U.S. patent application number 13/826103 was filed with the patent office on 2014-05-29 for compositions and methods for diagnosing and treating cancer.
The applicant listed for this patent is OncoMed Pharmaceuticals, Inc.. Invention is credited to Fumiko Takada Axelrod, Austin L. GURNEY, Timothy Charles Hoey, Sanjeev H. Satyal.
Application Number | 20140147443 13/826103 |
Document ID | / |
Family ID | 39268983 |
Filed Date | 2014-05-29 |
United States Patent
Application |
20140147443 |
Kind Code |
A9 |
GURNEY; Austin L. ; et
al. |
May 29, 2014 |
COMPOSITIONS AND METHODS FOR DIAGNOSING AND TREATING CANCER
Abstract
An isolated antibody that specifically binds to an extracellular
domain of human DLL4 and affects growth of a tumor comprising
cancer stem cells is described. Also described is a method of
treating cancer comprising administering a therapeutically
effective amount of an anti-DLL4 antibody.
Inventors: |
GURNEY; Austin L.; (San
Francisco, CA) ; Axelrod; Fumiko Takada; (Palo Alto,
CA) ; Hoey; Timothy Charles; (Hillsborough, CA)
; Satyal; Sanjeev H.; (San Carlos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OncoMed Pharmaceuticals, Inc. |
Redwood City |
CA |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130266569 A1 |
October 10, 2013 |
|
|
Family ID: |
39268983 |
Appl. No.: |
13/826103 |
Filed: |
March 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12782429 |
May 18, 2010 |
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13826103 |
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11905392 |
Sep 28, 2007 |
7750124 |
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12782429 |
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60847904 |
Sep 29, 2006 |
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60886260 |
Jan 23, 2007 |
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60942542 |
Jun 7, 2007 |
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Current U.S.
Class: |
424/136.1 ;
424/133.1; 424/139.1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61N 5/10 20130101; A61P 43/00 20180101; C07K 2317/565 20130101;
C07K 2317/73 20130101; C07K 16/3069 20130101; A61K 31/4745
20130101; C07K 16/30 20130101; C07K 16/3046 20130101; C07K 2317/734
20130101; A61K 2039/505 20130101; C07K 2317/24 20130101; C07K
2317/76 20130101; A61K 31/513 20130101; C07K 16/303 20130101; C07K
2317/34 20130101; A61K 39/39558 20130101; A61K 39/39558 20130101;
A61K 2300/00 20130101; A61P 35/00 20180101; C07K 2317/21 20130101;
C07K 2317/56 20130101; C07K 2317/732 20130101; A61K 2039/507
20130101; C07K 16/3015 20130101; C07K 16/3023 20130101 |
Class at
Publication: |
424/136.1 ;
424/133.1; 424/139.1 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395 |
Claims
1-64. (canceled)
65. A method of inhibiting tumor growth in a subject, comprising
administering to the subject a therapeutically effective amount of
an antibody that specifically binds human delta-like ligand 4
(DLL4), wherein the antibody is selected from the group consisting
of: a) an antibody comprising: i. a heavy chain variable region
comprising CDR amino acid sequences CDR1 (SEQ ID NO: 1); CDR2 (SEQ
ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4); and CDR3 (SEQ ID NO: 5),
and ii. a light chain variable region comprising CDR amino acid
sequences CDR1 (SEQ ID NO: 9); CDR2 (SEQ ID NO: 10); and CDR3 (SEQ
ID NO: 11); b) an antibody comprising: i. a heavy chain variable
region comprising amino acids selected from the group consisting
of: SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or ii. a light chain
variable region comprising SEQ ID NO:12; c) an antibody comprising
the same heavy and light chain polypeptide sequences as an antibody
encoded by a plasmid deposited with ATCC having deposit no.
PTA-8427 or PTA-8425; and d) an antibody containing the heavy chain
CDR amino acid sequences and the light chain CDR amino acid
sequences that are contained in the 21M18 antibody produced by the
hybridoma deposited with ATCC, having deposit no. PTA-8670.
66. A method of treating cancer in a subject, comprising
administering to the subject a therapeutically effective amount of
an antibody that specifically binds DLL4, wherein the antibody is
selected from the group consisting of: a) an antibody comprising:
i. a heavy chain variable region comprising CDR amino acid
sequences CDR1 (SEQ ID NO: 1); CDR2 (SEQ ID NO: 2, SEQ ID NO: 3, or
SEQ ID NO: 4); and CDR3 (SEQ ID NO: 5), and ii. a light chain
variable region comprising CDR amino acid sequences CDR1 (SEQ ID
NO: 9); CDR2 (SEQ ID NO: 10); and CDR3 (SEQ ID NO: 11); b) an
antibody comprising: i. a heavy chain variable region comprising
amino acids selected from the group consisting of: SEQ ID NO:6, SEQ
ID NO:7, and SEQ ID NO:8, or ii. a light chain variable region
comprising SEQ ID NO:12; c) an antibody comprising the same heavy
and light chain polypeptide sequences as an antibody encoded by a
plasmid deposited with ATCC having deposit no. PTA-8427 or
PTA-8425; and d) an antibody containing the heavy chain CDR amino
acid sequences and the light chain CDR amino acid sequences that
are contained in the 21M18 antibody produced by the hybridoma
deposited with ATCC, having deposit no. PTA-8670.
67. The method of claim 66, wherein the antibody comprises: a) a
heavy chain variable region comprising CDR amino acid sequences
CDR1 (SEQ ID NO:1), CDR2 (SEQ ID NO:3), and CDR3 (SEQ ID NO:5), and
b) a light chain variable region comprises CDR amino acid sequences
CDR1 (SEQ ID NO: 9); CDR2 (SEQ ID NO: 10); and CDR3 (SEQ ID NO:
11).
68. The method of claim 67, wherein the antibody comprises a heavy
chain variable region comprising SEQ ID NO:7.
69. The method of claim 67, wherein the antibody comprises a light
chain variable region comprising SEQ ID NO: 12.
70. The method of claim 67, wherein the antibody comprises a heavy
chain variable region comprising SEQ ID NO:7, and a light chain
variable region comprising SEQ ID NO: 12.
71. The method of claim 66, wherein the antibody is a monoclonal
antibody.
72. The method of claim 71, wherein the antibody is an antibody
fragment.
73. The method of claim 71, wherein the antibody is a chimeric,
bispecific, human, or humanized antibody.
74. The method of claim 73, wherein the antibody is a humanized
antibody.
75. The method of claim 71, wherein the antibody is an IgG
antibody.
76. The method of claim 75, wherein the antibody is an IgG.sub.2
antibody.
77. The method of claim 74, wherein the antibody is an IgG.sub.2
antibody.
78. The method of claim 75, wherein the antibody is an IgG.sub.1
antibody.
79. The method of claim 74, wherein the antibody is an IgG.sub.1
antibody.
80. The method of claim 66, wherein the cancer is colorectal
cancer, breast cancer, lung cancer, ovarian cancer, liver cancer,
prostate cancer, kidney cancer, or pancreatic cancer.
81. The method of claim 66, further comprising administering a
second therapeutic agent to the subject.
82. The method of claim 81, wherein the second therapeutic agent is
a chemotherapeutic agent.
83. The method of claim 81, wherein the second therapeutic agent is
a second therapeutic antibody.
84. The method of claim 81, wherein the second therapeutic agent is
radiation therapy.
85. A method of treating cancer in a subject, comprising
administering to the subject a therapeutically effective amount of
an antibody that specifically binds DLL4, wherein the antibody is
an antibody comprising the same heavy and light chain polypeptide
sequences as an antibody encoded by a plasmid deposited with ATCC
having deposit no. PTA-8425.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
12/782,429, filed May 18, 2010, which is a continuation of U.S.
application Ser. No. 11/905,392, filed Sep. 28, 2007, now U.S. Pat.
No. 7,750,124, which claims the benefit of U.S. Provisional Appl.
Nos. 60/847,904, filed Sep. 29, 2006; 60/886,260, filed Jan. 23,
2007; and 60/942,542, filed Jun. 7, 2007; each of which is
incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA
EFS-WEB
[0002] The content of the electronically submitted sequence listing
(Name: 22930250007SequenceListingascii.txt, Size: 29 KB; and Date
of Creation: Mar. 11, 2013) is herein incorporated by reference in
its entirety.
DESCRIPTION OF THE INVENTION
[0003] 1. Field
[0004] 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.
[0005] 2. Background
[0006] 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).
[0007] 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.
[0008] 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.
[0009] 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).
[0010] 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).
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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).
[0015] 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).
[0016] 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.
[0017] 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 and colon 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 in breast tumors 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).
Similarly, the ESA+, CD44+ subpopulation in colorectal tumors was
found to uniquely include tumorigenic cells, and the addition of
CD166 to this profile was able to further enrich for colon cancer
stem cells (CoCSC) (Dalerba et al. 2007 Proc Nat'l Acad Sci
104:10158-63). 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.
SUMMARY
[0018] Provided are antibodies that specifically bind to a human
Delta-like ligand 4 (DLL4) epitope formed by a combination of the
human DLL4 N-terminal region (SEQ ID NO: 27) and human DSL domain
(SEQ ID NO: 26), wherein the antibody affects tumor growth. Also
provided is a pharmaceutical composition comprising an antibody of
the present disclosure and a pharmaceutically acceptable vehicle.
Further provided is a method of treating cancer comprising
administering a therapeutically effective amount of a DLL4 antibody
of the present disclosure.
[0019] 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
[0020] FIG. 1: Specific Binding of anti-DLL4 21M18 Antibodies to
Native Cell-Surface DLL4 Protein. HEK 293 cells co-transfected with
full-length DLL4 and GFP were incubated with anti-DLL4 antibodies
and sorted by FACS. Anti-DLL4 antibodies 21M14 and 21M18 show
specific binding to cells expressing DLL4 as revealed by the linear
relationship between DLL4 antibody binding and GFP expression.
[0021] FIG. 2: DLL4 Antibodies Block the Interaction of Human DLL4
with the Notch Receptor. A) HEK 293 cells expressing DLL4 were
incubated with Notch-Fc or control Fc protein in the presence of
DLL4 or control antibodies. High fluorescence intensity indicates
the presence of Notch and DLL4 binding in the presence of a control
antibody (line 2) and 21M12 anti-DLL4 antibodies (line 5). Low
fluorescence intensity indicates the absence of Notch and DLL4
interactions in the absence of Notch (line 1) and the disruption of
Notch and DLL4 interactions in the presence of anti-DLL4 antibodies
21M18 (line 3) and 21M14 (line 4). B) HEK 293 cells expressing
Notch1 were incubated with either human or murine DLL4-Fc. Binding
was detected by fluorescently labeled anti-Fc and analyzed by FACS,
with high fluorescence intensity indicative of binding between DLL4
and Notch1 expressing cells. 21M18 blocks binding of human DLL4
(gray squares) but not murine DLL4 (black circles) to the Notch
receptor.
[0022] FIG. 3: Epitope Mapping of Anti-DLL4 Antibodies. A) Fusion
proteins with nested deletions of the extracellular domain of human
DLL4 were incubated in an ELISA assay with 21M14 and 21M18
anti-DLL4 antibodies. No binding above background was detected in
the presence of fusion proteins containing between amino acids 1 to
154 (aa 1-96, white bar with black dots; aa 1-154, black bar with
white dots). In contrast, binding was detected between anti-DLL4
antibodies and all fusion proteins containing between amino acids 1
to 217, including the DSL domain, of DLL4 (aa 1-217, horizontal
striped bar; aa 1-251, diagonal striped bar; aa 1-283, hatched bar;
aa 1-323, gray bar with white dots). B) Western blots show
expression of human DLL4 (h-DLL4) C-terminal deletion proteins and
murine-human DLL4 chimeric fusion proteins (anti-hFc; top). The
DLL4 fusion proteins comprise one or more of domains 1 to 6, where
domains 1 and 2 are N-terminal amino acids 1 to 154; domain 3 is
the DSL domain from amino acids 155 to 217; and domains 4, 5, and 6
are each an EGF domain as depicted graphically in C. 21M18
antibodies recognize h-DLL4 protein only in the presence of amino
acids 1-217 (hDLL4dom1-3). In contrast to the human protein, fusion
proteins comprising murine DLL4 (m-DLL4) amino acids 1-217 (dom1-3)
are not recognized by 21M18 (m-DLL4 dom1-3:h-DLL4dom4-6). Yet
fusion proteins comprising h-DLL4 amino acids 1-154 (dom1-2) in the
presence of murine dom3 are recognized by 21M18 (h-DLL4
dom1-2:mDLL4dom3-6). C) A schematic summary of the binding data of
B is shown. The domain structure of DLL4 is shown at top with the
DLL4 fusion proteins listed and shown schematically on the left
side with human protein represented by light gray and mouse protein
represented by dark gray. 21M18 binding to each DLL4 fragment is
indicated by a "+" versus a "-". D) ELISA analysis of 21M18 binding
to DLL4 protein fragments containing substitution of corresponding
murine residues for human residues at select positions. 21M18
displays impaired binding to DLL4 protein fragments with
substitutions at amino acids 68, 69, and 71 (replacement of valine,
valine, and proline) or at amino acids 142 and 144 (replacement of
lysine and alanine) E) ELISA analysis of the binding of antibodies
21M18 and 21M21 to DLL4 protein fragments containing substitution
of corresponding murine residues for human residues at select
positions within the DSL domain. Antibody 21M21 displays impaired
binding to human DLL4 protein fragments containing amino acid
substitutions at amino acids 161 and 162 (replacement of threonine
and serine). As 21M21 does not impair DLL4 function in signaling
assays (see FIG. 6), this demonstrates that not all antibodies that
bind to the DSL region impact DLL4 function.
[0023] FIG. 4: Sequence alignment of the heavy chain variable
region. A) Parental murine 21M18 antibody sequence (m-21M18-Vh,
top) (SEQ ID NO:28) human expressed framework sequence
(h-EST-framework, middle) (SEQ ID NO:29) and the humanized 21M18
heavy chain variable region sequence (21M18-H7, bottom) (SEQ ID
NO:30) are shown with conserved amino acid residues shaded in
black. The three CDRs are marked showing retention of parental
murine sequences in the humanized 21M18 antibody. The cysteine
residue at Kabat position 52a in CDR2 has been changed to a serine
and a valine residue without loss of specific binding to Dll4 in
21M18 H7 and 21M18 H9, respectively. Substitutions within the
framework region shown in 4A are numbered 1-6 with corresponding
Kabat positions in the Vh chain 16, 20, 27, 28, 38, 48. B) Parental
murine 21M18 antibody sequence (m-21M18-Vh, top) (SEQ ID NO:31),
human germline Vh sequence (h-germline-Vh, middle) (SEQ ID NO:32),
and the humanized 21M18 heavy chain variable region sequence
(21M18-H2, bottom) (SEQ ID NO:33) are shown with conserved amino
acid residues shaded in black. The three CDRs are marked showing
retention of parental murine sequences in the humanized 21M18
antibody. The cysteine residue at Kabat position 52a in CDR2 has
been changed to a serine and a valine residue without loss of
specific binding to Dll4 in 21M18 H7 and 21M18 H9, respectively.
The five retained murine residues within the variable framework
region of all heavy chain variants are numbered 1-5 at their
corresponding Kabat positions 20, 28, 38, 48, and 69.
[0024] FIG. 5: Sequence alignment of the light chain variable
region. Parental murine 21M18 antibody sequence (m-21M18-Vk, top)
(SEQ ID NO:34), human germline sequence (h-germline Vk, middle)
(SEQ ID NO:35), and humanized 21M18 light chain variable region
sequence (21M18-L2, bottom) (SEQ ID NO:36) are shown with conserved
amino acid residues shaded in black. The three CDRs are marked
showing retention of parental murine sequences in the humanized
21M18 antibody. The two retained murine residues within the
variable framework region are numbered 1-2 at their corresponding
Kabat positions 22 and 36.
[0025] FIG. 6: DLL4 Antibodies Block Notch Signaling. HeLa cells
co-transfected with Hes1-Luc reporter and Renilla luciferase
reporter vectors were incubated with DLL4-Fc protein in the
presence or absence of anti-DLL4 antibodies. Decreased luciferase
levels demonstrate loss of DLL4 Notch pathway activation by 21M14
and 21M18 antibodies.
[0026] FIG. 7: DLL4 Antibodies Modulate Expression of Notch Target
Genes in Colon Tumors. A) C8 colon tumors treated with anti-DLL4
21M18 antibodies or PBS (Control) were isolated and expression of
HES1 and ATOH-1 determined by quantitative RT-PCR. Relative gene
expression (y-axis) compared to control treated cells shows that
treatment with anti-DLL4 antibodies decreased expression of HES1
and increased expression of ATOH-1. B) Relative expression ratio
(y-axis) of HES1 versus ATOH1 in mouse lineage-depleted OMP-C11
colon tumor cell colonies is shown. C11 colonies overlaid with 3T3
cells overexpressing DLL4 (3T3+DLL4) showed an increased in the
HES1/ATOH1 expression ratio compared to colon cells overlaid with
3T3 cells (3T3) or not exposed to cell overlay (Control). This
increase in the HES1/ATOH1 expression ratio was eliminated by
incubation with 10 ug/mL 21M18 antibodies (21M18) or 5 uM-secretase
inhibitor DBZ (5 uM GSI).
[0027] FIG. 8: DLL4 Antibodies Reduce Tumor Growth. NOD/SCID mice
were injected with dissociated UM-C4 cells and treated with
anti-DLL4 21M18 antibodies (n=5) or PBS (n=10). Treatment with
21M18 antibodies (diamonds) reduced tumor growth starting on day
23, and up to 54% reduction was observed by day 48 compared to PBS
injected controls (black squares).
[0028] FIG. 9: Treatment with DLL4 Antibodies Reduces the Number of
Proliferating Tumor Cells in Vivo. C8 Colon tumors treated with
anti-DLL4 21M18 antibodies or control Ab were isolated.
Immunocytochemistry with an antibody against Ki67 showed a
reduction in the number of proliferating cells in 21M18 treated
tumors compared to control.
[0029] FIG. 10: Treatment with DLL4 Antibodies in Combination with
Fluorouracil (5-FU) Reduces Tumor Growth. NOD/SCID mice were
injected with dissociated UM-C4 cells and treated with anti-DLL4
antibodies or PBS in the presence or absence of 5-FU. A) Treatment
with 21M18 antibodies in combination with 5-FU (circles, dashed
line) reduced tumor growth 46 days after injection of tumor cells
to a greater degree than treatment with either 5-FU (triangles,
solid line) or 21M18 antibodies (diamonds, dotted line) alone and
to a greater degree than PBS injected controls (squares, solid
line). Tumor volume in mm.sup.3 is indicated on the y-axis. B)
Plots of tumor measurements on day 46 from individual animals. Each
dot represents one animal. Treatment with 21M18 antibodies or 5-FU
each reduced tumor size (mm.sup.3) compared to control.
Furthermore, combination treatment with 21M18 antibodies and 5-FU
had an additive effect, reducing tumor size to 1/5 the size of
control.
[0030] FIG. 11: Treatment with DLL4 Antibodies in Combination with
anti-EGFR Antibodies Reduces Tumor Growth. NOD/SCID mice were
injected with dissociated UM-C4 cells and treated with anti-DLL4
antibodies or PBS in the presence or absence of anti-EGFR
antibodies. Plots of tumor measurements on day 46 from individual
animals are shown. Each dot represents one animal. Treatment with
21M18 antibodies or anti-EGFR antibodies each reduced tumor size
(mm.sup.3) compared to control. Furthermore, combination treatment
with 21M18 and anti-EGFR antibodies had an additive effect,
reducing tumor size to less than 1/5 the size of control.
[0031] FIG. 12: Anti-DLL4 mAb 21M18 and Irinotecan Act
Synergistically to Inhibit Colon Tumor Growth. NOD/SCID mice were
injected with dissociated C8 cells and treated with anti-DLL4
antibodies or control antibody in the presence or absence of
Irinotecan. A) Treatment with murine 21M18 antibodies (circles) or
Irinotecan (triangles) alone each reduced tumor volume (y-axis
mm.sup.3) compared to control treated animals (black squares).
However, combination treatment with 21M18 and Irinotecan (inverse
triangles) had a synergistic effect, completely eliminating tumor
growth for up to 55 days post-cell injection. B) Treatment with
humanized 21M18 (h21M18) in combination with Irinotecan (irtcn)
(circles) has similar efficacy as murine 21M18 (m21M18) (triangles)
as compared to control antibody (black squares) or control antibody
with Irinotecan (triangles).
[0032] FIG. 13: Combination Anti-DLL4 21M18 and Irinotecan
Treatment Prevents Colon Tumor Re-Growth. NOD/SCID mice were
injected with dissociated C8 cells and treated with Irinotecan or
Irinotecan in combination with anti-DLL4 21M18 antibodies (n=10 per
group). A) Treatment with Irinotecan alone slowed colon tumor
growth, but growth continued after cessation of treatment on day 56
(* arrow) in all but two treated animals. B) In contrast, treatment
with a combination of Irinotecan and anti-DLL4 21M18 antibodies
eliminated colon tumor growth both during treatment and for up to
five weeks following cessation of treatment on day 56 in all ten
treated animals. Each line represents the growth curve for an
individual animal.
[0033] FIG. 14: Combination Anti-DLL4 21M18 and Irinotecan
Treatment Inhibits the Growth of Established Colon Tumors More
Effectively than Single Therapy Treatment. NOD/SCID mice were
injected with dissociated C8 cells and treated with anti-DLL4
antibodies or control antibody in the presence or absence of
Irinotecan. Treatment with 21M18 antibodies (diamonds) or
Irinotecan (triangles) alone each reduced tumor volume (y-axis
mm.sup.3) compared to control treated animals (black squares).
However, combination treatment with 21M18 plus Irinotecan (inverse
triangles) inhibited tumor growth more effectively than either
21M18 or Irinotecan treatment alone.
[0034] FIG. 15: Tumors Treated with Anti-DLL4 Antibodies Show
Decreased Numbers of Tumorigenic Cells. Immunocompromised mice
(n=10 per group) were injected with decreasing dosages of tumor
cells from the experiment shown in FIG. 14 that had been treated
with either control antibody, Irinotecan plus control antibody,
DLL4 21M18 antibodies alone, or a combination of DLL4 21M18
antibodies and Irinotecan (Combination). A) Results of tumor take
rates on day 81. Tumor volume (mm.sup.3) was graphed compared to
the number of human tumor cells injected: 900, 300, 100, and 50 for
each treatment group. The number of animals with detectable tumors
over the ten injected animals for each tumor cell dose is recorded
below the graph of tumor volume for each cell dose with control
treated tumor cells on the left (filled circles), anti-DLL4 21M18
antibody treated tumor cells second to the left (open squares),
Irinotecan treated tumor cells second to the right (filled
triangles), and Combination treated tumor cells on the right (open
circles). B) The stem cell frequency on day 81 was calculated. The
proportion of cancer stem cells (y-axis) from control treated
(left) compared to anti-DLL4 treated (second from left), Irinotecan
only treated (second from right), and Combination treated (right)
tumor cells is plotted with the 95% confidence interval. The
anti-DLL4 treated group has a statistically significant difference
versus the control group (*) and the combination group is
significantly different versus both the control (*) and Irinotecan
alone groups (**).
[0035] FIG. 16: Anti-DLL4 21M18 and Irinotecan Combination
Treatment Delays Tumor Recurrence. Immunocompromised mice were
injected with dissociated C8 cells and established tumors of
approximately 150 mm.sup.3 were treated with a combination of
Irinotecan (45 mg/kg, dosed twice a week) with either anti-DLL4
21M18 antibodies or control antibodies for 32 days after which
Irinotecan treatment was halted. Treatment with the either the
control antibody or 21M18 continued. Reoccurrence of tumors by
tumor volume (y-axis) was delayed in 21M18 treated animals
(triangles) as compared to controls (circles).
[0036] FIG. 17: Anti-DLL4 21M18 and Irinotecan Combination
Treatment Delays Tumor Recurrence. The individual animals from the
experiment shown in FIG. 16 are shown. The total tumor volume
(y-axis) of each animal is shown 47 days after termination of
Irinotecan treatment.
[0037] FIG. 18. Anti-DLL4 21M18 and anti-VEGF combination reduces
tumor growth. C17 tumor cells were implanted and treatment was
initiated two day later with either control antibody (black
squares, solid line), 21M18 (triangles, dashed line), anti-VEGF
(diamonds, solid line), or the combination of both antibodies
(circles, dotted line). Each anti-body was dosed at 10 mg/kg, given
twice a week and there were 10 animals per group. Both 21M18 and
anti-VEGF reduced tumor growth and the combination was more
effective than either single antibody.
DESCRIPTION OF THE EMBODIMENTS
[0038] 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 recognition 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.
[0039] 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.
[0040] 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 complementarity 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.
[0041] 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.
[0042] 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 complementarity determining regions (CDRs) within the
antigen determination region (or hypervariable region) of the
variable region of an antibody chain or chains are replaced by
residues from the CDR of a non-human species (e.g. mouse, rat,
rabbit, hamster) that have the desired specificity, affinity, and
capability. In some instances, residues from the variable chain
framework region (FR) of a human immunoglobulin are replaced with
the corresponding residues in an antibody from a non-human species
that has the desired specificity, affinity, and capability. The
humanized antibody can be further modified by the substitution of
additional residue either in the variable framework region and/or
within the replaced non-human residues to refine and optimize
antibody specificity, affinity, and/or capability. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two or three or four, variable domains containing all
or substantially all of the CDR regions that correspond to the
non-human immunoglobulin whereas all or substantially all of the FR
regions are those of a human immunoglobulin consensus sequence. The
humanized antibody can also comprise at least a portion of an
immunoglobulin constant region or domain (Fc), typically that of a
human immunoglobulin. Examples of methods used to generate
humanized antibodies are described in U.S. Pat. No. 5,225,539.
[0043] 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.
[0044] "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.
[0045] 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.
[0046] 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.
[0047] Competition between antibodies is determined by an assay in
which the immunoglobulin under test inhibits specific binding of a
reference antibody to a common antigen. Numerous types of
competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay (EIA), sandwich competition assay (see
Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid phase
direct biotin-avidin EIA (see Kirkland et al., J. Immunol.
137:3614-3619 (1986)); solid phase direct labeled assay, solid
phase direct labeled sandwich assay (see Harlow and Lane,
"Antibodies, A Laboratory Manual," Cold Spring Harbor Press
(1988)); solid phase direct label RIA using 1-125 label (see Morel
et al., Molec. Immunol. 25(1):7-15 (1988)); solid phase direct
biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and
direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82
(1990)). Typically, such an assay involves the use of purified
antigen bound to a solid surface or cells bearing either of these,
an unlabeled test immunoglobulin and a labeled reference
immunoglobulin. Competitive inhibition is measured by determining
the amount of label bound to the solid surface or cells in the
presence of the test immunoglobulin. Usually the test
immunoglobulin is present in excess. Antibodies identified by
competition assay (competing antibodies) include antibodies binding
to the same epitope as the reference antibody and antibodies
binding to an adjacent epitope sufficiently proximal to the epitope
bound by the reference antibody for steric hindrance to occur.
Usually, when a competing antibody is present in excess, it will
inhibit specific binding of a reference antibody to a common
antigen by at least 50 or 75%.
[0048] That an antibody "selectively binds" or "specifically binds"
means that the antibody reacts or associates more frequently, more
rapidly, with greater duration, with greater affinity, or with some
combination of the above to an epitope than with alternative
substances, including unrelated proteins. "Selectively binds" or
"specifically binds" means, for instance, that an antibody binds to
a protein with a K.sub.D of at least about 0.1 mM, but more usually
at least about 1 .mu.M. "Selectively binds" or "specifically binds"
means at times that an antibody binds to a protein at times with a
K.sub.D of at least about 0.1 .mu.M or better, and at other times
at least about 0.01 .mu.M or better. Because of the sequence
identity between homologous proteins in different species, specific
binding can include an antibody that recognizes a cancer stem cell
marker protein in more than one species.
[0049] 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).
[0050] 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.
[0051] 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.
[0052] The terms "proliferative disorder" and "proliferative
disease" refer to disorders associated with abnormal cell
proliferation such as cancer.
[0053] "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.
[0054] "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.
[0055] 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.
[0056] 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).
[0057] 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.
[0058] 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).
[0059] 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.
[0060] 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.
[0061] "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.
[0062] "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.
[0063] "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.
[0064] "Pharmaceutically acceptable vehicle" refers to a diluent,
adjuvant, excipient, or carrier with which at least one antibody of
the present disclosure is administered.
[0065] "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.
[0066] The term "therapeutically effective amount" refers to an
amount of an antibody, polypeptide, polynucleotide, small organic
molecule, or other drug effective to "treat" a disease or disorder
in a subject or mammal. In the case of cancer, the therapeutically
effective amount of the drug can reduce the number of cancer cells;
reduce the tumor size; inhibit or stop cancer cell infiltration
into peripheral organs including, for example, the spread of cancer
into soft tissue and bone; inhibit and stop tumor metastasis;
inhibit and stop tumor growth; relieve to some extent one or more
of the symptoms associated with the cancer, reduce morbidity and
mortality; improve quality of life; or a combination of such
effects. To the extent the drug prevents growth and/or kills
existing cancer cells, it can be referred to as cytostatic and/or
cytotoxic.
[0067] As used herein, "providing a diagnosis" or "diagnostic
information" refers to any information, including for example the
presence of cancer stem cells, that is useful in determining
whether a patient has a disease or condition and/or in classifying
the disease or condition into a phenotypic category or any category
having significance with regards to the prognosis of or likely
response to treatment (either treatment in general or any
particular treatment) of the disease or condition. Similarly,
diagnosis refers to providing any type of diagnostic information,
including, but not limited to, whether a subject is likely to have
a condition (such as a tumor), whether a subject's tumor comprises
cancer stem cells, information related to the nature or
classification of a tumor as for example a high risk tumor or a low
risk tumor, information related to prognosis and/or information
useful in selecting an appropriate treatment. Selection of
treatment can include the choice of a particular chemotherapeutic
agent or other treatment modality such as surgery or radiation or a
choice about whether to withhold or deliver therapy.
[0068] As used herein, the terms "providing a prognosis",
"prognostic information", or "predictive information" refer to
providing information, including for example the presence of cancer
stem cells in a subject's tumor, regarding the impact of the
presence of cancer (e.g., as determined by the diagnostic methods
of the present invention) on a subject's future health (e.g.,
expected morbidity or mortality, the likelihood of getting cancer,
and the risk of metastasis).
[0069] Terms such as "treating" or "treatment" or "to treat" or
"alleviating" or "to alleviate" refer to both 1) therapeutic
measures that cure, slow down, lessen symptoms of, and/or halt
progression of a diagnosed pathologic condition or disorder and 2)
prophylactic or preventative measures that prevent and/or slow the
development of a targeted pathologic condition or disorder. Thus
those in need of treatment include those already with the disorder;
those prone to have the disorder; and those in whom the disorder is
to be prevented. A subject is successfully "treated" according to
the methods of the present invention if the patient shows one or
more of the following: a reduction in the number of or complete
absence of cancer cells; a reduction in the tumor size; inhibition
of or an absence of cancer cell infiltration into peripheral organs
including, for example, the spread of cancer into soft tissue and
bone; inhibition of or an absence of tumor metastasis; inhibition
or an absence of tumor growth; relief of one or more symptoms
associated with the specific cancer; reduced morbidity and
mortality; improvement in quality of life; or some combination of
effects.
[0070] As used herein, the terms "polynucleotide" or "nucleic acid"
refer to a polymer composed of a multiplicity of nucleotide units
(ribonucleotide or deoxyribonucleotide or related structural
variants) linked via phosphodiester bonds, including but not
limited to, DNA or RNA. The term encompasses sequences that include
any of the known base analogs of DNA and RNA. including, but not
limited to, 4 acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinylcytosine, pseudoisocytosine, 5 (carboxyhydroxylmethyl)
uracil, 5-fluorouracil, 5 bromouracil, 5-carboxymethylaminomethyl 2
thiouracil, 5 carboxymethylaminomethyluracil, dihydrouracil,
inosine, N6 isopentenyladenine, 1 methyladenine,
1-methylpseudouracil, 1 methylguanine, 1 methylinosine,
2,2-dimethylguanine, 2 methyladenine, 2 methylguanine,
3-methylcytosine, 5 methylcytosine, N6 methyladenine, 7
methylguanine, 5 methylaminomethyluracil, 5-methoxyaminomethyl 2
thiouracil, beta D mannosylqueosine, 5'
methoxycarbonylmethyluracil, 5 methoxyuracil, 2 methylthio N6
isopentenyladenine, uracil 5 oxyacetic acid methylester, uracil 5
oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2
thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil, 4 thiouracil,
5-methyluracil, N-uracil 5 oxyacetic acid methylester, uracil 5
oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6
diaminopurine.
[0071] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (Tm) for the specific sequence at a defined
ionic strength pH. The Tm is the temperature (under defined ionic
strength, pH, and nucleic concentration) at which 50% of the probes
complementary to the target hybridize to the target sequence at
equilibrium (as the target sequences are present in excess, at Tm,
50% of the probes are occupied at equilibrium). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. For selective or specific hybridization,
a positive signal is at least two times background, preferably 10
times background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or, 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C.
[0072] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA).
The polypeptide can be encoded by a full length coding sequence or
by any portion of the coding sequence so long as the desired
activity or functional properties (e.g., enzymatic activity, ligand
binding, signal transduction, immunogenicity, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends for a
distance of about 1 kb or more on either end such that the gene
corresponds to the length of the full-length mRNA. Sequences
located 5' of the coding region and present on the mRNA are
referred to as 5' non-translated sequences. Sequences located 3' or
downstream of the coding region and present on the mRNA are
referred to as 3' 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.
[0073] The term "recombinant" when used with reference to a cell,
nucleic acid, protein or vector indicates that the cell, nucleic
acid, protein or vector has been modified by the introduction of a
heterologous nucleic acid or protein, the alteration of a native
nucleic acid or protein, or that the cell is derived from a cell so
modified. Thus, e.g., recombinant cells express genes that are not
found within the native (non-recombinant) form of the cell or
express native genes that are overexpressed or otherwise abnormally
expressed such as, for example, expressed as non-naturally
occurring fragments or splice variants. By the term "recombinant
nucleic acid" herein is meant nucleic acid, originally formed in
vitro, in general, by the manipulation of nucleic acid, e.g., using
polymerases and endonucleases, in a form not normally found in
nature. In this manner, operably linkage of different sequences is
achieved. Thus an isolated nucleic acid, in a linear form, or an
expression vector formed in vitro by ligating DNA molecules that
are not normally joined, are both considered recombinant for the
purposes of this invention. It is understood that once a
recombinant nucleic acid is made and introduced into a host cell or
organism, it will replicate non-recombinantly, i.e., using the in
vivo cellular machinery of the host cell rather than in vitro
manipulations; however, such nucleic acids, once produced
recombinantly, although subsequently replicated non-recombinantly,
are still considered recombinant for the purposes of the invention.
Similarly, a "recombinant protein" is a protein made using
recombinant techniques, i.e., through the expression of a
recombinant nucleic acid as depicted above.
[0074] As used herein, the term "heterologous gene" refers to a
gene that is not in its natural environment. For example, a
heterologous gene includes a gene from one species introduced into
another species. A heterologous gene also includes a gene native to
an organism that has been altered in some way (e.g., mutated, added
in multiple copies, linked to non-native regulatory sequences,
etc). Heterologous genes are distinguished from endogenous genes in
that the heterologous gene sequences are typically joined to DNA
sequences that are not found naturally associated with the gene
sequences in the chromosome or are associated with portions of the
chromosome not found in nature (e.g., genes expressed in loci where
the gene is not normally expressed).
[0075] As used herein, the term "vector" is used in reference to
nucleic acid molecules that transfer DNA segment(s) from one cell
to another. The term "vehicle" is sometimes used interchangeably
with "vector." Vectors are often derived from plasmids,
bacteriophages, or plant or animal viruses.
[0076] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments. Unless
otherwise provided, ligation can be accomplished using known
buffers and conditions with 10 units to T4 DNA ligase ("ligase")
per 0.5 ug of approximately equimolar amounts of the DNA fragments
to be ligated. Ligation of nucleic acid can serve to link two
proteins together in-frame to produce a single protein, or fusion
protein.
[0077] As used herein, the term "gene expression" refers to the
process of converting genetic information encoded in a gene into
RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of
the gene (e.g., via the enzymatic action of an RNA polymerase), and
for protein encoding genes, into protein through "translation" of
mRNA. Gene expression can be regulated at many stages in the
process. "Up-regulation" or "activation" refers to regulation that
increases the production of gene expression products (e.g., RNA or
protein), while "down-regulation" or "repression" refers to
regulation that decrease production. Molecules (e.g., transcription
factors) that are involved in up-regulation or down-regulation are
often called "activators" and "repressors," respectively.
[0078] The terms "polypeptide," "peptide," "protein," and "protein
fragment" are used interchangeably herein to refer to a polymer of
amino acid residues. The terms apply to amino acid polymers in
which one or more amino acid residue is an artificial chemical
mimetic of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers and non-naturally
occurring amino acid polymers.
[0079] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
Amino acid analogs refers to compounds that have the same basic
chemical structure as a naturally occurring amino acid, e.g., an
alpha carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide, methionine methyl sulfonium. Such analogs can
have modified R groups (e.g., norleucine) or modified peptide
backbones, but retain the same basic chemical structure as a
naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions
similarly to a naturally occurring amino acid.
[0080] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. "Amino acid variants" refers to
amino acid sequences. With respect to particular nucleic acid
sequences, conservatively modified variants refers to those nucleic
acids which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical or associated (e.g., naturally
contiguous) sequences. Because of the degeneracy of the genetic
code, a large number of functionally identical nucleic acids encode
most proteins. For instance, the codons GCA, GCC, GCG and GCU all
encode the amino acid alanine. Thus, at every position where an
alanine is specified by a codon, the codon can be altered to
another of the corresponding codons described without altering the
encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified
variations. Every nucleic acid sequence herein which encodes a
polypeptide also describes silent variations of the nucleic acid.
One of skill will recognize that in certain contexts each codon in
a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, silent variations of a nucleic acid which
encodes a polypeptide is implicit in a described sequence with
respect to the expression product, but not with respect to actual
probe sequences. As to amino acid sequences, one of skill will
recognize that individual substitutions, deletions or additions to
a nucleic acid, peptide, polypeptide, or protein sequence which
alters, adds or deletes a single amino acid or a small percentage
of amino acids in the encoded sequence is a "conservatively
modified variant" including where the alteration results in the
substitution of an amino acid with a chemically similar amino acid.
Conservative substitution tables providing functionally similar
amino acids are well known in the art. Such conservatively modified
variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles of the invention.
Typically conservative substitutions include: 1) Alanine (A),
Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine
(N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0081] The 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.
[0082] The present invention provides compositions and methods for
studying, diagnosing, characterizing, and treating cancer. In
particular, the present invention provides antibodies against solid
tumor stem cell markers and methods of using these antibodies to
inhibit tumor growth and treat cancer in human patients. In certain
embodiments, antibodies of the present invention include antagonist
antibodies that specifically bind to a cancer stem cell marker
protein and interfere with, for example, ligand binding, receptor
dimerization, expression of a cancer stem cell marker protein,
and/or signaling of a cancer stem cell marker protein. In certain
embodiments, disclosed antibodies include agonist antibodies that
specifically bind to a cancer stem cell marker protein and promote,
for example, ligand binding, receptor dimerization, and/or
signaling by a cancer stem cell marker protein. In certain
embodiments, disclosed antibodies do not interfere with or promote
the biological activity of a cancer stem cell marker protein but
inhibit tumor growth by, for example, internalization and/or
recognition by the immune system. In certain embodiments, the
antibodies specifically recognize more than one solid tumor tem
cells marker protein.
[0083] Provided is an isolated antibody that specifically binds to
a human DLL4 epitope formed by a combination of the human DLL4
N-terminal region (SEQ ID NO: 27) and human DSL (SEQ ID NO: 26),
wherein the antibody affects growth of a tumor. 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. Further provided is a
pharmaceutical composition comprising an antibody of the present
disclosure and a pharmaceutically acceptable vehicle.
[0084] Further provided is a method of treating cancer comprising
administering an therapeutically effective amount of an antibody or
a pharmaceutical composition of the present disclosure. 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.
[0085] Like the tissues in which they originate, solid tumors
consist of a heterogeneous population of cells. That the majority
of these cells lack tumorigenicity suggested that the development
and maintenance of solid tumors also relies on a small population
of stem cells (i.e., tumorigenic cancer cells) with the capacity to
proliferate and efficiently give rise both to additional tumor stem
cells (self-renewal) and to the majority of more differentiated
tumor cells that lack tumorigenic potential (i.e., non-tumorigenic
cancer cells). The concept of cancer stem cells was first
introduced soon after the discovery of hematopoietic stem cells
(HSC) and was established experimentally in acute myelogenous
leukemia (AML) (Park et al., 1971, J. Natl. Cancer Inst. 46:411-22;
Lapidot et al., 1994, Nature 367:645-8; Bonnet & Dick, 1997,
Nat. Med. 3:730-7; Hope et al., 2004, Nat. Immunol. 5:738-43). Stem
cells from solid tumors have more recently been isolated based on
their expression of a unique pattern of cell-surface receptors and
on the assessment of their properties of self-renewal and
proliferation in culture and in xenograft animal models. An
ESA+CD44+CD24-/low Lineage-population greater than 50-fold enriched
for the ability to form tumors relative to unfractionated tumor
cells was discovered (Al-Hajj et al., 2003, Proc. Nat'l. Acad. Sci.
100:3983-8). The ability to isolate tumorigenic cancer stem cells
from the bulk of non-tumorigenic tumor cells has led to the
identification of cancer stem cell markers, genes with differential
expression in cancer stem cells compared to non-tumorigenic tumor
cells or normal breast epithelium, using microarray analysis. The
present invention employs the knowledge of these identified cancer
stem cell markers to diagnosis and treat cancer.
[0086] The cancer stem cell markers of the present invention relate
to human DLL4, a Notch receptor ligand. The Notch signaling pathway
is one of several critical regulators of embryonic pattern
formation, post-embryonic tissue maintenance, and stem cell
biology. More specifically, Notch signaling is involved in the
process of lateral inhibition between adjacent cell fates and plays
an important role in cell fate determination during asymmetric cell
divisions. Unregulated Notch signaling 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 (Brennan and Brown, 2003, Breast Cancer Res. 5:69). Thus
carcinogenesis can proceed by usurping homeostatic mechanisms
controlling normal development and tissue repair by stem cell
populations (Beachy et al., 2004, Nature 432:324).
[0087] The Notch receptor was first identified in Drosophila
mutants. Haploinsufficiency of Drosophila Notch results in notches
at the wing margin whereas loss-of-function produces an embryonic
lethal "neurogenic" phenotype where cells of the epidermis switch
fate to neural tissue (Moohr, 1919, Genet. 4:252; Poulson, 1937,
PNAS 23:133; Poulson, 1940, J. Exp. Zool. 83:271). The Notch
receptor is a single-pass transmembrane receptor containing
numerous tandem epidermal growth factor (EGF)-like repeats and
cysteine-rich Notch/LIN-12 repeats within a large extracellular
domain (Wharton et al., 1985, Cell 43:567; Kidd et al., 1986, Mol.
Cell. Biol. 6:3094; reviewed in Artavanis et al., 1999, Science
284:770). Four mammalian Notch proteins have been identified
(NOTCH1, NOTCH2, NOTCH3, and NOTCH4), and mutations in these
receptors invariably result in developmental abnormalities and
human pathologies including several cancers as described in detail
below (Gridley, 1997, Mol. Cell. Neurosci. 9:103; Joutel &
Tournier-Lasserve, 1998, Semin. Cell Dev. Biol. 9:619-25).
[0088] The Notch receptor is activated by single-pass transmembrane
ligands of the Delta, Serrated, Lag-2 (DSL) family. The known Notch
ligands in mammals, Delta-like 1 (Dll1), Delta-like 3 (Dll3),
Delta-like 4 (Dll4), Jagged 1 and Jagged 2, are characterized by a
DSL domain and tandem EGF-like repeats within the extracellular
domain. The extracellular domain of the Notch receptor interacts
with that of its ligands, typically on adjacent cells, resulting in
two proteolytic cleaveages of Notch, an extracellular cleavage
mediated by an ADAM protease and a cleavage within the
transmembrane domain mediated by gamma secretase. This latter
cleavage generates the Notch intracellular domain (NICD). The NICD
then enters the nucleus where it activates the CBF1, Suppressor of
Hairless [Su(H)], Lag-2 (CSL) family of transcription factors as
the major downstream effectors to increase transcription of nuclear
basic helix-loop-helix transcription factors of the Hairy and
Enhancer of Split [E(spl)] family (Artavanis et al., 1999, Science
284:770; Brennan and Brown, 2003, Breast Cancer Res. 5:69; Iso et
al., 2003, Arterioscler. Thromb. Vasc. Biol. 23:543). Alternative
intracellular pathways involving the cytoplasmic protein Deltex
identified in Drosophila may also exist in mammals (Martinez et
al., 2002, Curr. Opin. Genet. Dev. 12:524-33), and this
Deltex-dependent pathway may act to suppress expression of Wnt
target genes (Brennan et al., 1999, Curr. Biol. 9:707-710; Lawrence
et al., 2001, Curr. Biol. 11:375-85).
[0089] Hematopoietic stem cells (HSCs) are the best understood stem
cells in the body, and Notch signaling is implicated both in their
normal maintenance as well as in leukemic transformation (Kopper
& Hajdu, 2004, Pathol. Oncol. Res. 10:69-73). 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. Constitutive activation of Notch1 signaling
in HSCs and progenitor cells establishes immortalized cell lines
that generate both lymphoid and myeloid cells in vitro and in
long-term reconstitution assays (Varnum-Finney et al., 2000, Nat.
Med. 6:1278-81), and the presence of Jagged 1 increases engraftment
of human bone marrow cell populations enriched for HSCs (Karanu et
al., 2000, J. Exp. Med. 192:1365-72). More recently, Notch
signaling has been demonstrated in HSCs in vivo and shown to be
involved in inhibiting HSC differentiation. Furthermore, Notch
signaling appears to be required for Wnt-mediated HSC self-renewal
(Duncan et al., 2005, Nat. Immunol. 6:314).
[0090] The Notch signaling pathway also plays a central role in the
maintenance of neural stem cells and is implicated both in their
normal maintenance as well as in brain cancers (Kopper & Hajdu,
2004, Pathol. Oncol. Res. 10:69-73; Purow et al., 2005, Cancer Res.
65:2353-63; Hallahan et al., 2004, Cancer Res. 64:7794-800). Neural
stem cells give rise to all neuronal and glial cells in the
mammalian nervous system during development, and more recently have
been identified in the adult brain (Gage, 2000, Science
287:1433-8). Mice deficient for Notch1; the Notch target genes
Hes1, 3, and 5; and a regulator of Notch signaling presenilinl
(PS1) show decreased numbers of embryonic neural stem cells.
Furthermore, adult neural stem cells are reduced in the brains of
PS 1 heterozygote mice (Nakamura et al., 2000, J. Neurosci.
20:283-93; Hitoshi et al., 2002, Genes Dev. 16:846-58). The
reduction in neural stem cells appears to result from their
premature differentiation into neurons (Hatakeyama et al., 2004,
Dev. 131:5539-50) suggesting that Notch signaling regulates neural
stem cell differentiation and self-renewal.
[0091] Aberrant Notch signaling is implicated in a number of human
cancers. The NOTCH1 gene in humans was first identified in a subset
of T-cell acute lymphoblastic leukemias as a translocated locus
resulting in activation of the Notch pathway (Ellisen et al., 1991,
Cell 66:649-61). Constitutive activation of Notch1 signaling in
T-cells in mouse models similarly generates T-cell lymphomas
suggesting a causative role (Robey et al., 1996, Cell 87:483-92;
Pear et al., 1996, J. Exp. Med. 183:2283-91; Yan et al., 2001,
Blood 98:3793-9; Bellavia et al., 2000, EMBO J. 19:3337-48).
Recently NOTCH1 point mutations, insertions, and deletions
producing aberrant NOTCH1 signaling have been found to be
frequently present in both childhood and adult T-cell acute
lymphoblastic leukemia/lymphoma (Pear & Aster, 2004, Curr.
Opin. Hematol. 11:416-33).
[0092] The frequent insertion of the mouse mammary tumor virus into
both the Notch1 and Notch4 locus in mammary tumors and the
resulting activated Notch protein fragments first implicated Notch
signaling in breast cancer (Gallahan & Callahan, 1987, J.
Virol. 61:66-74; Brennan & Brown, 2003, Breast Cancer Res.
5:69; Politi et al., 2004, Semin. Cancer Biol. 14:341-7). Further
studies in transgenic mice have confirmed a role for Notch in
ductal branching during normal mammary gland development, and a
constitutively active form of Notch4 in mammary epithelial cells
inhibits epithelial differentiation and results in tumorigenesis
(Jhappan et al., 1992, Genes & Dev. 6:345-5; Gallahan et al.,
1996, Cancer Res. 56:1775-85; Smith et al., 1995, Cell Growth
Differ. 6:563-77; Soriano et al., 2000, Int. J. Cancer 86:652-9;
Uyttendaele et al., 1998, Dev. Biol. 196:204-17; Politi et al.,
2004, Semin. Cancer Biol. 14:341-7). Currently the evidence for a
role for Notch in human breast cancer is limited to the expression
of Notch receptors in breast carcinomas and their correlation with
clinical outcome (Weijzen et al., 2002, Nat. Med. 8:979-86; Parr et
al., 2004, Int. J. Mol. Med. 14:779-86). Furthermore,
overexpression of the Notch pathway has been observed in cervical
cancers (Zagouras et al., 1995, PNAS 92:6414-8), renal cell
carcinomas (Rae et al., 2000, Int. J. Cancer 88:726-32), head and
neck squamous cell carcinomas (Leethanakul et al., 2000, Oncogene
19:3220-4), endometrial cancers (Suzuki et al., 2000, Int. J.
Oncol. 17:1131-9), and neuroblastomas (van Limpt et al., 2000, Med.
Pediatr. Oncol. 35:554-8) indicative of a potential role for Notch
in the development of a number of neoplasms. Interestingly, Notch
signaling might play a role in the maintenance of the
undifferentiated state of Apc-mutant neoplastic cells of the colon
(van Es & Clevers, 2005, Trends Mol. Med. 11:496-502).
[0093] The Notch pathway is also involved in multiple aspects of
vascular development including proliferation, migration, smooth
muscle differentiation, angiogenesis and arterial-venous
differentiation (Iso et al., 2003, Arterioscler. Thromb. Vasc.
Biol. 23:543). For example, homozygous null mutations in Notch-1/4
and Jagged-1 as well as heterozygous loss of Dll4 result in severe
though variable defects in arterial development and yolk sac
vascularization. Furthermore, Dll1-deficient and
Notch-2-hypomorphic mice embryos show hemorrhage that likely
results from poor development of vascular structures (Gale et al.,
2004, PNAS, 101:15949-54; Krebs et al., 2000, Genes Dev.
14:1343-52; Xue et al., 1999, Hum. MeI Genet. 8:723-30; Hrabe de
Angelis et al., 1997, Nature 386:717-21; McCright et al., 2001,
Dev. 128:491-502). In humans, mutations in JAGGED1 are associated
with Alagille syndrome, a developmental disorder that includes
vascular defects, and mutations in NOTCH3 are responsible for an
inherited vascular dementia (CADASIL) in which vessel homeostasis
is defective (Joutel et al., 1996, Nature 383:707-10).
[0094] The identification of DLL4 as expressed in cancer stem cells
compared to normal breast epithelium suggested targeting the Notch
pathway to eliminate not only the majority of non-tumorigenic
cancer cells, but also the tumorigenic cells responsible for the
formation and reoccurrence of solid tumors. Furthermore, because of
the prominent role of angiogenesis in tumor formation and
maintenance, targeting the Notch pathway via antibodies against
DLL4 can also effectively inhibit angiogenesis, starving a cancer
of nutrients and contributing to its elimination.
[0095] Thus, present invention provides a cancer stem cell marker,
the expression of which can be analyzed to diagnosis or monitor a
disease associated with cancer. 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, for 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.
[0096] 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).
[0097] 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.
[0098] 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 the Notch receptor ligand, DLL4.
[0099] 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.
[0100] 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.
[0101] 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, promote differentiation
of tumor cells into a less tumorigenic cell type, 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.
[0102] 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).
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] The antibodies of the invention can be prepared by any
conventional means known in the art. For example, polyclonal
antibodies can be prepared by immunizing an animal (e.g. a rabbit,
rat, mouse, donkey, etc) by multiple subcutaneous or
intraperitoneal injections of the relevant antigen (a purified
peptide fragment, full-length recombinant protein, fusion protein,
etc) optionally conjugated to keyhole limpet hemocyanin (KLH),
serum albumin, etc. diluted in sterile saline and combined with an
adjuvant (e.g. Complete or Incomplete Freund's Adjuvant) to form a
stable emulsion. The polyclonal antibody is then recovered from
blood, ascites and the like, of an animal so immunized. Collected
blood is clotted, and the serum decanted, clarified by
centrifugation, and assayed for antibody titer. The polyclonal
antibodies can be purified from serum or ascites according to
standard methods in the art including affinity chromatography,
ion-exchange chromatography, gel electrophoresis, dialysis,
etc.
[0108] 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.
[0109] 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).
[0110] 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.
[0111] 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.
[0112] Humanized antibodies can be produced using various
techniques known in the art. An antibody can be humanized by
substituting the CDRs of a human antibody with that of a non-human
antibody (e.g. mouse, rat, rabbit, hamster, etc.) having the
desired specificity, affinity, and capability following the methods
of (Jones et al., 1986, Nature, 321:522-525; Riechmann et al.,
1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science,
239:1534-1536). The humanized antibody can be further modified by
the substitution of additional residue either in the variable human
framework region and/or within the replaced non-human residues to
refine and optimize antibody specificity, affinity, and/or
capability.
[0113] The choice of human heavy and/or light chain variable
domains to be used in making humanized antibodies can be important
for reducing antigenicity. According to the "best-fit" method, the
sequence of the variable domain of a rodent antibody is screened
against the entire library of known human variable-domain amino
acid sequences. Thus in certain embodiments, the human amino acid
sequence which is most homologous to that of the rodent antibody
from which the CDRs are taken is used as the human framework region
(FR) for the humanized antibody (Sims et al., 1993, J. Immunol.,
151: 2296; Chothia et al., 1987, J. Mol. Biol., 196: 901). Another
method uses a particular FR derived from the consensus sequence of
all human antibodies of a particular subgroup of light or heavy
chains and can be used for several difference humanized antibodies
(Carter et al., 1992, PNAS, 89; 4285; Presta et al., 1993, J.
Immunol., 151: 2623). In certain embodiments, a combination of
methods is used to pick the human variable FR to use in generation
of humanized antibodies.
[0114] It is further understood that antibodies (e.g. rodent) to be
humanized must retain high affinity for the antigen as well as
other favorable biological properties. To achieve this goal,
humanized antibodies can be prepared by a process of analysis of
the parental sequence from the rodent antibody to be humanized and
the various candidate humanizing sequences. Three-dimensional
immunoglobulin models are available and familiar to those skilled
in the art. Computer programs can be used to illustrate and display
probable three-dimensional conformational structures of selected
candidate antibody sequences. Use of such models permits analysis
of the likely role of the residues in the function of the antibody
to be humanized, i.e., the analysis of residues that influence the
ability of the candidate antibody to bind its antigen. In this way,
FR residues can be selected and combined from the parental antibody
to the recipient humanized antibody so that the desired antibody
characteristics are achieved. In general, the residues in the CDRs
of the antigen determination region (or hypervariable region) are
retained from the parental antibody (e.g. the rodent antibody with
the desired antigen binding properties) in the humanized antibody
for antigen binding. In certain embodiments, at least one
additional residue within the variable FR is retained from the
parental antibody in the humanized antibody. In certain
embodiments, up to six additional residues within the variable FR
are retained from the parental antibody in the humanized
antibody.
[0115] Amino acids from the variable regions of the mature heavy
and light chains of immunoglobulins are designated Hx and Lx
respectively, where x is a number designating the position of an
amino acid according to the scheme of Kabat, Sequences of Proteins
of Immunological Interest, U.S. Department of Health and Human
Services, 1987, 1991. Kabat lists many amino acid sequences for
antibodies for each subgroup, and lists the most commonly occurring
amino acid for each residue position in that subgroup to generate a
consensus sequence. Kabat uses a method for assigning a residue
number to each amino acid in a listed sequence, and this method for
assigning residue numbers has become standard in the field. Kabat's
scheme is extendible to other antibodies not included in his
compendium by aligning the antibody in question with one of the
consensus sequences in Kabat by reference to conserved amino acids.
The use of the Kabat numbering system readily identifies amino
acids at equivalent positions in different antibodies. For example,
an amino acid at the L50 position of a human antibody occupies the
equivalent position to an amino acid position L50 of a mouse
antibody. Moreover, any two antibody sequences can be uniquely
aligned, for example to determine percent identity, by using the
Kabat numbering system so that each amino acid in one antibody
sequence is aligned with the amino acid in the other sequence that
has the same Kabat number. After alignment, if a subject antibody
region (e.g., the entire mature variable region of a heavy or light
chain) is being compared with the same region of a reference
antibody, the percentage sequence identity between the subject and
reference antibody regions is the number of positions occupied by
the same amino acid in both the subject and reference antibody
region divided by the total number of aligned positions of the two
regions, with gaps not counted, multiplied by 100 to convert to
percentage.
[0116] Example 1 below describes the production of exemplary
humanized anti-DLL4 antibodies which specifically bind human DLL4,
a cancer stem cell marker of the present disclosure (21M18 H9L2,
ATCC deposit no. PTA-8427 and 21M18 H7L2, ATCC deposit no.
PTA-8425, deposited May 10, 2007 under the terms of the Budapest
Treaty with American Type Culture Collection (ATCC), located at
10801 University Blvd., Manassas, Va. 20110-2209). In certain
embodiments, the humanized antibodies comprise nonhuman antigen
determination regions derived from murine monoclonal antibody
21M18. Specifically, in certain embodiments, one or more of the
heavy chain CDRs from the parental rodent antibody, CDR1 (SEQ ID
NO: 1), CDR2 (SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4, which
vary at Kabat position 52a), and CDR3 (SEQ ID NO: 5) are retained
in the humanized 21M18 antibody. In certain embodiments, one or
more of the light chain CDRs from the parental rodent antibody,
CDR1 (SEQ ID NO: 9), CDR2 (SEQ ID NO: 10), and CDR3 (SEQ ID NO:
11), are retained in the humanized 21M18 antibody. In certain
embodiments, the humanized antibodies further comprise at least one
FR substitution within either the heavy or light chain human
variable region.
[0117] In certain embodiments, the present invention provides a
humanized antibody which specifically binds to a human DLL4 epitope
formed by a combination of the human DLL4 N-terminal region (SEQ ID
NO: 27) and human DSL (SEQ ID NO: 26), wherein the antibody affects
growth of a tumor. In certain embodiments, the humanized antibody
is an intact IgG antibody. In certain embodiments, the humanized
antibody is an intact IgG.sub.2 antibody. In certain embodiments,
the humanized antibody is an antibody fragment. In certain
embodiments, the humanized antibody is a Fab fragment.
[0118] In certain embodiments, the humanized antibody of the
present invention comprises a heavy chain variable (V.sub.H) region
comprising a nonhuman antigen determination region and a human
variable framework region. In certain embodiments, the nonhuman
antigen determination region comprises complementarity
determination regions (CDRs) of rodent origin. In certain
embodiments, the nonhuman antigen determination region comprises
CDRs from a mouse antibody. In certain embodiments, the rodent CDRs
derive from monoclonal antibody 21M18, wherein 21M18 comprises a
heavy chain variable region designated SEQ ID NO: 6. In certain
embodiments, wherein the humanized antibody comprises a V.sub.H
region comprising an amino acid sequence of (a) CDR1 (SEQ ID NO:
1), CDR2 (SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4), and CDR3
(SEQ ID NO: 5) or (b) SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:
8.
[0119] In certain embodiments, the human heavy chain variable
framework region comprises expressed human sequences. In certain
embodiments, at least one residue in the human variable framework
region is substituted. In certain embodiments, at least one residue
in the human heavy chain variable framework region is at a position
selected from the group consisting of 16, 20, 27, 28, 38, and 48
based on the Kabat numbering system. In certain embodiments,
positions 16, 20, 27, 28, 38, and 48 are substituted based on the
Kabat numbering system. In certain embodiments, at least one
residue in the human variable framework region is substituted with
a residue occupying the corresponding position in an antibody
comprising the nonhuman antigen determination region.
[0120] In certain embodiments, the human heavy chain variable
framework region comprises IGH(V)1-18. In certain embodiments, at
least one residue in the human variable framework region is
substituted. In certain embodiments, at least one residue in the
human heavy chain variable framework region is at a position
selected from the group consisting of 20H, 28H, 38H, 48H, and 69H
based on the Kabat numbering system. In certain embodiments,
positions 20H, 28H, 38H, 48H, and 69H are substituted based on the
Kabat numbering system. In certain embodiments, at least one
residue in the human variable framework region is substituted with
a residue occupying the corresponding position in an antibody
comprising the nonhuman antigen determination region.
[0121] In certain embodiments, the humanized antibody of the
present invention comprises a light chain variable (V.sub.L) region
comprising a nonhuman antigen determination region and a human
variable framework region. In certain embodiments, the nonhuman
antigen determination region comprises CDRs of rodent origin. In
certain embodiments, the nonhuman antigen determination region
comprises CDRs from a mouse antibody. In certain embodiments, the
CDRs derive from monoclonal antibody 21M18, wherein 21M18 comprises
a V.sub.L region designated SEQ ID NO: 12. In certain embodiments,
the V.sub.L region comprises an amino acid sequence of (a) CDR1
(SEQ ID NO: 9), CDR2 (SEQ ID NO: 10), and CDR3 (SEQ ID NO: 11) or
(b) SEQ ID NO: 12.
[0122] In certain embodiments, the human light chain variable
framework region comprises IGK(V)4-1. In certain embodiments, at
least one residue in the human light chain variable framework
region is substituted. In certain embodiments, at least one residue
in the human variable framework region is at a position selected
from the group consisting of 22L and 36L based on the Kabat
numbering system. In certain embodiments, positions 22L and 36L are
substituted based on the Kabat numbering system. In certain
embodiments, at least one residue from the human variable framework
region is substituted with a residue occupying the corresponding
position in an antibody comprising the nonhuman antigen
determination region.
[0123] In certain embodiments, the antibody of the present
invention is an antibody that competes with the antibody 21M18 for
specific binding to human DLL4, wherein the 21M18 antibody
comprises: (a) a heavy chain with a variable region designated SEQ
ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and (b) a light chain with
a variable region designated SEQ ID NO: 12. In certain embodiments,
the antibody is a humanized antibody or a human antibody.
[0124] In certain embodiments, the humanized antibody that
specifically binds to a human DLL4 epitope formed by a combination
of the human DLL4 N-terminal region (SEQ ID NO: 27) and human DSL
domain (SEQ ID NO: 26), wherein the antibody comprises a heavy
chain variable region having at least 90% sequence identity to SEQ
ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and a light chain variable
region having at least 90% sequence identity to SEQ ID NO: 12. In
some embodiments, the heavy chain variable region has at least 95%
sequence identity to SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8
and the light chain variable region has at least 95% sequence
identity to SEQ ID NO: 12. In some embodiments, the heavy chain
variable region has at least 99% sequence identity to SEQ ID NO: 6,
SEQ ID NO: 7, or SEQ ID NO: 8 and the light chain variable region
has at least 99% sequence identity to SEQ ID NO: 12.
[0125] In certain embodiments, the present invention provides an
isolated polynucleotide molecule encoding a humanized antibody that
specifically binds to a human DLL4 epitope formed by a combination
of the human DLL4 N-terminal region (SEQ ID NO: 27) and human DSL
(SEQ ID NO: 26), wherein the antibody comprises a V.sub.H region
that comprises a nonhuman antigen determination region encoding
CDR1 (SEQ ID NO: 1); CDR2 (SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID
NO: 4); and CDR3 (SEQ ID NO: 5) and a human variable framework
region encoding IGH(V)1-18. In certain embodiments, the present
invention provides an isolated polynucleotide molecule encoding a
humanized antibody that specifically binds to a human DLL4 epitope
formed by a combination of the human DLL4 N-terminal region (SEQ ID
NO: 27) and human DSL (SEQ ID NO: 26), wherein the polynucleotide
molecule is selected from the group consisting of: (a) a
polynucleotide molecule encoding the amino acid sequence of SEQ ID
NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and (b) a polynucleotide
molecule which hybridizes to the complement of the polynucleotide
molecule according to (a) under stringent hybridization conditions.
In certain embodiments, the present invention provides an isolated
polynucleotide molecule encoding a humanized antibody that
specifically binds to a human DLL4 epitope formed by a combination
of the human DLL4 N-terminal region (SEQ ID NO: 27) and human DSL
(SEQ ID NO: 26), wherein the polynucleotide molecule is selected
from the group consisting of (a) SEQ ID NO: 13, SEQ ID NO: 14, or
SEQ ID NO: 15 and (b) a polynucleotide molecule which hybridizes to
the complement of the polynucleotide molecule according to (a)
under stringent hybridization conditions.
[0126] In certain embodiments, the present invention provides an
isolated polynucleotide molecule encoding a humanized antibody that
specifically binds to a human DLL4 epitope formed by a combination
of the human DLL4 N-terminal region (SEQ ID NO: 27) and human DSL
(SEQ ID NO: 26), wherein the antibody comprises a V.sub.L region
that comprises a nonhuman antigen determination region encoding
CDR1 (SEQ ID NO: 9); CDR2 (SEQ ID NO: 10); and CDR3 (SEQ ID NO: 11)
and a human variable framework region comprising IGK(V)4-1. In
certain embodiments, the present invention provides an isolated
polynucleotide molecule encoding a humanized antibody that
specifically binds to a human DLL4 epitope formed by a combination
of the human DLL4 N-terminal region (SEQ ID NO: 27) and human DSL
(SEQ ID NO: 26), wherein the polynucleotide molecule is selected
from the group consisting of: (a) a polynucleotide molecule
encoding the amino acid sequence of SEQ ID NO: 12 and (b) a
polynucleotide molecule which hybridizes to the complement of the
polynucleotide molecule according to (a) under stringent
hybridization conditions. In certain embodiments, the present
invention provides an isolated polynucleotide molecule encoding a
humanized antibody that specifically binds to a human DLL4 epitope
formed by a combination of the human DLL4 N-terminal region (SEQ ID
NO: 27) and human DSL (SEQ ID NO: 26), wherein the polynucleotide
molecule is selected from the group consisting of (a) SEQ ID NO: 16
and (b) a polynucleotide molecule which hybridizes to the
complement of the polynucleotide molecule according to (a) under
stringent hybridization conditions.
[0127] In certain embodiments is provided an expression vector
comprising an isolated polynucleotide molecule of the present
invention. In certain embodiments is provided a host cell
comprising an expression vector comprising an isolated
polynucleotide molecule of the present invention
[0128] In certain embodiments, the present invention provides a
method of treating cancer in a patient comprising administering to
the patient a therapeutically effective amount of a humanized
antibody of the present disclosure. In certain embodiments, the
cancer comprises breast cancer, colorectal cancer, lung cancer,
pancreatic cancer, prostate cancer, or head and neck cancer.
[0129] In certain embodiments, the present invention provides a kit
comprising a container and a composition contained therein, wherein
the composition comprises a humanized antibody of the present
disclosure, and further comprises a package insert indicating that
the composition can be used to treat cancer.
[0130] In addition, fully human antibodies can be directly prepared
using various techniques known in the art. Immortalized human B
lymphocytes immunized in vitro or isolated from an immunized
individual that produce an antibody directed against a target
antigen can be generated (See, e.g., Cole et al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner
et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No.
5,750,373). Also, the human antibody can be selected from a phage
library, where that phage library expresses human antibodies
(Vaughan et al., 1996, Nat. Biotech., 14:309-314; Sheets et al.,
1998, Proc. Nat'l. Acad. Sci., 95:6157-6162; Hoogenboom and Winter,
1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol.,
222:581). Human antibodies can also be made in transgenic mice
containing human immunoglobulin loci that are capable upon
immunization of producing the full repertoire of human antibodies
in the absence of endogenous immunoglobulin production. This
approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016.
[0131] 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.
[0132] 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))
[0133] 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.
[0134] According to the present invention, techniques can be
adapted for the production of single-chain antibodies specific to a
polypeptide of the invention (see U.S. Pat. No. 4,946,778). In
addition, methods can be adapted for the construction of Fab
expression libraries (Huse, et al., Science 246:1275-1281 (1989))
to allow rapid and effective identification of monoclonal Fab
fragments with the desired specificity for the Notch receptor
ligand DLL4, or derivatives, fragments, or homologs thereof.
Antibody fragments that contain the idiotypes to a polypeptide of
the invention may be produced by techniques in the art including,
but not limited to: (a) an F(ab')2 fragment produced by pepsin
digestion of an antibody molecule; (b) an Fab fragment generated by
reducing the disulfide bridges of an F(ab')2 fragment, (c) an Fab
fragment generated by the treatment of the antibody molecule with
papain and a reducing agent, and (d) Fv fragments.
[0135] 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).
[0136] 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.
[0137] For the purposes of the present invention, it should be
appreciated that modified antibodies can comprise any type of
variable region that provides for the association of the antibody
with the polypeptides of human DLL4. 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.
[0138] 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.
[0139] 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.
[0140] Besides their configuration, it is known in the art that the
constant region mediates several effector functions. For example,
binding of the C1 component of complement to antibodies activates
the complement system. Activation of complement is important in the
opsonisation and lysis of cell pathogens. The activation of
complement also stimulates the inflammatory response and can also
be involved in autoimmune hypersensitivity. Further, antibodies
bind to cells via the Fc region, with a Fc receptor site on the
antibody Fc region binding to a Fc receptor (FcR) on a cell. There
are a number of Fc receptors which are specific for different
classes of antibody, including IgG (gamma receptors), IgE (eta
receptors), IgA (alpha receptors) and IgM (mu receptors). Binding
of antibody to Fc receptors on cell surfaces triggers a number of
important and diverse biological responses including engulfment and
destruction of antibody-coated particles, clearance of immune
complexes, lysis of antibody-coated target cells by killer cells
(called antibody-dependent cell-mediated cytotoxicity, or ADCC),
release of inflammatory mediators, placental transfer and control
of immunoglobulin production. Although various Fc receptors and
receptor sites have been studied to a certain extent, there is
still much which is unknown about their location, structure and
functioning.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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. In some embodiments,
the antibodies can be conjugated to radioisotopes, such as
.sup.90Y, .sup.125I, .sup.131I, .sup.123I, .sup.111In, .sup.105Rh,
.sup.153Sm, .sup.67Cu, .sup.67Ga, .sup.166Ho, .sup.177Lu,
.sup.186Re and .sup.188Re using anyone of a number of well known
chelators or direct labeling. In other embodiments, the disclosed
compositions can comprise antibodies coupled to drugs, prodrugs or
lymphokines such as interferon. Conjugates of the antibody and
cytotoxic agent are made using a variety of bifunctional
protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)
propionate (SPDP), iminothiolane (IT), bifunctional derivatives of
imidoesters (such as dimethyl adipimidate HCL), active esters (such
as disuccinimidyl suberate), aldehydes (such as glutareldehyde),
bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). Conjugates of an antibody and
one or more small molecule toxins, such as a calicheamicin,
maytansinoids, a trichothene, and CC1065, and the derivatives of
these toxins that have toxin activity, can also be used. In some
embodiments, the modified antibodies can be complexed with other
immunologically active ligands (e.g. antibodies or fragments
thereof) wherein the resulting molecule binds to both the
neoplastic cell and an effector cell such as a T cell.
[0146] Regardless of how useful quantities are obtained, the
antibodies of the present invention can be used in any one of a
number of conjugated (i.e. an immunoconjugate) or unconjugated
forms. Alternatively, the antibodies of this invention can be used
in a nonconjugated or "naked" form to harness the subject's natural
defense mechanisms including complement-dependent cytotoxicity
(CDC) and antibody dependent cellular toxicity (ADCC) to eliminate
the malignant cells. The selection of which conjugated or
unconjugated modified antibody to use will depend of the type and
stage of cancer, use of adjunct treatment (e.g., chemotherapy or
external radiation) and patient condition. It will be appreciated
that one skilled in the art could readily make such a selection in
view of the teachings herein.
[0147] 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).
[0148] 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).
[0149] The binding affinity of an antibody to a cancer stem cell
marker antigen and the off-rate of an antibody-antigen interaction
can be determined by competitive binding assays. One example of a
competitive binding assay is a radioimmunoassay comprising the
incubation of labeled antigen (e.g. .sup.3H or .sup.125I), or
fragment or variant thereof, with the antibody of interest in the
presence of increasing amounts of unlabeled antigen followed by the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody against a cancer stem cell marker and the
binding off-rates can be determined from the data by scatchard plot
analysis. In some embodiments, BIAcore kinetic analysis is used to
determine the binding on and off rates of antibodies against a
cancer stem cell marker. BIAcore kinetic analysis comprises
analyzing the binding and dissociation of antibodies from chips
with immobilized cancer stem cell marker antigens on their
surface.
[0150] In certain embodiments, the invention encompasses isolated
polynucleotides that encode a polypeptide comprising an antibody,
or fragment thereof, against human DLL4. 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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
human DLL4.
[0155] 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.
[0156] 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.
[0157] 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).
[0158] The polypeptides of the present invention can be recombinant
polypeptides, natural polypeptides, or synthetic polypeptides
comprising an antibody, or fragment thereof, against human DLL4. 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
human DLL4 protein. Such mutants include deletions, insertions,
inversions, repeats, and type substitutions.
[0159] 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).
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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).
[0167] 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.
[0168] 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.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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).
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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).
[0178] 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.
[0179] 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).
[0180] In certain embodiments of the invention, the treatment
involves the combined administration of an antibody of the present
invention and a second therapeutic agent. As used herein, "a second
therapeutic agent" includes, but is not limited to,
chemotherapeutic agent, radiation therapy, cytokine and antibody
against other tumor associated antigen.
[0181] In other embodiments, the treatment involves the combined
administration of an antibody of the present invention and
radiation therapy. Treatment with the antibody can occur prior to,
concurrently with, or subsequent to administration of radiation
therapy. Any dosing schedules for such radiation therapy can be
used as determined by the skilled practitioner.
[0182] In other embodiments, the treatment can involve the combined
administration of antibodies of the present invention with other
antibodies against additional tumor associated antigens including,
but not limited to, antibodies that bind to the EGF receptor (EGFR)
(Erbitux.RTM.), the erbB2 receptor (HER2) (Herceptin.RTM.), and
vascular endothelial growth factor (VEGF) (Avastin.RTM.).
Furthermore, treatment can include administration of one or more
cytokines, can be accompanied by surgical removal of cancer cells
or any other therapy deemed necessary by a treating physician.
[0183] For the treatment of the disease, the appropriate dosage of
an antibody of the present invention depends on the type of disease
to be treated, the severity and course of the disease, the
responsiveness of the disease, whether the antibody is administered
for therapeutic or preventative purposes, previous therapy,
patient's clinical history, and so on all at the discretion of the
treating physician. The antibody can be administered one time or
over a series of treatments lasting from several days to several
months, or until a cure is effected or a diminution of the disease
state is achieved (e.g. reduction in tumor size). Optimal dosing
schedules can be calculated from measurements of drug accumulation
in the body of the patient and will vary depending on the relative
potency of an individual antibody. The administering physician can
easily determine optimum dosages, dosing methodologies and
repetition rates. In general, dosage is from 0.01 .mu.g to 100 mg
per kg of body weight, and can be given once or more daily, weekly,
monthly or yearly. The treating physician can estimate repetition
rates for dosing based on measured residence times and
concentrations of the drug in bodily fluids or tissues.
[0184] The present invention provides kits comprising the
antibodies described herein and that can be used to perform the
methods described herein. In certain embodiments, a kit comprises
at least one purified antibody against a cancer stem cell marker in
one or more containers. In some embodiments, the kits contain all
of the components necessary and/or sufficient to perform a
detection assay, including all controls, directions for performing
assays, and any necessary software for analysis and presentation of
results. One skilled in the art will readily recognize that the
disclosed antibodies of the present invention can be readily
incorporated into one of the established kit formats which are well
known in the art.
[0185] Embodiments of the present disclosure can be further defined
by reference to the following examples, which describe in detail
preparation of antibodies of the present disclosure and methods for
using antibodies of the present disclosure. It will be apparent to
those skilled in the art that many modifications, both to materials
and methods, may be practiced without departing from the scope of
the present disclosure. Wherever possible, the same reference
numbers will be used throughout the drawings to refer to the same
or like parts. As used herein and in the appended claims, the
singular forms "a," "or," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "an antibody" includes a plurality of such antibodies
or one or more antibodies and equivalents thereof known to those
skilled in the art. Furthermore, all numbers expressing quantities
of ingredients, reaction conditions, purity, polypeptide and
polynucleotide lengths, and so forth, used in the specification,
are modified by the term "about," unless otherwise indicated.
Accordingly, the numerical parameters set forth in the
specification and claims are approximations that may vary depending
upon the desired properties of the present invention.
EXAMPLES
Example 1
Production of Monoclonal and Humanized DLL4 Antibodies
Antigen Production
[0186] A recombinant polypeptide fragment of the extracellular
domain of human DLL4 was generated as an antigen for antibody
production. Standard recombinant DNA technology was used to isolate
a polynucleotide encoding amino acids 1-522 of DLL4 (SEQ ID NO:
25). This polynucleotide was 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 DLL4 polypeptide (O'Reilley et al., Baculovirus
expression vectors: A Laboratory Manual, Oxford: Oxford University
Press (1994)).
[0187] Cleavage of the endogenous signal sequence of human DLL4 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 DLL4 is
between amino acids 1 and 26, thus DLL4 antigen protein comprises
approximately amino acid 27 through amino acid 522. Antigen protein
was purified from insect cell conditioned medium using Protein A
and Ni.sup.++-chelate affinity chromatography. Purified antigen
protein was then dialyzed against PBS (pH=7), concentrated to
approximately 1 mg/ml, and sterile filtered in preparation for
immunization.
Immunization
[0188] Mice (n=3) were immunized with purified DLL4 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.
FACS Analysis
[0189] To select monoclonal antibodies produced by hybridomas
clones that recognize native cell-surface DLL4 protein, FACs
analysis was used. HEK293 cells were co-transfected with expression
vectors encoding a full-length cDNA clone of DLL4 and the
transfection marker GFP. Twenty-four to forty-eight hours
post-transfection, cells were collected in suspension and incubated
on ice with anti-DLL4 antibodies 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-DLL4 antibodies that specifically recognize
cell surface expression of native cell-surface DLL4 protein.
Monoclonal antibodies 21M14 and 21M18 recognized DLL4 on
transfected cells (FIG. 1). Murine antibody 21M18 was deposited on
Sep. 28, 2007 with American Tissue Culture Collection (No.
PTA-8670), located at 10801 University Blvd., Manassas, Va.
20110-2209, under the terms of the Budapest Treaty.
[0190] The ability of antibodies directed towards DLL4 to interfere
with the interaction between DLL4 and Notch was next determined
using flow cytometry. HEK 293 cells stably transduced with DLL4
cDNA were incubated with either Notch1-EGF10-15-Fc or control
protein-Fc in the presence of anti-DLL4 or control antibodies.
Binding of Fc fusion proteins to cells expressing DLL4 was detected
by PE-conjugated goat anti-Fc antibody and flow cytometry. The
ability of anti-DLL4 antibodies inhibit the binding of Notch to
DLL4 was thus determined by a decrease in fluorescence intensity.
As shown in FIG. 2A, inhibition of Notch binding was observed with
murine antibodies 21M14 and 21M18, but not 21M12. Furthermore,
murine 21M18 specifically binds human not murine DLL4, and blocks
binding of human DLL4 but not murine DLL4 binding to cells
expressing Notch1 (FIG. 2B). These data indicate that 21M18 in
xenograft experiments targets human DLL4 expressed on the tumor
cells and not murine DLL4 expressed on the vasculature.
Epitope Mapping
[0191] To identify antibodies that recognize specific regions of
the DLL4 extracellular domain, epitope mapping was performed.
Mammalian expression plasmid vectors comprising a CMV promoter
upstream of polynucleotides that encode a nested series of deletion
fragments of the extracellular domain of DLL4 fused to Fc protein
were generated using standard recombinant DNA technology.
Additional constructs that encoded fragments of DLL4 that were
chimera of human and mouse DLL4 fused to Fc protein were also
generated using standard recombinant DNA technology. A further
series of DLL4-fc fusion proteins were designed that included
specific amino acid substitutions. These recombinant fusion
proteins were expressed in transiently transfected HEK 293 cells
from which conditioned medium was collected twenty-four to
forty-eight hours post-transfection for ELISA. The DLL4 fusion
protein fragments were captured on plates coated with anti-human Fc
antibodies. Anti-DLL4 antibodies were then allowed to interact with
the bound DLL4 fragments and binding was measured by subsequent
incubation with HRP conjugated anti-mouse antibody and detection of
HRP activity (FIG. 3A). As shown in FIG. 3, monoclonal murine
antibodies 21M14 and 21M18 recognize the epitope contained within
amino acids 1-217 of DLL4. This region contains a motif termed the
"DSL (Delta/Serrate/lag-2)" domain present in several Notch ligands
(Tax et al., 1994, Nature 368:150-4). Additionally, anti-DLL4 mAbs
were examined for binding to DLL4 fusion protein fragments by
western blot analysis (FIG. 3B). This work demonstrates the human
specific binding of 21M18 to DLL4 within amino acids 1-154 in the
presence of a DSL domain present within amino acids 155-217. This
demonstrates a previously unappreciated importance of this
N-terminal sequence to DLL4 function. This work is summarized in
schematic form (FIG. 3C) with 21M18 binding or lack of binding
denoted by a "+" or "-" respectively. The binding of 21M18 was
further characterized by examination of binding of 21M18 to a
series of DLL4 protein fragments (DLL4dom1-6) containing specific
amino acid substitutions swapping the human DLL4 amino acids for
the corresponding murine amino acids. These fusion proteins were
screened for binding to 21M18 by ELISA. Several positions were
identified as important for 21M18 binding as shown (FIG. 3D). 21M18
displays impaired binding to DLL4 protein fragments with
substitutions at amino acids 68, 69, and 71 (replacement of valine,
valine, and proline) or at amino acids 142 and 144 (replacement of
lysine and alanine). In contrast a distinct antibody 21M21 binds to
an epitope contained within the DSL region (FIG. 3E), but this
antibody does not impact DLL4 function as shown in FIG. 6,
demonstrating that binding to DSL does not predict a functional
antibody.
Chimeric Antibodies
[0192] After monoclonal antibodies that specifically recognize DLL4
are identified, these antibodies are modified to overcome the human
anti-mouse antibody (HAMA) immune response when rodent antibodies
are used as therapeutics agents. In certain embodiments, 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 IgG.sub.1 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 IgG.sub.1 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
[0193] In certain embodiments, humanized antibodies against DLL4
are generated. The variable domains of the murine monoclonal
antibody 21M18 were isolated and sequenced from the hybridoma line
using degenerate PCR essentially as described in Larrick, J. M., et
al., 1989, Biochem. Biophys. Res. Comm. 160: 1250 and Jones, S. T.
& Bendig, M. M., 1991, Bio/Technology 9: 88. Human heavy and
light chain variable framework regions likely to be structurally
similar to the parental 21M18 antibody amino acid sequences are
then chosen as the human framework regions for humanization. To
identify the candidate human framework regions, the predicted
protein sequences encoded by the V.sub.H and V.sub.L murine
variable domains of 21M18 are compared with human antibody
sequences encoded by expressed human cDNA using BLAST searches for
human sequence deposited in Genbank. Using this method, expressed
human cDNA sequences (e.g. genbank AY393019, DC295533) are selected
for further analysis in designing a heavy chain framework.
[0194] The amino acid differences between candidate humanized
framework heavy chains and the parent murine monoclonal antibody
21M18 heavy chain are evaluated for likely importance, and a
judgment made as to whether each difference in position contributes
to proper folding of the 21M18 antibody. This analysis is guided by
examination of solved crystal structures of other antibody
fragments (e.g. the structure of fab 2E8 as described in Trakhanov
et al, Acta Crystallogr D Biol Crystallogr, 1999, 55:122-28).
Structures are modeled using computer software including Jmol,
quick PDB, and Pymol. Consideration is given to the potential
impact of an amino acid at a given position on the packing of the
.beta.-sheet framework, the interaction between the heavy and light
chain variable domains, the degree of solvent exposure of the amino
acid side chain, and the likelihood that an amino acid would impact
the positioning of the CDR loops. From this analysis, five
candidate V.sub.H chains fused in-frame to the human IgG2 constant
region are chemically synthesized. The candidate heavy chains
comprise: i) of a functional human framework containing selected
substitutions within the synthetic framework region based on
analysis of likely impact on 21M18 binding function and ii) the
parental 21M18 murine antibody CDRs (SEQ ID NOs: 1, 2, and 5).
[0195] Similarly, amino acid differences between a selected human
framework IGK(V)4-1 light chain and the parent murine monoclonal
antibody 21M18 light chain are identified, and a judgment is then
made as to whether each difference in position contributes to
proper folding of the 21M18 antibody. From this analysis, five
candidate V.sub.L chains are chemically synthesized. The first
candidate light chain comprises: i) a fully IGK(V)4-1 human
framework and ii) the parental 21M18 murine antibody CDRs (SEQ ID
NOs: 9, 10, and 11). The four additional candidate light chains
comprise: i) the IGK(V)4-1 human framework region with an
increasing number of 21M18 murine residues retained in the
framework region and ii) the parental 21M18 murine antibody CDRs
(SEQ ID NOs: 9, 10, and 11).
[0196] The functionality of each candidate variant humanized heavy
and light chain is tested by cotransfection into mammalian cells.
Each of the five candidate humanized 21M18 heavy chains described
above is cotransfected with the murine 21M18 light chain cDNA into
HEK 293 cells, and conditioned media is assayed for DLL4 antigen
binding activity by ELISA. The 21M18 heavy chain variant exhibiting
the most robust binding is selected. This variant--"21M18
H2"--contains, in addition to murine CDRs, substitutions at 6
framework positions within the Vh framework, Kabat positions 16,
20, 27, 28, 38, and 48 (FIG. 4A). The 21M18 H2 humanized heavy
chain is then cotransfected with each of the five candidate
humanized light chains into HEK293 cells, and conditioned media is
again assayed for antigen binding by ELISA. A single light chain
variant is found to exhibit better binding than the other
candidates--"21M18 L2"--retaining murine residues at Kabat
positions 22 and 36 (FIG. 5).
[0197] Next, the isolated cysteine residue in CDR2H2 (SEQ ID NO: 2)
is altered. Specifically, two heavy chain variants of H2 are
synthesized with the cysteine residue at Kabat position 52a
modified to a serine (variant H7; SEQ ID NO: 3) or a valine
(variant H9; SEQ ID NO: 4) residue. These heavy chains are
cotransfected into HEK293 cells with L2, and conditioned media
again assayed. Both variants (21M18 H7L2 and 21M18 H9L2)
demonstrate specific antigen binding by ELISA. Thus 21M18 heavy
chain CDR2 comprises SEQ ID NO: 2, 3, or 4 in which the residue at
Kabat position 52a comprises a cysteine, serine, or valine
residue.
[0198] In certain embodiments, humanized antibodies against DLL4
were generated. The variable domains of the murine monoclonal
antibody 21M18 were isolated and sequenced from the hybridoma line
using degenerate PCR essentially as described in Larrick, J. M., et
al., 1989, Biochem. Biophys. Res. Comm. 160: 1250 and Jones, S. T.
& Bendig, M. M., 1991, Bio/Technology 9: 88. Human heavy and
light chain variable framework regions most similar to the parental
21M18 antibody amino acid sequences were then chosen as the human
framework regions for humanization. To identify the most similar
human framework regions, the predicted protein sequences encoded by
the V.sub.H and V.sub.L murine variable domains of 21M18 were
compared with Ig variable domains encoded by the human genome using
BLAST searches for human genomic sequence deposited in Genbank.
Using this method, IGH(V)1-18 was chosen as the human heavy chain
framework region and IGK(V)4-1 was chosen as the human light chain
framework region.
[0199] The amino acid differences between the selected human
framework IGH(V)1-18 heavy chain and the parent murine monoclonal
antibody 21M18 heavy chain were identified, and a judgment was then
made as to whether each difference in position contributed to
proper folding of the 21M18 antibody. This analysis was guided by
examination of solved crystal structures of other antibody
fragments (e.g. the structure of fab 2E8 as described in Trakhanov
et al, Acta Crystallogr D Biol Crystallogr, 1999, 55:122-28).
Structures were modeled using computer software including Jmol,
quick PDB, and Pymol. Consideration was given to the potential
impact of an amino acid at a given position on the packing of the
.beta.-sheet framework, the interaction between the heavy and light
chain variable domains, the degree of solvent exposure of the amino
acid side chain, and the likelihood that an amino acid would impact
the positioning of the CDR loops. From this analysis, five
candidate V.sub.H chains fused in-frame to the human IgG2 constant
region were chemically synthesized. The first candidate heavy chain
comprised: i) a fully IGH(V)1-18 human framework and ii) the
parental 21M18 murine antibody CDRs (SEQ ID NOs: 1, 2, and 5). The
four additional candidate heavy chains comprised: i) the IGH(V)1-18
human framework region with an increasing number of 21M18 murine
residues retained in the framework region and ii) the parental
21M18 murine antibody CDRs (SEQ ID NOs: 1, 2, and 5).
[0200] Similarly, amino acid differences between the selected human
framework IGK(V)4-1 light chain and the parent murine monoclonal
antibody 21M18 light chain were identified, and a judgment was then
made as to whether each difference in position contributed to
proper folding of the 21M18 antibody. From this analysis, five
candidate V.sub.L chains were chemically synthesized. The first
candidate light chain comprised: i) a fully IGK(V)4-1 human
framework and ii) the parental 21M18 murine antibody CDRs (SEQ ID
NOs: 9, 10, and 11). The four additional candidate light chains
comprised: i) the IGK(V)4-1 human framework region with an
increasing number of 21M18 murine residues retained in the
framework region and ii) the parental 21M18 murine antibody CDRs
(SEQ ID NOs: 9, 10, and 11).
[0201] The functionality of each candidate variant humanized heavy
and light chain was tested by cotransfection into mammalian cells.
Each of the five candidate humanized 21M18 heavy chains described
above was cotransfected with the murine 21M18 light chain cDNA into
HEK 293 cells, and conditioned media was then assayed for DLL4
antigen binding activity by ELISA. The 21M18 heavy chain variant
exhibiting the most robust binding was selected. This
variant--"21M18 H2"--contained, in additional to murine CDRs,
murine residues at five framework positions, Kabat positions 20,
28, 38, 48, and 69 (FIG. 4). The 21M18 H2 humanized heavy chain was
then cotransfected with each of the five candidate humanized light
chains into HEK293 cells, and conditioned media was again assayed
for antigen binding by ELISA. A single light chain variant was
found to exhibit better binding than the other candidates--"21M18
L2"--retaining murine residues at Kabat positions 22 and 36 (FIG.
5).
[0202] Next, the isolated cysteine residue in CDR2H2 (SEQ ID NO: 2)
was altered. Specifically, two heavy chain variants of H2 were
synthesized with the cysteine residue at Kabat position 52a
modified to a serine (variant H7; SEQ ID NO: 3) or a valine
(variant H9; SEQ ID NO: 4) residue. These heavy chains were
cotransfected into HEK293 cells with L2, and conditioned media was
again assayed. Both variants (21M18 H7L2 and 21M18 H9L2)
demonstrated specific antigen binding by ELISA. Thus 21M18 heavy
chain CDR2 comprises SEQ ID NO: 2, 3, or 4 in which the residue at
Kabat position 52a comprises a cysteine, serine, or valine
residue.
[0203] The humanized 21M18 antibodies were then further
characterized. Specifically, the binding affinity of humanized
21M18 antibodies purified by protein A chromatography was
determined using Biacore. Affinity was determined to be
approximately 0.33 nM for 21M18 variant H2L2.
[0204] The humanized 21M18 antibodies were deposited with ATCC,
located at 10801 University Blvd., Manassas, Va. 20110-2209, under
the terms of the Budapest Treaty (21M18 H9L2, ATCC deposit no.
PTA-8427 and 21M18 H7L2, ATCC deposit no. PTA-8425, deposited May
10, 2007).
Human Antibodies
[0205] In some embodiments, human antibodies that specifically
recognize the extracellular domain of DLL4 are isolated using phage
display technology. A synthetic antibody library containing human
antibody variable domains is screened for specific and high
affinity recognition of the DLL4 antigen described above. CDR
cassettes in the library are specifically exchanged via unique
flanking restriction sites for antibody optimization. Optimized
human variable regions are then cloned into an Ig expression vector
containing human IgG.sub.1 heavy-chain and kappa light-chain for
expression of human antibodies in mammalian CHO cells.
Example 2
In Vitro Assays to Evaluate Antibodies Against DLL4
[0206] This example describes representative in vitro assays to
test the activity of antibodies generated against DLL4 on cell
proliferation, Notch pathway activation, and cytotoxicity.
Proliferation Assay
[0207] The expression of DLL4 by different cancer cell lines is
quantified using Taqman analysis. Cell lines identified as
expressing DLL4 are plated at a density of 10.sup.4 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-DLL4 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-DLL4 antibodies to
inhibit cell proliferation compared to control antibodies is
determined.
Pathway Activation Assay
[0208] In certain embodiments, the ability of antibodies against
DLL4 to block activation of the Notch signaling pathway is
determined in vitro. HeLa cells cultured in DMEM supplemented with
antibiotics and 10% FCS were co-transfected with 1) Hes1-Luc
reporter vector containing the Hes1 promoter upstream of a firefly
luciferase reporter gene to measure Notch signaling levels
(Jarriault et al., 1995, Nature 377:355-8) in response to DLL4
ligand and 2) a Renilla luciferase reporter (Promega; Madison,
Wis.) as an internal control for transfection efficiency.
Transfected cells were then added to cultures plates coated
overnight with 10 .mu.g/ml DLL4-Fc protein. Antibodies to DLL4 were
then added to the cell culture medium. Forty-eight hours following
transfection, luciferase levels were measured using a dual
luciferase assay kit (Promega; Madison, Wis.) with firefly
luciferase activity normalized to Renilla luciferase activity. The
ability of antibodies to inhibit DLL4 induced Notch pathway
activation was thus determined. Inhibition of DLL4 activation of
Notch pathway activation was observed with anti-DLL4 murine
antibodies 21M14 and 21M18 (FIG. 6). In contrast, anti-DLL4
antibody 21M21 did not inhibit Notch binding (FIG. 6) despite
binding to the DSL domain of DLL4 (FIG. 3E).
[0209] In certain embodiments, the ability of anti-DLL4 antibodies
to modulate downstream gene activation was determined. C8 colon
tumor cells from animals treated with murine 21M18 antibodies
(described in detail below) were isolated and expression of Notch
pathway genes HES1 and ATOH-1 was determined by RT-PCR. Total RNA
from tumor tissue was isolated with RNeasy Fibrous Tissue kit
(Qiagen, Valencia, Calif.) according to manufacturer's
instructions. The quantity of RNA samples was determined by the
ratio of 260 nm/280 nm. The integrity of RNA was determined by
running an aliquot of the RNA sample on a denaturing agarose gel
stained with ethidium bromide (EtBr). The ratio of 28s to 18s rRNA
on the gel was visualized using a Fluor Chem camera delivered with
the AphaEasa FC software. RNA samples were eluted in RNase-free
water and stored at -80.degree. C. The real-time RT-PCR was done
with a dual-fluorescent nonextendable probe containing 3'-TAMRA FAM
(6-carboxyfluorescein) reporter dye and a 3'-TAMRA
(6-carboxy-tetramethylrhodamine). One hundred micrograms of total
RNA was used for real-time PCR in a final volume of 25 uL
containing reverse transcriptase, 1.times. Tagman buffer (Applied
Biosystems, Foster City, Calif.) and the primer/probe mixture.
Reactions were carried out in an ABI 7900 HT Fast Real Time PCR
System (Applied Biosystems, Foster City, Calif.): 30 min at
48.degree. C., 10 min at 95.degree. C. and 40 cycles of 15 sec at
95.degree. C. and 1 min at 60.degree. C. The results were analyzed
using the SDS2.3 software (Applied Biosystems). All primer and
probe sets were obtained from Applied Biosystems (Foster City,
Calif.). The level of expression of target genes were normalized to
the expression level of the house keeping gene Gus B and expressed
as relative quantity. Treatment with anti-DLL4 murine 21M18
antibodies reduced expression of HES1 and increased expression of
ATOH-1 as compared to control treated tumors (FIG. 7A).
[0210] In some embodiments, mouse lineage-depleted OMP-C11 tumor
cell colonies were established using culture conditions known to
maintain tumorigenic cells in vitro. These tumor cell colonies were
overlaid with 3T3 cells without (3T3) or including human DLL4
(DLL4) overexpressed on the cell surface in the presence or absence
of 10 .mu.g/mL murine 21M18 or 5 .mu.M gamma-secretase inhibitor
(GSI; i.e. DBZ). A no overlay control was also included. While
3T3-DLL4 cells induced HEST and suppressed ATOH1 gene expression
(shown as ratio of HES1:ATOH1), either 21M18 or GSI alone inhibited
DLL4-induced Notch target gene changes.
Complement-Dependent Cytotoxicity Assay
[0211] In certain embodiments, cancer cell lines expressing DLL4 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 DLL4. Cells are suspended in 200
.mu.l RPMI 1640 culture medium supplemented with antibiotics and 5%
FBS at 10.sup.6 cells/ml. Suspended cells are then mixed with 200
.mu.l serum or heat-inactivated serum with antibodies against DLL4
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 DLL4 compared to heat-inactivated serum and control
antibodies. The ability of anti-DLL4 antibodies to mediated
complement-dependent cytotoxicity is thus determined.
Antibody-Dependent Cellular Cytotoxicity Assay
[0212] In certain embodiments, cancer cell lines expressing DLL4 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 DLL4. 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 DLL4 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 DLL4 to mediated antibody dependent cellular
cytotoxicity is thus determined.
Example 3
In Vivo Prevention of Tumor Growth Using Anti-DLL4 Antibodies
[0213] This example describes the use of anti-DLL4 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 1-4 hours with pipetting up and down through a 10-mL pipette
every 15-20 minutes. Digested cells are filtered through a 40 .mu.M
nylon mesh, washed with Hank's buffered saline solution (HBSS)
containing 2% heat-inactivated calf serum (HICS) and 25 mM HEPES
(pH 7.4). Dissociated tumor cells are then injected subcutaneously
into the mammary fat pads of NOD/SCID mice to elicit tumor
growth.
[0214] 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 10.sup.6
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 (Miltenyi Biotec, Auburn, Calif.),
anti-CD44, anti-CD24, and Lineage markers anti-CD2, -CD3, -CD10,
-CD16, -CD18, -CD31, -CD64, and -CD140b (collectively referred to
as Lin; BD Biosciences, 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 H2 Kd+ and murine CD45+ cells, and dead cells are
eliminated by using the viability dye DAPI. Flow cytometry is
performed on a FACSAria (BD Biosciences, San Jose, Calif.). 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.
[0215] In certain embodiments, anti-DLL4 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 right flank region of 6-8 week old NOD/SCID
mice. The day after tumor cell injection, animals were injected
intraperitoneal (i.p.) with 10 mg/kg murine 21M18 anti-DLL4
antibodies (n=5) or PBS (n=10) two times per week for the duration
of the experiment. 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 with 21M18 antibody
reduced tumor growth by 54% compared to PBS injected controls (FIG.
8).
[0216] The ability of anti-DLL4 antibodies to affect proliferation
in vivo was then determined. C8 colon tumors from animals treated
with murine 21M18 antibodies or control antibodies were isolated
and expression of Ki67, a marker of cell proliferation, determined
by immunocytochemistry. Specifically, formalin-fixed,
paraffin-embedded tumors were cut into 4-um thick sections.
Sections were deparaffinized in xylene and rehydrated in distilled
water. Immunohistochemistry was performed according to standard
methods. Briefly, sections were immersed in citrate buffer (pH 6)
in a water bath for 20 minutes in the Decoking chamber to retrieve
antigens. The slides were cooled for about 45 minutes and rinsed in
PBS. Sections were incubated with hydrogen peroxide (Sigma-Aldrich,
St Louis, Mo.) for 10 minutes at room temperature to remove
endogenous peroxidase prior to addition of primary antibody. The
rabbit anti-human Ki67 (Vector Laboratories Inc., Burlingame,
Calif.) at 1:50 dilution in horse dilution buffer (1% NHS, 1% BSA,
0.1% Tx-100, 0.05% NaN3 in PBS) was added to each section and
incubated for 1 hour or overnight at 4.degree. C. Slides were
rinsed 3 times in washing buffer (Gelatine 10%, Tx-100 10%, in PBS)
for 5 minutes each. The anti-rabbit secondary antibody conjugated
with HRP solution (Immpress anti-Rabbit pre-diluted, Vector
Laboratories Inc., Burlingame, Calif.) was added to the slides and
incubated for 30 minutes. After extensive wash with washing buffer,
Vector Nova Red (Vector Laboratories Inc., Burlingame, Calif.) was
added. The slides were rinsed with water, counterstained with
hematoxilin and mounted with permanent mounting medium (Vectamount,
Vector Laboratories Inc., Burlingame, Calif.). Treatment with
anti-DLL4 murine 21M18 antibodies reduced the number of cells
expressing Ki67 as compared to control treated tumors (FIG. 9).
Example 4
In Vivo Prevention and Treatment of Tumor Growth Using Anti-DLL4
Antibodies in Combination Therapy
[0217] DLL-4 Antibodies in Combination with Fluorouracil
[0218] In certain embodiments, anti-DLL4 antibodies were analyzed
in combination with chemotherapy for the ability to reduce growth
of UM-C4 colon tumor cells in vivo. Dissociated UM-C4 cells (10,000
per animal) were injected subcutaneously into the right flank
region of 6-8 week old NOD/SCID mice. The day after tumor cell
injection, animals were injected intraperitoneal (i.p.) with 10
mg/kg murine 21M18 anti-DLL4 antibodies or PBS two times per week
for the duration of the experiment with or without concurrent
treatment with the anti-metabolite chemotherapy agent fluorouracil
(5-FU) administered one time 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 with
anti-DLL4 murine 21M18 antibodies in combination with 5-FU reduced
tumor growth to a greater degree than either treatment alone (FIG.
10).
DLL-4 Antibodies in Combination with EGFR or VEGF Antibodies
[0219] In certain embodiments, anti-DLL4 antibodies were tested in
combination with anti-EGF receptor (EGFR) antibodies for the
ability to affect tumor take frequency in vivo. Dissociated UM-C4
cells (10,000 per animal) were injected subcutaneously into the
right flank region of 6-8 week old NOD/SCID mice. The day after
tumor cell injection, animals (n=10) were injected intraperitoneal
(i.p.) with 10 mg/kg murine 21M18 anti-DLL4 antibodies, anti-EGFR
antibodies, a combination of anti-DLL4 and anti-EGFR antibodies, or
PBS. Tumors were detected in all animals treated with anti-DLL4 or
anti-EGFR antibodies and 9 out of 10 control animals. In contrast,
only 2 out of 10 animals treated with a combination of anti-DLL4
and anti-EGFR antibodies had detectable tumors several weeks after
treatment (FIG. 11). Furthermore, treatment with anti-DLL4 murine
21M18 antibodies in combination with anti-EGFR antibodies reduced
the frequency of tumorigenesis versus either treatment alone (FIG.
11).
[0220] In certain embodiments, anti-DLL4 antibodies were tested in
combination with anti-EGF receptor (EGFR) antibodies for the
ability to affect tumor take frequency in vivo. C17 tumor cells
were implanted in mice (n=10 per group) and treatment was initiated
two day later with either control antibody, murine 21M18, anti-VEGF
antibodies, or the combination of both antibodies. Each antibody
was dosed at 10 mg/kg, given twice a week. Both 21M18 and anti-VEGF
reduced tumor growth, and the combination was more effective than
either antibody alone (FIG. 18).
DLL-4 Antibodies in Combination with Irinotecan
[0221] In certain embodiments, anti-DLL4 antibodies were tested in
combination with the chemotherapeutic Irinotecan. In some
embodiments, dissociated OMP-C8 tumor cells (10,000 per animal)
were injected subcutaneously into the right flank region of 6-8
week old NOD/SCID mice. The day after tumor cell injection, animals
were injected intraperitoneal (i.p.) with 10 mg/kg murine 21M18
anti-DLL4 antibodies or control antibody two times per week for the
duration of the experiment with or without concurrent treatment
with the chemotherapy agent Irinotecan administered one time per
week at a dosage of 7.5 mg/kg. Tumor growth was monitored weekly
until growth was detected, after which point tumor growth was
measured twice weekly. Treatment with anti-DLL4 21M18 antibodies in
combination with Irinotecan reduced tumor growth to a greater
degree than either treatment alone (FIG. 12A). And, while tumor
growth continued or accelerated in most animals after cessation of
weekly treatment with 7.5 mg/kg Irinotecan alone, the combination
of 10 mg/kg, twice per week, anti-DLL4 21M18 and 7.5 mg/kg weekly
Irinotecan prevented further colon tumor growth after treatment
cessation on day 56 for over five weeks (FIG. 13).
[0222] In certain embodiments, humanized H7L2 21M18 anti-DLL4
antibodies were tested in combination with Irinotecan. In some
embodiments, dissociated C8 tumor cells (10,000 per animal) were
injected subcutaneously into the right flank region of 6-8 week old
NOD/SCID mice. The day after tumor cell injection, animals were
injected intraperitoneal (i.p.) with 10 mg/kg humanized 21M18
anti-DLL4 antibodies, murine 21M18 antibodies, or control
antibodies two times per week for the duration of the experiment
with or without concurrent treatment with the chemotherapy agent
Irinotecan administered one time per week at a dosage of 7.5 mg/kg.
Tumor growth was monitored weekly until growth was detected, after
which point tumor growth was measured twice weekly. Treatment with
humanized anti-DLL4 21M18 antibodies in combination with Irinotecan
showed similar efficacy against tumor growth as murine 21M18 (FIG.
12B).
[0223] In some embodiments, combination anti-DLL4 murine 21M18 and
Irinotecan treatment was used to treat established colon tumors.
Dissociated C8 cells (10,000 per animal) were injected
subcutaneously into the right flank region of 6-8 week old NOD/SCID
mice. When the injected cells produced tumors of approximately 60
mm.sup.3, treatment was commenced. Animals were injected
intraperitoneal (i.p.) with 10 mg/kg murine 21M18 anti-DLL4
antibodies or a control two times per week for the duration of the
experiment with or without concurrent treatment with the
chemotherapy agent Irinotecan administered one time per week at a
dosage of 7.5 mg/kg. Treatment with anti-DLL4 murine 21M18
antibodies in combination with Irinotecan reduced the growth of
established colon tumors to a greater degree than either treatment
alone (FIG. 14).
[0224] In some embodiments, combination therapy followed by
antibody treatment delayed tumor recurrence. Dissociated C8 cells
(10,000 per animal) were injected subcutaneously into the right
flank region of 6-8 week old NOD/SCID mice. When the injected cells
produced tumors of approximately 150 mm.sup.3, treatment was
commenced. Animals were administered intraperitoneal (i.p.) 10
mg/kg murine 21M18 anti-DLL4 or control antibodies two times per
week in combination with Irinotecan at 7.5 mg/kg once weekly for a
total of 32 days. Combination treatment was then discontinued and
antibodies were treated with DLL4 21M18 or control antibodies for
the remainder of the experiment. Treatment with anti-DLL4 21M18
antibodies following combination therapy significantly delayed the
recurrence of tumor growth compared to control treated animals
(FIG. 16). Individual tumor volume is also shown at 47 days after
termination of Irinotecan treatment (FIG. 17).
DLL-4 Antibody Reduction of Cancer Stem Cell Frequency Is Enhanced
by Irinotecan
[0225] In certain embodiments, the ability of anti-DLL4 21M18
antibodies alone or in combination with Irinotecan to reduce the
frequency of cancer stem cells was determined using a limiting
dilution analysis. C8 colon tumors from mice treated with either
control or DLL4 murine 21M18 antibodies, Irinotecan, or DLL4 murine
21M18 antibodies in combination with Irinotecan as described above
were isolated after 38 days of treatment. Isolated tumors (n=3 per
experimental group) were processed as described below. Tumors were
removed, and minced with a sterile razor blade. To obtain single
cell suspensions, a digestion solution containing
Collagenase/Hyaluronidase:Dispase (1:1:8 of 10.times.) in MEBM
medium (Cambrex, East Rutherford, N.J.) with a 1:100 dilution of
DNAseI (Worthington, Lakewood, N.J.) was mixed with the tumor
suspension and incubated for 1 hour at 37.degree. C. Cells were
centrifuged and resuspended in 1 ml of ACK medium (0.15M
NH.sub.4C1, 10 mM KHCO.sub.3, 0.1 mM Na.sub.2EDTA in distilled
water) on ice for 2 minutes to remove red blood cells. Cells were
centrifuged and resuspended at a concentration of 1.times.10.sup.7
cells/ml in FACS buffer and then incubated with biotinylated mouse
antibodies (.alpha.-mouse CD45-biotin 1:200 dilution and rat
.alpha.-mouse H.sub.2Kd-biotin 1:100 dilution, BioLegend, San
Diego, Calif.) on ice for 30 minutes followed by addition of
strepavadin magnetic beads (Invitrogen, Carlsbad, Calif.) to remove
mouse cells. The remaining human cells in the suspension were
collected, counted and diluted to the desired concentration for
further use. Serial dilutions of human cells were then re-injected
into immuno-compromised mice. Specifically, mice were injected with
900, 300, 100, or 50 isolated human tumor cells in the right flank
region (n=10 per group). Tumor volume was assessed twice per
week.
[0226] Upon termination of the study on day 81, the percentage of
mice with detectable tumors was decreased in all groups injected
with DLL4 21M18 antibody treated tumor cells and even further
decreased in DLL4 21M18-Irinotecan treated tumor cells compared to
those treated with either control or Irinotecan alone (FIG. 15A).
Using these tumor generation frequencies, the stem cell frequency
was calculated using Poisson statistics provided by L-Calc.TM.
software. Briefly, based on Poisson distribution statistics,
exactly one stem cell exists among the known number of injected
cells if 37% of the animals fail to develop tumors. Treatment of
tumors with Irinotecan alone increased the number of cancer stem
cells from 1:93 in control treated tumors to 1:82. In contrast,
anti-DLL4 antibodies reduced the cancer stem cell frequency from
1:93 in control treated tumors to 1:238 in DLL4 antibody treated
tumors and to 1:573 in combination DLL4 21M18-Irinotecan treated
tumor cells (FIG. 15B).
Example 5
In Vivo Treatment of Tumors Using Anti-DLL4 Antibodies
[0227] This example describes the use of humanized anti-DLL4 21M18
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.
[0228] Following tumor cell injection, animals are monitored for
tumor growth. Once tumors reach an average size of approximately
100 mm.sup.3, antibody treatment begins. Each animal receives 100
.mu.g DLL4 21M18 humanized 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 DLL4
humanized antibodies to prevent further tumor growth or to reduce
tumor size compared to control antibodies is thus determined.
[0229] 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-DLL4 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-DLL4 receptor or control antibodies
present in the tumor biopsy. Furthermore antibodies that detect
different tumor and tumor-recruited cell types such as, for
example, anti-VE cadherin (CD144) or anti-PECAM-1 (CD31) antibodies
to detect vascular endothelial cells, anti-smooth muscle
alpha-actin antibodies to detect vascular smooth muscle cells,
anti-Ki67 antibodies to detect proliferating cells, TUNEL assays to
detect dying cells, anti-intracellular domain (ICD) Notch fragment
antibodies to detect Notch signaling can be used to assess the
effects of antibody treatment on, for example, angiogenesis, tumor
growth and tumor morphology.
[0230] In certain embodiments, the effect of anti-DLL4 humanized
antibody treatment on tumor cell gene expression is also assessed.
Total RNA is extracted from a portion of each harvested tumor from
DLL4 antibody treated and control antibody treated mice and used
for quantitative RT-PCR. Expression levels of DLL4, Notch
receptors, components of the Notch signaling pathway, as well as
addition cancer stem cell markers previously identified (e.g. CD44)
are analyzed relative to the house-keeping gene GAPDH as an
internal control. Changes in tumor cell gene expression upon DLL4
antibody treatment are thus determined.
[0231] In addition, the effect of anti-DLL4 antibody treatment on
the presence of cancer stem cells in a tumor is assessed. Tumor
samples from DLL4 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.
[0232] The tumorigenicity of cells isolated based on ESA+, CD44+,
CD24-/low, Lin-expression following anti-DLL4 antibody treatment
can then assessed. ESA+, CD44+, CD24-/low, Lin-cancer stem cells
isolated from DLL4 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 Humanized Anti-DLL4 Antibodies
[0233] This example describes methods for treating cancer using
humanized antibodies against DLL4 to target tumors comprising
cancer stem cells and/or tumor cells in which Notch receptor or
Notch receptor ligand 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 DLL4 to detect protein expression.
[0234] 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 -DLL4 antibodies to detect cancer
stem cells, and the presence of ESA+, CD44+, CD24-/low, Lin-, DLL4+
tumor stem cells is determined by flow cytometry as described in
detail above.
[0235] Cancer patients whose tumors are diagnosed as expressing a
Notch receptor or Notch receptor ligand are treated with humanized
anti-DLL4 antibodies. In certain embodiments, humanized anti-DLL4
antibodies generated as described above are purified and formulated
with a suitable pharmaceutical vehicle for injection. In some
embodiments, patients are treated with the DLL4 antibodies at least
once a month for at least 10 weeks. In some embodiments, patients
are treated with the DLL4 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-DLL4 antibody is
administered. In some embodiments, between about 5 to about 40
mg/ml of an anti-DLL4 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.
[0236] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims. All publications, patents and patent applications
cited herein are incorporated by reference in their entirety into
the disclosure.
TABLE-US-00001 SEQUENCES SEQ ID NO: 1 (Heavy chain CDR1) TAYYIH SEQ
ID NO: 2 (Heavy chain CDR2, H2) YISCYNGATNYNQKFKG SEQ ID NO: 3
(Heavy chain CDR2 H7) YISSYNGATNYNQKFKG SEQ ID NO: 4 (Heavy chain
CDR2 H9) YISVYNGATNYNQKFKG SEQ ID NO: 5 (Heavy chain CDR3)
RDYDYDVGMDY SEQ ID NO: 6 (Heavy chain variable region, H2)
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISCYNGATNYNQ
KFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS SEQ ID
NO: 7 (Heavy chain variable region, H7)
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATNYNQ
KFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS SEQ ID
NO: 8 (Heavy chain variable region, H9)
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISVYNGATNYNQ
KFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS SEQ ID
NO: 9 (Light chain CDR1) RASESVDNYGISFMK SEQ ID NO: 10 (Light chain
CDR2) AASNQGS SEQ ID NO: 11 (Light chain CDR3) QQSKEVPWTFGG SEQ ID
NO: 12 (Light chain variable region)
DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGV
PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIK SEQ ID NO: 13
(Nucleotide sequence of heavy chain, H2, variable region)
ATGGACTGGACCTGGAGCATCCTGTTCCTGGTGGCTGCTGCTACAGGAGCTCACTCCCAGGT
TCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTAGCGTGAAGATCAGCTGCA
AGGCTAGCGGATACTCCTTTACAGCTTACTACATCCACTGGGTGAAGCAGGCCCCTGGACAA
GGGCTGGAGTGGATCGGATATATCAGCTGTTACAACGGAGCTACAAACTACAACCAGAAGTT
CAAGGGCAGGGTCACCTTCACAACAGACACAAGCACAAGCACAGCCTACATGGAGCTGAGGA
GCCTGAGAAGCGACGACACAGCCGTGTACTACTGTGCTAGGGACTACGACTACGACGTGGGG
ATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCAGCTCA SEQ ID NO: 14
(Nucleotide sequence of heavy chain, H7, variable region)
ATGGACTGGACCTGGAGCATCCTGTTCCTGGTGGCTGCTGCTACAGGAGCTCACTCCCAGGT
TCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTAGCGTGAAGATCAGCTGCA
AGGCTAGCGGATACTCCTTTACAGCTTACTACATCCACTGGGTGAAGCAGGCCCCTGGACAA
GGGCTGGAGTGGATCGGATATATCAGCTCCTACAACGGAGCTACAAACTACAACCAGAAGTT
CAAGGGCAGGGTCACCTTCACAACAGACACAAGCACAAGCACAGCCTACATGGAGCTGAGGA
GCCTGAGAAGCGACGACACAGCCGTGTACTACTGTGCTAGGGACTACGACTACGACGTGGGG
ATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCAGCTCA SEQ ID NO: 15
(Nucleotide sequence of heavy chain, H9)
ATGGACTGGACCTGGAGCATCCTGTTCCTGGTGGCTGCTGCTACAGGAGCTCACTCCCAGGT
TCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTAGCGTGAAGATCAGCTGCA
AGGCTAGCGGATACTCCTTTACAGCTTACTACATCCACTGGGTGAAGCAGGCCCCTGGACAA
GGGCTGGAGTGGATCGGATATATCAGCGTCTACAACGGAGCTACAAACTACAACCAGAAGTT
CAAGGGCAGGGTCACCTTCACAACAGACACAAGCACAAGCACAGCCTACATGGAGCTGAGGA
GCCTGAGAAGCGACGACACAGCCGTGTACTACTGTGCTAGGGACTACGACTACGACGTGGGG
ATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCAGCTCA SEQ ID NO: 16
(Nucleotide sequence of light chain)
ATGGTGCTCCAGACCCAGGTCTTCATTTCCCTGCTGCTCTGGATCAGCGGAGCCTACGGGGA
CATCGTGATGACCCAGTCCCCTGACTCCCTGGCTGTGTCCCTGGGCGAGAGGGCCACCATCT
CCTGCAGAGCCAGCGAATCCGTCGATAATTATGGCATTTCCTTTATGAAGTGGTTCCAGCAG
AAACCAGGACAGCCTCCTAAGCTGCTCATTTACGCTGCATCCAACCAAGGGTCCGGGGTCCC
TGACAGGTTCTCCGGCAGCGGGTCCGGAACAGATTTCACTCTCACCATCAGCAGCCTGCAGG
CTGAAGATGTGGCTGTCTATTACTGTCAGCAAAGCAAGGAGGTGCCTTGGACATTCGGAGGA
GGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCCCCCTCCGTCTTCATCTTCCCCCCCAG
CGATGAGCAGCTGAAAAGCGGCACTGCCAGCGTGGTGTGCCTGCTGAATAACTTCTATCCCC
GGGAGGCCAAAGTGCAGTGGAAGGTGGATAACGCCCTCCAAAGCGGCAACTCCCAGGAGAGC
GTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAA
AGCCGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCAGCC
CCGTCACAAAGAGCTTCAACAGGGGCGAGTGTTGA SEQ ID NO: 17 (Nucleotide
sequence of heavy chain CDR1) ACAGCTTACTACATCCAC SEQ ID NO: 18
(Nucleotide sequence of heavy chain CDR2, H2)
ATATCAGCTGTTACAACGGAGCTACAAACTACAACCAGAAGTTCAAGGGC SEQ ID NO: 19
(Nucleotide sequence of heavy chain CDR2, H7)
ATATCAGCTCCTACAACGGAGCTACAAACTACAACCAGAAGTTCAAGGGC SEQ ID NO: 20
(Nucleotide sequence of heavy chain CDR2, H9)
ATATCAGCGTCTACAACGGAGCTACAAACTACAACCAGAAGTTCAAGGGC SEQ ID NO: 21
(Nucleotide sequence of heavy chain CDR3)
AGGGACTACGACTACGACGTGGGGATGGACTAC SEQ ID NO: 22 (Nucleotide
sequence of light chain CDR1)
AGAGCCAGCGAATCCGTCGATAATTATGGCATTTCCTTTATGAAG SEQ ID NO: 23
(Nucleotide sequence of light chain CDR2) GCTGCATCCAACCAAGGGTCC SEQ
ID NO: 24 (Nucleotide sequence of light chain CDR3)
CAGCAAAGCAAGGAGGTGCCTTGGACATTCGGAGGA SEQ ID NO: 25 (Human DLL4
Antigen)
MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCL
KHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHA
PGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSR
LCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGR
LCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTC
TCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCF
NGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTG
TYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCY
TDLSTDTFVCNCPYGFVGSRCEFPVG SEQ ID NO: 26 (Human DLL4 DSL Domain)
WLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEY C
SEQ ID NO: 27 (Human DLL4 N-Terminal Region)
MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCL
KHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHA
PGDDLRPEALPPDALISKIAIQGSLAVGQN
Sequence CWU 1
1
3616PRTArtificial SequenceMurine Heavy Chain CDR1 1Thr Ala Tyr Tyr
Ile His 1 5 217PRTArtificial SequenceMurine Heavy Chain CDR2, H2
2Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Gln Lys Phe Lys 1
5 10 15 Gly 317PRTArtificial SequenceMurine Heavy Chain CDR2, H7
3Tyr Ile Ser Ser Tyr Asn Gly Ala Thr Asn Tyr Asn Gln Lys Phe Lys 1
5 10 15 Gly 417PRTArtificial SequenceMurine Heavy Chain CDR2, H9
4Tyr Ile Ser Val Tyr Asn Gly Ala Thr Asn Tyr Asn Gln Lys Phe Lys 1
5 10 15 Gly 511PRTArtificial SequenceMurine Heavy Chain CDR3 5Arg
Asp Tyr Asp Tyr Asp Val Gly Met Asp Tyr 1 5 10 6119PRTArtificial
SequenceMurine Heavy Chain Variable Region, H2 6Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ala Tyr 20 25 30 Tyr
Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Gln Lys Phe
50 55 60 Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp Tyr Asp Val Gly Met
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
7119PRTArtificial SequenceMurine Heavy Chain Variable Region, H7
7Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ala
Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Tyr Ile Ser Ser Tyr Asn Gly Ala Thr Asn
Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Phe Thr Thr Asp
Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Asp
Tyr Asp Val Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 8119PRTArtificial SequenceMurine Heavy Chain
Variable Region, H9 8Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Ala Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ser Val
Tyr Asn Gly Ala Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg
Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Tyr Asp Tyr Asp Val Gly Met Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 915PRTArtificial
SequenceMurine Light Chain CDR1 9Arg Ala Ser Glu Ser Val Asp Asn
Tyr Gly Ile Ser Phe Met Lys 1 5 10 15 107PRTArtificial
SequenceMurine Light Chain CDR2 10Ala Ala Ser Asn Gln Gly Ser 1 5
1112PRTArtificial SequenceMurine Light Chain CDR3 11Gln Gln Ser Lys
Glu Val Pro Trp Thr Phe Gly Gly 1 5 10 12111PRTArtificial
SequenceMurine Light Chain Variable Region 12Asp Ile Val Met Thr
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala
Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly
Ile Ser Phe Met Lys Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Asp
50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys
Gln Gln Ser Lys 85 90 95 Glu Val Pro Trp Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 110 13414DNAArtificial SequenceMurine
Nucleotide Sequence of Heavy Chain, H2 Variable Region 13atggactgga
cctggagcat cctgttcctg gtggctgctg ctacaggagc tcactcccag 60gttcagctgg
tgcagtctgg agctgaggtg aagaagcctg gggctagcgt gaagatcagc
120tgcaaggcta gcggatactc ctttacagct tactacatcc actgggtgaa
gcaggcccct 180ggacaagggc tggagtggat cggatatatc agctgttaca
acggagctac aaactacaac 240cagaagttca agggcagggt caccttcaca
acagacacaa gcacaagcac agcctacatg 300gagctgagga gcctgagaag
cgacgacaca gccgtgtact actgtgctag ggactacgac 360tacgacgtgg
ggatggacta ctggggccaa ggaaccctgg tcaccgtcag ctca
41414414DNAArtificial SequenceMurine Nucleotide Sequence of Heavy
Chain, H7 Variable Region 14atggactgga cctggagcat cctgttcctg
gtggctgctg ctacaggagc tcactcccag 60gttcagctgg tgcagtctgg agctgaggtg
aagaagcctg gggctagcgt gaagatcagc 120tgcaaggcta gcggatactc
ctttacagct tactacatcc actgggtgaa gcaggcccct 180ggacaagggc
tggagtggat cggatatatc agctcctaca acggagctac aaactacaac
240cagaagttca agggcagggt caccttcaca acagacacaa gcacaagcac
agcctacatg 300gagctgagga gcctgagaag cgacgacaca gccgtgtact
actgtgctag ggactacgac 360tacgacgtgg ggatggacta ctggggccaa
ggaaccctgg tcaccgtcag ctca 41415414DNAArtificial SequenceMurine
Nucleotide Sequence of Heavy Chain, H9 15atggactgga cctggagcat
cctgttcctg gtggctgctg ctacaggagc tcactcccag 60gttcagctgg tgcagtctgg
agctgaggtg aagaagcctg gggctagcgt gaagatcagc 120tgcaaggcta
gcggatactc ctttacagct tactacatcc actgggtgaa gcaggcccct
180ggacaagggc tggagtggat cggatatatc agcgtctaca acggagctac
aaactacaac 240cagaagttca agggcagggt caccttcaca acagacacaa
gcacaagcac agcctacatg 300gagctgagga gcctgagaag cgacgacaca
gccgtgtact actgtgctag ggactacgac 360tacgacgtgg ggatggacta
ctggggccaa ggaaccctgg tcaccgtcag ctca 41416717DNAArtificial
SequenceMurine Nucleotide Sequence of Light Chain 16atggtgctcc
agacccaggt cttcatttcc ctgctgctct ggatcagcgg agcctacggg 60gacatcgtga
tgacccagtc ccctgactcc ctggctgtgt ccctgggcga gagggccacc
120atctcctgca gagccagcga atccgtcgat aattatggca tttcctttat
gaagtggttc 180cagcagaaac caggacagcc tcctaagctg ctcatttacg
ctgcatccaa ccaagggtcc 240ggggtccctg acaggttctc cggcagcggg
tccggaacag atttcactct caccatcagc 300agcctgcagg ctgaagatgt
ggctgtctat tactgtcagc aaagcaagga ggtgccttgg 360acattcggag
gagggaccaa ggtggaaatc aaacgtacgg tggctgcccc ctccgtcttc
420atcttccccc ccagcgatga gcagctgaaa agcggcactg ccagcgtggt
gtgcctgctg 480aataacttct atccccggga ggccaaagtg cagtggaagg
tggataacgc cctccaaagc 540ggcaactccc aggagagcgt cacagagcag
gacagcaagg acagcaccta cagcctcagc 600agcaccctga ccctgagcaa
agccgactac gagaaacaca aagtctacgc ctgcgaagtc 660acccatcagg
gcctgagcag ccccgtcaca aagagcttca acaggggcga gtgttga
7171718DNAArtificial SequenceMurine Nucleotide Sequence of Heavy
Chain CDR1 17acagcttact acatccac 181850DNAArtificial SequenceMurine
Nucleotide Sequence of Heavy Chain CDR2, H2 18atatcagctg ttacaacgga
gctacaaact acaaccagaa gttcaagggc 501950DNAArtificial SequenceMurine
Nucleotide Sequence of Heavy Chain CDR2, H7 19atatcagctc ctacaacgga
gctacaaact acaaccagaa gttcaagggc 502050DNAArtificial SequenceMurine
Nucleotide Sequence of Heavy Chain CDR2, H9 20atatcagcgt ctacaacgga
gctacaaact acaaccagaa gttcaagggc 502133DNAArtificial SequenceMurine
Nucleotide Sequence of Heavy Chain CDR3 21agggactacg actacgacgt
ggggatggac tac 332245DNAArtificial SequenceMurine Nucleotide
Sequence of Light Chain CDR1 22agagccagcg aatccgtcga taattatggc
atttccttta tgaag 452321DNAArtificial SequenceMurine Nucleotide
Sequence of Light Chain CDR2 23gctgcatcca accaagggtc c
212436DNAArtificial SequenceMurine Nucleotide Sequence of Light
Chain CDR3 24cagcaaagca aggaggtgcc ttggacattc ggagga 3625522PRTHomo
sapiens 25Met Ala Ala Ala Ser Arg Ser Ala Ser Gly Trp Ala Leu Leu
Leu Leu 1 5 10 15 Val Ala Leu Trp Gln Gln Arg Ala Ala Gly Ser Gly
Val Phe Gln Leu 20 25 30 Gln Leu Gln Glu Phe Ile Asn Glu Arg Gly
Val Leu Ala Ser Gly Arg 35 40 45 Pro Cys Glu Pro Gly Cys Arg Thr
Phe Phe Arg Val Cys Leu Lys His 50 55 60 Phe Gln Ala Val Val Ser
Pro Gly Pro Cys Thr Phe Gly Thr Val Ser 65 70 75 80 Thr Pro Val Leu
Gly Thr Asn Ser Phe Ala Val Arg Asp Asp Ser Ser 85 90 95 Gly Gly
Gly Arg Asn Pro Leu Gln Leu Pro Phe Asn Phe Thr Trp Pro 100 105 110
Gly Thr Phe Ser Leu Ile Ile Glu Ala Trp His Ala Pro Gly Asp Asp 115
120 125 Leu Arg Pro Glu Ala Leu Pro Pro Asp Ala Leu Ile Ser Lys Ile
Ala 130 135 140 Ile Gln Gly Ser Leu Ala Val Gly Gln Asn Trp Leu Leu
Asp Glu Gln 145 150 155 160 Thr Ser Thr Leu Thr Arg Leu Arg Tyr Ser
Tyr Arg Val Ile Cys Ser 165 170 175 Asp Asn Tyr Tyr Gly Asp Asn Cys
Ser Arg Leu Cys Lys Lys Arg Asn 180 185 190 Asp His Phe Gly His Tyr
Val Cys Gln Pro Asp Gly Asn Leu Ser Cys 195 200 205 Leu Pro Gly Trp
Thr Gly Glu Tyr Cys Gln Gln Pro Ile Cys Leu Ser 210 215 220 Gly Cys
His Glu Gln Asn Gly Tyr Cys Ser Lys Pro Ala Glu Cys Leu 225 230 235
240 Cys Arg Pro Gly Trp Gln Gly Arg Leu Cys Asn Glu Cys Ile Pro His
245 250 255 Asn Gly Cys Arg His Gly Thr Cys Ser Thr Pro Trp Gln Cys
Thr Cys 260 265 270 Asp Glu Gly Trp Gly Gly Leu Phe Cys Asp Gln Asp
Leu Asn Tyr Cys 275 280 285 Thr His His Ser Pro Cys Lys Asn Gly Ala
Thr Cys Ser Asn Ser Gly 290 295 300 Gln Arg Ser Tyr Thr Cys Thr Cys
Arg Pro Gly Tyr Thr Gly Val Asp 305 310 315 320 Cys Glu Leu Glu Leu
Ser Glu Cys Asp Ser Asn Pro Cys Arg Asn Gly 325 330 335 Gly Ser Cys
Lys Asp Gln Glu Asp Gly Tyr His Cys Leu Cys Pro Pro 340 345 350 Gly
Tyr Tyr Gly Leu His Cys Glu His Ser Thr Leu Ser Cys Ala Asp 355 360
365 Ser Pro Cys Phe Asn Gly Gly Ser Cys Arg Glu Arg Asn Gln Gly Ala
370 375 380 Asn Tyr Ala Cys Glu Cys Pro Pro Asn Phe Thr Gly Ser Asn
Cys Glu 385 390 395 400 Lys Lys Val Asp Arg Cys Thr Ser Asn Pro Cys
Ala Asn Gly Gly Gln 405 410 415 Cys Leu Asn Arg Gly Pro Ser Arg Met
Cys Arg Cys Arg Pro Gly Phe 420 425 430 Thr Gly Thr Tyr Cys Glu Leu
His Val Ser Asp Cys Ala Arg Asn Pro 435 440 445 Cys Ala His Gly Gly
Thr Cys His Asp Leu Glu Asn Gly Leu Met Cys 450 455 460 Thr Cys Pro
Ala Gly Phe Ser Gly Arg Arg Cys Glu Val Arg Thr Ser 465 470 475 480
Ile Asp Ala Cys Ala Ser Ser Pro Cys Phe Asn Arg Ala Thr Cys Tyr 485
490 495 Thr Asp Leu Ser Thr Asp Thr Phe Val Cys Asn Cys Pro Tyr Gly
Phe 500 505 510 Val Gly Ser Arg Cys Glu Phe Pro Val Gly 515 520
2663PRTHomo sapiens 26Trp Leu Leu Asp Glu Gln Thr Ser Thr Leu Thr
Arg Leu Arg Tyr Ser 1 5 10 15 Tyr Arg Val Ile Cys Ser Asp Asn Tyr
Tyr Gly Asp Asn Cys Ser Arg 20 25 30 Leu Cys Lys Lys Arg Asn Asp
His Phe Gly His Tyr Val Cys Gln Pro 35 40 45 Asp Gly Asn Leu Ser
Cys Leu Pro Gly Trp Thr Gly Glu Tyr Cys 50 55 60 27154PRTHomo
sapiens 27Met Ala Ala Ala Ser Arg Ser Ala Ser Gly Trp Ala Leu Leu
Leu Leu 1 5 10 15 Val Ala Leu Trp Gln Gln Arg Ala Ala Gly Ser Gly
Val Phe Gln Leu 20 25 30 Gln Leu Gln Glu Phe Ile Asn Glu Arg Gly
Val Leu Ala Ser Gly Arg 35 40 45 Pro Cys Glu Pro Gly Cys Arg Thr
Phe Phe Arg Val Cys Leu Lys His 50 55 60 Phe Gln Ala Val Val Ser
Pro Gly Pro Cys Thr Phe Gly Thr Val Ser 65 70 75 80 Thr Pro Val Leu
Gly Thr Asn Ser Phe Ala Val Arg Asp Asp Ser Ser 85 90 95 Gly Gly
Gly Arg Asn Pro Leu Gln Leu Pro Phe Asn Phe Thr Trp Pro 100 105 110
Gly Thr Phe Ser Leu Ile Ile Glu Ala Trp His Ala Pro Gly Asp Asp 115
120 125 Leu Arg Pro Glu Ala Leu Pro Pro Asp Ala Leu Ile Ser Lys Ile
Ala 130 135 140 Ile Gln Gly Ser Leu Ala Val Gly Gln Asn 145 150
28138PRTArtificial Sequencem-21M18-Vh 28Met Gly Trp Ser Trp Ile Phe
Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Ser Glu Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Thr Gly Ala
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45 Thr
Ala Tyr Tyr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55
60 Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn
65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser
Ser Ser 85 90 95 Thr Ala Phe Met Gln Phe Asn Ser Leu Thr Ser Glu
Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Tyr Asp Tyr Asp
Val Gly Met Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Ser Val Thr Val
Ser Ser 130 135 29127PRTArtificial SequenceEST-Framework 29Met Asp
Trp Thr Trp Ser Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15
Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20
25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr
Phe 35 40 45 Gly Thr Tyr Ala Val Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu 50 55 60 Glu Trp Met Gly Arg Ile Ile Pro Met Leu Glu
Arg Pro Asn Tyr Ala 65 70 75 80 Gln Lys Phe Gln Gly Arg Val Thr Phe
Thr Thr Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg
Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125
30138PRTArtificial SequenceH7 30Met Asp Trp Thr Trp Ser Ile Leu Phe
Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45 Thr Ala Tyr
Tyr Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu
Trp Ile Gly Tyr Ile Ser Ser Tyr Asn Gly Ala Thr Asn Tyr Asn 65 70
75 80 Gln Lys Phe Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr
Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp
Thr Ala Val 100 105
110 Tyr Tyr Cys Ala Arg Asp Tyr Asp Tyr Asp Val Gly Met Asp Tyr Trp
115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135
31138PRTArtificial Sequencem-21M18-Vh 31Met Gly Trp Ser Trp Ile Phe
Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Ser Glu Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Thr Gly Ala
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45 Thr
Ala Tyr Tyr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55
60 Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn
65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser
Ser Ser 85 90 95 Thr Ala Phe Met Gln Phe Asn Ser Leu Thr Ser Glu
Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Tyr Asp Tyr Asp
Val Gly Met Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Ser Val Thr Val
Ser Ser 130 135 32127PRTArtificial Sequenceh-Germline-Vh 32Met Asp
Trp Thr Trp Ser Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15
Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20
25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe 35 40 45 Thr Ser Tyr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu 50 55 60 Glu Trp Met Gly Trp Ile Ser Ala Tyr Asn Gly
Asn Thr Asn Tyr Ala 65 70 75 80 Gln Lys Leu Gln Gly Arg Val Thr Met
Thr Thr Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg
Ser Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125
33138PRTArtificial Sequence21M18-H2 33Met Asp Trp Thr Trp Ser Ile
Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45 Thr
Ala Tyr Tyr Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu 50 55
60 Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn
65 70 75 80 Gln Lys Phe Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser
Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp
Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Tyr Asp Tyr Asp
Val Gly Met Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 130 135 34131PRTArtificial Sequencem-21M18-Vk 34Met Glu Ser
Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly
Ser Lys Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala 20 25
30 Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser
35 40 45 Val Asp Asn Tyr Gly Ile Ser Phe Met Lys Trp Phe Gln Gln
Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser
Asn Gln Gly Ser 65 70 75 80 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Ser 85 90 95 Leu Asn Ile His Pro Met Glu Glu
Asp Asp Thr Ala Met Tyr Phe Cys 100 105 110 Gln Gln Ser Lys Glu Val
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys 130
35134PRTArtificial Sequenceh-germline-Vk 35Met Val Leu Gln Thr Gln
Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser
Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser 35 40 45
Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50
55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
Asp Val Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro
Pro Trp Thr Phe Gly Gln Gly 115 120 125 Thr Lys Val Glu Ile Lys 130
36131PRTArtificial Sequence21M18-L2 36Met Val Leu Gln Thr Gln Val
Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp
Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser Leu
Gly Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser 35 40 45 Val
Asp Asn Tyr Gly Ile Ser Phe Met Lys Trp Phe Gln Gln Lys Pro 50 55
60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser
65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr 85 90 95 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys 100 105 110 Gln Gln Ser Lys Glu Val Pro Trp Thr Phe
Gly Gly Gly Thr Lys Val 115 120 125 Glu Ile Lys 130
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