Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPOSITIONS AND METHODS FOR REGULATING
TUMOR-ASSOCIATED ANTIGEN EXPRESSION
Field of the Invention
This invention relates to methods and compositions for the treatment of
tumors. In
particular, the invention relates to methods and agents for the treatment of
tumors expressing a
tumor-antigen [also known as a tumor-associated antigen (TAA), lineage
specific,
differentiation, or self -antigen]. and more specifically to methods and
agents for the treatment
of melanomas expressing the Melan-A/MART-1 antigen. The invention also relates
to
rn methods for identifying agents that regulate expression of tumor-antigens.
Background of the Invention
Many solid tumors are presently known to involve the infiltration of
autologous
lymphocytes. These autologous lymphocytes. known in the art as tumor-
infiltrating
is lymphocytes (TIL), have been shown to recognize specific antigens expressed
by cells of the
solid tumor. Expression of such tumor-associated antigens (TAA) in combination
with
appropriate accessory signals leads to a specific cytolytic (cytotoxic)
reactivity of the TILs
toward the solid tumors.
Several tumor antigens have been identified in association with a variety of
tumors
~o Boon T, et al., Ann Rev Immunol, 1994, 12:337-65: Kawakami Y. et al., Proc
Natl Acad Sci
USA, 1994, 91:315-9; Bakker A, et al., J Exp Med, 1994, 179:1005-9). In
addition to the
identification of TAAs, immunodominant epitopes recognized by TILs have also
been
described for widely-expressed lineage-specific antigens. for example, the HLA-
A2-restricted
Melan-A/MART-1 in melanomas (Sensi M, et al., Proc Natl Acad Sci USA, 1995.
92:5674-8;
s Kawakami Y, et al., JExp Med, 1994, 180:347-52).
Melanomas are aggressive, frequently metastatic tumors derived from either
melanocytes or melanocyte related nevus cells (Cellular and Molecular
Immunology, 1991,
(eds) Abbas A. K., Lechtman. A. H., Pober, J. S.; W. B. Saunders Company,
Philadelphia:
pages 340-341 ). In some melanoma patients. TILs show strong in vitro lytic
activity which is
30 often directed against targets expressing HLA-A2 and the immunodominant
peptide of Melan-
A/MART-1 (AAGIGILTV, SEQ ID NO:1) (Kawakami Y, and Rosenberg SA, Int Rev
Immunol. 1997. 14:173-92; Zhai, Y., et al.,) J. Immunoh 1996, 16:700-710;
Stevens. E., et
al., J. Immunoh 1995, 154:762-71; Rivoltini, L., et al.. J Imnaunol, 1995,
154:2257-2260. In
such cases, the tumor cells which evoke a cytolytic immune response must also
be able to
3a implement a mechanism to elude the immune response. In other reported
melanoma cases.
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however. TILs have been unable to recognize the melanoma cells leading to
tumor
progression. In these latter cases. loss of MHC surface molecules as a result
of defective
expression of MHC Class I heavy chain, (32-microglobulin or TAP genes. has
been suggested
as an escape mechanism from the host's immune defenses (Maeurer MJ, et al., J
Clin Invest,
1996, 98:1633-41; Rivoltini, L., et al., Cancer Res, 1995, X5:3149-~7;
Ferrone, S. &
Marincola. F., Immunol. Today. 1995, 16:487-94; Garrido, F., et al., Immunol.
Today, 1997,
18:89-95). Recently, the proto-oncogene PML-1 was reported to modulate MHC
expression
through control of TAP and LMP genes, thereby participating in tumor escape
from TIL
recognition (Zheng, P., et al., Nature, 1998. 396:373-6). In addition.
selective loss of nominal
m antigen reportedly also results in immune escape (deVries T. et al., Cancer
Res, 1997,
57:3223-9; Labarriere, N., et al.. 1997, J. Immunol, 158:1238-45).
There exists a need to identify additional mechanisms for tumor recognition
escape by
the TILs and other cells of the immune system.
There also exists a need to identify agents that prevent such immune system
rs recognition escape.
Summary of the Invention
The invention provides methods for identifying agents that modulate expression
of
tumor-associated antigens in tumor cells. The invention also provides agents
and
~n pharmaceutical compositions containing such agents that modulate expression
of tumor
associated antigens in tumor cells. The invention, therefore, is particularly
useful. inter alia,
for treating subjects with autologous. solid tumors having cells that express.
or that can be
induced to express, tumor-associated antigens.
One category of materials according to the invention is tumor-antigen
expression
down-regulating agents. These agents include tumor cell isolates and isolated
or substantially
purified materials. Another category of materials according to the invention
is inhibitors of
such agents that down-regulate tumor-antigen expression.
According to one aspect of the invention, a malignant melanoma cell isolate is
provided that down-regulates Melan-A/MART-1 expression in malignant melanoma
cells
3~ when contacted with malignant melanoma cells. The cell isolate can be
derived from lVlelan
A/MART-1 antigen -expressing and/or -nonexpressing malignant melanoma cells.
Preferably,
the malignant melanoma cells are low-Melan-A/MART-1 antigen-expressing cells.
In certain
embodiments, the cell isolate comprises a polypeptide. In further embodiments.
the cell isolate
is a substantially pure polypeptide. In important embodiments. the
substantially pure
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polypeptide is a polypeptide selected from the group consisting of Oncostatin
M.
Stanniocalcin, and Tissue Factor Pathway Inhibitor-2. In one embodiment. the
cell isolate is a
supernatant, or fraction thereof. of Melan-A/MART-1 antigen-expressing
malignant
melanoma cells. In some embodiments. the cell isolate comprises at least one
polypeptide
selected from the group consisting of Oncostatin M. Stanniocalcin, and Tissue
Factor Pathway
Inhibitor-2. In certain embodiments, the malignant melanoma cells express low
levels of
Melan-A/MART-1 antigen. In yet further embodiments, the malignant melanoma
cells express
high levels of Melan-A/MART-1 antigen.
According to another aspect of the invention, a substantially pure organic
agent is
rn provided that down-regulates Melan-A/MART-1 expression in malignant
melanoma cells at
effective concentrations. The agent is present at effective concentrations in
the supernatant of
confluent malignant melanoma cells cultured under standard conditions (e.g.,
flatbed, static
culture) for a period of at least one hour. In certain embodiments. the
culture conditions
include a ratio of 5 x 106 cells/ml medium, and the agent is heat sensitive at
80°C, proteinase K
~s sensitive, and binds to and elutes-off blue- and/or red-
Sepharose~(Pharmacia Biotech, Inc.,
Piscataway, NJ). In some embodiments. the organic agent is a polypeptide. In
important
embodiments, the polypeptide is selected from the group consisting of
Oncostatin M,
Stanniocalcin, and Tissue Factor Pathway Inhibitor-2. In certain embodiments.
the malignant
melanoma cells express low levels of Melan-A/MART-1 antigen. In further
embodiments,
~n the malignant melanoma cells express high levels of Melan-A/MART-1 antigen.
According to another aspect of the invention, an isolated organic agent is
provided that
binds selectively to a malignant melanoma cell isolate that down-regulates
~Melan-A/MART-1
expression in malignant melanoma cells. and inhibits down-regulation of Melan-
A/MART-1
expression in malignant melanoma cells. In some embodiments, the malignant
melanoma cell
>; isolate comprises at least one polypeptide. In certain embodiments, the
malignant melanoma
cell isolate is a substantially pure polypeptide. In important embodiments.
the substantially
pure polypeptide is a polypeptide selected from the group consisting of
Oncostatin M,
Stanniocalcin, and Tissue Factor Pathway Inhibitor-2. In further embodiments,
the isolated
binding organic agent is a polypeptide. In yet further embodiments, when the
isolated binding
3n organic agent is a polypeptide, the polypeptide can be an antibody or an
antibody fragment
selected from the group consisting of a Fab fragment, a F(ab)~ fragment. or a
fragment
including a CDR3 region selective for the polypeptide. The antibody or
fragment thereof may
be chimeric or humanized. In further important embodiments, the antibody can
be selected
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from the group consisting of an anti-Oncostatin-M antibody, an anti-
Stanniocalcin antibody.
and an anti-Tissue Factor Pathway Inhibitor-2 antibody.
According to another aspect of the invention, an isolated binding organic
agent is
provided which binds selectively to a substantially pure organic agent that
down-regulates
Melan-A/MART-1 expression in malignant melanoma cells, and inhibits down-
regulation of
Melan-A/MART-1 expression in malignant melanoma cells. In some embodiments,
the
substantially pure organic agent comprises at least one polypeptide having
Melan-A/MART-1
expression down-regulating properties in malignant melanoma cells. In certain
embodiments,
the substantially pure organic agent is a substantially pure polypeptide. In
important
m embodiments. the substantially pure polypeptide is a polypeptide selected
from the group
consisting of Oncostatin M, Stanniocalcin, and Tissue Factor Pathway Inhibitor-
2. In further
embodiments. the isolated binding organic agent is a polypeptide. In yet
further embodiments,
when the isolated binding organic agent is a polypeptide, the polypeptide can
be an antibody
or an antibody fragment selected from the group consisting of a Fab fragment,
a F(ab)2
~s fragment, or a fragment including a CDR3 region selective for the
polypeptide. The antibody
or fragment thereof may be chimeric or humanized. In further important
embodiments. the
antibody can be selected from the group consisting of an anti-Oncostatin-M
antibody, an anti-
Stanniocalcin antibody, and an anti-Tissue Factor Pathway Inhibitor-2
antibody.
According to a further aspect of the invention, a pharmaceutical composition
is
zn provided. The composition includes an isolated binding organic agent which
binds selectively
to a substantially pure organic agent that down-regulates Melan-A/MART-1
expression in
malignant melanoma cells. The isolated binding organic agent that binds
selectively the
substantially pure organic agent inhibits such down-regulation, and is present
in an effective
amount to inhibit such down-regulation. The composition also includes a
pharmaceutically
acceptable carrier.
According to another aspect of the invention, a pharmaceutical composition is
provided. The composition includes an isolated binding organic agent which
binds selectively
a malignant melanoma cell isolate that down-regulates Melan-A/MART-1
expression in
malignant melanoma cells. The isolated binding organic agent that binds
selectively the
;n malignant melanoma cell isolate inhibits such down-regulation. and is
present in an effective
amount to inhibit such down-regulation. The composition also includes a
pharmaceutically
acceptable carrier.
The invention in another aspect provides a method for isolating a tumor cell-
derived
tumor-antigen expression down-regulating agent. The method involves (a)
preparing a culture
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of tumor cells that have down-regulated tumor-antigen expression. (b)
isolating a supernatant
or cell isolate suspected of containing a tumor-antigen expression down-
regulating agent from
the culture of step (a), (c) fractionating the supernatant or cell isolate
into a pluralit<~ of
fractions, (d) contacting a fraction from the plurality of fractions with a
tumor-antigen
expressing tumor cell, (e) measuring tumor-antigen expression on the tumor-
antigen
expressing cell. and (f) determining whether tumor-antigen expression on the
tumor cell is
down-regulated as a result of such contacting, for example, by comparison to a
control. In
some embodiments, the origin of the tumor cells may be of: biliary tract
cancer; brain cancer,
including glioblastomas and medulloblastomas; breast cancer; cervical
carcinoma;
m choriocarcinoma; colon cancer; endometrial cancer: esophageal cancer;
gastric cancer;
hematological neoplasms, including acute lymphocytic and myelogenous leukemia:
multiple
myeloma: AIDS associated leukemias and adult T-cell leukemia lymphoma;
intraepithelial
neoplasms, including Bowen's disease and Paget"s disease; liver cancer: lung
cancer;
lymphomas, including Hodgkin's disease and lymphocvtic lymphomas;
neuroblastomas; oral
~s cancer, including squamous cell carcinoma; ovarian cancer, including those
arising from
epithelial cells, stromal cells, germ cells and mesenchymal cells: pancreas
cancer; prostate
cancer; rectal cancer: sarcomas, including leiomyosarcoma, rhabdomyosarcoma,
liposarcoma,
fibrosarcoma and osteosarcoma; skin cancer, including melanoma. Kaposi's
sarcoma,
basocellular cancer and squamous cell cancer; testicular cancer, including
germinal tumors
~n (seminoma. non-seminoma[teratomas, choriocarcinomas]), stromal tumors and
germ cell
tumors; thyroid cancer, including thyroid adenocarcinoma and medullar
carcinoma; and renal
cancer including adenocarcinoma and Wilms tumor. In certain embodiments, the
tumor-
antigen can be Melan-A/MART-1. Dipeptidyl peptidase IV (DPPIV), adenosine
deaminase-
binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)--
C017-
=, lA/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-l
and
CAP-2, etv6, amll, Prostate Specific Antigen (PSA) and its immunogenic
epitopes PSA-1,
PSA-2, and PSA-3. prostate-specific membrane antigen (PSMA), T-cell
receptor/CD3-zeta
chain, MAGE-family of tumor antigens, GAGE-1.2, BAGE, RAGE. GnT-V, MUM-1.
CDK4,
tyrosinase. p~3, MUC family, HER2/neu, p2lras, RCAS1, a-fetoprotein, E-
cadherin. a.-
3n catenin, (3-catenin and y-catenin, p120ctn, gpl00Pmem~, pRAME, NY-ESO-1.
cdc27,
adenomatous polyposis coli protein (APC), fodrin, Smad family of tumor
antigens. lmp-l,
EBV-encoded nuclear antigen (EBNA)-1, or c-erbB-2. In any of the foregoing
embodiments
the fraction from the plurality of fractions can be undiluted or concentrated.
In important
embodiments. cancers or tumors escaping immune recognition and tumor-antigens
associated
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with such tumors (but not exclusively), include acute lymphoblastic leukemia
(etv6; amll;
cyclophilin b), glioma (E-cadherin; a-catenin; ~3-catenin; y-catenin; p I
20ctn), bladder cancer
(p2lras), billiary cancer (p2lras). breast cancer (MUC family; HER2/neu; c-
erbB-2), cervical
carcinoma (p53; p2lras), colon carcinoma (p2lras; HER2/neu: c-erbB-2; MUC
family),
s colorectal cancer (Colorectal associated antigen (CRC)--C017-lA/GA733; APC),
choriocarcinoma (CEA), epithelial cell-cancer (cyclophilin b), gastric cancer
(HER2/neu; c-
erbB-2; ga733 glycoprotein), hepatocellular cancer (a-fetoprotein), hodgkins
lymphoma (lmp-
1; EBNA-I), lung cancer (CEA; MAGE-3: NY-ESO-I), lymphoid cell-derived
leukemia
(cyclophilin b), myeloma (MUC family; p2lras), non-small cell lung carcinoma
(HER2/neu;
m c-erbB-2), nasopharyngeal cancer (lmp-1: EBNA-1), ovarian cancer cancer (MUC
family;
HER2/neu; c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA) and its
immunogenic
epitopes PSA-l, PSA-2. and PSA-3; PSMA: HER2/neu; c-erbB-2), pancreatic cancer
(p2lras;
MUC family; HER2/neu; c-erbB-2; ga733 glycoprotein), renal (HER2/neu; c-erbB-
2),
testicular cancer (NY-ESO-1 ). T cell leukemia (HTLV-1 epitopes), and melanoma
(Melan
> > A/MART-1; cdc27; MAGE-3 : p21 ras; gp 1 OOP"'ei i ").
The invention in a further aspect provides a method of screening for tumor-
antigen
expression modulating agents. The method involves (a) contacting an agent
suspected of
being a tumor antigen expression modulating agent with a tumor-antigen
expressing tumor
cell, (b) measuring tumor-antigen expression of the tumor cell, and (c)
determining whether
~n tumor-antigen expression on the tumor cell is modulated as a result of such
contacting, for
example, by comparison to a control. Both up-modulating and down-modulating
agents can
be identified. Tumor cells and tumor-antigens expressed by the tumor cells are
as described
above. In some embodiments. the agent suspected of being a tumor-antigen
expression
modulating agent is an agent present in a tumor cell-culture supernatant,
tumor cell eluate, or
~s tumor cell lysate.
According to another aspect of the invention, a method is provided for
isolating an
agent that up-regulates tumor-antigen expression. The method includes (a)
providing a tumor-
antigen expression down-regulating agent which may be isolated according to
any of the
foregoing methods of the invention. (b) preparing a culture of tumor cells.
wherein the tumor
3n cells may be identical to those used in the isolation of the tumor-antigen
expression down-
regulating agent of (a), (c) contacting the isolated tumor-antigen expression
down-regulating
agent of (a) and a putative inhibitory agent of the isolated tumor-antigen
expression down-
regulating agent of (a) with the culture cells of (b), (d) determining tumor-
antigen expression
in the culture cells, and (e) comparing the tumor-antigen expression
determined in (d) with a
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control. Tumor cells and tumor-antigens expressed by the tumor cells are as
described above.
In preferred embodiments the tumor cells are melanoma cells and the tumor-
antigen is Melan-
A/MART-1. In some embodiments. the control tumor-antigen expression is
determined in the
presence of an agent of (a) and in absence of the putative inhibitory agent of
(a).
According to another aspect of the invention, a method is provided for
enhancing a
melanoma-specific immune response in a subject with melanoma. The method
involves
administering to a subject in need of such treatment an isolated binding
organic agent which
binds selectively to: (i) a substantially pure organic agent, or (ii) a
malignant melanoma cell
isolate, either of which down-regulates Melan-A/MART-1 expression in malignant
melanoma
rn cells when contacted with malignant melanoma cells, in an amount effective
to inhibit down-
regulation of Melan-A/MART-1 expression in malignant melanoma cells and
enhance a
melanoma-specific immune response in the subject. In some embodiments. the
isolated
binding organic agent is at least one polypeptide. In important embodiments.
the polypeptide
is selected from the group consisting of Oncostatin M, Stanniocalcin, and
Tissue Factor
~s Pathway Inhibitor-2. In certain embodiments, the method further comprises
co-administering
to the subject an anti-tumor agent other than the agents of the invention.
According to a further aspect. methods for preparing medicaments useful in
enhancing
a melanoma-specific immune response in a subject with melanoma, are also
provided.
These and other aspects of the invention, as well as various advantages and
utilities,
zn will be more apparent with reference to the detailed description of the
preferred embodiments.
Brief Description of the Drawings
Figure 1 shows graphs depicting Melan-A/MART-1 expression on different
melanoma
tumor lines and the differences culture density has on such expression: Fig.
1A on MU-tumor;
Fig.lB on MA-tumor; Fig.lC on MO-tumor; Fig.lD control, HLA-I expression on MU-
tumor;
Fig.lE control, HLA-A2 expression on MU-tumor.
lure 2 shows graphs depicting the effect of MART-1/Melan-A expression on
target
cell recognition and lysis by TIL; Fig. 2A shows MU-TIL lysis of autologous MU-
tumor; Fig.
2B shows MU-TIL lysis of Melan-A/MART-1-negative targets; and Fig. 2C shows
anti-HLA-
A2-specific lysis of targets.
3n Brief Description of the Secruences
SEQ ID NO:1 is the immunodominant amino acid sequence of the Melan-A/MART-1
peptide.
SEQ ID N0:2 is a melanocyte lineage-derived peptide for Tyrosinase.
SEQ ID N0:3 is a melanocyte lineage-derived peptide for Tyrosinase.
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SEQ ID N0:4 is a melanocvte lineage-derived peptide for MAGE-3.
Detailed Description of the Invention
We describe herein, methods for identifying agents that modulate expression of
tumor
associated antigens in tumor cells. We also describe herein, agents and
pharmaceutical
compositions containing such agents that modulate expression of tumor-
associated antigens in
tumor cells. The foregoing can be used, inter alia, in vivo or in vitro, for
the purpose of
inhibiting growth of a tumor having cells expressing tumor-associated
antigens, and in a
variety of screening assays in order to identify additional agents that
modulate expression of
rn tumor-associated antigens in tumor cells.
We have discovered, unexpectedly, that tumor cells which normally express or
present
TAAs (intracellularly or on their surface) "lose" (or down-modulate) such TAA
expression/
presentation when cultured at high density. We also discovered, unexpectedly,
that such TAA
down-modulation is mediated through an agent secreted by the tumor cells
(i.e., autocrine
rs secretion/down-modulation). The terms ''down-modulation'' and "up-
modulation" are used
interchangeably with the terms "down-regulation" and "up-regulation."
respectively,
throughout this application.
"Down-modulating (down-regulating)," as used herein, refers to inhibition of
tumor
antigen (or TAA) expression. Inhibition of tumor-antigen expression refers to
inhibiting (i.e.,
~n reducing to a detectable extent) expression/presentation of the specific
antigen intracellularly
and/or at the surface of a tumor cell. Such inhibition of tumor-antigen
expression can be
directly determined by detecting a decrease in the level of mRNA for the gene
encoding the
antigen, or the level of peptide expression of the tumor-antigen, using any
suitable means
known to the art, such as nucleic acid hybridization or, preferably, antibody
detection
methods, respectively (see Examples). Inhibition of tumor-antigen expression
can also be
determined indirectly, for example. by detecting a change in tumor-cell lysis
ability by TILs
that specifically recognize the tumor-antigen.
The present invention relates in one aspect to a malignant melanoma cell
isolate with
Melan-A/MART-1 expression down-regulating activity. Therefore, in one
important
3n embodiment. the malignant melanoma cell isolate is useful in screening for
binding agents that
inhibit its activity. Inhibition of the isolates activity is desirable in
melanomas. where the
proliferating cells secrete the isolate and down-modulate expression of the
Melan-A/MART-1
antigen, to escape immune recognition, and continue to grow and expand.
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As used herein with respect to malignant melanoma cells, ''isolate" means
separated
from the environment of the cells, such as a supernatant, a lysate, a fraction
thereof; etc.
"Isolated" means separated from its native environment and present in
sufficient quantity to
permit its identification or use according to the invention. ''Isolated'',
when referring to a
s protein or polypeptide, means. for example: (i) selectively produced by
expression cloning. or
(ii) partially purified as by chromatography or electrophoresis. Isolated
proteins or
polypeptides may, but need not be, substantially pure. Because an isolated
protein may be
admixed with a pharmaceutically acceptable carrier in a pharmaceutical
preparation. the
protein may comprise only a small percentage by weight of the preparation. The
protein is
m nonetheless isolated in that it has been separated from many of the
substances with which it
may be associated in living systems. i.e. isolated from certain other
proteins.
In important embodiments of the present invention. the agent that down-
regulates
Melan-A/MART-1 expression in malignant melanoma cells when contacted with
malignant
melanoma cells, is a substantially pure. native, biological. organic agent.
Preferably, the agent
is is heat sensitive at 80°C, proteinase K sensitive, and binds to and
elutes-off blue- and/or Red
Sepharose~. Preferably, the substantially pure organic agent is at least one
polypeptide. In
important embodiments, the at least one polypeptide with Melan-A/MART-1
expression
down-regulating properties in malignant melanoma cells has a molecular weight
between
about 20kD and about 30 kD, and preferably at about 25kD. In further important
~n embodiments, the agent that down-regulates Melan-A/MART-1 expression in
malignant
melanoma cells when contacted with malignant melanoma cells, is at least one.
at least two. or
at least three polypeptides. each polypeptide selected from the group
consisting of Oncostatin
M, Starmiocalcin, and Tissue Factor Pathway Inhibitor-2.
Oncostatin M is the subject of United States patent 5,618.71 to Shoyab et al.,
which is
s expressly incorporated herein by reference.
Stanniocalcin is the subject of United States patents 5.994,301, 5,877,290,
and
5,837,498, and of WO 9524411, all of which are expressly incorporated herein
by reference.
Tissue Factor Pathway Inhibitor-2 is the subject of United States patents
5,849.875,
5,773,251, 5,76,294, 5,466.783, x.106.833, and 4.966.852. all of which are
expressly
3u incorporated herein by reference.
The malignant melanoma cell isolate can be obtained from a non-homogenous
proteinaceous solution such as a cell culture supernatant or cell homogenate.
Malignant
melanoma cells can be isolated from a subject using a tumor biopsy, by
disaggregating the
biopsy sample. and forming cell suspensions. These malignant melanoma cell
suspensions
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can be cultured according to standard cell culture techniques. In small scale.
the cultures can
be contained in culture plates, flasks, and dishes. In important embodiments.
under standard
culture conditions (e.g., on typical culture plates, flasks. and dishes that
are 'static' -i.e.
nonperfused), the culture conditions include a ratio of 5 x 106 cells/ml of
medium. In larger
scale, the cultures can be contained in roller bottles, spinner flasks (i.e.
'nonstatic') and other
large scale culture vessels such as fermenters. Culturing in a three-
dimensional. porous, solid
matrices, as well as constant perfusion of media conditions may also be used.
Conveniently. the malignant melanoma cell isolate can be obtained from the
supernatants of the above-described cell cultures, although the entire culture
can be
m homogenized and subjected to the steps described below for isolation of a
malignant
melanoma cell isolate that down-regulates Melan-A/MART-1 expression in
malignant
melanoma cells when contacted with malignant melanoma cells. Typically the
supernatant is
removed by aspiration or by centrifugation of the cell culture to remove the
cells. The cultures
can also be filtered to remove cells and cell debris. In important
embodiments. the collected
supernatant is (in its entirety) the malignant melanoma cell isolate.
The malignant melanoma cell supernatant can be fractionated according to
standard
chromatographic procedures to facilitate further isolation of the desired
agent. One of
ordinary skill in the art will be familiar with such procedures that include.
but are not limited
to, size-exclusion chromatography. FPLC, HPLC, gel filtration chromatography,
ion-exchange
~n chromatography, hydrophobic chromatography, immune-affinity chromatography,
electrophoresis, etc.
In preferred embodiments, the fractions of malignant melanoma cell isolate-
containing
supernatant then are used to down-regulate Melan-A/MART-1 expression in
malignant
melanoma cells when contacted with malignant melanoma cells. The down-
regulating
activity of the fraction can be measured as described above, or according to
the assays
described in greater detail in the Examples. Other suitable methods will be
known to one of
ordinary skill in the art and can be employed using routine experimentation.
The fractions which are positive for the malignant melanoma cell isolate can
be
subjected to additional rounds of screening using the foregoing methodology.
The purity of
3n the fraction can be assessed after each round of culture stimulation by
subjecting an aliquot of
the fraction to SDS-PAGE or other analytical methods for visualizing the
mixture of
constituents in the fraction. The nature of the malignant melanoma cell
isolate as a protein,
nucleic acid, lipid, carbohydrate etc., can be confirmed at any time by
treating an aliquot of a
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positive fraction with non-specific degradative enzymes for the foregoing
classes of molecules
and testing the treated fraction in the same assays detailed above.
The malignant melanoma cell isolate can then be further purified for the
active down
regulating agent. if desired, using immunological and molecular biological
methods (see, e.g.
_ Molecular Cloning: A Laboratory .Vanual. J. Sambrook, et al.. eds., Second
Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current
P~°otocols
in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons. Inc., New
York). For
example, a fraction positive for one active agent (e.g., the agent that is
heat sensitive at 80°C,
proteinase K sensitive, binds to and elutes-off blue-Sepharose~, and has Melan-
A/MART-1
m expression down-modulating activity) which is sufficiently purified can be
subjected to protein
sequencing according to standard methods. For example, the fraction can be
subjected to
SDS-PAGE, transferred to a membrane such as polyvinylidene fluoride by
electroblotting, and
N-terminal amino sequence determined by Edman degradation. mass spectroscopy,
etc. Any
sequence information can be used to screen databases for homology to existing
proteins and
also to generate degenerate nucleic acids useful for screening a cDNA library
by standard
methods such as colony hybridization or polymerase chain reaction.
Alternatively, the
positive fraction can be used to generate antibodies which recognize the
malignant melanoma
cell isolate. Such antibodies can then be used in expression cloning
protocols, Western blots,
and other techniques useful in isolation of the malignant melanoma cell
isolate. In the
n foregoing methods, any cDNA libraries, expression libraries, etc., are
preferably created from
malignant melanoma cells.
In important embodiments. the malignant melanoma cell isolate, comprises at
least
one, at least two. or at least three polypeptides, each polypeptide selected
from the group
consisting of Oncostatin M, Stanniocalcin, and Tissue Factor Pathway Inhibitor-
2.
The invention also makes it possible to isolate agents which bind to the
malignant
melanoma cell isolate as disclosed herein, including antibodies and cellular
binding partners of
the malignant melanoma cell isolate, such as receptors or ligands. Once the
malignant
melanoma cell isolate or active agent with down-regulating activity is
substantially pure, it can
be used, for example, to generate polyclonal or monoclonal antibodies
according to standard
3n methods (see e.g., Harlow and Lane, eds., .-Intibodies: A Laboratory
Manual. Cold Spring
Harbor Press. Cold Spring Harbor. NY, 1988)
The organic agents which bind to the active agent in the malignant melanoma
cell
isolate can be used, for example, in screening assays to detect the presence
or absence of the
active agent in the malignant melanoma cell isolate and complexes of the
active agents and
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their respective binding partners, and in purification protocols to isolate
the active agent in the
malignant melanoma cell isolate. The binding organic agents also can be used
to block the
effects of the agent down-regulating TAA expression in malignant melanoma
cells.
The invention. therefore, embraces organic binding agents which. for example,
can be
antibodies or fragments of antibodies having the ability to selectively bind
the active agents)
in malignant melanoma cell isolates and inhibit the Melan-A/MART-1 expression
down-
modulating properties of the agents) in the isolate. In important embodiments,
the organic
binding agents are peptides. In further important embodiments, the peptide
binding agents
bind selectively to the -~-25kD polypeptide with Melan-A/MART-1 expression
down-
m modulating activity and inhibit down-modulation of Melan-A/MART-1 expression
in
malignant melanoma cells, thereby allowing TILs and other cells of the immune
system to
recognize the malignant melanoma cells as foreign leading to their
elimination. The organic
binding peptides can be antibodies, including polyclonal and monoclonal
antibodies, prepared
according to conventional methodology. In important embodiments, the organic
binding
~s peptides are antibodies that bind to one or more polypeptides, each
polypeptide selected from
the group consisting of Oncostatin M, Stanniocalcin, and Tissue Factor Pathway
Inhibitor-2.
Anti-Oncostatin-M antibodies are known in the art. and include Mouse and Goat
anti-
human Oncostatin M from Research Diagnostics. Flanders. NJ.
Anti-starmiocalcin antibodies and methods of preparing such antibodies are
known in
~n the art, some of which are described in detail in United States patent
5.877.290 to Olsen.
Anti-Tissue Factor Pathway Inhibitor antibodies are also known in the art, and
include
sheep anti-human Tissue Factor Pathway Inhibitor antibodies from Enzyme
Research
Laboratories. South Bend, IN.
Significantly, as is well-known in the art. only a small portion of an
antibody molecule.
's the paratope, is involved in the binding of the antibody to its epitope
(see. in general, Clark.
W.R: (1986) The Experimental Foundations of Modern Immunolo~y Wiley & Sons.
Inc., New
York; Roitt. I. (1991) Essential Immunolo~v. 7th Ed.. Blackwell Scientific
Publications.
Oxford). The pFc' and Fc regions, for example. are effectors of the complement
cascade but
are not involved in antigen binding. An antibody from which the pFc' region
has been
;n enzymaticallv cleaved. or which has been produced without the pFc" region.
designated an
F(ab')2 fragment, retains both of the antigen binding sites of an intact
antibody. Similarly. an
antibody from which the Fc region has been enzymatically cleaved, or which has
been
produced without the Fc region, designated an Fab fragment, retains one of the
antigen
binding sites of an intact antibody molecule. Proceeding further. Fab
fragments consist of a
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covalently bound antibody light chain and a portion of the antibody heavy
chain denoted Fd.
The Fd fragments are the major determinant of antibody specificity (a single
Fd fragment may
be associated with up to ten different light chains without altering antibody
specificity) and Fd
fragments retain epitope-binding ability in isolation.
Within the antigen-binding portion of an antibody. as is well-known in the
art. there
are complementarity determining regions (CDRs). which directly interact with
the epitope of
the antigen. and framework regions (FRs), which maintain the tertian'
structure of the paratope
(see. in general, Clark, 1986: Roitt, 1991 ). In both the heavy chain Fd
fragment and the light
chain of IgG immunoglobulins, there are four framework regions (FR1 through
FR4)
m separated respectively by three complementaritv determining regions (CDR1
through CDR3).
The CDRs. and in particular the CDR3 regions. and more particularly the heavy
chain CDR3,
are largely responsible for antibody specificity.
It is now well-established in the art that the non-CDR regions of a mammalian
antibody may be replaced with similar regions of cospecific or heterospecific
antibodies while
retaining the epitopic specificity of the original antibody. This is most
clearly manifested in
the development and use of "humanized" antibodies in which non-human CDRs are
covalently
joined to human FR and/or Fc/pFc' regions to produce a functional antibody.
Thus. for
example. PCT International Publication Number WO 92/04381 teaches the
production and use
of humanized murine RSV antibodies in which at least a portion of the murine
FR regions
=n have been replaced by FR regions of human origin. Such antibodies.
including fragments of
intact antibodies with antigen-binding ability. are often referred to as
"chimeric'' antibodies.
Thus, as will be apparent to one of ordinary skill in the art. the present
invention also
provides for F(ab')Z, Fab, Fv and Fd fragments: chimeric antibodies in which
the Fc and/or FR
and/or CDRI and/or CDR2 and/or light chain CDR3 regions have been replaced by
homologous human or non-human sequences: chimeric F(ab')~ fragment antibodies
in which
the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been
replaced by
homologous human or non-human sequences: chimeric Fab fragment antibodies in
which the
FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced
by
homologous human or non-human sequences: and chimeric Fd fragment antibodies
in which
3n the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous
human or non
human sequences. The present invention also includes so-called single chain
antibodies.
Thus, the invention involves polypeptides of numerous size and type that bind
specifically to the active agent in the malignant melanoma cell isolate. and
complexes of both
the active agent in the malignant melanoma cell isolate and its binding
partners. These
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specifically binding polypeptides may be derived also from sources other than
antibody
technology. For example. such polypeptide binding agents can be provided by
degenerate
peptide libraries which can be readily prepared in solution, in immobilized
form or as phage
display libraries. Combinatorial libraries also can be synthesized of peptides
containing one or
more amino acids. Libraries further can be synthesized of peptides and non-
peptide synthetic
moieties.
As detailed herein. the foregoing antibodies and other binding molecules may
be used
for example to identify tissues expressing protein or to purify protein.
Antibodies also may be
coupled to specific diagnostic labeling agents for imaging of cells and
tissues that express the
m active agent in the malignant melanoma cell isolate.
The agent which is a tumor-antigen expression down-regulating agent is an
agent
present in a tumor cell culture supernatant. tumor cell eluate, and/or tumor
cell lysate. The
tumor cell may be of a cancer or tumor type thought to escape immune
recognition. Such
cancers or tumors may be of the folowing origin: biliary tract cancer; brain
cancer, including
malioblastomas and medulloblastomas; breast cancer; cervical carcinoma:
choriocarcinoma;
colon cancer: endometrial cancer; esophageal cancer; gastric cancer:
hematological
neoplasms, including acute lymphocytic and myelogenous leukemia; multiple
myeloma; AIDS
associated leukemias and adult T-cell leukemia lymphoma: intraepithelial
neoplasms,
including Bowen's disease and Paget's disease; liver cancer; lung cancer;
lymphomas,
.o including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral
cancer,
including squamous cell carcinoma: ovarian cancer, including those arising
from epithelial
cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer;
prostate cancer; rectal
cancer: sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma,
fibrosarcoma
and osteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma.
basocellular cancer
a and squamous cell cancer, testicular cancer, including germinal tumors
(seminoma, non-
seminoma[teratomas: choriocarcinomas]), stromal tumors and germ cell tumors;
thyroid
cancer. including thyroid adenocarcinoma and medullar carcinoma; and renal
cancer including
adenocarcinoma and Wilms tumor. In certain embodiments. the tumor-antigen can
be Melan-
A/MART-1, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein
3n (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)--C017-lA/GA733,
Carcinoembr~~onic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2,
etv6,
amll; Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1. PSA-
2, and
PSA-3. prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta
chain, MAGE-
family of tumor antigens. GAGE-1.2. BAGS, RAGE. GnT-V, MUM-1. CDK4,
tyrosinase,
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p53, MUC family, HER2/neu, p21 ras, RCAS 1, a-fetoprotein. E-cadherin, a-
catenin. (3-catenin
and y-catenin, p 120ctn, gp 1 OOP"'e~ ~ ~ ~, PRAME, NY-ESO-1, cdc27,
adenomatous polyposis coli
protein (APC), fodrin, Smad family of tumor antigens, lmp-l, EBV-encoded
nuclear antigen
(EBNA)-l, or c-erbB-2. In any of the foregoing embodiments the fraction from
the plurality
of fractions can be undiluted or concentrated.
In important embodiments. cancers or tumors escaping immune recognition and
tumor-
antigens associated with such tumors (but not exclusively), include acute
lymphoblastic
leukemia (etv6amll; cyclophilin b), glioma (E-cadherin; oc-catenin; (3-
catenin; ~-catenin;
p120ctn), bladder cancer (p2lras), billiary cancer (p2lras), breast cancer
(MUC family;
mHER2/neu; c-erbB-2), cervical carcinoma (p53; p2lras), colon carcinoma
(p2lras; HER2/neu;
c-erbB-2; MUC family). colorectal cancer (Colorectal associated antigen (CRC)--
C017-
lA/GA733; APC), choriocarcinoma (CEA), epithelial cell-cancer (cyclophilin b),
gastric
cancer (HER2/neu; c-erbB-2; ga733 glycoprotein), hepatocellular cancer (a-
fetoprotein),
hodgkins lymphoma (lmp-l; EBNA-1), lung cancer (CEA; MAGE-3; NY-ESO-1),
lymphoid
~s cell-derived leukemia (cyclophilin b), myeloma (MUC family; p2lras), non-
small cell lung
carcinoma (HER2/neu; c-erbB-2), nasopharyngeal cancer (lmp-l; EBNA-1), ovarian
cancer
cancer (MUC family; HER2/neu; c-erbB-2), prostate cancer (Prostate Specific
Antigen (PSA)
and its immunogenic epitopes PSA-l, PSA-2, and PSA-3; PSMA; HER2/neu; c-erbB-
2),
pancreatic cancer (p2lrasMUC family; HER2/neu; c-erbB-2, ga733 glycoprotein),
renal
.n (HER2/neu; c-erbB-2), testicular cancer (NY-ESO-1), T cell leukemia (HTLV-1
epitopes),
and melanoma (Melan-A/MART-1; cdc27; MAGE-3; p21 ras; gp 1 OOp"'e" ~ ~ ).
Given the
teachings of the present invention, a person of ordinary skill in the art can
easily identify such
tumor-antigens and apply the methods of the invention to identify agents which
modulate
expression of such antigens.
As used herein, a subject is a human, non-human primate, cow, horse, pig.
sheep. goat,
dog, cat or rodent. In all embodiments. human subjects are preferred.
When used therapeutically, the isolated Melan-A/MART-1 expression down-
modulation inhibitors of the invention are administered in therapeutically
effective amounts.
In general, a therapeutically effective amount means that amount necessary to
delay the onset
o of, inhibit the progression of, or halt altogether the particular condition
being treated.
Generally, a therapeutically effective amount will vary with the subject's
age. condition. and
sex, as well as the nature and extent of the disease in the subject, all of
which can be
determined by one of ordinary skill in the art. The dosage may be adjusted by
the individual
physician or veterinarian, particularly in the event of any complication. A
therapeutically
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effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg,
preferably from about
0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg//kg to
about 20 mg/kg,
in one or more dose administrations daily, for one or more days.
The therapeutically effective amount of the isolated Melan-A/MART-1 expression
down-modulation inhibitors of the invention is that amount effective to
inhibit Melan-
A/MART-1 antigen expression down-modulation. and can be determined using. for
example,
standard tests known in the art. For example. a direct way to measure tumor-
antigen (e.g.,
Melan-A/MART-1) expression the tumor cell (e.g., melanoma) is to use
antibodies specific for
the tumor-antigen and a number of immunocyto- and immunohisto- chemical
protocols well
m known in the art. Antibodies specific for the Melan-A/MART-1 antigen, for
example, are
fully described in U.S. Patent 5,674,749 to Chen et al., entitled: "Monoclonal
antibodies which
bind to tumor rejection antigen precursor Melan-A. and uses thereof.''
Additionally, the isolated Melan-A/MART-1 expression down-modulation
inhibitors
of the invention (i.e.. tumor-antigen(TAA) expression upregulating molecules
of the
~s invention) may be co-administered with an anti-cancer agent other than an
agent of the
invention (e.g., other than an isolated Melan-A/MART-1 expression down-
modulation
inhibitor), that can act cooperatively, additively or synergistically with an
agent of the
invention, for treating or preventing cancers that express (and, it is
believed. through autocrine
secretions down-modulate) tumor-antigens. The term ''co-administered," means
administered
n substantially simultaneously with another agent (e.g., in different or same
compositions/formulations). By substantially simultaneously, it is meant that
a Melan
A/MART-1 expression down-modulation inhibitor of the invention is administered
to the
subject close enough in time with the administration of the other agent (e.g.,
an anti-cancer
agent) whereby the two agents may exert an additive or even synergistic effect
to upregulate
s Melan-A/MART-1 expression and inhibit growth and/or proliferation of the
cancer.
Anti-cancer agents other than agents of the invention include, but are not
limited to:
Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin;
Aldesleukin;
AltretamineAmbomycinAmetantrone Acetate: Aminoglutethimide; Amsacrine;
AnastrozoleAnthramycin; Asparaginase; Asperlin; Azacitidine: Azetepa;
Azotomycin;
~o Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide
Dimesylate;
Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;
Cactinomycin;
Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine: Carubicin
Hydrochloride;
Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine;
Crisnatol Mesylate;
Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin
Hydrochloride;
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Decitabine: Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone;
Docetaxel;
Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate;
Dromostanolone
Propionate: Duazomycin; Edatrexate; Eflornithine Hydrochloride: Elsamitrucin;
Enloplatin:
Enpromate: Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin
Hydrochloride;
Estramustine; Estramustine Phosphate Sodium: Etanidazole; Etoposide; Etoposide
Phosphate;
Etoprine; Fadrozole Hydrochloride; Fazarabine: Fenretinide; Floxuridine;
Fludarabine
Phosphate: Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium;
Gemcitabine;
Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;
Ilmofosine;
Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-
n3; Interferon
mBeta-I a; Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride:
Lanreotide Acetate;
Letrozole: Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;
Lomustine;
Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine
Hydrochloride;
Megestrol Acetate: Melengestrol Acetate; I~Melphalan; Menogaril;
Mercaptopurine;
MethotrexateMethotrexate Sodium; Metoprine; Meturedepa; Mitindomide;
Mitocarcin;
m Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane;
Mitoxantrone
Hydrochloride: Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin;
Oxisuran;
Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;
Perfosfamide;
Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane;
Podofilox;
Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride;
Puromycin;
.n Puromycin Hydrochloride; Pvrazofurin; Riboprine; Rogletimide; Safingol;
Safingol
Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin;
Spirogermanium
Hydrochloride: Spiromustine: Spiroplatin; Streptonigrin; Streptozocin;
Sulofenur;
Talisomycin; Taxotere; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride;
Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine;
Thiotepa;
Tiazofurin; Tirapazamine: Topotecan Hydrochloride; Toremifene Citrate,
Trestolone Acetate;
Triciribine Phosphate; TrimetrexateTrimetrexate Glucuronate; Triptorelin;
Tubulozole
Hydrochloride; Uracil Mustard: Uredepa; Vapreotide; Verteporfin; Vinblastine
Sulfate;
Vincristine Sulfate; Vindesine: Vindesine Sulfate: Vinepidine Sulfate;
Vinglycinate Sulfate;
Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine
Sulfate;
3n Vorozole; Zeniplatin; Zinostatin: Zorubicin Hydrochloride.
The above-described drug therapies are well known to those of ordinary skill
in the art
and are administered by modes known to those of skill in the art. The drug
therapies are
administered in amounts which are effective to achieve the physiological goals
in combination
with the isolated Melan-A/MART-1 expression down-modulation inhibitors of the
invention.
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An isolated Melan-A/MART-1 expression down-modulation inhibitor (tumor-antigen
(TAA) expression upregulating molecule j may be administered alone or in
combination with
the above-described drug therapies as part of a pharmaceutical composition.
Such a
pharmaceutical composition may include the isolated Melan-A/MART-1 expression
down-
_ modulation inhibitor in combination with any standard physiologically and/or
pharmaceutically acceptable carriers which are known in the art. The
compositions can be
sterile and contain a therapeutically effective amount of the isolated Melan-
A/MART-1
expression down-modulation inhibitor in a unit of weight or volume suitable
for
administration to a patient. The term "pharmaceutically-acceptable earner'' as
used herein
m means one or more compatible solid or liquid filler, diluents or
encapsulating substances
which are suitable for administration into a human or other animal. The term
"carrier" denotes
an organic or inorganic ingredient. natural or synthetic, with which the
active ingredient is
combined to facilitate the application. The components of the pharmaceutical
compositions
also are capable of being co-mingled with the molecules of the present
invention, and with
m each other, in a manner such that there is no interaction which would
substantially impair the
desired pharmaceutical efficacy. Pharmaceutically acceptable further means a
non-toxic
material that is compatible with a biological system such as a cell, cell
culture, tissue, or
organism. The characteristics of the carrier will depend on the route of
administration.
Physiologically and pharmaceutically acceptable carriers include diluents,
fillers, salts,
o buffers. stabilizers, solubilizers, and other materials which are well known
in the art.
A variety of administration routes are available. The particular mode selected
will
depend. of course, upon the particular drug selected, the severity of the
condition being
treated. and the dosage required for therapeutic efficacy. The methods of the
invention,
generally speaking, may be practiced using any mode of administration that is
medically
__ acceptable, meaning any mode that produces effective levels of the active
compounds without
causing clinically unacceptable adverse effects. Such modes of administration
include oral,
rectal. topical, nasal, intradermal. or parenteral routes. The term
"parenteral" includes
subcutaneous. intravenous, intramuscular, or infusion. Intravenous or
intramuscular routes are
not particularly suitable for long-term therapy and prophylaxis. They could.
however, be
~n preferred in emergency situations. Oral administration will be preferred
for prophylactic
treatment because of the convenience to the patient as well as the dosing
schedule.
Compositions suitable for parenteral administration conveniently comprise a
sterile
aqueous preparation of the isolated Melan-A/MART-1 expression down-modulation
inhibitor,
which is preferably isotonic with the blood of the recipient. This aqueous
preparation may be
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formulated according to known methods using suitable dispersing or wetting
agents and
suspending agents. The sterile injectable preparation also may be a sterile
injectable solution
or suspension in a non-toxic parenterally-acceptable diluent or solvent. for
example. as a
solution in 1.3-butane diol. Among the acceptable vehicles and solvents that
may be
employed are water, Ringer's solution, and isotonic sodium chloride solution.
In addition.
sterile. fixed oils are conventionally employed as a solvent or suspending
medium. For this
purpose, any bland fixed oil may be employed including synthetic mono- or di-
glycerides. In
addition, fatty acids such as oleic acid may be used in the preparation of
injectables. Carrier
formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc.
administrations
m can be found in Remington's Pharmaceutical Sciences. Mack Publishing Co..
Easton, PA.
The pharmaceutical compositions may conveniently be presented in unit dosage
form
and may be prepared by any of the methods well-known in the art of pharmacy.
All methods
include the step of bringing the isolated Melan-A/MART-1 expression down-
modulation
inhibitor into association with a carrier which constitutes one or more
accessory ingredients.
i > In general, the compositions are prepared by uniformly and intimately
bringing the isolated
Melan-A/MART-1 expression down-modulation inhibitor into association with a
liquid
carrier, a finely divided solid carrier, or both, and then. if necessary,
shaping the product.
Compositions suitable for oral administration may be presented as discrete
units, such
as capsules, tablets, lozenges, each containing a predetermined amount of the
isolated Melan
zo A/MART-1 expression down-modulation inhibitor. Other compositions include
suspensions
in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an
emulsion.
Other delivery systems can include time-release. delayed release or sustained
release
delivery systems. Such systems can avoid repeated administrations of the
isolated Melan-
A/MART-1 expression down-modulation inhibitors described above, increasing
convenience
,; to the subject and the physician. Many types of release delivery systems
are available and
known to those of ordinary skill in the art. They include the above-described
polymeric
systems, as well as polymer base systems such as poly(lactide-glycolide),
copolyoxalates,
polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid.
and
polyanhydrides. Microcapsules of the foregoing polymers containing drugs are
described in,
~n for example, L'.S. Patent 5,075,109. Delivery systems also include non-
polymer systems that
are: lipids including sterols such as cholesterol. cholesterol esters and
fatty acids or neutral fats
such as mono- di- and tri-glycerides; hydrogel release systems; sylastic
systems: peptide based
systems; wax coatings; compressed tablets using conventional binders and
excipients; partially
fused implants: and the like. Specific examples include. but are not limited
to: (a) erosional
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systems in which the isolated Melan-A/MART-1 expression down-modulation
inhibitor is
contained in a form within a matrix such as those described in U.S. Patent
Nos. 4,452,775,
4,675.189, and 5,736,152, and (b) diffusional systems in which an active
component
permeates at a controlled rate from a polymer such as described in U.S. Patent
Nos. 3,854,480,
5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can
be used.
some of which are adapted for implantation.
Use of a long-term sustained release implant may be particularly suitable for
treatment
of chronic conditions. Long-term release, are used herein, means that the
implant is
constructed and arranged to delivery therapeutic levels of the active
ingredient for at least 30
m days, and preferably 60 days. Long-term sustained release implants are well-
known to those
of ordinary skill in the art and include some of the release systems described
above.
The invention also embraces methods for isolating tumor cell-derived tumor-
antigen
expression down-regulating agents. A method according to this aspect of the
invention
typically involves: (a) preparing a culture of tumor cells that have down-
regulated tumor-
m antigen expression, (b) isolating a supernatant or cell isolate suspected of
containing a tumor-
antigen expression down-regulating agent from the culture of tumor cells that
have down-
regulated tumor-antigen expression, (c) fractionating the supernatant or cell
isolate into a
plurality of fractions, (d) contacting a fraction from the plurality of
fractions with a tumor-
antigen expressing tumor cell, (e) measuring tumor-antigen expression on the
tumor-antigen
'n expressing cell, and (f) determining whether tumor-antigen expression on
the tumor cell is
down-regulated as a result of such contacting, for example, by comparison to a
control.
Typically, the tumor-antigen expressing cells contacted in step (d) with the
supernatants (cell
isolates, or fractions thereof) of the cultures of tumor cells that have down-
regulated tumor-
antigen expression cells of step (a), are of the same origin (i.e.,
patient/tissue/cell line source)
., with the tumor cells that have down-regulated tumor-antigen expression
cells of step (a), the
only difference being that the tumor-antigen expressing cells still express
the tumor-antigen.
In some embodiments, the origin of the tumor cells may be of: biliary tract
cancer; brain
cancer, including glioblastomas and medulloblastomas; breast cancer; cervical
carcinoma;
choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric
cancer;
3n hematological neoplasms, including acute lymphocytic and myelogenous
leukemia: multiple
myeloma; AIDS associated leukemias and adult T-cell leukemia lymphoma;
intraepithelial
neoplasms; including Bowen's disease and Paget's disease; liver cancer: lung
cancer:
lymphomas. including Hodgkin's disease and lymphocytic lymphomas;
neuroblastomas; oral
cancer, including squamous cell carcinoma; ovarian cancer, including those
arising from
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epithelial cells, stromal cells. germ cells and mesenchymal cells; pancreas
cancer; prostate
cancer; rectal cancer; sarcomas. including leiomyosarcoma, rhabdomyosarcoma,
liposarcoma,
fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi's
sarcoma.
basocellular cancer and squamous cell cancer; testicular cancer, including
germinal tumors
(seminoma, non-seminoma[teratomas, choriocarcinomas]), stromal tumors and germ
cell
tumors, thyroid cancer, including thyroid adenocarcinoma and medullar
carcinoma: and renal
cancer including adenocarcinoma and Wilms tumor. In certain embodiments, the
tumor-
antigen can be Melan-A/MART-1, Dipeptidyl peptidase IV (DPPIV), adenosine
deaminase-
binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)--
C017-
m lA/GA733. Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1
and
CAP-2, etv6, amll, Prostate Specific Antigen (PSA) and its immunogenic
epitopes PSA-1,
PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell
receptor/CD3-zeta
chain, MAGE-family of tumor antigens. GAGE-1,2, BAGE, RAGE. GnT-V. MUM-l,
CDK4,
tyrosinase, p53, MUC family, HER2/neu, p2lras, RCAS1, a-fetoprotein. E-
cadherin, a-
mcatenin, (3-catenin and y-catenin, p120ctn, gpl00P°'em~, PRAMS, NY_ESO-
1, cdc27,
adenomatous polyposis coli protein (APC), fodrin, Smad family of tumor
antigens, lmp-1,
EBV-encoded nuclear antigen (EBNA)-1, or c-erbB-2. In any of the foregoing
embodiments
the fraction from the plurality of fractions can be undiluted or concentrated.
In important embodiments, cancers or tumors escaping immune recognition and
tumor
s antigens associated with such tumors (but not exclusively), include acute
lymphoblastic
leukemia (etv6; amll; cyclophilin b), glioma (E-cadherin; a-catenin; (3-
catenin: y-catenin;
p 120ctn), bladder cancer (p21 ras), billiary cancer (p21 ras), breast cancer
(MUC family;
HER2/neu; c-erbB-2), cervical carcinoma (p53; p2lras), colon carcinoma
(p2lras; HER2/neu;
c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRC)--
C017
=, lA/GA733; APC), choriocarcinoma (CEA), epithelial cell-cancer (cyclophilin
b), gastric
cancer (HER2/neu; c-erbB-2; ga733 glycoprotein), hepatocellular cancer (a-
fetoprotein),
hodgkins lymphoma (lmp-l; EBNA-1), lung cancer (CEA; MAGE-3; NY-ESO-1),
lymphoid
cell-derived leukemia (cyclophilin b), myeloma (MUC family; p2lras), non-small
cell lung
carcinoma (HER2/neu; c-erbB-2), nasopharyngeal cancer (lmp-1: EBNA-1 ),
ovarian cancer
;mcancer (MUC family: HER2/neu; c-erbB-2), prostate cancer (Prostate Specific
Antigen (PSA)
and its immunogenic epitopes PSA-l, PSA-2, and PSA-3; PSMA: HER2/neu; c-erbB-
2),
pancreatic cancer (p2lras; MUC family; HER2/neu; c-erbB-2: ga733
glycoprotein). renal
(HER2/neuc-erbB-2), testicular cancer (NY-ESO-1), T cell leukemia (HTLV-1
epitopes),
and melanoma (Melan-A/MART-l; cdc27: MAGE-3; p2lras; gp100~me~"')~ In any of
the
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foregoing embodiments the fraction from the plurality of fractions can be
undiluted or
concentrated.
The tumor cell isolate can be obtained from a non-homogenous proteinaceous
solution
such as a cell culture supernatant or a cell homogenate. Tumor cells can be
isolated from a
_ subject using a tumor biopsy, by disaggregating the biopsy sample. and
forming cell
suspensions. These tumor cell suspensions can be cultured according to
standard cell culture
techniques. In small scale, the cultures can be contained in culture plates.
flasks, and dishes.
Under standard culture conditions (e.g., on typical culture plates, flasks.
and dishes that are
'static" -i.e. nonperfused and/or nonmoving), a ratio of ~ x 106 cells/ml of
medium is typically
n~ sufficient to yield an active agent according to the invention, enough so
that its effects could
be demonstrated. The foregoing "typical" ratio of cells/ml of medium can of
course vary
according to the cell type, the stage of the tumor, etc., and a person of
ordinary skill in the art
can easily determine the optimal culture conditions on a per individual cell
type basis utilizing
routine experimentation. Such conditions will also vary when larger scale cell
cultures are
m employed (e.g., use of roller bottles, spinner flasks, fermenters, three-
dimensional, porous,
solid matrices. as well as constant perfusion of media conditions).
Conveniently, the tumor cell isolate can be obtained from the supernatants of
the
above-described cell cultures [i.e.. of the cultured cells of step (a)],
although the entire culture
can be homogenized and subjected to the steps described below for isolation of
a tumor cell
n isolate that down-regulates tumor-antigen expression when contacted with
tumor cells that
express a tumor antigen. Typically the supernatant is removed by aspiration or
by
centrifugation of the cell culture to remove the cells. The cultures can also
be filtered to
remove cells and cell debris. In important embodiments, the collected
supernatant is (in its
entirety) the tumor cell isolate.
=, The tumor cell supernatant can be fractionated according to standard
chromatographic
procedures to facilitate further isolation of the desired agent. One of
ordinary skill in the art
will be familiar with such procedures that include, but are not limited to,
size-exclusion
chromatography, FPLC, HPLC, gel filtration chromatography, ion-exchange
chromatography,
hydrophobic chromatography, immune-affinity chromatography, etc.
3u In preferred embodiments. the fractions of tumor cell isolate-containing
supernatant
then are used to down-regulate tumor-antigen expression in tumor cells when
contacted with
the tumor cells. The down-regulating activity of the fraction can be measured
according to
assays described elsewhere herein. Other suitable methods will be known to one
of ordinary
skill in the art and can be employed using routine experimentation.
Determining whether
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tumor-antigen expression on the tumor cell is down-regulated as a result of
such contacting, is
facilitated by comparison to a control. Typical controls include identically
isolated and
cultured cells. with the exception that the supernatant medium in the control
cultures is
removed at regular intervals during the culture period (e.g. every 2-6 hours).
being replaced
with fresh culture medium. This media change effectively eliminates any down-
regulatory
effects a control-tumor cell isolate may exert on the control-tumor cell tumor-
antigen
expression.
The fractions which are positive for the tumor cell isolate can be subjected
to
additional rounds of screening using the foregoing methodology. The purity of
the fraction
m can be assessed after each round of culture stimulation by subjecting an
aliquot of the fraction
to SDS-PAGE or other analytical methods for visualizing the mixture of
constituents in the
fraction. The nature of the tumor cell isolate as a protein. nucleic acid.
lipid, carbohydrate
etc., can be confirmed at any time by treating an aliquot of a positive
fraction with non
specific degradative enzymes for the foregoing classes of molecules and
testing the treated
i ~ fraction in the same assays detailed above. The tumor cell isolate can
then be further purified
for the active down-regulating agent, if desired, using immunological and
molecular biological
methods (see, e.g. Molecular Cloning.' A Laboratory Manual, J. Sambrook, et
al., eds., Second
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
1989, or
Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley
& Sons, Inc.,
n New York).
The invention in a further aspect provides a method of screening for tumor-
antigen
expression modulating agents. The method involves (a) contacting an agent
suspected of
being a tumor antigen expression modulating agent with a tumor-antigen
expressing tumor
cell, (b) measuring tumor-antigen expression of the tumor cell, and (c)
determining whether
=, tumor-antigen expression on the tumor cell is modulated as a result of such
contacting, for
example, by comparison to a control. Both up-modulating and down-modulating
agents can
be identified using such methods. Tumor cells and tumor-antigens expressed by
the tumor
cells are as described above. In some embodiments. the agent suspected of
being a tumor
antigen expression modulating agent is an agent present in a tumor cell-
culture supernatant.
3n tumor cell eluate, or tumor cell lysate.
According to another aspect of the invention. a method is provided for
isolating an
agent that up-regulates tumor-antigen expression. The method involves (a)
providing a tumor-
antigen expression down-regulating agent which may be isolated according to
any of the
foregoing methods of the invention, (b) preparing a culture of tumor cells.
wherein the tumor
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cells may be identical (as to the patient/tissue/cell line source) to those
used in the isolation of
the tumor-antigen expression down-regulating agent which may be isolated
according to any
of the foregoing methods of the invention, (c) contacting the isolated tumor-
antigen expression
down-regulating agent and its putative inhibitory agent with the culture cells
of step (b), (d)
determining tumor-antigen expression in the culture cells, and (e) comparing
the tumor-
antigen expression determined in (d) with a control. Tumor cells and tumor-
antigens
expressed by the tumor cells are as described above. In preferred embodiments
the tumor cells
are melanoma cells and the tumor-antigen is Melan-A/MART-I. In some
embodiments. the
control tumor-antigen expression is determined in the presence of an agent of
(a) and in
m absence of the putative inhibitory agent of (a). Controls typically include
cultures similar to
the foregoing control cultures described earlier.
As described in the foregoing paragraph, the method involves contacting the
isolated
tumor-antigen expression down-regulating agent and its putative inhibitory
agent with a
culture of tumor cells. wherein the tumor cells may be identical (as to the
patient/tissue/cell
is line source) to those used in the isolation of the tumor-antigen expression
down-regulating
agent. Typically, a plurality of cultures are run in parallel, each culture
containing different
concentrations of the putative inhibitory agent in order to obtain a different
level of tumor-
antigen expression. Typically, one of these concentrations serves as a
negative control, i.e., at
zero concentration of agent or at a concentration of agent below the limits of
assay detection.
~o Putative inhibitory agents encompass numerous chemical classes. although
typically they are
organic compounds. In certain embodiments, the putative inhibitory agents are
small organic
compounds. i.e.. those having a molecular weight of more than ~0 yet less than
about 2500.
preferably less than about 1000 and, more preferably. less than about X00.
Putative inhibitory
agents comprise functional chemical groups necessary for structural
interactions with
polypeptides and/or nucleic acids, and typically include at least an amine.
carbonyl, hydroxyl
or carboxyl group, preferably at least two of the functional chemical groups
and more
preferably at least three of the functional chemical groups. The putative
inhibitory agents can
comprise cyclic carbon or heterocyclic structure and/or aromatic or
polyaromatic structures
substituted with one or more of the above-identified functional groups.
Putative inhibitory
3n agents also can be biomolecules such as peptides. saccharides, fatty acids.
sterols, isoprenoids,
purines, pyrimidines, derivatives or structural analogs of the above. or
combinations thereof
and the like. In some embodiments, the putative inhibitory agents are
polypeptides that bind
the isolated tumor-antigen expression down-regulating agent (e.g.,
antibodies). Where the
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agent is a nucleic acid. the agent typically is a DNA or RNA molecule.
although modified
nucleic acids as defined herein are also contemplated.
Putative inhibitory agents are obtained from a wide variety of sources
including
libraries of synthetic or natural compounds. For example. numerous means are
available for
random and directed synthesis of a wide variety of organic compounds and
biomolecules,
including expression of randomized oligonucleotides, synthetic organic
combinatorial
libraries, phage display libraries of random peptides, and the like.
Alternatively, libraries of
natural compounds in the form of bacterial, fungal. plant and animal extracts
are available or
readily produced. Additionally. natural and synthetically produced libraries
and compounds
m can be readily be modified through conventional chemical, physical, and
biochemical means.
Further, known pharmacological agents may be subjected to directed or random
chemical
modifications such as acylation, alkylation, esterification. amidification,
etc. to produce
structural analogs of the agents.
A variety of other reagents also can be included in the culture media. These
include
reagents such as neutral proteins (e.g., albumin). etc., which may be used to
facilitate optimal
protein-protein and/or protein-nucleic acid binding. Such a reagent may also
reduce non
specific or background interactions of the reaction components. Other reagents
that improve
the efficiency of the culture assay such as protease inhibitors, nuclease
inhibitors,
antimicrobial agents, and the like may also be used, provided that the
reagents do not
~n adversely affect the growth of the cells in the culture.
The order of addition of components. incubation temperature, time of
incubation. and
other parameters of the assay may be readily determined. Such experimentation
merely
involves optimization of the assay parameters. not the fundamental composition
of the assay.
Preferably, the isolated tumor-antigen expression down-regulating agent and
its putative
', inhibitory agent may be contacted together prior to their addition to the
cell culture. Under
such circumstances, incubation temperatures typically are between 4°C
and 40°C. Incubation
times preferably are minimized to facilitate rapid. high throughput screening.
and typically are
between 0.1 and 10 hours. After incubation. the isolated tumor-antigen
expression down-
regulating agent and its putative inhibitory agent are added to the tumor cell
culture and after
3n further incubation at cell culture conditions (typically between 1 and 48
hours). tumor-antigen
expression is detected by any convenient method available to the user, supra.
The invention will be more fully understood by reference to the following
examples.
These examples, however, are merely intended to illustrate the embodiments of
the invention
and are not to be construed to limit the scope of the invention.
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Examples
Experimental procedures
Materials and Methods
Tumor and Tumor Infiltrating Lymphocytes (TIL)
Melanomas and tumor infiltrating lymphocytes (TIL) from the melanoma tissues
were
obtained according to approved Massachusetts General Hospital guidelines. and
propagated in
vitro as previously described (Hishii M, et al., Proc. Natl. Acad Sci L'S=I.
1997, 94:1378-
1383). Briefly, tumors were cultured in DMEM medium supplemented with 10% FBS,
and
!u TIL were propagated in RPMI 1640 supplemented with 5% Human Serum
containing
recombinant human IL-2 at 100 Units/ml (fetus, Emoryville, CA). TIL clones
were isolated
by limiting dilution as previously described (Hishii M, et al., id). Briefly,
TIL were cloned by
limiting dilution using irradiated mononuclear feeder cells together with PHA
as a polyclonal
stimulus as previously described. Limiting dilution was performed on TIL which
had been in
culture for two weeks prior to cloning. A minimum of 5 x 10~ cells were
utilized for
functional assays and PCR analyses.
Tumor cell lines MU, MO, MA and EW were obtained from cutaneous metastatic
melanoma deposits and some (MU, MO and MA), were previously described
(Pandolfi F, et
al., Clin. Exp. Immunol, 1994, 95:141-7; Pandolfi F, et al., Cancer Res, 1991,
51:3164-3170).
o Melan-A/MART-1 negative variant MU-X were obtained by culture of MU tumor
cells at high
density (>SxlO~ cells/ml) for several days prior to immunoselection with anti-
Melan-
A/MART-1 specific TIL. After 1 week of co-culture of tumor and lymphocytes in
the
presence of recombinant human IL-2, the tumor cells which propagate were
collected and
maintained in culture in the absence of T cells. The melanoma origin of the
lines was
__ confirmed using antibodies to melanoma-associated antigen S-100 and HMB-45
(Ordonez N,
et al., Am J Clin Pathol, 1988. 90:385-90). Both MU and MA tumor s expressed
HLA-A2;
MU tumor cells were also HLA-A1 positive. MO tumor was derived from a patient
whose
leukocytes expressed HLA-A2, while MO tumor cells did not express this class I
MHC
antigen. Tumor cells were cultured either in DMEM supplemented with 10% human
serum
;n (MU). or in RPMI 1640 supplemented with 5% human serum (MA and MO).
Expression of Melan-AIMART 1 by tumor cells
To evaluate the expression of Melan-A/MART-1 antigen in the cytoplasm of
melanoma tumor cell lines, the cells were first fixed for 1 Omin in 1 %
paraformaldehyde,
followed by Smin in 0.1% saponin prior to addition of monoclonal antibody
specific for
Melan-A/MART-1, A-103, (Castelli C, et al., J Exp Med, 1995. 181:363-8)(a kind
gift of E.
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Stockert and L. Old. Ludwig Institute. New York, NY) for 4~min at 22°C.
Following two
washes. the cells were stained for 30min with FITC-conjugated goat-anti-mouse
Ig antibody
(DAKO, Carpenteria, CA) prior to fixation in 1% paraformaldehyde and analysis
by flow
cytometry (FACScan, Becton-Dickinson, Mt. View, CA). Histograms of
fluorescence
staining were generated for comparison of anti-Melan-A~'vIART-1 staining of
various cell
populations. Mean channel fluorescence was calculated using the "Consort 30"
software
provided by the manufacturer.
Generation of Conditioned Medium
Conditioned medium from melanoma tumor Iines were generated by culturing cells
at
m a starting concentration of 5 x 105 cells/ml in DMEM medium supplemented
with between 1
and 10% FBS. Supernatants were collected after 72 hours by centrifugation of
the cell
cultures and filtration of the medium through a 0.2 micron filter (Millipore.
Bedford, MA).
Conditioned medium containing 1% FBS was concentrated between 10 and 20 fold
by
collecting the retentate from a nominal 30kD YM membrane (Centriprep,
Millipore, Bedford,
~s MA).
Cytotoxicity Assays
s~
TIL were assayed for the ability to lyse melanoma target cells in 4 hour Cr-
release
assays as previously described (Hishii M, et al., Proc. :Vatl. Acad Sci USA.
1997, 94:1378-
1383). The melanoma target cells with high constitutive expression of Melan-
A/MART-1
~n were generated by low density culture ( 1-2 x 1 OS/ml) were compared with
respect to their
susceptibility to cytolysis with the same cells cultured for 3 to 6 days in
the presence of
conditioned medium from the Melan-A/MART-1 negative variant, MU-X. to derive
target
cells with low Melan-A/MART-1 expression. Low Melan-A/MART-1 expressing cells
were
further assayed after pulsing with Melan-A/MART-1 peptide 27-35, (AAGIGILTV,
SEQ ID
NO:1)( Boon T. et al., Ann Rev Immunol, 1994, 12:337-6~: Sensi M, et al., Proc
Natl Acad Sci
USA, 1995, 92:5674-8; Mackensen A, et al., Cancer Res, 1993, 53:3569-73;
Peoples G, et al.,
J Immunoh 1993, 151:5472-80), by culturing these target cells at 37o for 2
hours in 1 ml of
medium containing ~~g of peptide prior to labeling with Cr for use in
cytolytic assays to
demonstrate renewed susceptibility to specific T cell recognition (essentially
as previously
3n described in Kurnick J. et al., Clin Immunol Immunopathol. 1986, 38:367-
380).
In further instances. bulk and cloned TIL progeny was also assayed against
autologous
tumor (MU). allogeneic melanomas, as well as NK (K~62) and LAK (Daudi), and
EBV-
transformed B lymphocyte targets: EBV-3 (HLA-A1. B8, DR3), EBV-19 (HLA-A2,
B18,
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DRS), using the foregoing ' Cr-release assay. Pulsing included the following
melanocyte
lineage-derived peptides: Tyrosinase (Rivoltini, L., et al., J. Immunol, 1995,
14:2257-2265):
MLLAVLYCL (SEQ ID N0:2) or YMNGTMSQV(SEQ ID N0:3), MAGE-3 (Gaugler B, et
al., J Exp Med, 1994, 179:921-30): EVDPIGHLY (SEQ ID N0:4). Clones were
screened for
cvtotoxic activity at effector to target ratios of 50:1 and below.
Example l: Melanoma-Antigen Recognition by Tumor Infiltrating T Lymphocytes
(TIL):
Effect of Differential Expression of lhlelan-AlMART 1
Results
Phenotype and Function of TIL Bulk and Clones:
~n The IL-2 responding TIL propagated from patient MU were over 98% CD3+,
CD8+,
(CD4-) T cells expressing the a.(3 TCR. As previously reported (Pandolfi F, et
al., Clin. Exp.
51
Immunol, 1994, 95:141-7), strong cytotoxic activity was noted. when tested in
a Cr-release
assay against the autologous MU-melanoma tumor cell line. This Bulk TIL line
showed
minimal NK or LAK activity, as it was only weakly lytic to K562 or Daudi
target cells (18,
~s Pandolfi F, et al., Clin. Exp. Immunoh 1994, 95:141-7; Pandolfi F, et al.,
Cancer Res, 1991,
51:3164-3170). The ability of Bulk MU-TIL to lyse autologous tumor targets was
blocked by
antibodies to HLA-Class I antigens (W6/32). Furthermore, the TIL did not lyse
the EBV-
transformed B cell lines, EBV-3 or EBV-19.
From limiting dilution of the Bulk culture (MU-TIL), a total of 145 MU-TIL
clones
~n were isolated as previously described (Pandolfi F, et al., Clin. Exp.
Immunoh 1994, 95:141-7).
As summarized in Table l, for further analysis we selected 8 of the clones
which showed
consistent anti-tumor cytotoxicity (at <25:1 effector to target ratio),
without NK activity.
Table 1. Summary of Functional Activities and TCR Usage By MU-TIL Clones
zs
MU-45 +++ V2S 1 V 19D1 Three clones
J41 J2S6
MU-63 +++ V2S 1 V 19D have identical
J41 1 J2
S6
MU-79 ++++ V2S1J41 V19DIJ2S6TCR
MU-9 +++++ V2SIJ35 V14D2J2S7Two clones have
identical
MU-11~ +++ V2S1J3~ V14D2J2S7TCR
MU57 +++++ V2S1J22 VSS8D1J1S TCRa.chainsimilarto
1 1 8B3
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-79_
MU-10 ++++ V2SSJ27 V3D2J2S3 Two clones have
V23J10 identical TCR~3
chains.
but different TCRAJ
MU-58 +++ V2SSJ23 V3D2J2S3 and additional
different
V 15J TCRAV
15
MU-135 ++++ V7S2J27 V6DIJ2S2 ~ NK killing only,
no melanoma killing
B. Tumour target lysis is shown a follows: <10% specific lysis: -; 10-20%: + :
'_'0-40%: ++ ;
C. 40-60%: +++ ; 60-80%: ++++ ; >80%: ++++-r .
s Peptide-Specificity of MU TIL:
The Bulk-MU culture and clones MU-4~, MU-57, MU-63, MU-79 and MU-115
showed strong reactivity against EBV-19 targets pulsed with Melan-A/:~MART- 1
peptide
(AAGIGILTV, SEQ ID NO:1). Two additional clones (MU-10 and MU-~8) failed to
lyse
these EBV-targets pulsed with the Melan-A/MART-1. None of the T cells lysed
the EBV
m targets alone, or these targets pulsed with tyrosinase or MAGE 3 peptides.
One of the clones,
MU-9, which lysed autologous melanoma targets could not be recovered from the
freezer for
testing for fine specificity with the melanoma peptides, although its TCR w-as
identical to MU-
11 S, indicating that MU-9 was also likely to be Melan-A/MART-1 peptide
specific. As noted,
clones MU-45, MU-63 and MU-79 were identical to one another as determined by
TCR gene
~s sequencing.
Modulation of Melan-AIMART 1 on Melanoma Cell Lines:
The ability of the Bulk MU-TIL and MU-TIL clones to lyse MU tumor targets
varied
considerably over time; particularly when tumor cells were grown for
protracted periods of
time in the same culture vessel without trypsinization of strongly adherent
cells. The ability of
o the TIL to lyse such tumor targets was notably diminished. We therefore
tested the tumor
cells for the presence of both the Melan-A/MART-1 antigen, (the target antigen
for six of the
clones), and HLA-class I antigen required for peptide antigen presentation.
As shown in Figures 1 and 2, the intensity of intracytoplasmic ~Melan-A/MART-1
antigen expression in tumor cells varied widely. depending on in vitro culture
conditions. The
ability of TIL to lyse the tumor cells was diminished in direct proportion to
the intensity of
cytoplasmic Melan-A/MART-1 expression (Figure 2).
As shown in Figure 1A, when the MU tumor cells were cultured at high density
(5 x
10, cells/ml) the mean channel of fluorescence of Melan-A/MART-1 was
approximately half
_;
that of the same tumor cells cultured at 1 x10 cells/ml. As shown in Figure
?A, the
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susceptibility to lysis of MIU-tumor cells by TIL was similarly reduced by
approximately half
on the low-expressors, (cultured at higher cell density), in comparison to the
high-expressors
(cultured at lower cell density). Further evidence that the diminished
cytotoxicity noted with
these high-density tumor cells was related to Melan-A/MART-1 expression was
the finding
that addition of Melan-A/MART-1 peptide to the low-expressor tumor targets. MU-
Lo and
MLT-X. restored high levels of cytotoxicity (Figure 2B). The specificity of
TIL in this assay
was further demonstrated by their ability to lyse HLA-A2-expressing EBV-
transformed B
cells only after they were pulsed with Melan-A/MART-1 peptide (Figure 2B).
Diminished Melan-A/MART-1-expression (Figure 1B) paralleled decreased target
cell
m lysis for a second tumor cell, (MA), which expressed both Melan-A/MART-1 and
HLA-A2.
A third melanoma tumor target, (MO), also showed diminished Melan-A/MART-1-
expression
with increasing culture density (Figure 1 C), although the absence of HLA-A2
on this tumor
rendered it non-susceptible to lysis by the TIL.
As shown in Figure 1D, while Melan-A/MART-1 decreased among cells cultured at
i ~ high density, the surface staining of HLA-class I antigen did not change
significantly under the
same conditions. The HLA-A2 staining, although weaker than that noted with the
broad-
specificity HLA-class I antibody. also did not change with culture conditions
(Figure 1E),
indicating that this restricting element was not the limiting factor in the
diminished lytic
susceptibility. The unaltered HLA-staining also indicates that not all
proteins were decreased
n in parallel with Melan-A/MART-1 when the tumor cells were cultured at higher
cell density.
Futhermore, the continued presence of HLA-A2, and its ability to serve as a
target antigen for
cytotoxic T cell recognition was reflected by the ability of HLA-A2-specific
alloreactive T
cells to lyse the low HLA-A2-expressing tumor cells, (Figure 2C), as well as a
Melan-
A/MART-1-deficient variant of the MU tumor, although the Melan-A/MART-1
specific T
., cells showed diminished target recognition with reduced Melan-A/MART-1
expression(see
Figure 2A).
The decrease in Melan-A/MART-1. in cells cultured at higher density, was
reversible
when the cells were passaged at lower density. Cells cultured at high density
showed
diminished Melan-A/MART-1 expression (Mean Channel of Fluorescence: 48.9). If
these
_;
;n cells were passaged at high density (5 x 10 cells/ml), they continued to
show low Melan
A/MART-1 (l~~iean Channel: 40.9), while culture of the same cells for 7 days
beginning at 1 x
10, cells/ml resulted in an approximate doubling of Melan-A/MART-1 expression
(Mean
_;
Channel: 108.9). Successive passage of cells at 1 x 10 cells/ml allowed higher
retention of
higher levels of Melan-A/MART-1 expression (Mean Channel: 112.1), but. when
these cells
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were allowed to grow to high density over a subsequent 7 day culture period.
the resulting
culture again showed decreased Melan-A/MART-1 intensity (Mean Channel: 46.9).
These
results indicate the cycling of Melan-A/MART-1 expression in relation to the
time in culture
and cell density. As noted in Figure 2. the decrease in Melan-A/MART-1
intensity was
a paralleled with a decreased susceptibility to lysis.
Example 2: .-Iutocrine down-modulation of tumor-antigen recognition
Results
Melan-A~'MART-1 expressing melanocarcinoma cell lines can spontaneously down
modulate expression of this cytoplasmic antigen, as demonstrated by staining
with the
m monoclonal antibody, A-103 (Chen Y, et al., Proc Natl Acad Sci USA, 1996,
93:5915-5919),
;_
when the cells are cultured at high concentration (~ x 10 cells/ml), as
compared to cells
cultured at lower density (1 x 10, cells/ml). Similarly. down-modulation of
Melan-A/MART-1
was also observed by the addition of supernatants collected from melanomas
with low, or
undetectable, Melan-A/MART-1 expression. Culture of tumor cells at low cell
concentration
is (1x105 cells/ml) in the presence of supernatants obtained from tumor cell
cultured at high
density which express low levels of Melan-A/MART-1. resulted in down-modulated
levels of
cytoplasmic Melan-A/MART-1 expression. Of particular note was that tumor
variants which
have constitutively low, or undetectable levels of expression of Melan-A/MART-
1 molecules,
such as MU-X and EW, produced the highest levels of "antigen silencing"
activity. This
~n down-modulation is reversible. as the cytoplasmic expression of Melan-
A/MART-1 is up-
modulated when the tumor cells are returned to culture at low cell numbers. At
the same time,
HLA class I expression was NOT down-regulated by high density growth of tumor
cells or
following exposure to supernatants from Melan-A/MART-1-negative tumor
variants.
Significantly, the diminution of expression of the Melan-A/MART-1 antigen
~s correlated with reduced susceptibility of the target cells to lysis by HLA-
A2-restricted, Melan
A/MART-1-specific CTL. Thus, as the level of Melan-A/MART-1 antigen expression
by
tumor cells diminished, while levels of HLA-A2 cell surface expression did not
decrease, the
T cell recognition of these targets decreased. However. when the target cells
were pulsed with
the Melan-A/MART-1 peptide (AAGIGILTV. SEQ ID NO:l) (Chen Y, et al.,
P~°oc Natl Acad
~n Sci USA, 1996. 93:5915-5919; Mattei, S., et al., Int. J. Cancer. 1994.
56:853-7: Paglia, D., et
al., J. Interferon Cytokine Res, 1995, 15:455-60. Armstrong. C. et al., Exp.
Dermatol. 199?,
1:37-45; Bennicelli, J. L. & Guerry, D. Exp Dermatol. 1993, 2:186-90; Francis,
C'~. M., et al.,
Melanoma Res, 1996, 6:191-201; Singly R. K., et al., J. Interferon Cytokine
Res, 1995, 15:81-
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7; Castelli C. et al., J Exp Med. 1995. 181:363-8), normal levels of cell
lysis were observed,
indicating that the tumor cells had not become resistant to cell-mediated
lysis.
Previously it was shown that the absence of Melan-A/MART-1 expression
correlated
with a lack of Melan-A/MART-1 gene promoter activity (Butterfield. L. H.. et
al., Gene. 1997,
191:129-34). This activity was found to be mediated through the 233bp minimal
promoter of
the Melan-A/MART-1 gene.
The Melan-A/MART-1 silencing activity could be heat inactivated by treatment
of the
supernatants at 80oC for 60 minutes. A similar treatment at 60°C did
not influence the
activity.
~n To define this silencing activity in more detail, an assessment of cytokine
and
chemokine production by tumor cells which lacked Melan-A/MART-1 protein
expression was
performed. These Melan-A/MART-1 silencing supernatants were found to contain
several
known cytokines, but they lacked TNFa. which has been demonstrated to have a
partial down-
regulatory activity directed at the Melan-A/MART-1 promoter (Butterfield, L.
H., et al., Gene,
m 1997, 191:129-34). Moreover. the most prevalent proteins identified. IL-6.
IL-8 and MCP-1
did not mimic the antigen silencing of the whole supernatant, either
individually over a 3 log
dosage, or in a "cocktail" approximating the levels detected in the active
supernatants. In all,
we have assessed over 20 proteins, including many known to be secreted by
melanomas
(Mattel; S., et al., Int. J. Cancer. 1994, 56:853-7: Paglia, D., et al.. J.
Interferon Cytokine Res,
=n 1995, 15:455-60, Armstrong, C. et al., Exp. Dermatol. 1992, 1:37-4~;
Bennicelli, J. L. &
Guerry, D. Exp Dermatol, 1993. 2:186-90; Francis. G. M., et al., alelanoma
Res, 1996. 6:191-
201; Singly R. K., et al., J. Interferon Cytokine Res, 1995, 1:81-7), for
their capacity to
mimic the antigen silencing activity. Other than TNFa, which was not present
in the active
supernatants, none of the following proteins impacted cytoplasmic Melan-
A,iMART-1
., expression: MSG/GROa, EGF. PDGFa, PDGFb, TGFa, TGF~3. NGF. RANTES. MIP-la,
LIF, PF-4. NAP-2. The following cytokines were not detected in the active
supernatants : IL-
1 (3, .IL-2, IL-12, IL-15, TNFa, IFNy. In addition. heparin, which is known to
bma to several
growth factors, did not affect Melan-A/MART-1 expression, and did not impact
the silencing
activity of active supernatants.
.n Example 3: Characterization of the Melan-AlMART 1 expression down-
modulating activity
(MASA)
Preliminarv data indicate that MASA was stable to low pH (3.0). It was
resistant to
repeated lyophilisation and to high temperature (60°C), but was labile
at 80°C. Recovery of
activity following membrane filtration was also possible (fully retained by 10
kD but only
_ _ partially retained by 50 kD cutoff membranes). It also binds to blue and
red Sepharose (and
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can be eluted off with 1.5M KCl). but not to heparin. The majority of the
activity binds to
Conconavalin A-sepharose (ConA) and can be eluted off with sugar and/or O.SM a-
methyl-
mannoside. By isolating the ConA-bound-eluted material, and further purifying
it by binding
and eluting it from blue and/or red Sepharose, we can delete 99% of the
protein. while
retaining approximately 10% of the original antigen silencing activity.
Material purified by binding and elution from Red Sepharose, followed by
binding and
elution to Con-A-Sepharose, can be size fractionated on G-~0 Sephadex. with
the activity
centering at approximately 25kD. The "activity" parallels the appearance of a
"band" on
polyacrylamide gel electrophoresis of material fractionated on G-50 Sephadex.
m Fractionation of cell supernatant concentrate on a High Q anion-exchanger at
pH 8.0
resulted in the majority of the MASA, eluting mostly in the unbound fraction
(QUB pH 8.0).
This activity was associated with less than ~0% of the total protein. A small
amount of MASA
was bound to the column and was eluted off in high salt ( 1 M NaCI (QB pH
8.0)) along with
the majority of the protein. These preliminary data indicated that the MASA
has a pI of about
8. MASA from the QUB pH8.0 fraction was refractionated on the High Q column at
pH 9.5
and the unbound (QUB pH 9.5) and bound (QB pH 9.5, eluted with IM NaCI)
fractions were
collected and assayed for activity. The activity bound at pH 9.5, suggesting
that it has a pI of
7.5-9.5. Further experiments confirmed that the activity elutes at a molecular
weight of about
25kD. Preliminary studies of the stability of MASA in reverse phase HPLC
buffers. 0.1
~n trifluoroacetic acid and 0.1 % trifluoroacetic acid / 30% acetonitrile,
show that the 25kD
molecular weight fraction retains its activity in these buffers, thus enabling
reverse phase
HPLC separation to be considered as a useful additional step in obtaining
highly purified
MASA.
Results
Sample '~ Total Protein (~.g/ml) ' Specific Activity
(Units/mg)**
500 ', 100
QUB 8 '~, 230 ~ 200
1.200 '
i QB 9.5 I 42 i~
~i 25kD* ~~ 0.4 I 120.000
a
*Fraction from Size Exclusion Column
* * 100 Units/mg total protein in internal laboratory "standard" active
supernatant that provides
maximal "antigen-silencing" activity in 72 hour Melan-A/MART-1 staining assay.
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Characterization of Activity (Summary):
Activity was found to be associated with an agent (melanoma isolate) that:
(i) binds to Blue and/or Red Sepharose dye resin (and can be eluted with 1.5M
KCl),
but does not bind to Heparin,
(ii) binds to Conconavalin-A-Sepharose, and can be eluted with O.SM alpha-
methyl
mannoside.
(iii) when purified by binding and elution from Red Sepharose, followed by
binding
and elution to Con-A-Sepharose, can be size fractionated on G-50 Sephadex,
with the activity
m centering at approximately 25kD, and
(iv) is sensitive to proteinase K digestion.
In addition. tryptic digest sequence analysis (performed at Harvard
MicroChemistry,
Cambridge, M.A) showed the presence of sequences corresponding to at least 7
different
~s human proteins. Some of these proteins include Oncostatin-M (OSM),
stanniocalcin (STC),
and tissue factor pathway inhibitor-2 (TFPI-2).
Recombinant Oncostatin-M (OSM) shows "antigen-silencing" activity when tested
on
the human melanoma cell line, "MU-89" (James T. Kurnick, Massachusetts General
Hospital).
At doses of 100-lOng/ml, recombinant OSM causes down-modulation of Melan-
A/MART-1
n expression comparable to that of "active" supernatants isolated from human
melanoma cell
lines, such as the EW tumor cell line (James T. Kurnick, Massachusetts General
Hospital). At
doses below 2 ng/ml, the down modulation of Melan-A/MART-1 on the MU-89 cell
line
minimal to undetectable. (Assay of Melan-A/MART-1 is by cytoplasmic staining
of
Melan-A/MART-1 using monoclonal antibody A-103 (Lloyd Old, Ludwig Institute.
NY),
~s together with FITC-labeled goat anti-mouse antibody analyzed by flow
cytometry for
fluorescence intensity per cell).
A commercially available polyclonal antibody (Research Diagnostics, Flanders,
NJ) to
OSM can block the activity of the recombinant OSM. The same polyclonal
antibody can
block the activity of the most purified fraction of EW-supernatant which had
been purified by
3n Red Sepharose binding and elution. followed by binding to and elution from
Con-A--
Sepharose, and fractionation on G-50 Sephadex, to isolate material of
approximately 25kD.
However, the same polyclonal anti-OSM antibody which blocks recombinant OSM
and
"purified" fractions from EW-supernatant, can only partially (approximately
50%) block the
unfractionated EW- supernatant. Furthermore OSM-depleted EW-supernatant fluid
(produced
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by passage over an anti-OSM immuno-affinity columny is still active in down-
modulating
Melan-A/MART-1 expression by MU-89 melanoma cells. OSM-depleted EW supernatant
shows that most of the residual MASA still binds to Red-Sepharose. The OSM
content of
EW-supernatants (as determined by quantitative ELISA) is approximately lng/ml,
yet the
antigen-silencing activity of this material is equivalent of recombinant OSM
at a dose of
10-20ng/ml. Supernatants from other melanoma cell lines. such as MU-96, which
are active in
antigen silencing, do not have detectable OSM.
In summary, the foregoing data indicate that Oncostatin-M, a known cytokine,
with
well-characterized structure, and several known functions, is capable of
mediating
m antigen-silencing in the melanoma system we have investigated, however. an
additional
molecule or molecules produced by melanoma cells are also able to manifest
antigen
silencing.
Antibodies to the receptor for Oncostatin-M (Research Diagnostics, Flanders,
NJ),
reported to neutralize the OSM activity in other assay systems, do not block
the antigen
>> silencing activity of either purified, recombinant OSM or of the EW-
supernatant.
The gp130 molecule is reported to be a shared chain in the OSM receptor, the
LIF-receptor, and IL-6 receptor. Antibodies to this molecule (also available
from Research
Diagnostics, Flanders, NJ), do not block the antigen- silencing activity of
either purified.
recombinant OSM, or of the EW-supernatant. Antibodies to the LIF-receptor(also
available
zn from Research Diagnostics. Flanders, NJ), which can act as a receptor and
signal transducer
for OSM in some assay systems, do not block the antigen-silencing activity of
either purified,
recombinant OSM, or of the EW-supernatant.
In summary, the foregoing data indicate that Oncostatin-M, and other molecules
capable of manifesting antigen-silencing activity may act through additional
receptors.
including, but not limited to, oncostatin-M binding receptor molecules.
Oncostatin-M is produced by at least one melanoma cell line (EW), (detectable
protein
was demonstrated by ELISA), and mRNA transcripts can be isolated from several
melanoma
cell lines. Melanomas and melanocytes have nto been previously known to
express oncostatin-
M.
3n Antigen-silencing activity can be demonstrated from many different melanoma
cell
lines, including Melan-A/MART-1 negative cell lines. and from PMA-stimulated
U937, a
pro-monocytic leukemic cell line which can be shown to produce Oncostatin-M.
Oncostatin-M
can be used at several fold the dose needed to manifest antigen-silencing (use
up to 100ng/ml)
without causing significant cellular toxicity, but EW supernatants contain
significant toxic
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activities, even in partially purified fractions, indicating that additional
factors are influencing
the cellular viability and phenotype. All isolates which have been shown to be
active at
down-modulating Melan-A/MART-1 expression also seem to alter the shape and
growth of
melanoma cells in 3 day assays. indicating that additional phenotypic changes.
including
cytoskeletal alterations, are being manifested by . MASA-containing materials.
Those skilled in the art will recognize. or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following claims.
All references disclosed herein are incorporated by reference in their
entirety.
~o What is claimed is presented below and is followed by a Sequence Listing.
We claim:
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SEQUENCE LISTING
<110> THE GENERAL HOSPITAL CORPORATION
Kurnick, James T.
<120> COMPOSITIONS AND METHODS FOR REGULATING
TUMOR-ASSOCIATED ANTIGEN EXPRESSION
<130> M0765/7032W0/ERG/KA
<150> U.S. 60/165,806
<151> 1999-11-16
<160> 4
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 9
<212> PRT
<213> Homo sapiens
<220>
<221> PEPTIDE
<222> (1)...(9)
<223> Melan-A/MART-1
<400> 1
Ala Ala Gly Ile Gly Ile Leu Thr Val
1 5
<210> 2
<211> 9
<212> PRT
<213> Homo sapiens
<220>
<221> PEPTIDE
<222> (1)...(9)
<223> Tyrosinase
<400> 2
Met Leu Leu Ala Val Leu Tyr Cys Leu
1 5
<210> 3
<211> 9
<212> PRT
<213> Homo sapiens
<220>
<221> PEPTIDE
<222> (1)...(9)
<223> Tyrosinase
<400> 3
Tyr Met Asn Gly Thr Met Ser Gln Val
1 5
CA 02388517 2002-04-19
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<210> 4
<211> 9
<212> PRT
<213> Homo Sapiens
<220>
<221> PEPTIDE
<222> (1)...(9)
<223> MAGE-3
<400> 4
Glu Val Asp Pro Ile Gly His Leu Tyr
1 5
