Labeled cells
were then sorted by FACS to identify anti-FZD antibodies that
specifically recognize cell surface expression of the corresponding
FZD receptor. Antibodies that recognize FZD10 (FIG. 1A); FZD7 (FIG.
1B); FZD5 (FIG. 1C); FZD6 (FIG. 1D); FZD4 (FIG. 1E); and FZD8 (FIG.
1F) were identified. Antibodies that recognize FZD1, FZD2, FZD3,
and FZD5 are similarly generated using the extracellular ligand
binding as an antigen for immunization of mice.
Chimeric Antibodies
[0162] After monoclonal antibodies that specifically recognize a
FZD receptor are identified, these antibodies are modified to
overcome the human anti-mouse antibody (HAMA) immune response when
rodent antibodies are used as therapeutics agents. The variable
regions of the heavy-chain and light-chain of the selected
monoclonal antibody are isolated by RT-PCR from hybridoma cells and
ligated in-frame to human IgG1 heavy-chain and kappa light chain
constant regions, respectively, in mammalian expression vectors.
Alternatively a human Ig expression vector such as TCAE 5.3 is used
that contains the human IgG1 heavy-chain and kappa light-chain
constant region genes on the same plasmid (Preston et al., 1998,
Infection & Immunity 66:4137-42). Expression vectors encoding
chimeric heavy- and light-chains are then co-transfected into
Chinese hamster ovary (CHO) cells for chimeric antibody production.
Immunoreactivity and affinity of chimeric antibodies are compared
to parental murine antibodies by ELISA and FACS.
Humanized Antibodies
[0163] As chimeric antibody therapeutics are still frequently
antigenic, producing a human anti-chimeric antibody (HACA) immune
response, chimeric antibodies against a FZD receptor can undergo
further humanization. To generate humanized antibodies, key aspects
of the specificity determining motifs of the antibody, potentially
including elements from both the three short hypervariable
sequences, or complementary determining regions (CDRs), and/or the
framework regions required to correctly position the CDR regions of
the antibody heavy- and light-chain variable domains described
above are engineered using recombinant DNA technology into the
germline DNA sequences of human heavy- and light-chain antibody
genes, respectively, and then cloned into a mammalian expression
vector for expression in CHO cells. The immunoreactivity and
affinity of the humanized antibodies are compared to parental
chimeric antibodies by ELISA and FACS. Additionally, site-directed
or high-density mutagenesis of the variable region can be used to
optimize specificity, affinity, etc. of the humanized antibody.
Human Antibodies
[0164] In some embodiments, human antibodies that specifically
recognize the extracellular domain of a FZD receptor are isolated
using phage display technology. A phage display antibody library
containing human antibody variable domains displayed as single
chain Fv or as fab domains is screened for specific and high
affinity recognition of a FZD receptor antigen described above. The
identified variable domain antibody sequences are then reformatted
into an Ig expression vector containing human IgG1 heavy-chain and
kappa light-chain for expression of human antibodies in CHO
cells.
Example 2
Production of Antibodies that Recognize Multiple FZD Family
Members
[0165] To target more than one human FZD receptor, antibodies that
specifically recognize multiple members of the FZD receptor family
are generated. Soluble proteins comprising the N-terminal Frizzled,
or Fri, ligand binding domains of either FZD4, FZD5, and FZD8 fused
to human Fc bind to and prevent signaling by all classes of Wnt
ligands that signal by mechanisms that include stabilization of
beta catenin including Wnt1, Wnt2, Wnt3, Wnt3a, and Wnt7b (FIG. 2).
Specifically, HEK 293 cells stably transfected with
8.times.TCF-luciferase reporter were incubated with increasing
amounts of FZD Fc soluble receptors in the presence of different
Wnt ligands including Wnt1, Wnt2, Wnt3, Wnt3a and Wnt7b. FZD4 Fc,
FZD5 Fc and FZD8 Fc fusion proteins inhibited Wnt signaling
mediated by all five Wnt ligands (FIG. 2). Thus in certain
embodiments, antibodies that specifically recognize two or more of
FZD4, FZD5, and FZD8 receptors are produced.
[0166] In certain embodiments, antibodies are generated as
described in detail in Example 1 by immunizing mice with one or
more of the FZD receptor antigens. Antibodies generated against
each FZD receptor are then tested for cross-reactivity with other
FZD receptors. Antibodies that specifically recognize FZD2 &
FZD6; FZD7 & FZD10; FZD4 & FZD5; FZD4 & FZD8; FZD5
& FZD8; and FZD4, FZD5, & FZD8 are then identified and
tested for the ability to prevent tumor cell growth as described in
detail below.
[0167] In certain embodiments, a phage display library is used to
identify antibodies that recognize multiple FZD family members. A
region of high homology among the N-terminal extracellular domains
of human FZD receptors is used to screen the library for phage
displaying an antigen-binding domain that specifically recognizes
two or more FZD receptors. For example, a homologues region of
human FZD receptors is expressed as a FZD-Fc protein, and the
recombinant protein is coated on an appropriate surface at 10
.mu.g/mL. A human phage library is then panned through two rounds
of enrichment (See e.g., Griffiths et al., EMBO J. 12:715-34).
Genes encoding the antigen binding domain are then recovered from
the phage and used to construct a complete human antibody molecule
by joining the antigen binding domain with constant regions for
expression in a suitable host cell line. In certain embodiments,
antibodies that recognize FZD2 & FZD6; FZD7 & FZD10; FZD4
& FZD5; FZD4 & FZD8; FZD5 & FZD8; and FZD4, FZD5, &
FZD8 are identified and tested for the ability to prevent tumor
cell growth as described in detail below.
[0168] In certain embodiments, antibodies that antagonize the Wnt
signaling pathway by interfering with signaling via the canonical
Wnt signaling pathway are developed. For example, antibodies that
specifically recognize two or more of FZD4, FZD5, and FZD8 are
identified via phage display. In certain embodiments, antibodies
that antagonize the Wnt signaling pathway by activating the
antagonistic non-canonical Wnt signaling pathway are developed. For
example, antibodies that specifically recognize FZD6 and FZD2 are
identified via phage display.
Example 3
In Vitro Assays to Evaluate Antibodies Against a FZD Receptor
[0169] This example describes representative in vitro assays to
test the activity of antibodies generated against a FZD receptor on
cell proliferation, pathway activation, and cytotoxicity.
Proliferation Assay
[0170] The expression of a FZD receptor by different cancer cell
lines is quantified using Taqman analysis. Cell lines identified as
expressing a FZD receptor are plated at a density of 104 cell per
well in 96-well tissue culture microplates and allowed to spread
for 24 hours. Subsequently cells are cultured for an additional 12
hours in fresh DMEM with 2% FCS at which point anti-FZD antibodies
versus control antibodies are added to the culture medium in the
presence of 10 .mu.mol/L BrdU. Following BrdU labeling, the culture
media is removed, and the cells fixed at room temperature for 30
minutes in ethanol and reacted for 90 minutes with
peroxidase-conjugated monoclonal anti-BrdU antibody (clone BMG 6H8,
Fab fragments). The substrate is developed in a solution containing
tetramethylbenzidine and stopped after 15 minutes with 25 .mu.l of
1 mol/L H.sub.2SO.sub.4. The color reaction is measured with an
automatic ELISA plate reader using a 450 nm filter (UV Microplate
Reader; Bio-Rad Laboratories, Richmond, Calif.). All experiments
are performed in triplicate. The ability of anti-FZD antibodies to
inhibit cell proliferation compared to control antibodies is
determined.
Pathway Activation Assay
[0171] In certain embodiments, the ability of antibodies against a
FZD receptor to block activation of the Wnt signaling pathway is
determined in vitro. For example, HEK 293 cells cultured in DMEM
supplemented with antibiotics and 10% FCS are co-transfected with
1) Wnt7B and FZD10 expression vectors to activate the Wnt signaling
pathway; 2) a TCF/Luc wild-type or mutant reporter vector
containing three or eight copies of the TCF-binding domain upstream
of a firefly luciferase reporter gene to measure canonical Wnt
signaling levels (Gazit et al., 1999, Oncogene 18:5959-66); and 3)
a Renilla luciferase reporter (Promega; Madison, Wis.) as an
internal control for transfection efficiency. Anti-FZD10 and
control antibodies are then added to the cell culture medium.
Forty-eight hours following transfection, luciferase levels are
measured using a dual luciferase assay kit (Promega; Madison, Wis.)
with firefly luciferase activity normalized to Renilla luciferase
activity. Three independent experiments are preformed in
triplicate. The ability of FZD10 antibodies to inhibit Wnt pathway
activation is thus determined.
[0172] In some embodiments, the ability of antibodies against human
FZD10 receptor to interfere with Wnt ligand binding is determined
in vitro. For example, HEK 293 cells were transfected with the Wnt
responsive luciferase reporter TOPFLASH (Upstate Group LLC; catalog
#21-170) and a Wnt3A expression vector to activate endogenous Wnt
signaling in transfected cells. Soluble FZD5 Fc containing the
extracellular domain (amino acids 1-233) of FZD5 linked in-frame to
human IgG1 Fc was added to the culture medium of transfected cells
to bind Wnt3A either alone (FIG. 3; HT medium Wnt3A, right bar) or
in the presence of various antibodies generated against FZD5 (FIG.
1C; 44M1-32). In the absence of FZD5 antibodies, FZD5 Fc completely
eliminated Wnt signaling in transfected cells as measured by
luciferase activity, whereas addition of FZD5 antibodies that
interfere with ligand binding by FZD5 Fc to Wnt3A, restored Wnt
signaling (FIG. 3).
[0173] In some embodiments, the ability of antibodies that
specifically bind to two or more human FZD receptors (e.g. FZD2 and
FZD6) to antagonize FZD-mediated canonical Wnt signaling by, for
example, acting as an agonist of non-canonical Wnt signaling is
determined in vitro. For example, HEK 293 cells are transfected
with the Wnt responsive luciferase reporter TOPFLASH. Forty-eight
hours post-transfection, antibodies that specifically recognize
human FZD2 and FZD6 or an isotype control are added to the culture
medium along with a Wnt ligand such as, for example, Wnt-3a.
Activation of canonical Wnt signaling in the presence and absence
of antibodies is then determined by measuring luciferase
activity.
Complement-dependent Cytotoxicity Assay
[0174] In certain embodiments, cancer cell lines expressing a FZD
receptor or cancer stem cells isolated from a patient sample
passaged as a xenograft in immunocompromised mice (as described in
detail below) are used to measure complement dependent cytotoxicity
(CDC) mediated by an antibody against a FZD receptor. Cells are
suspended in 200 ul RPMI 1640 culture medium supplemented with
antibiotics and 5% FBS at 106 cells/ml. Suspended cells are then
mixed with 200 .mu.l serum or heat-inactivated serum with
antibodies against a FZD receptor or control antibodies in
triplicate. Cell mixtures are incubated for 1 to 4 hours at
37.degree. C. in 5% CO.sub.2. Treated cells are then collected,
resuspended in 100 .mu.l FITC-labeled annexin V diluted in culture
medium and incubated at room temperature for 10 minutes. One
hundred microliters of a propidium iodide solution (25 .mu.g/ml)
diluted in HBSS is added and incubated for 5 minutes at room
temperature. Cells are collected, resuspended in culture medium and
analyzed by flow cytometry. Flow cytometry of FITC stained cells
provides total cell counts, and propidium iodide uptake by dead
cells as a percentage of total cell numbers is used to measure cell
death in the presence of serum and antibodies against a FZD
compared to heat-inactivated serum and control antibodies. The
ability of anti-FZD antibodies to mediated complement-dependent
cytotoxicity is thus determined.
Antibody-Dependent Cellular Cytotoxicity Assay
[0175] In certain embodiments, cancer cell lines expressing a FZD
receptor or cancer stem cells isolated from a patients sample
passaged as a xenograft in immunocompromised mice (as described in
detail below) are used to measure antibody dependent cellular
cytotoxicity (ADCC) mediated by an antibody against a FZD receptor.
Cells are suspended in 200 .mu.l phenol red-free RPMI 1640 culture
medium supplemented with antibiotics and 5% FBS at 10.sup.6
cells/ml. Peripheral blood mononuclear cells (PBMCs) are isolated
from heparinized peripheral blood by Ficoll-Paque density gradient
centrifugation for use as effector cells. Target cells (T) are then
mixed with PBMC effector cells (E) at E/T ratios of 25:1, 10:1, and
5:1 in 96-well plates in the presence of at least one FZD receptor
antibody or a control antibody. Controls include incubation of
target cells alone and effector cells alone in the presence of
antibody. Cell mixtures are incubated for 1 to 6 hours at
37.degree. C. in 5% CO.sub.2. Released lactate dehydrogenase (LDH),
a stable cytosolic enzyme released upon cell lysis, is then
measured by a colorimetric assay (CytoTox96 Non-radioactive
Cytotoxicity Assay; Promega; Madison, Wis.). Absorbance data at 490
nm are collected with a standard 96-well plate reader and
background corrected. The percentage of specific cytotoxicity is
calculated according to the formula: %
cytotoxicity=100.times.(experimental LDH release-effector
spontaneous LDH release-target spontaneous LDH release)/(target
maximal LDH release-target spontaneous LDH release). The ability of
antibodies against a FZD receptor to mediated antibody dependent
cellular cytotoxicity is thus determined.
Example 4
In Vivo Prevention of Tumor Growth Using Anti-FZD Receptor
Antibodies
[0176] This example describes the use of anti-FZD receptor
antibodies to prevent tumor growth 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
are prepared for repassaging into experimental animals. Tumor
tissue is removed under sterile conditions, cut up into small
pieces, minced completely using sterile blades, and single cell
suspensions obtained by enzymatic digestion and mechanical
disruption. Specifically, pleural effusion cells or the resulting
tumor pieces are mixed with ultra-pure collagenase III in culture
medium (200-250 units of collagenase per mL) and incubated at
37.degree. C. for 3-4 hours with pipetting up and down through a
10-mL pipette every 15-20 minutes. Digested cells are filtered
through a 45 .mu.M nylon mesh, washed with RPMI/20% FBS, and washed
twice with HBSS. Dissociated tumor cells are then injected
subcutaneously into the mammary fat pads of NOD/SCID mice to elicit
tumor growth.
[0177] In certain embodiments, dissociated tumor cells are first
sorted into tumorigenic and non-tumorigenic cells based on cell
surface markers before injection into experimental animals.
Specifically, tumor cells dissociated as described above are washed
twice with Hepes buffered saline solution (HBSS) containing 2%
heat-inactivated calf serum (HICS) and resuspended at 106 cells per
100 .mu.l. Antibodies are added and the cells incubated for 20
minutes on ice followed by two washes with HBSS/2% HICS. Antibodies
include anti-ESA (Biomeda, Foster City, Calif.), anti-CD44,
anti-CD24, and Lineage markers anti-CD2, -CD3, -CD10, -CD16, -CD18,
-CD31, -CD64, and -CD140b (collectively referred to as Lin;
PharMingen, San Jose, Calif.). Antibodies are directly conjugated
to fluorochromes to positively or negatively select cells
expressing these markers. Mouse cells are eliminated by selecting
against H2Kd+ cells, and dead cells are eliminated by using the
viability dye 7AAD. Flow cytometry is performed on a FACSVantage
(Becton Dickinson, Franklin Lakes, N.J.). Side scatter and forward
scatter profiles are used to eliminate cell clumps. Isolated ESA+,
CD44+, CD24-/low, Lin- tumorigenic cells are then injected
subcutaneously into NOD/SCID mice to elicit tumor growth.
[0178] In certain embodiments anti-FZD6 and anti-FZD5 antibodies
were analyzed for their ability to reduce the growth of UM-C4 colon
tumor cells. Dissociated UM-C4 cells (10,000 per animal) were
injected subcutaneously into the flank region of 6-8 week old
NOD/SCID mice. Two days after tumor cell injection, animals were
injected intraperitoneal (i.p.) with 10 mg/kg either anti-FZD6 or
anti-FZD5 receptor antibodies two times per week. Tumor growth was
monitored weekly until growth was detected, after which point tumor
growth was measured twice weekly for a total of 8 weeks. Treatment
of animals with anti-FZD6 antibody 23M2 and anti-FZD5 antibody
44M13 significantly reduced tumor growth as compared to PBS
injected controls (FIG. 4).
Example 5
In Vivo Treatment of Tumors Using Anti-FZD Receptor Antibodies
[0179] This example describes the use of anti-FZD receptor
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
are prepared for repassaging into experimental animals. Tumor
tissue is 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 into the flank, for non-breast 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.
[0180] Following tumor cell injection, animals are monitored for
tumor growth. Once tumors reach an average size of approximately
150 to 200 mm, antibody treatment begins. Each animal receives 100
.mu.g FZD receptor antibodies or control antibodies i.p. two to
five times per week for a total of 6 weeks. Tumor size is assessed
twice a week during these 6 weeks. The ability of FZD receptor
antibodies to prevent further tumor growth or to reduce tumor size
compared to control antibodies is thus determined.
[0181] 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-FZD receptor 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.m 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-FZD 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-.beta.-catenin antibodies to detect Wnt
signaling, and 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.
[0182] In certain embodiments, the effect of anti-FZD receptor
antibody treatment on tumor cell gene expression is also assessed.
Total RNA is extracted from a portion of each harvested tumor from
FZD antibody treated and control antibody treated mice and used for
quantitative RT-PCR. Expression levels of FZD receptors, components
of Wnt signaling pathway including, for example, Wnt1 and
.beta.-catenin, 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 FZD receptor antibody treatment are thus
determined.
[0183] In addition, the effect of anti-FZD receptor antibody
treatment on the presence of cancer stem cells in a tumor is
assessed. Tumor samples from FZD 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.
[0184] The tumorigenicity of cells isolated based on ESA+, CD44+,
CD24-/low, Lin- expression following anti-FZD antibody treatment
can then assessed. ESA+, CD44+, CD24-/low, Lin- cancer stem cells
isolated from FZD 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 6
Treatment of Human Cancer Using Anti-FZD Receptor Antibodies
[0185] This example describes methods for treating cancer using
antibodies against a FZD receptor to target tumors comprising
cancer stem cells and/or tumor cells in which FZD receptor
expression has been detected. 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. Sections are incubated with antibodies against a FZD
receptor to detect protein expression.
[0186] 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, -Lin, and -FZD antibodies to detect cancer
stem cells, and the presence of ESA+, CD44+, CD24-/low, Lin-, FZD+
tumor stem cells is determined by flow cytometry as described in
detail above.
[0187] Cancer patients whose tumors are diagnosed as expressing a
FZD receptor are treated with anti-FZD receptor antibodies. In
certain embodiments, humanized or human monoclonal anti-FZD
receptor antibodies generated as described above are purified and
formulated with a suitable pharmaceutical vehicle for injection. In
some embodiments, patients are treated with the FZD antibodies at
least once a month for at least 10 weeks. In some embodiments,
patients are treated with the FZD antibodies at least once a week
for at least about 14 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 anti-FZD antibody is
administered. In some embodiments, and between about 5 to about 40
mg/ml of an anti-FZD 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, for
example, based on 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.
[0188] All publications and patents mentioned in the above
specification are herein incorporated by reference. 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.
TABLE-US-00001 SEQ ID NO: 1 FZD10 N-terminal extracellular domain
MQRPGPRLWLVLQVMGSCAAISSMDMERPGDGKCQPIEIPMCKDIGYNM
TRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQ
VSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLC
MEAPNNGSDEPTRGSGLFPPLFRPQRPHSAQEHPLKDGGPGRGGCDNPG
KFHHVEKSASCAPLCTPGVDVYWSREDKRFA SEQ ID NO: 2 FZD7 N-terminal
extracellular domain
MRDPGAAAPLSSLGLCALVLALLGALSAGAGAQPYHGEKGISVPDHGFC
QPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPEL
RFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLR
CENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLPDLPFTALPPG
ASDGRGRPAFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRANGLMYF KEEERRFARL SEQ ID
NO: 3 FZD5 N-terminal extracellular domain
MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLT
HMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDY
HKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLC
MDYNRSEATTAPPRPFPAKPTLPGPPGAPASGGECPAGGPFVCKCREPF
VPILKESHPLYNKVRTGQVPNCAVPCYQPSFSADERT SEQ ID NO: 4 FZD6 N-terminal
extracellular domain
MEMFTFLLTCIFLPLLRGHSLFTCEPITVPRCMKMAYNMIFFPNLMGHY
DQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKL
CEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHTEFLGPQ
KKTEQVQRDIGFWCPRHLKTSGGQGYKFLGIDQCAPPCPNMYFKSDELE FAKSFIGTVSI SEQ
ID NO: 5 FZD4 N-terminal extracellular domain
MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCD
PIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQ
FFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLN
CSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEECHSVGTNSDQYIW
VKRSLNCVLKCGYDAGLYSRSAKEFTDI SEQ ID NO: 6 FZD8 Fri domain
MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYN
YTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLE
DYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTL CMDYNRTDLTT
Sequence CWU 1
1
61226PRTArtificial Sequencerecombinant human FZD10 N-terminal
extracellular domain 1Met Gln Arg Pro Gly Pro Arg Leu Trp Leu Val
Leu Gln Val Met Gly 1 5 10 15 Ser Cys Ala Ala Ile Ser Ser Met Asp
Met Glu Arg Pro Gly Asp Gly 20 25 30 Lys Cys Gln Pro Ile Glu Ile
Pro Met Cys Lys Asp Ile Gly Tyr Asn 35 40 45 Met Thr Arg Met Pro
Asn Leu Met Gly His Glu Asn Gln Arg Glu Ala 50 55 60 Ala Ile Gln
Leu His Glu Phe Ala Pro Leu Val Glu Tyr Gly Cys His 65 70 75 80 Gly
His Leu Arg Phe Phe Leu Cys Ser Leu Tyr Ala Pro Met Cys Thr 85 90
95 Glu Gln Val Ser Thr Pro Ile Pro Ala Cys Arg Val Met Cys Glu Gln
100 105 110 Ala Arg Leu Lys Cys Ser Pro Ile Met Glu Gln Phe Asn Phe
Lys Trp 115 120 125 Pro Asp Ser Leu Asp Cys Arg Lys Leu Pro Asn Lys
Asn Asp Pro Asn 130 135 140 Tyr Leu Cys Met Glu Ala Pro Asn Asn Gly
Ser Asp Glu Pro Thr Arg 145 150 155 160 Gly Ser Gly Leu Phe Pro Pro
Leu Phe Arg Pro Gln Arg Pro His Ser 165 170 175 Ala Gln Glu His Pro
Leu Lys Asp Gly Gly Pro Gly Arg Gly Gly Cys 180 185 190 Asp Asn Pro
Gly Lys Phe His His Val Glu Lys Ser Ala Ser Cys Ala 195 200 205 Pro
Leu Cys Thr Pro Gly Val Asp Val Tyr Trp Ser Arg Glu Asp Lys 210 215
220 Arg Phe 225 2255PRTArtificial Sequencerecombinant human FZD7
N-terminal extracellular domain 2Met Arg Asp Pro Gly Ala Ala Ala
Pro Leu Ser Ser Leu Gly Leu Cys 1 5 10 15 Ala Leu Val Leu Ala Leu
Leu Gly Ala Leu Ser Ala Gly Ala Gly Ala 20 25 30 Gln Pro Tyr His
Gly Glu Lys Gly Ile Ser Val Pro Asp His Gly Phe 35 40 45 Cys Gln
Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala Tyr Asn Gln 50 55 60
Thr Ile Leu Pro Asn Leu Leu Gly His Thr Asn Gln Glu Asp Ala Gly 65
70 75 80 Leu Glu Val His Gln Phe Tyr Pro Leu Val Lys Val Gln Cys
Ser Pro 85 90 95 Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr Ala Pro
Val Cys Thr Val 100 105 110 Leu Asp Gln Ala Ile Pro Pro Cys Arg Ser
Leu Cys Glu Arg Ala Arg 115 120 125 Gln Gly Cys Glu Ala Leu Met Asn
Lys Phe Gly Phe Gln Trp Pro Glu 130 135 140 Arg Leu Arg Cys Glu Asn
Phe Pro Val His Gly Ala Gly Glu Ile Cys 145 150 155 160 Val Gly Gln
Asn Thr Ser Asp Gly Ser Gly Gly Pro Gly Gly Gly Pro 165 170 175 Thr
Ala Tyr Pro Thr Ala Pro Tyr Leu Pro Asp Leu Pro Phe Thr Ala 180 185
190 Leu Pro Pro Gly Ala Ser Asp Gly Arg Gly Arg Pro Ala Phe Pro Phe
195 200 205 Ser Cys Pro Arg Gln Leu Lys Val Pro Pro Tyr Leu Gly Tyr
Arg Phe 210 215 220 Leu Gly Glu Arg Asp Cys Gly Ala Pro Cys Glu Pro
Gly Arg Ala Asn 225 230 235 240 Gly Leu Met Tyr Phe Lys Glu Glu Glu
Arg Arg Phe Ala Arg Leu 245 250 255 3233PRTArtificial
Sequencerecombinant human FZD5 N-terminal extracellular domain 3Met
Ala Arg Pro Asp Pro Ser Ala Pro Pro Ser Leu Leu Leu Leu Leu 1 5 10
15 Leu Ala Gln Leu Val Gly Arg Ala Ala Ala Ala Ser Lys Ala Pro Val
20 25 30 Cys Gln Glu Ile Thr Val Pro Met Cys Arg Gly Ile Gly Tyr
Asn Leu 35 40 45 Thr His Met Pro Asn Gln Phe Asn His Asp Thr Gln
Asp Glu Ala Gly 50 55 60 Leu Glu Val His Gln Phe Trp Pro Leu Val
Glu Ile Gln Cys Ser Pro 65 70 75 80 Asp Leu Arg Phe Phe Leu Cys Ser
Met Tyr Thr Pro Ile Cys Leu Pro 85 90 95 Asp Tyr His Lys Pro Leu
Pro Pro Cys Arg Ser Val Cys Glu Arg Ala 100 105 110 Lys Ala Gly Cys
Ser Pro Leu Met Arg Gln Tyr Gly Phe Ala Trp Pro 115 120 125 Glu Arg
Met Ser Cys Asp Arg Leu Pro Val Leu Gly Arg Asp Ala Glu 130 135 140
Val Leu Cys Met Asp Tyr Asn Arg Ser Glu Ala Thr Thr Ala Pro Pro 145
150 155 160 Arg Pro Phe Pro Ala Lys Pro Thr Leu Pro Gly Pro Pro Gly
Ala Pro 165 170 175 Ala Ser Gly Gly Glu Cys Pro Ala Gly Gly Pro Phe
Val Cys Lys Cys 180 185 190 Arg Glu Pro Phe Val Pro Ile Leu Lys Glu
Ser His Pro Leu Tyr Asn 195 200 205 Lys Val Arg Thr Gly Gln Val Pro
Asn Cys Ala Val Pro Cys Tyr Gln 210 215 220 Pro Ser Phe Ser Ala Asp
Glu Arg Thr 225 230 4207PRTArtificial Sequencerecombinant human
FZD6 N-terminal extracellular domain 4Met Glu Met Phe Thr Phe Leu
Leu Thr Cys Ile Phe Leu Pro Leu Leu 1 5 10 15 Arg Gly His Ser Leu
Phe Thr Cys Glu Pro Ile Thr Val Pro Arg Cys 20 25 30 Met Lys Met
Ala Tyr Asn Met Thr Phe Phe Pro Asn Leu Met Gly His 35 40 45 Tyr
Asp Gln Ser Ile Ala Ala Val Glu Met Glu His Phe Leu Pro Leu 50 55
60 Ala Asn Leu Glu Cys Ser Pro Asn Ile Glu Thr Phe Leu Cys Lys Ala
65 70 75 80 Phe Val Pro Thr Cys Ile Glu Gln Ile His Val Val Pro Pro
Cys Arg 85 90 95 Lys Leu Cys Glu Lys Val Tyr Ser Asp Cys Lys Lys
Leu Ile Asp Thr 100 105 110 Phe Gly Ile Arg Trp Pro Glu Glu Leu Glu
Cys Asp Arg Leu Gln Tyr 115 120 125 Cys Asp Glu Thr Val Pro Val Thr
Phe Asp Pro His Thr Glu Phe Leu 130 135 140 Gly Pro Gln Lys Lys Thr
Glu Gln Val Gln Arg Asp Ile Gly Phe Trp 145 150 155 160 Cys Pro Arg
His Leu Lys Thr Ser Gly Gly Gln Gly Tyr Lys Phe Leu 165 170 175 Gly
Ile Asp Gln Cys Ala Pro Pro Cys Pro Asn Met Tyr Phe Lys Ser 180 185
190 Asp Glu Leu Glu Phe Ala Lys Ser Phe Ile Gly Thr Val Ser Ile 195
200 205 5224PRTArtificial Sequencerecombinant human FZD4 N-terminal
extracellular domain 5Met Leu Ala Met Ala Trp Arg Gly Ala Gly Pro
Ser Val Pro Gly Ala 1 5 10 15 Pro Gly Gly Val Gly Leu Ser Leu Gly
Leu Leu Leu Gln Leu Leu Leu 20 25 30 Leu Leu Gly Pro Ala Arg Gly
Phe Gly Asp Glu Glu Glu Arg Arg Cys 35 40 45 Asp Pro Ile Arg Ile
Ser Met Cys Gln Asn Leu Gly Tyr Asn Val Thr 50 55 60 Lys Met Pro
Asn Leu Val Gly His Glu Leu Gln Thr Asp Ala Glu Leu 65 70 75 80 Gln
Leu Thr Thr Phe Thr Pro Leu Ile Gln Tyr Gly Cys Ser Ser Gln 85 90
95 Leu Gln Phe Phe Leu Cys Ser Val Tyr Val Pro Met Cys Thr Glu Lys
100 105 110 Ile Asn Ile Pro Ile Gly Pro Cys Gly Gly Met Cys Leu Ser
Val Lys 115 120 125 Arg Arg Cys Glu Pro Val Leu Lys Glu Phe Gly Phe
Ala Trp Pro Glu 130 135 140 Ser Leu Asn Cys Ser Lys Phe Pro Pro Gln
Asn Asp His Asn His Met 145 150 155 160 Cys Met Glu Gly Pro Gly Asp
Glu Glu Val Pro Leu Pro His Lys Thr 165 170 175 Pro Ile Gln Pro Gly
Glu Glu Cys His Ser Val Gly Thr Asn Ser Asp 180 185 190 Gln Tyr Ile
Trp Val Lys Arg Ser Leu Asn Cys Val Leu Lys Cys Gly 195 200 205 Tyr
Asp Ala Gly Leu Tyr Ser Arg Ser Ala Lys Glu Phe Thr Asp Ile 210 215
220 6158PRTArtificial Sequencerecombinant human FZD8 Fri domain
6Met Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu Leu Ala Ala Leu 1
5 10 15 Ala Leu Leu Gln Arg Ser Ser Gly Ala Ala Ala Ala Ser Ala Lys
Glu 20 25 30 Leu Ala Cys Gln Glu Ile Thr Val Pro Leu Cys Lys Gly
Ile Gly Tyr 35 40 45 Asn Tyr Thr Tyr Met Pro Asn Gln Phe Asn His
Asp Thr Gln Asp Glu 50 55 60 Ala Gly Leu Glu Val His Gln Phe Trp
Pro Leu Val Glu Ile Gln Cys 65 70 75 80 Ser Pro Asp Leu Lys Phe Phe
Leu Cys Ser Met Tyr Thr Pro Ile Cys 85 90 95 Leu Glu Asp Tyr Lys
Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu 100 105 110 Arg Ala Lys
Ala Gly Cys Ala Pro Leu Met Arg Gln Tyr Gly Phe Ala 115 120 125 Trp
Pro Asp Arg Met Arg Cys Asp Arg Leu Pro Glu Gln Gly Asn Pro 130 135
140 Asp Thr Leu Cys Met Asp Tyr Asn Arg Thr Asp Leu Thr Thr 145 150
155
* * * * *