U.S. patent application number 10/268882 was filed with the patent office on 2003-05-01 for treatment of prostate cancer by inhibitors of ncam2.
Invention is credited to Green, Jennifer McPhate, Tso, J. Yun.
Application Number | 20030082188 10/268882 |
Document ID | / |
Family ID | 26986685 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082188 |
Kind Code |
A1 |
Tso, J. Yun ; et
al. |
May 1, 2003 |
Treatment of prostate cancer by inhibitors of NCAM2
Abstract
The present invention is directed to a method of treating or
detecting prostate cancer or breast cancer in a subject in need of
such a treatment or detection by administering an inhibitor of
NCAM2 in a pharmaceutically effective amount. The present invention
also provides for a pharmaceutical composition comprising an
inhibitor of NCAM2.
Inventors: |
Tso, J. Yun; (Menlo Park,
CA) ; Green, Jennifer McPhate; (Belmont, CA) |
Correspondence
Address: |
HOWREY SIMON ARNOLD & WHITE, LLP
BOX 34
301 RAVENSWOOD AVE.
MENLO PARK
CA
94025
US
|
Family ID: |
26986685 |
Appl. No.: |
10/268882 |
Filed: |
October 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60329178 |
Oct 11, 2001 |
|
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60331965 |
Nov 21, 2001 |
|
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Current U.S.
Class: |
424/155.1 ;
530/388.26 |
Current CPC
Class: |
C07K 16/3015 20130101;
G01N 2405/00 20130101; C07K 2317/73 20130101; C07K 16/30 20130101;
C07K 16/40 20130101; A61K 2039/505 20130101; C07K 16/2833 20130101;
C07K 16/3069 20130101; G01N 33/57492 20130101 |
Class at
Publication: |
424/155.1 ;
530/388.26 |
International
Class: |
A61K 039/395; C07K
016/30 |
Claims
1. A method of treating prostate cancer in a subject in need of the
treating comprising administering to said subject an inhibitor of
NCAM2 or RNCAM in a pharmaceutically effective amount.
2. The method according to claim 1, wherein said inhibitor is a
protein that directly interacts with NCAM2.
3. The method according to claim 1, wherein said inhibitor
down-regulates biological activities of NCAM2.
4. The method according to claim 1, wherein said inhibitor inhibits
protein expression of NCAM2.
5. The method according to claim 4, wherein said inhibitor is an
anti-sense nucleic acid of a nucleic acid sequence encoding part or
full NCAM2.
6. The method according to claim 4, wherein said inhibitor is a
transcriptional factor.
7. The method according to claim 2, wherein said inhibitor is an
antibody that binds to NCAM2 or neutralizes biological activities
of NCAM2.
8. The method according to claim 7, wherein said antibody inhibits
prostate cancer cell proliferation by at least 10%.
9. The method according to claim 7, wherein said antibody inhibits
prostate cancer cell proliferation by at least 35%.
10. The method according to claim 7, wherein said antibody is a
monoclonal antibody.
11. The method according to claim 10, wherein said monoclonal
antibody is a humanized antibody or a fully human antibody.
12. The method according to claim 10, wherein said monoclonal
antibody is a chimeric antibody.
13. The method according to claim 10, wherein said antibody is an
antibody tetramer, Fab, (Fab').sub.2, or Fv.
14. The method according to claim 1, wherein said inhibitor is an
antibody conjugate comprising an anti-NCAM2 antibody.
15. The method according to claim 14, wherein said anti-NCAM2
antibody is conjugated to a cytotoxin agent.
16. The antibody conjugate according to claim 15, wherein said
cytotoxin agent is a protein cytotoxin or a Fc domain of a
monoclonal antibody.
17. The method according to claim 1, further comprising
administering a chemotherapeutic agent to the subject, wherein said
treating is formulated in a manner allowing it to be administered
serially or in combination with another agent for treatment of
cancer.
18. The method according to claim 1, further comprising
administering radiation therapy to the subject, wherein said
treating is formulated in a manner allowing it to be administered
serially or in combination with another agent for treatment of
cancer.
19. A method of detecting prostate cancer comprising detecting
presence of NCAM2 in prostate cells of a subject in need of the
detecting.
20. A method of detecting breast cancer comprising detecting
presence of NCAM2 in breast cells of a subject in need of the
detecting.
21. The method according to claim 19 or claim 20, wherein said
detecting comprising using an antibody conjugate, wherein said
antibody conjugate comprises an antibody or an antibody fragment
that binds to at least one epitope of human NCAM2, wherein said
antibody or antibody fragment is conjugated to a diagnostic imaging
agent.
22. A pharmaceutical composition comprising an inhibitor of NCAM2
and a pharmaceutical carrier.
23. The pharmaceutical composition according to claim 22, wherein
said inhibitor is an anti-NCAM2 antibody.
24. The pharmaceutical composition according to claim 23, wherein
said anti-NCAM2 antibody is a humanized or human anti-NCAM2
antibody.
25. An antibody that binds to or neutralizes NCAM2.
26. The antibody according to claim 25, wherein said antibody
inhibit tumor cell proliferation by more than 10%
27. The antibody according to claim 25, wherein said antibody
inhibit tumor cell proliferation by more than 30%
28. The antibody according to claim 25, wherein said tumor cell is
a prostate cancer cell.
29. The antibody according to claim 25, wherein said antibody
inhibits prostate cancer cell colony formation.
30. The antibody according to claim 25, wherein said antibody
inhibits prostate cancer cell colony formation by more than
20%.
31. The antibody according to claim 25, wherein said antibody
inhibits prostate cancer cell colony formation by more than
40%.
32. The antibody according to claim 25, wherein said antibody is a
humanized anti-NCAM2 antibody.
33. The antibody according to claim 25, wherein said antibody is a
fully human anti-NCAM2 antibody.
34. The antibody according to claim 25, wherein said antibody is an
antibody tetramer Fab, (Fab').sub.2, or Fv.
35. A method of treating breast cancer in a subject in need of the
treating comprising administering to said subject an inhibitor of
NCAM2 or RNCAM in a pharmaceutically effective amount.
36. A hybridoma cell line P9-64 deposited with American Type
Culture Collection (ATCC) as accession number PTA-4734.
37. A monoclonal antibody produced by the hybridoma cell line
according to claim 36.
38. A method of inhibiting growth of a cancer cell comprising
contacting an inhibitor of NCAM2 with said cancer cell.
39. The method according to claim 38, wherein said cancer cell is a
prostate cancer cell.
40. The method according to claim 38, wherein said inhibitor is an
antibody that binds to or neutralizes NCAM2.
41. The method according to claim 40, wherein said antibody
inhibits prostate cancer cell proliferation.
42. The method according to claim 41, wherein said antibody
inhibits prostate cancer cell proliferation by at least 30%.
Description
[0001] This application claims the benefit of priority of the U.S.
provisional application U.S. Ser. No. 60/329,178 filed Oct. 11,
2001 and the U.S. provisional application U.S. Ser. No. 60/331,965,
filed Nov. 21, 2001, each of which is incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0002] This invention concerns methods for treating prostate cancer
or breast cancer by inhibitors of RNCAM, preferably, NCAM2.
BACKGROUND OF THE INVENTION
[0003] Prostate cancer is the most common cancer in men with an
estimated 244,000 cases in 1995 in the United States. It is the
second leading cause among men who die from neoplasia with an
estimated 44,000 deaths per year. Prompt detection and treatment is
needed to limit mortality caused by prostate cancer. As described
in W. J. Catalona, "Management of Cancer of the Prostate," (New
Engl. J. Med. 331(15): 996-1004 (1994)), the management of prostate
cancer can be achieved by watchful waiting, curative treatment, and
palliation.
[0004] A number of approaches have been developed to treat prostate
cancer. Where prostate cancer is localized and the patient's life
expectancy is 10 years or more, radical prostatectomy offers the
best chance for eradication of the disease. Historically, the
drawback of this procedure is that most cancers had spread beyond
the bounds of the operation by the time they were detected. After
surgery, if there are detectable serum prostate-specific antigen
concentrations (PSA), persistent cancer is indicated. In many
cases, prostate-specific antigen concentrations can be reduced by
radiation treatment. However, this concentration often increases
again within two years.
[0005] Cytotoxic chemotherapy is largely ineffective in treating
prostate cancer. Its toxicity makes such therapy unsuitable for
elderly patients. In addition, prostate cancer is relatively
resistant to cytotoxic agents.
[0006] In view of the deficiency of the existing treatment
approaches, it is of great significance to pursue new methods of
treatment that particularly target the prostate tumor cells, such
as anti-prostate tumor agents. The present invention has identified
a novel tumor marker for prostate cancer, RNCAM, and is intended to
use the inhibitors of this molecule as such anti-tumor agents.
[0007] NCAM (neural cell adhesion molecule) is the first molecule
mediating cell-cell adhesion to be identified on the basis of
functional criteria (Thiery, et al., J. Biol. Chem. 252: 6841-45
(1977)). Common features of these proteins are immunoglobulin-like
domains in their NH2-terminal extracellular parts and sequences
similar to fibronectin type III repeats carboxyl-terminal to them.
Originally identified in chick embryo nervous system, NCAM is now
known to be expressed in a great variety of tissues and cell types
of all vertebrate species, and play important roles in axonal
pathfinding and neurite outgrowth (Goridis, et al., Seminars in
Cell Biology, Vol. 3, pp189-197 (1992); Baldwin et al., J. of
Cellular Biochemistry 61: 502-513 (1996)).
[0008] Neural cell adhesion molecule 2 (NCAM2, NCBI protein
accession number 4758764, see also Paoloni-Giacobino et al,
Genomics 43, 43-51 (1997)) is a homologue of a murine protein
called Rb-8 neural adhesion molecule (RNCAM, NCBI protein accession
number 3334269, see also Alenius, M. and Bohm, S., J. Biol. Chem.
272, 26083-26083 (1997)). RNCAM is a novel molecule in NCAM family.
Its amino acid sequence and protein expression pattern are
different from the existing NCAM molecules. (Alenius, M. and Bohm,
S., J. (1997)) The sequence of RNCAM predicted molecules having an
extracellular region of 5 immunoglobulin C2-type domains followed
by two fibronectin type III domains. Alternative splicing of the
NCAM2 and RNCAM transcripts generate two isoforms: the long form
containing a transmembrane domain and the short form containing a
glycosylphosphatidylinositol-anchor attached to the membrane. The
expression of RNCAM is restricted to the olfactory neurons in the
brain and in the nasal vomeronasal organ. The transcript of RNCAM
is not detectable in lung, gut, liver, heart, testis and kidney.
The function of NCAM2 or RNCAM is not known, but the molecule may
play a role in selective axon projection.
[0009] So far no investigation has been conducted to explore the
possible therapeutic application of inhibitors of RNCAM (NCAM2) in
prostate cancer treatment. This invention is directed to methods
for treating or preventing prostate cancer or breast cancer by
using the inhibitors of RNCAM, preferably, NCAM2.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to a method of treating or
preventing prostate cancer or breast cancer in a subject in need of
such a treatment by administering an inhibitor of NCAM2 in a
pharmaceutically effective amount.
[0011] The present invention also provides a pharmaceutical
composition comprising an inhibitor of NCAM2.
[0012] The present invention further provides for a method of
detecting a cancer derived from a type of cells comprising
detecting presence of NCAM2 in said type of cells.
[0013] Preferably, the present invention provides for a method of
detecting prostate cancer or breast cancer comprising detecting
presence of NCAM2 in prostate cells or breast cancer cells of a
subject in need of the detecting.
[0014] The present invention also provides for a method of
inhibiting growth of a cancer cell comprising contacting an
inhibitor of NCAM2 with said cancer cell. Preferably, said cancer
cell is a prostate cancer cell or a breast cancer cell.
[0015] The cancers can be treated or detected by the methods of the
present invention include, but are not limited to, Hodgkin's
Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma,
breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma,
primary thrombocytosis, primary macroglobulinemia, small-cell lung
tumors, primary brain tumors, stomach cancer, colon cancer,
malignant pancreatic insulanoma, malignant carcinoid, urinary
bladder cancer, premalignant skin lesions, testicular cancer,
lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,
genitourinary tract cancer, malignant hypercalcemia, cervical
cancer, endometrial cancer, adrenal cortical cancer, and
leukemia.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. Flow chart summarizing how the tumor-specific
hybridomas were obtained from the LNCaP lipid raft
immunization.
[0017] FIG. 2. Antigen grouping by immunoprecipitation. .sup.125I
labeled LNCaP lysate was incubated individually with 20 hybridoma
supernatants (see Table 1). Antibody-antigen complexes were
captured by Gamma Bind Plus Sepharose and analyzed by SDS-PAGE.
Lane 1, P1-42; Lane 2, P2-23; Lane 3, P3-53; Lane 4, P4-79; Lane 5,
P6-49; Lane 6, P9-65; Lane 7, P8-2; Lane 8, P8-11; Lane 9, P8-14;
Lane 10, P8-35; Lane 11, P8-74; Lane 12, P9-32; Lane 13, P9-64;
Lane 14, P10-2; Lane 15, P10-28, Lane 16, P10-29; Lane 17, P10-62;
Lane 18, P10-70; Lane 19, P10-82; and Lane 20, P12-22. Molecular
weight standards (MW) are in kD.
[0018] FIG. 3. Anti-NCAM2 inhibits LNCaP cell proliferation. LNCaP
cells (20,000 cells/well) were plated into a 96 well tissue culture
plate. After cells were allowed to grow undisturbed for two days, 4
different NCAM2-specific antibodies (5 .mu.g/ml) were added and
incubated with the cells for 24 hours. AlamarBlue reagent was added
to assess cell proliferation. Fluorescence was detected at
.lambda.ex=530 nm, .lambda.em=590 nm. Data are expressed as the
mean +/- SEM of 4 replicates.
[0019] FIG. 4. Anti-NCAM2 inhibits LNCaP colony formation in soft
agar. LNCaP cells were plated in soft agar and treated with 4
different NCAM2-specific antibodies (5 .mu.g/ml) for up to 20 days.
Colonies were counted under an inverted phase-contrast microscope
and a group of 10 or more cells were counted as a colony.
[0020] FIG. 5. Table 1 shows the reactivity profiles of 20
anti-LNCaP lipid raft hybridomas that showed limited binding to
other cancer cell lines.
[0021] FIG. 6. Table 3 shows the reactivity profiles of 1
anti-LNCaP lipid raft hybridomas that showed broad binding to other
cancer cell lines.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] Definitions:
[0023] As used herein, the term "antibody" or "immunoglobulin"
refers to a protein consisting of one or more polypeptides
substantially encoded by immunoglobulin genes. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma
(IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4), delta, epsilon and mu
constant region genes, as well as the myriad immunoglobulin
variable region genes. Full-length immunoglobulin "light chains"
(about 25 Kd or 214 amino acids) are encoded by a variable region
gene at the NH2-terminus (about 110 amino acids) and a kappa or
lambda constant region gene at the COOH--terminus. Full-length
immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids), are
similarly encoded by a variable region gene (about 116 amino acids)
and one of the other aforementioned constant region genes, e.g.,
gamma (encoding about 330 amino acids).
[0024] One form of immunoglobulin constitutes the basic structural
unit of an antibody. This form is a tetramer and consists of two
identical pairs of immunoglobulin chains, each pair having one
light and one heavy chain. In each pair, the light and heavy chain
variable regions are together responsible for binding to an
antigen, and the constant regions are responsible for the antibody
effector functions. In addition to antibodies, immunoglobulins may
exist in a variety of other forms including, for example, Fv, Fab,
and (Fab').sub.2, as well as bifunctional hybrid antibodies (e.g.,
Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single
chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85,
5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988),
which are incorporated herein by reference). (See, generally, Hood
et al., "Immunology", Benjamin, N.Y., 2nd ed. (1984), and
Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are
incorporated herein by reference).
[0025] The preferred antibody is the monoclonal antibody that binds
to or neutralizes RNCAM, preferably NCAM2.
[0026] By "a pharmaceutically effective" amount of a drug or
pharmacologically active agent or pharmaceutical formulation is
meant a nontoxic but sufficient amount of the drug, agent or
formulation to provide the desired effect.
[0027] A "subject," "individual" or "patient" is used
interchangeably herein, which refers to a vertebrate, preferably a
mammal, more preferably a human.
[0028] The term "inhibit growth of cancer (tumor) cells" refers to
any action that may decrease the growth of a cancer cell. The
inhibition may reduce the growth rate or the size of cancer cells,
or inhibit or prevent proliferation, growth, or migration of cancer
cells. The inhibition may inhibit the colony formation of cancer
cells due to the inhibition of anchorage-independent growth.
Preferably, such an inhibition at the cellular level may reduce the
size, deter the growth, reduce the aggressiveness, or prevent or
inhibit metastasis of a tumor in a patient.
[0029] The term "colony formation" refers to the number of cancer
(tumor) cell colonies formed due to the anchorage-independent
cancer (tumor) cell growth. A variety of methods can be used to
measure the "colony formation", such as counting the number of the
formed colonies (see Examples).
[0030] The term "lipid raft" refers to a lipid raft or a portion
thereof in a clustered state or a non-clustered state, including
"lipid raft", "clustered lipid rafts", and "DRM", each of which has
been described in detail in Simons, K., et al., Nature
Reviews/Molecular Cell Biology: Vol. 1 pp 31-39 (2000). In
particular, "lipid raft" contains a given set of proteins that can
change size and composition in response to intra- or extracellular
stimuli. This favors specific protein-protein interactions,
resulting in the activation of signally cascade. Sometimes, the
lipid rafts may be clustered together. It has been reported that
clustering is used both artificially and physiologically to trigger
signally cascades. DRMs (detergent-resistant membranes) are the
rafts that remain insoluble after treatment on ice with detergents,
such as Triton X-100 or NP-40. They are believed to be non-native
aggregated rafts.
[0031] The term "epitope" includes any protein determinant capable
of specific binding to an immunoglobulin or an antibody. Epitopic
determinants usually consist of active surface groupings of
molecules such as amino acids or sugar side chains and usually have
specific three-dimensional structural characteristics, as well as
specific charge characteristics. Two antibodies are said to bind to
the same epitope of a protein if amino acid mutations in the
protein that reduce or eliminate binding of one antibody also
reduce or eliminate binding of the other antibody, and/or if the
antibodies compete for binding to the protein, i.e., binding of one
antibody to the protein reduces or eliminates binding of the other
antibody.
[0032] The term "derived from" means "obtained from" or "produced
by".
[0033] The term "genetically altered antibodies" means antibodies
wherein the amino acid sequence has been varied from that of a
native antibody. Because of the relevance of recombinant DNA
techniques to this invention, one need not be confined to the
sequences of amino acids found in natural antibodies; antibodies
can be redesigned to obtain desired characteristics. The possible
variations are many and range from the changing of just one or a
few amino acids to the complete redesign of, for example, the
variable or constant region. Changes in the constant region will,
in general, be made in order to improve or alter characteristics,
such as complement fixation, interaction with membranes and other
effector functions. Changes in the variable region will be made in
order to improve the antigen binding characteristics.
[0034] The term "humanized antibody" or "humanized immunoglobulin"
refers to an immunoglobulin comprising a human framework, at least
one and preferably all complimentarity determining regions (CDRs)
from a non-human antibody, and in which any constant region present
is substantially identical to a human immunoglobulin constant
region, i.e., at least about 85-90%, preferably at least 95%
identical. Hence, all parts of a humanized immunoglobulin, except
possibly the CDRs, are substantially identical to corresponding
parts of one or more native human immunoglobulin sequences. See,
e.g. Queen et al., U.S. Pat. Nos. 5,5301,101; 5,585,089; 5,693,762;
and 6,180,370 (each of which is incorporated by reference in its
entirety).
[0035] The term "chimeric antibody" refers to an antibody in which
the constant region comes from an antibody of one species
(typically human) and the variable region comes from an antibody of
another species (typically rodent).
[0036] The present invention provides a method of treating prostate
cancer or breast cancer.
[0037] In a preferred embodiment of the present invention, an
inhibitor of RNCAM is administered to a subject in need of such a
treatment in a pharmaceutically effective amount. Preferably, said
RNCAM is a human NCAM2.
[0038] The present invention also provide for a method of
inhibiting growth of a cancer cell comprising contacting an
inhibitor of RNCAM, preferably NCAN2 with said cancer cell.
Preferably, said cancer cell is a prostate cancer cell or a breast
cancer cell. Preferably, said inhibiting reduces the proliferation
of a cancer cell by more than 10%, more preferably, by more than
30%, even more preferably, by about 39%. In addition, said
inhibiting can reduce the colony formation of a cancer cell,
preferably, a prostate cancer cell, by more than 20%, more
preferably, by more than 40%, even more preferably, by about
42%.
[0039] Preferably, the inhibitor is a protein.
[0040] Preferably, the protein directly interacts with RNCAM,
preferably NCAM2.
[0041] Preferably, the protein binds to RNCAM, preferably
NCAM2.
[0042] More preferably, the inhibitor is an anti-RNCAM (preferably
anti-NCAM2) antibody or antibody fragment thereof. More preferably,
said antibody or the fragment thereof neutralizes RNCAM
(preferably, NCAM2) biological activity.
[0043] In addition, the inhibitor can also be a compound that
inhibits the biological activities of RNCAM (preferably, NCAM2).
More examples include molecules that may interact with RNCAM
(preferably, NCAM2) signaling pathway and down-regulates the
activity of RNCAM (preferably, NCAM2). The understanding of NCAM
signaling pathway provides the useful information for the search of
this kind of compounds. It has been reported that antagonists of
calcium channels can inhibit NCAM activities (NCAM-dependent
neurite outgrowth). In additions, growth factors such as FGF may
regulate the activities of NCAM (Baldwin et al.). Such
understanding will provide the starting point for the search of the
molecules down-regulating NCAM2. The inhibitors can also be mutant
RNCAM (preferably, NCAM2) derived from a wild-type RNCAM
(preferably, NCAM2) by terminal truncation or amino acid
substitution. Preferably such mutant NCAM2 can retain the binding
of the NCAM2 to it signaling molecule but lose the ability of
triggering the signaling cascade of NCAM2 and thus blocking the
biological activity of NCAM2. The suitable compounds can be sought
by using the conventional techniques known to a skilled artisan in
the field of molecular biology, organic chemistry and
biochemistry.
[0044] In addition, the inhibitor can inhibit the protein
expression of RNCAM (preferably, NCAM2). RNCAM expression can be
regulated at the level of alternative splicing of RNCAM mRNA, or by
a transcription factor (Gorisdis, C., et al.). The inhibitor in the
present invention includes the down-regulating molecules at each
level. The inhibitor can also be a nucleic acid, including but not
limited to, an anti-sense nucleic acid of the nucleic acid sequence
encoding part or full or having substantial sequence similarity of
RNCAM (preferably, NCAM2). The DNA sequence of NCAM2 is known in
the art. Subsequently, anti-sense nucleic acid probe of DNA of
NCAM2, and the optimal condition of the anti-sense blocking can be
developed by using the related techniques known to a skilled
artisan in the field of molecular biology.
[0045] The present invention also provides for a pharmaceutical
composition comprising an inhibitor of RNCAM (preferably, NCAM2).
The pharmaceutical composition can further comprise a
pharmaceutically acceptable carrier. Preferably, such a composition
comprises anti-RNCAM antibodies, more preferably, anti-NCAM2
antibodies.
[0046] The effective treatment of prostate cancer by the inhibitors
of RNCAM (preferably, NCAM2) includes various stages, such as
androgen-dependent prostate cancer and androgen-independent
prostate cancer.
[0047] The anti-NCAM2 antibodies may be in a polyclonal or
monoclonal form and may bind to any epitope or subunit of NCAM2).
Preferably, the anti-NCAM2 antibody is an isolated monoclonal
antibody. Anti-NCAM2 antibodies of all species of origins are
included. Non-limiting exemplary anti-NCAM2 antibodies include
antibodies derived from human, chicken, goats, and rodents (e.g.,
rats, mice, hamsters and rabbits), including transgenic rodents
genetically engineered to produce human antibodies (see, e.g.,
Lonberg et al., WO93/12227 (1993) and Kucherlapati, et al.,
WO91/10741 (1991)), which are herein incorporated by reference in
their entirety).
[0048] Preferably, the anti-NCAM2 antibody is an isolated
monoclonal antibody that binds to or neutralizes NCAM2.
[0049] Preferably, the antibodies can inhibit the proliferation of
a cancer cell by at least 10%, preferably at least 15%, and more
preferably at least 30% or 35%, or as high as 39%. Preferably, such
a cancer is a malignant solid tumor, more preferably, a prostate
cancer.
[0050] Preferably, the antibodies inhibit prostate cancer cell
colony formation, more preferably, by more than 20% or by more than
40%.
[0051] In one aspect of the present invention, the anti-NCAM2
antibodies can also bind to an isolated lipid raft. Preferably, the
isolated lipid raft is derived from a cancer cell, and more
preferably a prostate cancer cell. The present invention for the
first time discovers the association of NCAM2 with lipid rafts of
cancer cells. Immunization of a host animal, for example mouse,
with the lipid rafts obtained from prostate cancer cells gives rise
to hybridoma antibodies that bind to NCAM2 as well as the lipid
raft. Accordingly, NCAM2 exists as a component of the lipid rafts
in cancer cells. These hybridoma antibodies include, but are not
limited to, the antibodies produced by the hybridoma cell line
P3-53, P9-64, P10-28, or P10-29.
[0052] Preferably, the present invention provides a hybridoma cell
line P9-64 deposited with American Type Culture Collection (ATCC)
as accession number PTA-4734. The deposit of this hybridoma cell
line (PTA-4734) was received by American Type Culture Collection
(ATCC) on Oct. 1, 2002.
[0053] More preferably, the present invention provides a monoclonal
antibody produced by the hybridoma cell line having an ATCC
Accession Number PTA-4734.
[0054] The polyclonal forms of these antibodies can be produced by
immunization of host animals by NCAM2. The polyclonal antibodies
are secreted into the bloodstream and can be recovered using known
techniques. Purified forms of these antibodies can, of course, be
readily prepared by standard purification techniques, preferably
including affinity chromatography with Protein A,
anti-immunoglobulin, or the antigen itself. In any case, in order
to monitor the success of immunization, the antibody levels with
respect to the antigen in serum will be monitored using standard
techniques such as ELISA, RIA and the like.
[0055] The monoclonal antibodies can be produced by conventional
hybridoma methodology known in the art. In particular, after the
immunization with NCAM2, the host animal may be sacrificed and the
lymphocytes of said animal are isolated. The lymphocytes can
produce or be capable of producing antibodies that specifically
bind to the protein used for immunization. Lymphocytes then are
fused with myeloma cells using suitable fusing agents to form
hybridomas cells that produce the desired monoclonal antibody.
[0056] The antibodies can also be produced by using the method of
lipid raft immunization, which is disclosed in Examples and U.S.
Ser. No. 60/331,965, hereby incorporated by reference in its
entirety. The present invention discovered the association of NCAM2
with the lipid rafts derived from cancer cells. Accordingly, the
anti-NCAM2 antibodies can be produced by immunizing host animals
with isolated lipid rafts from cancer cells.
[0057] Antibodies useful in the present invention also may be made
using phage display methods (see, e.g., Dower et al., WO91/17271
and McCafferty et al., WO92/01047, which are herein incorporated by
reference in their entirety.
[0058] The present invention also includes genetically altered
anti-NCAM2 antibodies that are functionally equivalent to above
antibodies and antibody fragments. Modified antibodies providing
improved stability and/or therapeutic efficacy are preferred.
Examples of modified antibodies include those with conservative
substitutions of amino acid residues, and one or more deletions or
additions of amino acids which do not significantly deleteriously
alter the antigen binding utility. Substitutions can range from
changing or modifying one or more amino acid residues to complete
redesign of a region as long as the therapeutic utility is
maintained. Antibodies of this invention can be can be modified
post-translationally (e.g., acetylation, and phosphorylation) or
can be modified synthetically (e.g., the attachment of a labeling
group). Fragments of these modified antibodies that retain the
binding specificity can also be used.
[0059] The genetically altered antibodies also include chimeric
antibodies that bind to and neutralize NCAM2. Preferably, the
chimeric antibodies comprise a variable region derived from a mouse
or rat and a constant region derived from a human so that the
chimeric antibody has a longer half-life and is less immunogenic
when administered to a human subject. The method of making chimeric
antibodies is known in the art.
[0060] Preferably, the genetically altered antibodies used in the
present invention include humanized antibodies that bind to or
neutralize NCAM2. More preferably, said humanized antibody
comprising CDRs of a mouse donor immunoglobulin and heavy chain and
light chain frameworks of a human acceptor immunoglobulin. The
method of making humanized antibody is disclosed in U.S. Pat. Nos.:
5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 each of
which is incorporated by reference in its entirety.
[0061] Anti-NCAM2 fully human antibody is also included in the
present invention. Fully human antibody against NCAM2 is produced
by a variety of techniques. First, trioma methodology may be used
to develop the fully human antibody. The basic approach and an
exemplary cell fusion partner, SPAZ-4, for use in this approach
have been described by Oestberg et al., Hybridoma 2:361-367 (1983);
Oestberg, U.S. Pat. No. 4,634,664;and Engleman et al., U.S. Pat.
No. 4,634,666 (each of which is incorporated by reference in its
entirety for all purposes).
[0062] Human antibodies against NCAM2 can also be produced from
non-human transgenic animals having transgenes encoding at least a
segment of the human immunoglobulin locus. Usually, the endogenous
immunoglobulin locus of such transgenic animals is functionally
inactivated. Preferably, the segment of the human immunoglobulin
locus includes unrearranged sequences of heavy and light chain
components. Both inactivation of endogenous immunoglobulin genes
and introduction of exogenous immunoglobulin genes can be achieved
by targeted homologous recombination, or by introduction of YAC
chromosomes. The transgenic animals resulting from this process are
capable of functionally rearranging the immunoglobulin component
sequences, and expressing a repertoire of antibodies of various
isotypes encoded by human immunoglobulin genes, without expressing
endogenous immunoglobulin genes. The production and properties of
animals having these properties are described in detail by, e.g.,
Lonberg et al., WO 93/12227 (1993); Kucherlapati, WO 91/10741
(1991) (each of which is incorporated by reference in its
entirety).
[0063] Phage display methods can also be used for obtaining human
anti-NCAM2 antibodies. The methods include the steps of screening a
DNA library from human B cells according to the general protocol
outlined by Huse et al., Science 246:1275-1281 (1989). Antibodies
binding to NCAM2 or a fragment thereof are selected. Sequences
encoding such antibodies (or binding fragments) are then cloned and
amplified. The protocol described by Huse is rendered more
efficient in combination with phage-display technology. See, e.g.,
Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047 (each
of which is incorporated by reference in its entirety).
[0064] The fragments of the above-described anti-NCAM2 antibodies,
which retain the binding specificity to NCAM2, are also included in
the present invention. Examples include, but are not limited to,
the heavy chains, the light chains, and the variable regions as
well as the truncated chains (truncated at the carboxyl end), which
is particularly useful for immunoscintigraphic procedures. Examples
of truncated chains include, but are not limited to Fab fragment
(consisting of the VL, VH, CL and CH1 domains): the Fd fragment
(consisting of the VH and CH1 domains); the Fv (consisting of VL
and VH domains of a single arm of an antibody); dab fragment
(consisting of a VH domain); isolated CDR regions; F(ab').sub.2
fragment, a bivalent fragment (comprising two Fab fragments linked
by a disulphide bridge at the hinge region). The truncated chains
can be produced by conventional biochemistry techniques, such as
enzyme cleavage, or recombinant DNA techniques, each of which is
known in the art.
[0065] The genes of the antibody fragments may be fused to
functional regions from other genes (e.g., enzymes, U.S. Pat. No.
5,004,692, which is incorporated by reference in its entirty) to
produce fusion proteins (e.g., immunotoxins) or conjugates having
novel properties.
[0066] When used therapeutically, the antibodies disclosed herein
may be used in unmodified form or may be modified with an effector
moiety that delivers a toxic effect, such as a drug, cytotoxin
(preferably, a protein cytotoxin or a Fc domain of the monoclonal
antibodies), radionuclide, etc (see, e.g., U.S. Pat. No. 6,086,900,
which is hereby incorporated by reference in its entirety).
[0067] Preferably, a pharmaceutical composition of the present
invention comprises the use of the subject antibodies in
immunotoxins. Conjugates that are immunotoxins including
conventional antibodies have been widely described in the art. The
toxins may be coupled to the antibodies by conventional coupling
techniques or immunotoxins containing protein toxin portions can be
produced as fusion proteins. The conjugates of the present
invention can be used in a corresponding way to obtain such
immunotoxins. Illustrative of such immunotoxins are those described
by Byers, B. S. et al. Seminars Cell Biol 2:59-70 (1991) and by
Fanger, M. W. et al. Immunol Today 12:51-54 (1991).
[0068] A variety of cytotoxic agents are suitable for use in
immunotoxins. Cytotoxic agents can include radionuclides, such as
Iodine-131 or other isotopes of iodine, Yttrium-90, Rhenium-188,
and Bismuth-212 or other alpha emitters; a number of
chemotherapeutic drugs, such as vindesine, methotrexate,
adriamycin, and cisplatinum; and cytotoxic proteins such as
ribosomal inhibiting proteins like pokeweed antiviral protein,
Pseudomonas exotoxin A, ricin, diphtheria toxin, ricin A chain,
etc., or an agent active at the cell surface, such as the
phospholipase enzymes (e.g., phospholipase C). (See, generally,
"Chimeric Toxins," Olsnes and Phil, Pharmac. Ther., 25, 355-381
(1982), and "Monoclonal Antibodies for Cancer Detection and
Therapy," eds. Baldwin and Byers, pp. 159-179, 224-266, Academic
Press (1985), all of which are incorporated herein by
reference.)
[0069] The delivery component of the immunotoxin will include the
antibodies described herein, including chimeric antibodies,
humanized antibodies, and human antibodies of the present
invention. Intact immunoglobulins or their binding fragments, such
as Fab, are preferably used. Typically, the antibodies in the
immunotoxins will be of the human IgM or IgG isotype, but other
mammalian constant regions may be utilized as desired.
[0070] There are various methods of administering the inhibitors.
The inhibitor may be administered to a patient intravenously as a
bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, topical,
inhalation routes, or other delivery means known to the people
skilled in the art.
[0071] Preferably, pharmaceutical compositions of the present
invention are useful for parenteral administration, i.e.,
subcutaneously, intramuscularly and particularly, intravenously.
The compositions for parenteral administration commonly comprise a
solution of the inhibitor, preferably the antibody, or a cocktail
thereof dissolved in an acceptable carrier, preferably an aqueous
carrier. A variety of aqueous carriers can be used, e.g., water,
buffered water, 0.4% saline, 0.3% glycine and the like. These
solutions are sterile and generally free of particulate matter. The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions such
as pH adjusting and buffering agents, toxicity adjusting agents and
the like, for example sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate, histidine and arginine.
The concentration of the inhibitors (preferably antibodies) in
these formulations can vary widely, i.e., from less than about
0.01%, usually at least about 0.1% to as much as 5% by weight and
are selected primarily based on fluid volumes, and solubilities in
accordance with the particular mode of administration selected.
[0072] Thus, a typical pharmaceutical composition for injection
could be made up to contain 1 ml sterile buffered water, and 1-100
mg of an inhibitor. A typical composition for intravenous infusion
can be made up to contain 250 ml of sterile Ringer's solution, and
10 mg of the inhibitor, such as anti-NCAM2 antibody. Actual methods
for preparing parentally administrable compositions are known or
apparent to those skilled in the art and are described in more
detail in, for example, Remington's Pharmaceutical Science (15th
Ed., Mack Publishing Company, Easton, Pa., 1980), which is
incorporated herein by reference.
[0073] The present invention provides for a pharmaceutical
composition comprising an inhibitor of NCAM2, preferably, an
antibody that binds to or neutralizes NCAM2.
[0074] For the purpose of treatment of disease, the appropriate
dosage of the above inhibitors will depend on the severity and
course of disease, the patient's clinical history and response, the
toxicity of the inhibitors, and the discretion of the attending
physician. The inhibitors are suitably administered to the patient
at one time or over a series of treatments. The initial candidate
dosage may be administered to a patient. The proper dosage and
treatment regime can be established by monitoring the progress of
therapy using conventional techniques known to the people skilled
of the art.
[0075] The amount of active ingredients that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. It will be understood, however, that the specific
dose level for any particular patient will depend upon a variety of
factors, including the activity of the specific inhibitor employed,
the age, body weight, general health, sex, diet, time of
administration, route of administration, and rate of excretion,
drug combination and the severity of the particular disease
undergoing therapy, and can be determined by those skilled in the
art.
[0076] The compositions can be administered for prophylactic and/or
therapeutic treatments, comprising preventing, inhibiting,
reversing tumor cell proliferation, and/or reducing tumor size. An
amount adequate to accomplish the desired effect without toxic
effect is defined as a "pharmaceutically effective amount" and will
generally range from about 0.01 to about 100 mg of antibody per
dose.
[0077] Additionally, the inhibitors can be utilized alone in
substantially pure form, or together with chemotherapeutic agents,
as are known to those of skill in the art (see, e.g., Cancer:
Principles and Practice of Oncology, 5.sup.th ed., Devita et al.,
Lippincott-Ravel Publishers, 1997). Other therapies that may be
used in conjunction with treatment with the antibodies include
administration of anti-sense nucleic acid molecules or biologicals,
such as additional therapeutic antibodies, as well as radiation
and/or surgery (see, e.g., WO0034337). Thus, the treatment of the
present invention is formulated in a manner allowing it to be
administered serially or in combination with another agent for the
treatment of cancer.
[0078] Antibodies disclosed herein are useful in diagnostic and
prognostic evaluation of diseases and disorders, particularly
cancers associated with NCAM2 expression. At each stage of disease,
monoclonal antibodies may be used to improve diagnostic accuracy
and facilitate treatment decisions. Unlike standard diagnostic
methods for tumors and cancer, such as computed topographic (CT)
scans, which depend on a change in size or architecture of organs
or lymph nodes, labeled monoclonals can detect abnormal cells at an
early stage, because of their expression of tumor antigens, such as
NCAM2. Once cancer is diagnosed, accurate staging is important in
deciding on the most appropriate therapy. Later, during follow-up
of surgery, rising serum levels of tumor antigens may indicate
recurrence before it can be detected by conventional methods.
[0079] Methods of diagnosis can be performed in vitro using a
cellular sample (e.g., blood sample, lymph node biopsy or tissue)
from a patient or can be performed by in vivo imaging.
[0080] In particular embodiments, the present invention provides a
antibody conjugate wherein the antibodies of the present invention
is conjugated to a diagnostic imaging agent. Compositions
comprising the antibodies of the present invention can be used to
detect NCAM2, for example, by radioimmunoassay, ELISA, FACS, etc.
One or more labeling moieties can be attached to the humanized
immunoglobulin. Exemplary labeling moieties include radiopaque
dyes, radiocontrast agents, fluorescent molecules, spin-labeled
molecules, enzymes, or other labeling moieties of diagnostic value,
particularly in radiologic or magnetic resonance imaging
techniques.
[0081] The present invention also provides for a method of
detecting prostate cancer or breast cancer, comprising detecting
the presence of NCAM2 in the prostate cells or breast cells of a
subject in need of such detection. Since our data show that NCAM2
is differentially expressed in the prostate tumor cells or breast
cancer cells but not normal prostate cells, an antibody against
NCAM2 can be used as a bio-marker for detecting the prostate cancer
or breast cancer.
[0082] The present invention also provides for a diagnostic kit
comprising anti-NCAM2 antibodies. Such a diagnostic kit further
comprises a packaged combination of reagents in predetermined
amounts with instructions for performing the diagnostic assay.
Where the antibody is labeled with an enzyme, the kit will include
substrates and co-factors required by the enzyme. In addition,
other additives may be included such as stablizers, buffers and the
like. The relative amounts of the various reagents may be varied
widely to provide for concentrations in solution of the reagents
that substantially optimize the sensitivity of the assay.
Particularly, the reagents may be provided as dry powders, usually
lyophilized, including excipients that, on dissolution, will
provide a reagent solution having the appropriate
concentration.
[0083] The inhibitors of the present invention may also be employed
for the inhibition of cancer cell growth, or for the treatment of
other types of cancer or neoplasm or malignant tumors found in
mammals, including carcinomas and sarcomas. Examples of cancers are
cancer of the brain, breast, cervix, bladder, colon, head &
neck, kidney, lung, non-small cell lung, melanoma, mesothelioma,
ovary, sarcoma, stomach, uterus, and Medulloblastoma. Preferably,
the inhibitors may be employed to detect or treat disorders
including, but not limited to, Hodgkin's Disease, Non-Hodgkin's
Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian
cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,
primary macroglobulinemia, small-cell lung tumors, primary brain
tumors, stomach cancer, colon cancer, malignant pancreatic
insulanoma, malignant carcinoid, urinary bladder cancer,
premalignant skin lesions, testicular cancer, lymphomas, thyroid
cancer, neuroblastoma, esophageal cancer, genitourinary tract
cancer, malignant hypercalcemia, cervical cancer, endometrial
cancer, adrenal cortical cancer, and leukemia.
[0084] Though the inhibitors of the present invention are primarily
concerned with the treatment of human subjects, they may also be
employed for the treatment of other mammalian subjects such as dogs
and cats for veterinary purposes.
[0085] The following examples are offered by way of illustration
and not by way of limitation. The disclosure of all citations in
the specification is expressly incorporated herein by
reference.
EXAMPLES
Example 1
[0086] This example describes identifying the anti-prostate tumor
agent by the immunization of lipid rafts from LNCaP cell lines.
[0087] Materials and Methods
[0088] a. Lipid Raft Preparation
[0089] Lipid rafts were prepared as described in Green et al, J.
Cell Biol. 146, 673-682 (1999). Briefly, cells (8.0.times.10.sup.6
cells/sample) were lysed in 0.1% vol/vol Brij-58, 20 mM Tris HCl,
pH 8.2, 140 mM NaCl, 2 mM EDTA, 25 .mu.g/ml aprotinin, 25 .mu.g/ml
leupeptin, and 1 mM phenylmethylsulfonyl fluoride for 10 minutes on
ice. Cells were homogenized using 10 strokes of a Dounce
homogenizer, then lysed 20 minutes more on ice. The resulting
lysate was adjusted to 40% wt/wt sucrose and applied onto a 60%
wt/wt sucrose cushion. A sucrose step-gradient consisting of 25%
wt/wt sucrose and 5% wt/wt sucrose were layered on top of the
lysate. Gradients were centrifuged 18 hours at 170,000.times.g at
4.degree. C. in a SW55 rotor. Fractions (0.2 ml) were taken from
the top of the gradient. Lipid rafts float to the interface of the
25% and 5% sucrose layers (Fractions 7 and 8). The amount of
protein in each fraction was determined using the BCA Protein Assay
Kit. Protein was concentrated by centrifugation at 2000.times.g in
Vivaspin 6 PES membrane columns (molecular weight cut off=10,000
kDa).
[0090] b. Lipid Raft Immunization
[0091] Lipid raft proteins (approximately 5 .mu.g) were mixed
together with 50 .mu.L Ribi.RTM., and then injected into the foot
pad of a BALB/c mouse. Mice were boosted with 50 .mu.L of lipid
raft proteins in Ribi.RTM. on day 7 and day 14. Three days after
the last boost, the mice were sacrificed and the hind leg lymph
node was harvested. The lymph node was washed in pre-warmed DMEM
and then ground using a Dounce homogenizer. After 5 gentle strokes,
the cell suspension was removed into the plastic tube. This process
was repeated four more times, each time adding 5 ml of fresh DMEM.
The lymphocytes were pooled and washed 3 times in DMEM. The
lymphocytes were mixed with the appropriate number of pre-washed
fusion partner NSO/BCL-2 (NS0 transfectant expressing the mouse
BCL-2 cDNA) to yield ratio of 2-3 lymphocytes for every 1 NS0. The
mixture was pelleted and warmed at 37.degree. C. for 1 min.
Pre-warmed 50% PEG was slowly added onto the pellet and then cells
were centrifuged at 300.times.g for 3 minutes at room temperature.
Five mL DMEM was added and then 10 mL DMEM with 10% FBS and 1% P/S
was added. The cells were then centrifuged 5 minutes at 300.times.g
at room temperature. The pellet was resuspended in HAT selection
medium (DMEM with 20% fetal bovine serum, 2 mM glutamine, 0.1 mM
non-essential amino acids, 1 mM sodium pyruvate, 0.1 mM sodium
hypoxanthine, 16 .mu.M thymidine, 20 .mu.M aminopterin,
2.times.Origen cloning factor, 10 mM HEPES, 50 .mu.M
beta-mercaptoethanol, 0.2 units/mL penicillin, 0.2 .mu.g/mL
streptomycin) to yield 0.25.times.10.sup.6 lymphocytes/mL. Cells
were aliquoted into ten 96-well flat bottom plates at 200 .mu.L per
well for the selection of hybridomas.
[0092] c. Flow Cytometry Screening
[0093] Flow cytometry was used to screen hybridoma supernatants for
the presence of cell surface binding antibodies. T he cells
(2.times.10.sup.5) were resuspended in 100 .mu.L ice cold PBS with
10 .mu.L tissue culture supernatant on ice for 1 hour. After
extensive washing, cells were incubated with
phycoerythrin-conjugated goat antibodies specific for mouse IgG for
30 minutes on ice. Cells were washed again and cell surface bound
antibody was detected using a Becton Dickenson FACScan.
Additionally, hybridoma supernatants were similarly screened on
many cancer cell lines or whole blood to test for specificity.
[0094] d. Affinity Purification of Antigen
[0095] Cells (5.times.10.sup.8) were lysed in 1% vol/vol NP-40,
0.5% wt/vol deoxycholate, 20 mM Tris HCl, pH 8.2, 150 mM NaCl, 1 mM
EDTA, 25 .mu.g/ml aprotinin, 25 .mu.g/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride for 1 hour on ice with frequent
mixing. Lysate was centrifuged for 20 minutes at 300.times.g to
remove nuclei and debris. Antigens were purified by standard
hybridoma affinity chromatography techniques as described in Hill
et al, J. Immunol. 152, 2890-2898 (1994).
[0096] e. Antigen Grouping
[0097] Cells (2.times.10.sup.7) were cell surface iodinated as
described (Landolfi and Cook, Mol. Immunol. 23, 297-309 (1986)).
Cells were then lysed in 1% NP-40, 0.5% deoxycholate, 50 mM
Tris-HCl , pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 .mu.g/ml aprotinin,
10 .mu.g/ml leupeptin, and 1 mM PMSF for 1 hour on ice. Cell lysate
was centrifuged at 14,000.times.g for 5 minutes to remove nuclei
and debris. Cell lysate was pre-cleared with rotation by incubation
with Gamma Bind Plus Sepharose beads for 2 hours at 4.degree. C.
The beads were spun down and the cell lysate was then aliquoted
into Eppendorf tubes containing Gamma Bind Plus Sepharose beads
that had been pre-incubated with hybridoma supernatant. The tubes
were rotated overnight at 4.degree. C. After extensive washing,
bound antigen was eluted from the beads by boiling in the presence
of 5% wt/vol SDS, 125 mM Tris-HCl, pH 6.8, and 4% vol/vol
.beta.-mercaptoethanol, and 50% vol/vol glycerol. Proteins were
then subjected to SDS-PAGE. After electrophoresis, the gel was
fixed for 30 minutes with 60% H.sub.2O/30% methanol/10% acetic
acid. The gel was then washed for 30 minutes with water then dried
down. The dried gel was put on film (Kodak.RTM. Biomax MS.RTM. film
with appropriate Biomax MS.RTM. screen) overnight.
[0098] f. Soft Agar Colony Formation Assay
[0099] For anchorage-independent cell growth, a soft agar colony
formation assay was performed in a six-well plate. Each well
contained 2 mL of 1% agar in complete medium as the bottom layer.
The top layer contained 2 mL 0.5% agar in complete medium,
1000-10000 LNCaP cells, and 5 .mu.g/mL mAb (anti-ATP synthase,
anti-NCAM2, or anti-Trop1). One mL complete medium was added and
the cultures were maintained at 37.degree. C. in a humidified 5%
CO.sub.2 atmosphere for up to 20 days. One mL complete medium was
added once a week. Media was removed and the colonies were stained
with 0.005% crystal violet in PBS for 2 hours. The number of
colonies was determined by counting them under an inverted
phase-contrast microscope at 100.times., and a group of 10 or more
cells were counted as a colony.
[0100] g. LNCaP Proliferation
[0101] LNCaP cells were plated at 20,000 cells/well into a 96 well
tissue culture plate. After cells were allowed to grow undisturbed
for two days, antibodies (5 .mu.g/ml anti-ATP synthase, anti-NCAM2,
anti-Trop-1, or anti-MHC class II) were added and incubated with
the cells for 24 hours. Cell proliferation was measured using the
AlamarBlue vital dye indicator assay. AlamarBlue reagent was added
to each well and the plates were incubated for 3 to 4 hours at
37.degree. C. to allow for fluorescence development. Fluorescence
was detected at .lambda.ex=530 nm, .lambda.em=590 nm. Data is
expressed as the mean +/- SEM of 4 replicates.
[0102] Results and Discussions
[0103] One BALB/c mouse was immunized with a lipid raft preparation
from the prostate cancer cell line LNCaP. After two boosts,
lymphocytes were isolated from the mouse lymph nodes and fused with
myeloma NSO cells to generate hybridomas. A total of about 700
hybridomas were generated and supernatant from each hybridoma was
screened by flow cytometry for binding to LNCaP. About 203
supernatants tested positive, and they were further tested for
binding to normal prostate cells. Thirty-four of these supernatants
tested negative for binding to normal prostate cells. Antibodies
from these 34 hybridomas were further tested against multiple
cancer lines by flow cytometry to determine whether they are LNCaP
specific, prostate cancer specific, or pan-cancer specific. The
cancer cell lines we used include: DU 145 (prostatic), PC-3
(prostatic), PANC-1 (pancreatic), RT4 (bladder), HT-29
(colorectal), NCI-H292 (lung), T-47D (breast), and NIH:OVCAR-3
(ovarian). In addition, a primary, non-transformed HUVEC line
(human umbilical vein endothelial cells) was used to ensure that
these antibodies do not cross-react with normal endothelial cells.
The 34 LNCaP reacting hybridomas can be divided into two main
groups based on their antigen expression profiles: antibodies from
20 of these hybridomas (Table 2) showed that their antigens are
expressed only in a limited number (1-3) of cancer cell lines and
14 (Table 3) showed that their antigens are expressed in multiple
lines. A flow chart describing how these tumor-associated, LNCaP
lipid raft-derived antigens were identified by the hybridoma
technology is shown in FIG. 1.
[0104] We performed an immunoprecipitation experiment to determine
the molecular weight of the antigens that showed limited expression
profiles. Of the twenty antibodies used, three predominant antigens
of MW 98 (recognized by 3 antibodies), 100 (recognized by 6
antibodies), and 120 kD (recognized by 4 antibodies) were
identified (FIG. 2, see also the last column of Table 1). Seven
antibodies were not able to immunoprecipitate antigen for molecular
weight determination. The antigen grouping results indicate the
hybridomas of Table 1 covered a minimum of four to a maximum of ten
antigens-.
[0105] a. Lipid Raft Tumor-associated Antigen NCAM2
[0106] The identity of the antigen defined by the hybridoma P3-53
had been determined. The monoclonal antibody produced by hybridoma
P3-53 (Table 1) immunoprecipitated a major protein band of 120 kD
and a minor band of 110 kD (see FIG. 2, Lane 3). The P3-53 antigen
is expressed only in the prostate cell line LNCaP and breast cell
line T47D. The monoclonal antibody from P3-53 was conjugated to
CNBr-activated Sepharose to generate an affinity column (20 mg
conjugated to a 2 ml column). LNCaP whole cell lysate was prepared
from 2.times.10.sup.8 cells as described in MATERIAL AND METHODS
and passed onto the P3-53 affinity column. After extensive washing,
the retained protein was eluted with low pH buffer. About 5 .mu.g
of the P3-53 antigen was purified. SDS-PAGE analysis followed by
silver staining revealed the P3-53 antigen consisted of a major
protein band of 120 kD and a minor band of 110 kD. The purified
antigen was subjected to microsequencing analysis and the result
showed that it had a NH2-terminal sequence of X-L-QV-T-I-S-L-S-K,
where X was probably "L", but might also be "G". This sequence was
searched against the entire NCBInr database using Protein
Prospector. Only one human protein with the NH.sub.2-terminal
sequence of L-L-Q-V-T-I-S-L-S-K matched the determined P3-53
antigen sequence. The human protein is called neural cell adhesion
molecule 2 (NCAM2, NCBI protein accession number 4758764, see also
Paoloni-Giacobino et al, Genomics 43, 43-51 (1997)), which is a
homologue of a murine protein called Rb-8 neural adhesion molecule
(RNCAM, NCBI protein accession number 3334269, see also Alenius, M.
and Bohm, S., J. Biol. Chem. 272, 26083-26083 (1997) and Yoshihara
et al. J. NeuroSci. 17: 5830-5842 (1997). The identification of the
antigen recognized by P3-53 was confirmed to be NCAM2 by MALDI-TOF
peptide-mass profiling as described in the MATERIALS AND METHODS in
Example 1.
[0107] The sequence of RNCAM predicted molecules having an
extracellular region of 5 immunoglobulin C2-type domains followed
by two fibronectin type III domains. Alternative splicing of the
NCAM2 and RNCAM transcripts generate two isoforms: the long form
containing a transmembrane domain and the short form containing a
glycosylphosphatidylinositol-anchor attached to the membrane. The
expression of RNCAM is restricted to the olfactory neurons in the
brain and in the nasal vomeronasal organ. The transcript of RNCAM
is not detectable in lung, gut, liver, heart, testis and kidney.
The function of NCAM2 or RNCAM is not known, but the molecule may
play a role in selective axon projection. NCAM2 was also shown to
be a homophilic adhesion molecule (see Yoshihara et al. J.
NeuroSci. 17: 5830-5842 (1997)). Thus we found that certain
prostate and breast cancer cell lines express this protein marker
that is neural in origin. Accordingly, because NCAM2 is expressed
in a substantial percentage of prostate and breast cancer cells, an
antibody to NCAM2 provides an effective treatment for prostate or
breast cancer. Antibodies are better drugs than small molecules
against cancer cells expressing neural markers because they do not
cross the blood-brain barrier to potentially have toxic effects on
normal neurons.
[0108] b. Inhibition of Prostate Cancer Cell Proliferation by
Anti-NCAM2 Antibodies
[0109] As shown in Table 2 and FIG. 2, P3-53, P9-64, P10-28, and
P10-29 all immunoprecipitated an antigen with a molecular weight of
120/110. The P3-53 antigen was identified as NCAM2. We tested the
anti-cancer activity of 4 anti-NCAM2 antibodies in a proliferation
assay using the prostate cancer cell line LNCaP. The results are
shown in FIG. 3. One of the 4 antibodies, namely P9-64, had
significant inhibition activity against proliferation of LNCaP
cells. This reduction in cellular proliferation by P9-64 was about
39%. P10-28 and P10-29 had about 16 and 11% inhibition activity,
respectively, and P3-53 was not effective. . This substantial
inhibition of cell proliferation by some anti-NCAM2 antibodies
suggests a potential of anti-ATP synthase antibody as an anti-tumor
agent for treating prostate cancer.
[0110] c. Inhibition of Prostate Cancer Cell Colony Formation by
Anti-NCAM2 Antibodies
[0111] Transformed cancer cells are resistant to
anchorage-independent growth inhibition and are able to growth in
soft agar without attaching to cell matrix. Formation of colonies
(three-dimensional growth under tissue culture growth conditions)
of cancer cells in soft agar is often correlated to the
aggressiveness of the tumor in vivo. To assess whether the four
anti-NCAM2 antibodies any anti-cancer activity, we used them
inhibit LNCaP colony formation in vitro. As shown in FIG. 4, LNCaP
cancer cell colony formation can be reduced by some anti-NCAM2
antibodies at 5 .mu.g/ml. As in the proliferation assay, the most
potent inhibitor is P9-64. It inhibited colony formation by 42%.
P10-28 and P10-29 inhibited about 22% and 36% respectively. The
inhibitory activity of P3-53 was again low, at about 10%
inhibition. The substantial inhibition of colony formation by some
anti-NCAM2 antibodies suggests a potential of anti-NCAM2 antibody
as an anti-tumor agent for treating prostate cancer.
[0112] The above experiments demonstrate that NCAM2 expression is
closely linked to some types of prostate cancer cells. It is
localized on the surface of cancer cells, and may play a key role
in the biological activities of prostate cancer cells. Blocking the
activity of NCAM2 by antibodies inhibits prostate cancer cell
growth, suggesting the possibility of clinical application of NCAM2
inhibitors as anti-tumor agents for treating prostate cancer.
Example 2
[0113] This example describes inhibition of tumor adhesion and
spreading by cell adhesion and migration assay
[0114] Antibody inhibition of adhesion and spreading is evaluated.
Tissue culture 12-well plates were coated 2 hours at room
temperature with components of the extracellular matrix, i.e.
vitronectin (VN), fibronectin (FN), collagen type I, type III and
type IV, laminin (LA), or hyaluronic acid (HA) in Hanks buffered
salt solution (HBSS). Plates are blocked for 2 hours with 1% BSA in
PBS. Cells are plated in HBSS with 1 mM CaCl.sub.2 and 1 mM
MgCl.sub.2 in the presence or absence of antibody. Cells are
allowed to spread for 30 minutes to 2 hours at 37.degree. C. prior
to photography.
[0115] Inhibition of cancer cell migratory activity of anti-tumor
agents is evaluated in a matrigel assay. Membranes with a pore size
of 8 .mu.m were coated with 50 .mu.l matrigel. The membranes were
inserted into 24 well plates that contain medium without
supplements. Cancer cells are resuspended in medium with 10% FCS in
the presence or absence of antibodies and then seeded on the
matrigel coated transwell plates. Plates are incubated for 48 hours
at 37.degree. C. Thereafter, cells at the bottom of the chamber are
counted using an inverted microscope.
Example 3
[0116] This example describes selection of anti-lipid raft
antibodies for cancer therapy based on their antigen expression
profiles and anti-cancer activities in vitro.
[0117] For solid tumors, monoclonal antibodies against the
identified antigens are used to stain by immunohistochemistry
normal or neoplastic human tissues to establish the expression
profiles of the tumor associated antigens. Valuable
tumor-associated tumor antigens should have low or no expression in
normal tissues and high expression in cancer cells. To be a good
targets for antibody therapy, tumor associated antigens should be
differentially expressed in substantial percentage (20% and above)
of certain cancer type. Valuable antibodies against these antigens
may have anti-cancer activities in vitro. These activities include
inhibition of cell proliferation, induction of apoptosis and
inhibition of cell migration.
[0118] For hematological malignancies, monoclonal antibodies
against the identified antigens are used to stain by flow cytometry
patient's leukemic cell as well as normal human blood and bone
marrow cells. Antigens that are expressed in hematopoietic stem
cells (within the CD34-positive population), T cells, platelets, or
granulocytes should be excluded because triggering or killing of
these cells by antibodies will cause severe toxicity in humans.
Antigens of interest may be expressed in B cells, macrophages or
monocytes but not in other normal tissues. The ideal
tumor-associated antigens are the ones that can be triggered to
induce cell death in leukemic cells.
Example 4
[0119] This example describes the treatment of prostate cancer by
antibodies specific for NCAM2 in well-established
androgen-dependent and androgen-independent prostate cancer
xenografts.
[0120] Six to ten week old male nude NCR nu/nu mice are inoculated
subcutaneously in the mid-scapular region with 5.times.10.sup.6
androgen-dependent LNCaP cells. Cells that are injected are
reconstituted with basement membrane in the form of Matrigel as
described (Sato et al Cancer Res. 5, 1584-1589 (1997)). To maintain
serum testosterone levels, male mice are implanted with 12.5 mg
sustained release testosterone pellets subcutaneously prior to
receiving the tumor cell inoculation. Antibodies specific for NCAM2
are given intraperitoneally on day 2 and 4. Tumors are measured
every three to four days with vernier calipers. Tumor volumes are
calculated by the formula .pi./6.times.(larger
diameter).times.(smaller diameter).sup.2. For the
androgen-independent prostate cancer xenograft studies, DU 145 or
PC-3 cells are used.
Example 5
[0121] This example describes the human prostate cancer therapeutic
regime by using the inhibitors NCAM2.
[0122] The patient's cancer biopsy sample is stained positively for
the expression of NCAM2 on the cancer cell surface by
immunohistochemistry for the patient to be eligible for treatment.
Anti-NCAM2 antibodies are administered either intravenously or
subcutaneously in a dose range from 0.05 to about 25 mg/kg.
Patients receive at least 4 weekly doses. Tumor size is monitored
by CAT scan or MRI prior to therapy and post therapy. Reduction of
the tumor size is the primary indication of the drug's efficacy.
Tumor shrinkage by 50% or more is considered as a partial response.
Complete disappearance of the tumor is considered as a complete
response. For prostate cancer patients, PSA level prior to therapy
and post therapy is also monitored as a secondary indication of
treatment efficacy.
[0123] Although the invention has been described with reference to
the presently preferred embodiments, it should be understood that
various modifications can be made without departing from the spirit
of the invention.
[0124] All publications, patents, patent applications, and web
sites are herein incorporated by reference in their entirety to the
same extent as if each individual patent, patent application, or
web site was specifically and individually indicated to be
incorporated by reference in its entirety.
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