WO2001036461A2

WO2001036461A2 - Compositions and methods for regulating tumor-associated antigen expression

Info

Publication number: WO2001036461A2
Application number: PCT/NL2000/000835
Authority: WO
Inventors: Petrus Josephus Van Den Elsen; James T. Kurnick
Original assignee: Academisch Ziekenhuis Leiden
Priority date: 1999-11-16
Filing date: 2000-11-15
Publication date: 2001-05-25
Also published as: WO2001035903A2; EP1232178A2; AU2043201A; CA2388517A1; AU2236201A; JP2003515535A; WO2001036461A3; AU1615701A; WO2001036472A3; WO2001036472A2

Abstract

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-antigens], and more specifically to methods and agents for the treatment of melanomas expressing the Melan-A/MART-1 antigen. The invention also relates to methods for identifying agents that regulate expression of tumor-antigens.

Description

₅ COMPOSITIONS AND METHODS FOR REGULATING

TUMOR-ASSOCIATED ANTIGEN EXPRESSION

Government Sαpport

This work was funded in part by grant number HL-43793 from the National Institutes in of Health. Accordingly, the United States Government may have certain rights to this invention.

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 is 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/M ART- 1 antigen. The invention also relates to methods for identifying agents that regulate expression of tumor-antigens.

2n Background of the Invention

Many solid tumors are presently known to involve the infiltration of autolσgous lymphocytes. These autologous lymphocytes, known in the art as tumor-infiltrating 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 :s appropriate accessory signals leads to a specific cytolytic (cytotoxic) reactivity of the TTLs toward the solid tumors.

Several tumor antigens have been identified in association with a variety of tumors (1- 3). In addition to the identification of TAAs, immunodominant epitopes recognized by TTLs have also been described for widely-expressed lineage-specific antigens, for example, the in HLA-A2-restricted Melan-A MART- 1 in melanomas (4, 5).

Melanomas are aggressive, frequently metastatic tumors derived from either melanocytes or melanocyte related nevus cells (Cellular and Molecular Immunology, 1991 , (e s) 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 ₃₅ often directed against targets expressing HLA-A2 and the immunodominant peptide of Melan- A/MART-1 (AAGIGILTV, SEQ ID NO:l) (6,-9). In such cases, the tumor cells which evoke a cytolytic immune response must also be able to implement a mechanism to elude the immune response. In other reported melanoma cases, 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, β2-microglobulin or s TAP genes, has been suggested as an escape mechanism from the host's immune defenses (10- 13). 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 (14). In addition, selective loss of nominal antigen reportedly also results in immune escape (15, 16). in 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 recognition escape.

is 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 pharmaceutical compositions containing such agents that modulate expression of tumor- associalcd antigens in tumor cells. The invention, therefore, is particularly useful, inter alia.

2t) 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 is 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 when contacted with malignant melanoma cells. The cell isolate can be derived from Melan- A/MART-1 antigen -expressing and or -nonexpressing malignant melanoma cells. Preferably, in 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 one embodiment, the cell isolate mediates its down- regulatory effects on Melan- A/MART- 1 expression through the Melan- A/MART- 1 gene promoter. In a further embodiment, the cell isolate is a supernatant, or fraction thereof, of Melan-A/MART-1 antigen-expressing malignant melanoma cells. 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.

5 According to another aspect of the invention, a substantially pure organic agent is 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 in include a ratio of 5 x 10⁶ cells/ml medium, and the agent is heat sensitive at 80°C, protcinase K sensitive, and binds to and elutes-off blue-Sepharose*(Pharmacia Biotech, Inc., Piscataway, N ). In some embodiments, the organic agent is a polypeptide. In certain embodiments, the malignant melanoma cells express low levels of Melan-A/MART-1 antigen. In further embodiments, the malignant melanoma cells express high levels of Melan-A/MART-1 antigen. is 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 ceil isolate comprises a polypeptide. In certain embodiments, the malignant melanoma cell isolate

2n is a substantially pure polypeptide. 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 fragment, or a fragment including a CDR3 region selective for the polypeptide. The antibody or fragment thereof may be chimeric or

25 humanized.

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

HI substantially pure organic agent comprises a 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 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 fragment, or a fragment including a CDR3 region selective for the polypeptide. The antibody or fragment thereof may be chimeric or humanized. According to a further aspect of the invention, a pharmaceutical composition is 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 in 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 is malignant melanoma cells. The isolated binding organic agent that binds selectively the 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 iι tumor-antigen expression down-regulating agent. The method involves (a) preparing a culture 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 plurality of fractions, (d) contacting a fraction from the plurality of fractions with a tumor-antigen

25 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 glioblasto as and medulloblastomas; breast cancer; cervical carcinoma; in choriocareinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer hematological neoplasms, including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS associated leuke ias and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer, lung cancer, lymphomas, including Hodgkiπ'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, s 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 Wil s tumor. In certain embodiments, the tumor- m antigen can be Melan-A/MART-1, Dipeptidyl peptidase IV (DPPIV), adcnosine deaminase- binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)-C017- 1A GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etvό, 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 is chain, MAGE-family of tumor antigens, GAGE- 1 ,2, BAGE, RAGE, GnT-V, MUM-1 , CDK4, tyrosinase, ρ53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α- catenin, β-catenin and γ-catenin, pl20ctn, gplOO¹'™"¹⁷, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Smad family of tumor antigens, lmp-1, EBV-encoded nuclear antigen (EBNA)-l, or c-erbB-2. In any of the foregoing embodiments

20 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 (etv6; amll; cyclophilin b), glioma (E-cadherin; α-catenin; β-catenin; γ-cateπin; pl20ctn), bladder cancer (p21ras), billiary cancer (p21ras), breast cancer (MUC family; HER2/neu; c-erbB-2), cervical

25 carcinoma (p53; p21ras), colon carcinoma (p21ras; HER2/neu; c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRC)~C017-1A/GA733; APC), choriocareinoma (CEA), epithelial cell-cancer (cyclophilin b), gastric cancer (HER2 neu; c- erbB-2; ga733 glycoprotein), hepatocellular cancer (α-fetoprotein), hodgkins lymphoma (lmp- 1; EBNA-1), lung cancer (CEA; MAGE-3; NY-ESO-1), lymphoid cell-derived leukemia in (cyclophilin b), myeloma (MUC family; p21ras), non-small cell lung carcinoma (HER2/neu; 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-1, PSA-2, and PSA-3; PSMA; HER2 neu; c-erbB-2), pancreatic cancer (p21ras; MUC family; HER2 neu; c-erbB-2; ga733 glycoprotein), renal (HER2/neu; c-erbB-2), testicular cancer (NY-ESO-l), T cell leukemia (HTLV-1 epitopes), and melanoma (Melan- A/MART-1; cdc27; MAGE-3; p21ras; gpl0O^Pmellπ).

The invention in a further aspect provides a method of screening for tumor-antigen

5 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 rumor- 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 in identified. Tumor cells and tumor-anugens 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 tumor cell lysate.

According to another aspect of the invention, a method is provided for isolating an is 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 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 n 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 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 io 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 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 melano a-specific immune response in the subject. In some embodiments, the isolated binding organic agent is a polypeptide. In certain embodiments, the method further comprises co-administering to the subject an anti-tumor agent other than the agents of the invention.

These and other aspects of the invention, as well as various advantages and utilities, s 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. in Figure 2 shows graphs depicting the effect of MART-1/Melan-A expression on target cell recognition and lysis by TIL; Figure 2A shows MU-TIL lysis of autologous MU-tumor; Figure 2B shows MU-TIL lysis of Melan-A MART-1 -negative targets; and Figure 2C shows anti-HLA-A2-specific lysis of targets.

FigureJ shows graphs depicting the down-modulation of Melan- A/MART- 1, but not is Class I HLA, in tumor conditioned-medium.

Figure 4 shows graphs depicting the loss of cytotoxic T cell Recognition of MU-tumor cells concomitant with down-modulation of Melan-A/MART-1 antigen.

Figure 5 shows bar graphs depicting the promoter activity for Melan-A/MART-1 (Figure 5 A), and a 230bp HLA-A (Figure 5B), in a MU tumor treated with supernatant (SUP) from Melan-A/MART-1 negative melanoma tumor cell lines.

Figure 6 shows a Coomassie Blue stain of an agent purified from a Melan-A/MART-1 melanoma tumor cell line supernatant associated with McIan-A/MART-1 downmodulating activity at different stages purification.

₂₅ Brief Description of the Sequences

SEQ ID NO:l is the immunodominant amino acid sequence of the Melan-A/MART-1 peptide.

Detailed Description of the Invention

_JO 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 tumor-associated antigens in tumor cells.

We have discovered, unexpectedly, that tumor cells which normally express or present s 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 secretion/down-modulation). The terms "down-modulation" and "up-modulation" are used interchangeably with the terms "down-regulauon" and "up-regulation," respectively, in 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., 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 is 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 ui 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 embodiment, the malignant melanoma cell isolate is useful in screening for binding agents that inhibit its activity. Inhibition of the isolate' s activity is desirable in melanomas, where the

25 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.

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

HI permit its identification or use according to the invention. "Isolated", when referring to a 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 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. s 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 heat sensitive at 80°C, protcinase K. sensitive, and binds to and elutes-off blue-Sepharose®. Preferably, the substantially pure organic agent is a polypeptide. In important embodiments, in the polypeptide with Melan-A MART-1 expression down-regulating properties in malignant melanoma cells at a molecular weight between about 20kD and about 30 kD, and preferably at about 25kD.

The malignant melanoma cell isolate can be obtained from a non-homogenous proteinaceous solution such as a cell culture supernatant or cell homogenate. Malignant is 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 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.

20 nonperfused), the culture conditions include a ratio of 5 x IO⁶ 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

25 supematants of the above-described cell cultures, although the entire culture can be 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 so 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 chromatography, hydrophobic chromatography, immune-affinity chromatography, electrophoresis, etc. s 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 m 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 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 is 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 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-

:n 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., New York). For example, a fraction positive for one active agent (e.g., the agent that is heat sensitive at 80°C, is proteiπase K. sensitive, binds to and elutes-off blue-Sepharose®, and has Melan- A/MART- 1 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 so 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. Altematively, 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 foregoing methods, any cDNA libraries, expression libraries, etc., are preferably created from malignant melanoma cells. s 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 in methods (see e.g., Harlow and Lane, eds., Antibodies: 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 is 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-rcgulatiπg 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 agent(s)

2<i in malignant melanoma cell isolates and inhibit the Melan-A/MART-1 expression down- modulating properties of the agent(s) 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- modulating activity and inhibit down-modulation of Melan-A/MART-1 expression in

25 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.

Significantly, as is well-known in the art, only a small portion of an antibody molecule,

30 the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) The Experimental Foundations of Modem Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology. 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 enzymatically 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 cieaved, or which has been s 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 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 in 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 tertiary structure of the paratope (sec, in general, Clark. 1986; Roitt, 1991 ). In both the heavy chain Fd fragment and the light is chain of IgG iπununoglobulins, there are four framework regions (FRl through FR4) separated respectively by three complementarity determining regions (CDRl 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 noπ-CDR regions of a mammalian antibody t) may be replaced with similar regions of cospecific or hetcrospecific 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/pFc¹ regions to produce a functional antibody. Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use 5 of humanized murine RSV antibodies in which at least a portion of the murine FR regions 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')₂, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR jo and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')₂ 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; 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 the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non- human sequences. The present invention also includes so-called singie chain antibodies. s 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 specifically binding polypeptides may be derived also from sources other than antibody technology. For example, such polypeptide binding agents can be provided by degenerate in peptide libraries which can be readily prepared in solution, in immobilized form or as hage 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 is 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 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

2o tumor cell may be of a cancer or tumor type thought to escape immune recognition. Such cancers or tumors may be of the folowiπg origin: biliary tract cancer; brain cancer, including glioblasto as and medulloblastomas; breast cancer; cervical carcinoma; choriocareinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms, including acute lymphocytic and myeiogenous leukemia; multiple myeloma; AIDS associated 5 leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen's disease and Pagct'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 epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer, sarcomas, so 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 -14-

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)~C017-1A/GA733, Carcinoembryonic Antigen (CEA) s and its immunogenic epitopes CAP-1 and CAP -2, etvό, amll, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMΛ), T-cell rcccplor/CD3-/.ctii chain, MAGE-family of tumor antigens, GAGIΪ-1.2. BAGE, RAGE, GnT-V, MUM-1, CD 4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCASl, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, pl20ctn, gplOϋ¹"™"¹⁷, in PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Smad family of tumor antigens, lmp-1, 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-

15 antigens associated with such tumors (but not exclusively), include acute lymphoblastic leukemia (etv6; amll; cyclophilin b), glioma (E-cadherin; α-catenin; β-catenin; γ-catenin; pl20ctn), bladder cancer (p21ras), billiary cancer (p21ras), breast cancer (MUC family HER2/neu; c-erbB-2), cervical carcinoma (ρ53; p2Iras), colon carcinoma (p21ras; HER2/neu c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRQ— C017- 0 1A/GA733; APC), choriocareinoma (CEA), epithelial cell-cancer (cyclophilin b), gastric cancer (HER2/neu c-erbB-2; ga733 glycoprotein), hepatocellular cancer (α-fetoprotein), hodgkins lymphoma (lmp-1 ; EBNA-1), lung cancer (CEA; MAGE-3; NY-ESO-1), lymphoid cell-derived leukemia (cyclophilin b), myeloma (MUC family; p21ras), non-small cell lung carcinoma (HER2/πeu; c-erbB-2), nasopharyngeal cancer (lmp-1; EBNA-1), ovarian cancer 5 cancer (MUC family; HER2/neu; c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3; PSMA; HER2/neu; c-erbB-2), pancreatic cancer (p21ras; 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; p21ras; gpl00^Pmclιπ). Given the teachings sn 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. s In general, a therapeutically effective amount means that amount necessary to delay the onset 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 o physician or veterinarian, particularly in the event of any complication. A therapeutically 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 s 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 n 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 Mclan-A/MART-1 expression down-modulation inhibitors of the invention (i.e., tumor-antigen(TAA) expression upregulatiπg molecules of the invention) 5 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 substantially I 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) wherεby the tWO gsniS may exert an additive or even synergistic effect to upregulate 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; j Altretamine; Ambomycin; A etantrone Acetate; Amiπoglutethimide; Amsacrine;

Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin;

Bati astat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate;

Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;

Calusterone; Caracemide; Carbetimer, Carboplatin; Carmustine; Carubicin Hydrochloride; in Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;

Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubiciπ Hydrochloride;

Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;

Doxorubicin; Doxorubiciπ Hydrochloride; Droloxifene; Droioxifene Citrate; Dromostanolone

Propionate; Duazomyciπ; Edatrexate; Eflomithine Hydrochloride; Elsamitxucin; Enloplatin; is 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; Idarubiciπ Hydrochloride; Ifosfamide; Ilmofosine; 20 Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon

Beta-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; Meleπgestrol Acetate; Melphalan; Menogaril; Mercaptopurine; 5 Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin;

Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantroπe

Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;

Paclitaxel; Pegaspargase; Pelio ycin; Pentamustine; Peplomycin Sulfate; Perfosfamide

Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Podofilox; so Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromyciπ;

Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol

Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium

Hydrochloride; Spiromustiπc; Spiroplatin: Streptonierin; Streptozocin; Sulofenur; Talisomycin; Taxotere; Tecogalan Sodium; Tegarur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofuπn; Tirapaza ine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole s Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate Vinleurosine Sulfate; Vinorelbine Tartrate; Viπrosidine Sulfate; Vinzolidine Sulfate; Vorozole Zeniplatin; Zinostatin; Zorubicin Hydrochloride.

The above-described drug therapies are well known to those of ordinary skill in the art in 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.

An isolated Melan-A/MART-1 expression down-modulation inhibitor (tumor-antigen (TAA) expression upregulating molecule) may be administered alone or in combination with is 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 D expression down-modulation inhibitor in a unit of weight or volume suitable for administration to a patient. The term "pharmaceutically-acceptable carrier" as used herein 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 5 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 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 ui characteristics of the carrier will depend on the route of admimstration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, 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, s 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 preferred m 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 is 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

.'ft 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 can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

₂₅ 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. In general, the compositions are prepared by uniformly and intimately bringing the isolated in Melan-A/MART-1 expression down- modulation inhibitor into association with a liquid earner, 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- 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 descπbed above, increasing convenience to the subject and the physician Many types of release delivery systems are available and known to those of ordinary skill m the art They include the above-described polymeric systems, as well as polymer base systems such as poly(lactide-glycohde), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, poiyhydroxybutyπc acid, and in polyanhydrides Microcapsuies of the foregoing polymers containing drugs are described in, for example, U.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 tπ-glyccπdes; hydrogel release systems; sylasuc systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially is fused implants; and the like Specific examples include, but are not limited to: (a) erosional systems in which the isolated Melan- A/M ART- 1 expression down-modulation inhibitor is contained in a form within a matrix such as those descπbed m 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

2o 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 therapeuttc levels of the active ingredient for at least 30

25 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 descπbed above

The mvenuon also embraces methods for isolating tumor cell-deπved tumor-anttgen expression down-regulating agents A method according to this aspect of the mvenuon typically involves- (a) prepaπng a culture of tumor cells that have down-regulated tumor- in 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 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 supematants (cell s 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 o cancer, including glioblastomas and medulloblastomas; breast cancer; cervical carcinoma; choriocareinoma; 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, s lymphomas, 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, 0 basocellular cancer and squamous cell cancer; testicular cancer, including germinal tumors (seminoma, non-semtnoma[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-

₂₅ binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)— CO 17- 1A/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-1, CDK4. in tyrosinase, p53, MUC family, HER2 neu, p21ras, RCAS1, -fetoprotcin, E-cadherin, α- catenin, β-catenin and γ-catenin, _P120ctn, gpl00^I'^,~¹"⁷, PRAME, 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- antigens associated with such tumors (but not exclusively), include acute lymphoblastic s leukemia (etv6; amll; cyclophilin b), glioma (E-cadherin; α-cateniπ; β-catenin; γ-catenin; pl20ctn), bladder cancer (p21ras), billiary cancer (p2Iras), breast cancer (MUC family; HER2/neu; c-erbB-2), cervical carcinoma (p53; ρ21ras), colon carcinoma (p21ras; HER2/neu; c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRC)-C017- 1A/GA733; APC), choriocareinoma (CEA), epithelial cell-cancer (cyclophilin b), gastric in cancer (HER2/neu; c-erbB-2; ga733 glycoprotein), hepatocellular cancer (α-fetoprotein), hodgkins lymphoma (lmp-1; EBNA-1), lung cancer (CEA; MAGE-3; NY-ESO-1), lymphoid cell-derived leukemia (cyclophilin b), myeloma (MUC family; p21ras), non-small cell lung carcinoma (HER2/neu; c-erbB-2), nasopharyngeal cancer (Imp- 1: EBNA-l), ovarian cancer cancer (MUC family; HER2/neu; c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA) is and its immunogenic epitopes PSA-1, PSA-2, and PSA-3; PSMA; HER2/neu; c-erbB-2), pancreatic cancer (p21ras; 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; p21ras; gpl00^Pme"¹⁷). In any of the foregoing embodiments the fraction from the plurality of fractions can be undiluted or 0 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

25 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 5 x IO⁶ cells/ml of medium is typically 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 in 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 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 supematants of the above-described cell cultures [i.e., of the cultured cells of step (a)], although the entire culture s can be homogenized and subjected to the steps described below for isolation of a tumor ceil 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 in 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, is hydrophobic chromatography, immune-affinity chromatography, etc.

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 o skill in the art and can be employed using routine experimentation. Determining whether 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 5 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 can be

HI 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 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 immuno logical and molecular biological methods (see, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second

5 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., 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 in 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 is 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 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- n 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 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 5 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-1. In some embodiments, the control tumor- m antigen expression is determined in the presence of an agent of (a) and in 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 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- anugen expression Typically, one of these concentrations serves as a negative control, i.e., at zero concentrauon of agent or at a concentration of agent below the limits of assay detection. Putauve 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 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500 Putative inhibitory agents compπse 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 agents also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, punnes, pyrimidmes, 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. , antibodies) Where the 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 libraπes of synthetic or natural compounds For example, numerous means are available for random and directed synthesis of a wide variety ol organic- compounds and biomolecules, including expression of randomized oligonucleotides, syntheuc organic combmatoπal libranes, phage display libraπes of random peptides, and the like. Alternatively, libraπes of natural compounds in the form of bacteπal, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libranes and compounds 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 5uch 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 s protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce nonspecific 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 adversely affect the growth of the cells in the culture. in 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 is such circumstances, incubation temperatures typically are between 4^βC and 40^βC. 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 further incubation at cell culture conditions (typically between 1 and 48 hours), tumor-antigen

20 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.

Examples

Experimental procedures Materials and Methods

Tumor and Tumor Infiltrating Lymphocytes (TIL) sn 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 (17). Briefly, tumors were cultured in DMEM medium supplemented with 10% FBS, and TIL were propagated in RPMI 1640 supplemented with 5% Human Serum containing recombinant human IL-2 at 100 Units/ml (Cetus, Emoryville, CA). TIL clones were isolated by limiting dilution as previously described (17). Briefly, TTL 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 s 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 (18,19) . Melan- A/MART-1 negative variant MU-X were obtained by culture of MU tumor cells at high density (>5xl0 cells/ml) for several days prior to immunoselection with anti-Melan- in 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 (20). Both MU and MA tumor s expressed HLA-A2; MU tumor cells were also HLA-Al positive. MO is 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 (MU), or in RPMI 1640 supplemented with 5% human serum (MA and MO).

:n Expression of Melan-A/MART-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 lOmin in 1% paraformaldehyde, followed by 5min in 0.1% saponin prior to addition of monoclonal antibody specific for Melan-A/MART-1, A-103, (34)(a kind gift of E. Stockert and L. Old, Ludwig Institute, New

2s York, NY) for 45min at 22°C. Following two washes, the cells were stained for 3Omin with FITC-conjugated goat-anti-mouse Ig antibody (DA O, 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/MART-1 staining of various cell populations. Mean channel fluorescence was jo calculated using the "Consort 30" software provided by the manufacturer.

Generation of Conditioned Medium Conditioned medium from melanoma tumor lines were generated by culturing cells at a starting concentration of 5 x 10 cells/ml in DMEM medium supplemented with between 1 and 10% FBS. Supematants 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 s medium containing 1% FBS was concentrated between 10 and 20 fold by collecting the retentate from a nominal 30kD YM membrane (Centriprep, Millipore, Bedford, MA).

Cytotoxicity Assays

TIL were assayed for the ability to lyse melanoma target cells in 4 hour Gr-release in assays as previously described (17). The melanoma target cells with high constitutive expression of Melan-A/MART-1 were generated by low density culture (1-2 x 10 /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 is expressing cells were further assayed after pulsing with Melan-A/MART-1 peptide 27-35, (AAGIGILTV)(1 , 4, 21 , 22), by culturing these target cells at 37° for 2 hours in 1 ml of medium containing 5μg of peptide prior to labeling with Cr for use in cytolytic assays to demonstrate renewed susceptibility to specific T cell recognition (essentially as previously described in 23).

2tι In further instances, bulk and cloned TIL progeny was also assayed against autologous tumor (MU), allogeneic melanomas, as well as NK (K562) and LAK (Daudi), and EBV- transformed B lymphocyte targets: EBV-3 (HLA-A1 , B8, DR3), EBV-19 (HLA-A2, B18,

DR5), using the foregoing Cr-release assay. Pulsing included the following meianocyte lineage-derived peptides: Tyrosinase(l l ): MLLAVLYCL or YMNGTMSQV, MAGE-3(24): 5 EVDPIGHLY. Clones were screened for cytotoxic activity at effector to target ratios of 50:1 and below.

Promoter Activity

Assessment of the promoter activity for Mclan-A/MART-1 in melanomas was made in

JO transient transfection experiments using the 233bp minimal promoter sequence coupled to the firefly luciferase reporter gene (25), (kindly provided by Lisa Butterfield, UCLA Medical

Center, Los Angeles, CA). A 230bρ HLA-A-firefly-luciferase construct (26) was transfected in parallel into the melanoma cells. An SV-40-renilla-luciferase reporter gene was co- transfected in order to provide an internal control, using the Dual-Luciferase Reporter Assay System, as described by the manufacturer (Promega, Madison, Wϊ). Transient transfections were carried out using the CaCb precipitation method as previously described (26). After 16 hours, the cultures were washed and after an additional 48 hours, the cells were lysed and luciferase assays were performed. All assays were performed in quadruplicate, with the SV40- renilla control used to "correct" for transfection efficiency. Data shown are means, (+/- standard deviation), of the corrected luciferase activity, standardized to 100% for optimal promoter activity of control Melan-A/MART-1 expressing tumor cells.

Example 1: Melanoma-Antigen Recognition by Tumor Infiltrating T Lymphocytes (TIL):

Effect of Differential Expression of Melan-A/MART-1 Results Phenotype and Function of TIL Bulk and Clones:

The IL-2 responding TIL propagated from patient MU were over 98% CD3+, CD8+, (CD4-) T cells expressing the αβ TCR. As previously reported (18), strong cytotoxic activity was noted, when tested in a Cr-release assay against the autologous MU-melaπoma 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, 19). The ability of Bulk MU-TIL to lyse autologous tumor targets was blocked by antibodies to HLA-Class I antigens (W6/32). Furthermore, the ΗL 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 were isolated as previously described ( 18). As summarized in Table 1 , 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

B. Tumour target lysis is shown a follows: <10% specific iysis: -; 10-20%: + ; 20-40%:

C. 40-60%: +-H- ; 60-80%: ++^■+-+ ; >80%: m n .

PeptideSpecificity of MU-TIL:

The Bulk-MU culture. and clones MU-45, MU-57, MU-63, MU-79 and MU-115 showed strong reactivity against EBV-19 targets pulsed with Melan-A/MART- 1 peptide (AAGIGILTV). Two additional clones (MU-10 and MU-58) failed to lyse these EBV-targets pulsed with the Melan-A/MART-1. None of the T cells lysed the EBV 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 was identical to MU-115, indicating that MU-9 was also likely to be Melan-A/M ART- 1 peptide specific. As noted, clones MU-45, MU-63 and MU-79 were identical to one another as determined by TCR gene sequencing.

Modulation of Melan-A/MART- 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 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 1 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 TTL 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 s that of the same tumor cells cultured at 1 xlO cells ml. As shown in Figure 2A, the susceptibility to lysis of MU-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 in that addition of Melan-A MART-1 peptide to the low-expressor tumor targets, MU-Lo and MU-X, restored high levels of cytotoxicity (Figure 2B). The specificity of ΗL 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 IB) paralleled decreased target cell is 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 ID, while Melan-A/MART-1 decreased among cells cultured at 0 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 IE), 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

25 in parallel with Melan-A/MART-1 when the tumor cells were cultured at higher cell density. Futheπnore, 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 jo cells showed diminished target recognition with reduced Mclan-A/MART-1 cxprcssion(sce Figure 2A).

The decrease in Melan-A/MART-1, in cells cultured ai 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 cells were passaged at high density (5 x 10 cells/ml), they continued to show low Melan- A/MART-1 (Mean 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

J 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 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 m and cell density. As noted in Figure 2, the decrease in Melan-A/MART-1 intensity was paralleled with a decreased susceptibility to lysis.

Example 2: Autocrine down-modulation of tumor-antigen recognition is Results

As shown in Figure 3, Melan-A/MART-1 expressing melanocarcinoma cell lines can spontaneously down-modulate expression of this cytoplasmic antigen, as demonstrated by staining with the monoclonal antibody, A-103 (27), when the cells are cultured at high s s concentration (5 10 cells/ml), as compared to cells cultured at lower density (1 x 10

20 cells/ml) (See Figure 3A). Similarly, down-modulation of Melan-A/MART-1 was also observed by the addition of supematants collected from melanomas with low, or undetectable, Melan-A/MART-1 expression. Culture of tumor cells at low cell concentration (1x10$ cells/ml) in the presence of supematants obtained from tumor cell cultured at high density which express low levels of Melan-A/MART-1, resulted in down-modulated levels of

25 cytoplasmic Melan-A/MART-1 expression (Figure 3B). Of particular note was that tumor variants which have constitutively low, or undetectable levels of expression of Melan- A/MART- I molecules, such as MU-X and EW, produced the highest levels of "antigen silencing" activity (Figure 3C). This down-modulation is reversible, as the cytoplasmic expression of Melan-A/MART-1 is up-modulated when the tumor cells are returned to culture io 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 supematants from Melan- A/MART-1 -negative tumor variants(Figure 3D). Significantiy, the diminution of expression of the Melan-A/MART-1 antigen correlated with reduced susceptibility of the target cells to lysis by HLA-A2-restricted, Melan-A/MART- l-specific CTL (See Figure 4). 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 s ceil recognition of these targets decreased. However, when the target cells were pulsed with the Melan-A/MART-1 peptide (27-35, AAGIGILTV), 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 (25). This activity was found to be o mediated through the 233bp minimal promoter of the Melan-A/MART-1 gene. Using this minimal promoter coupled to the luciferase reporter gene, the impact of the antigen silencing activity on this gene promoter was assessed. As shown in Figure 5A, in a series of transient transfection experiments, there was baseline promoter activity in Melan-A/MART-1 negative tumor cells (MU-X), whereas this promoter showed abundant activity in tumor cells which s expressed Melan-A/MART-1, as described by Butterfield et al. (25). Of particular note, the addition of conditioned medium from Melan-A/MART-1 negative tumor cells abolished the high level of constitutive Melan-A MART-1 promoter activity in the MU tumor cells which express high levels of cytoplasmic Melan-A/MART-1. The promoter activity was down- modulated in a dose dependent fashion. Although the soluble factors) displayed rather modest activity, as it quickly diluted out from these supematants, the activity could be concentrated, as it was retained by a nominal 10,000 Dalton cutoff-membrane (Millipore, Bedford MA, USA). This concentrated material ( ASA) retained virtually all of the activity in the starting material, as a 10 fold concentrate could be diluted 10, 20 or 40 fold and showed activity almost identical to 100%, 50% and 25% supernatant fluids. The fact that the activity S could be diluted, so that it constituted only a small proportion of the original cell culture medium, demonstrated that the antigen silencing was not due simply to nutrient deprivation. The reversibility of the promoter down-modulation was demonstrated by the return of this activity when the cells were removed from the presence of the silencing activity. Furthermore, promoter activity for HLA class I molecules required for T cell recognition of the Melan- so A/MART-1 derived peptide (AAGIGILTV). was not down-modulated (Figure 5B).

Together these results clearly show that some cell-associated proteins are not down- regulated by the supematants which silence Melan-A/MART-1 promoter activity and protein expression. The Melan-A MART-1 silencing activity could be heat inactivated by treatment of the supematants at 80°C for 60 minutes. A similar treatment at 60°C did not influence the activity.

To define this silencing activity in more detail, an assessment of cytokiπe and chemokine production by tumor cells which lacked Melan-A/MART-1 protein expression was

5 performed. These Melan-A/MART-1 silencing supematants were found to contain several known cytokines, but they lacked TNFα, which has been demonstrated to have a partial down- regulatory activity directed at the Melan-A/MART-1 promoter (25). 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 m levels detected in the active supematants. In all, we have assessed over 20 proteins, including many known to be secreted by melanomas (28-33), for their capacity to mimic the antigen silencing activity. Other than TNFα, which was not present in the active supematants, none of the following proteins impacted cytoplasmic Melan- A/MART- 1 expression and promoter activity: MSG/GROα, EGF, PDGFa, PDGFb, TGFα, TGFβ, NGF, RANTES, MlP-lα, L1F, is PF-4, NAP-2. The following cytokines were not detected in the active supematants : IL-lβ, IL-2, IL- 12, IL-15, TNFα, IFNγ. In addition, heparin, which is known to bind to several growth factors, did not affect Melan-A/MART-1 expression, and did not impact the silencing activity of active supematants.

2o Example 3: Characterization of the Melan-A/MART-1 expression down-modulating activity (MASA)

Preliminary 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

2S 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 Sepharose hut not to heparin. Some of the activity binds to Conconavaiin A (ConA) and can be eluted with sugar, and some of the activity docs not bind to ConA. By isolating the ConA-bound-eluted material, and further purifying it by binding and eluting it from blue Sepharose, we can delete 99% of

HI the protein, while retaining approximately 10% of the original antigen silencing activity.

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 50% of the total protein. A small amount of MASA was bound to the column and was eluted off in high salt (1M NaCl (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 1M NaCl) 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 suggest 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% 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 m MASA.

Results

^•Fraction from Size Exclusion Column

15 ^••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.

Characterization of Activity (Summary):

Activity was found to be associated with an agent (melanoma isolate) that: 0 (i) binds to Sepharose-Blue dye resin but not to Heparin,

(ii) when enriched by Ion Exchange Chromatography appears of -25kD molecular weight, (see Figure 6),

(iii) is sensitive to proteinase K digestion, and

(iv) is variably glycosylated, since - 2/3 rds of the agent's activity does no bind to

25 ConA and the remaining - l/3rd does. References

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Immunol Immuπopathol 1986; 38:367-380. 24. Gaugler B, Van den Eynde B, van der Bruggen P, et al. Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Journal of Experimental Medicine 1994; 179:921-30.

25. Butterfield, L. H., Stoll, T. C, Lau, R. & Economou, J. S. (1997) Gene 191, 129-34. s 26. Gobin, S„ Wilson, L., Keijser, V. &. van den Elsen, P. (1997) J. Immunol. 158.

27. Chen Y, Stockert E, Jungbluth A, Tsang S, Coplan K, Scanlan M, Old L. Serological analysis of Melan-A(MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas. Proc Natl Acad Sci U S A 93 (12): 1996; 93:5915- 5919. in 28. Mattei, S., Colombo, M., Melani, C, Silvani, A., Parmiani, G. & Herlyn, M. (1994) Int. J. Cancer 56, 853-7.

29. Paglia, D., Oran, A., Lu, C, Kerbel, R. S., Sauder, D. N. & McKenzie, R. C. (1995) J. Interferon Cytokine Res. 15, 455-60.

30. Armstrong, C. A., Tara, D. C, Hart, C. E., Kock, A., Luger, T. A. & Ansel, J. C. is (1992) Exp. Dermatol. 1, 37-45.

31. Bennicelli, J. L. & Guerry, D. t. (1993) Exp Dermatol 2, 186-90.

32. Francis, G. M., Krohn, E. G., Woods, K. V., Buzaid, A. C. & Grimm, E. A. (1996) Melanoma Res. 6, 191-201.

33. Singh, R. K., Gutman, M. & Radinsky, R. (1995) J. Interferon Cytokine Res. 15, 81-7. :o 34. Castelli C, Storkus W, Maeurer M, et al. Mass spectrometric identification of a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes. J Exp Med 1995; 181:363-8.

Those skilled in the art will recognize, or be able to ascertain using no more than 25 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. What is claimed is presented below and is followed by a Sequence Listing. We claim: o

Claims

1. A malignant melanoma cell isolate that down-regulates Melan-A/MART-1 expression in malignant melanoma cells when contacted with malignant melanoma ceils.

s 2. The malignant melanoma cell isolate of claim 1, wherein the isolate is derived from Melan-A/MART-1 antigen -expressing or -nonexpressing malignant melanoma cells.

3. The malignant melanoma cell isolate of claim 1, wherein the isolate comprises a polypeptide and wherein the polypeptide down-regulates Melan-A/MART-1 expression in in malignant melanoma cells.

4. The malignant melanoma cell isolate of claim 1, wherein the isolate is a substantially pure polypeptide.

is 5. The malignant melanoma cell isolate of claim I , wherein the isolate mediates its down- regulatory effects on Melan-A/MART-1 expression through the Melan-A/MART-1 gene promoter.

6. The malignant melanoma cell isolate of claim 1 , wherein the isolate is a supernatant, or 2tι fraction thereof, of Melan-A/MART-1 antigen-expressing malignant melanoma cells.

7. A substantially pure organic agent that down-regulates Melan-A/MART-1 expression in malignant melanoma cells, at effective concentrations, wherein the agent is present at effective concentrations in the supernatant of confluent Mclan-A/MART-1 antigen-

2s expressing malignant melanoma cells cultured under standard conditions for a period of at least one hour, wherein the culture conditions include a ratio of 5 x I O⁶ cells/ml medium, and wherein the agent is heat sensitive at 80°C, proteinase K sensitive, and binds to and elutes-off blue-Sepharose.

so

8. The organic agent of claim 7, wherein the agent is a polypeptide.

9. An isolated binding organic agent which binds selectively to a malignant melanoma cell isolate of claim 1 and inhibits down-regulation of Melan-A/MART-1 expression in malignant melanoma cells.

s 10. The isolated binding organic agent of claim 9, wherein the isolated binding organic agent is a polypeptide.

11. The isolated binding organic agent of claim 9, wherein the malignant melanoma cell isolate of claim 1 comprises a polypeptide and wherein the polypeptide down-regulates in Melan-A/MART-1 expression in malignant melanoma cells.

12. The isolated binding organic agent of claim 9, wherein the malignant melanoma cell isolate of claim 1 is a substantially pure polypeptide.

is 13. The polypeptide of claim 10, wherein the polypeptide is an antibody or an antibody fragment selected from the group consisting of a Fab fragment, a F(ab)₂ fragment, or a fragment including a CDR3 region selective for the polypeptide.

14. An isolated binding organic agent which binds selectively to a substantially pure 2n organic agent of claim 7, and inhibits down-regulation of Melan-A/MART-1 expression in malignant melanoma cells.

15. The isolated binding organic agent of claim 14, wherein the isolated binding organic agent is a polypeptide.

2S

16. The isolated binding organic agent of claim 14, wherein the substantially pure organic agent of claim 7 comprises a polypeptide and wherein the polypeptide down-regulates Melan-A/MART-1 expression in malignant melanoma cells.

_in 17. The isolated binding organic agent of claim 14, wherein the substantially pure organic agent of claim 7 is a substantially pure polypeptide.

18. The polypeptide of claim 15, wherein the polypeptide is an antibody or an antibody fragment selected from the group consisting of a Fab fragment, a F(ab)2 fragment, or a fragment including a CDR3 region selective for the polypeptide.

s 19. A pharmaceutical composition comprising: an agent comprising an isolated binding organic agent of claim 9, in a pharmaceutically effective amount to inhibit down-regulation of Melan-A/MART-1 expression in malignant melanoma cells by a malignant melanoma cell isolate that down-regulates Melan-A/MART-1 expression in malignant melanoma cells when in contacted with malignant melanoma cells, and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition comprising: an agent comprising an isolated binding organic agent of claim 14, in a is pharmaceutically effective amount to inhibit down-regulation of Melan-A/MART-1 expression in malignant melanoma cells by a substantially pure organic agent that down-regulates Melan-A/MART-1 expression in malignant melanoma cells when contacted with malignant melanoma cells, and a pharmaceutically acceptable carrier.

20

21. A method for isolating a tumor cell-derived tumor-antigen expression down-regulating agent, comprising:

(a) preparing a culture of tumor cells,

(b) isolating a supernatant suspected of containing a tumor-antigen expression down-regulating agent from the culture of step (a),

(c) fractionating the supernatant 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 cell, and

Ml (f) determining whether tumor-antigen expression on the tumor cell is down- regulated compared to a control.

22. The method of claim 21, wherein the tumor cells are selected from the group consisting of acute lymphoblastic leukemia cells, glioma cells, bladder cancer cells, billiary cancer cells, breast cancer cells, cervical carcinoma cells, colon carcinoma cells, colorectal cancer cells, choriocareinoma cells, epithelial cancer cells, gastric cancer cells, s hepatocellular cancer ceils, hodgkins lymphoma cells, lung cancer cells, lymphoid cell-derived leukemia cells, myeloma cells, non-small cell lung carcinoma cells, nasopharyngeal cancer cells, ovarian cancer cancer cells, prostate cancer cells, pancreatic cancer cells, renal cancer cells, testicular cancer cells, T cell leukemia cells, and melanoma cells, and the tumor-cell antigen is selected from the group consisting of o Melan-A/MART-1, Dipeptidyl peptidase IV (DPPIV), adenosine deamiπase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)-C017- 1A/GA733, Carciπoembryonic 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 s receptor/CD3-zeta chain, MAGE-family of tumor antigens, GAGE-1,2, BAGE, RAGE,

GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α- fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, pl20ctπ, gpl00^Prnc"¹ . PRAME, NY-ESO-1 , cdc27, adenomatous polyposis coli protein (APC), fodriπ, Smad family of tumor antigens, lmp-1, EBV-encoded nuclear antigen (EBNA)-l, or c-erbB- 0 2.

23. The method of claim 21, wherein the tumor cells are melanoma cells and the tumor- antigen is Melan-A/MART-1.

₅ 24. The method according to any one of claims 21-23, wherein the fraction from the plurality of fractions is undiluted or concentrated.

25. A method of screening for tumor-antigen expression modulating agents, comprising:

(a) contacting an agent suspected of being a tumor-antigen expression _HI modulating agent with a tumor-antigen expressing tumor cell,

(b) measuring tumor-antigen expression on the tumor cell, and

(c) determining whether tumor-antigen expression on the tumor cell is modulated compared to a control.

26. The method of claim 25, wherein the tumor cells are selected from the group consisting of of acute lymphoblastic leukemia cells, glioma cells, bladder cancer cells, billiary cancer cells, breast cancer cells, cervical carcinoma cells, colon carcinoma cells, s colorectal cancer cells, choriocareinoma cells, epithelial cancer cells, gastric cancer cells, hepatocellular cancer cells, hodgkins lymphoma cells, lung cancer cells, lymphoid cell-derived leukemia cells, myeloma cells, non-small cell lung carcinoma cells, nasopharyngeal cancer cells, ovarian cancer cancer cells, prostate cancer cells, pancreatic cancer cells, renal cancer cells, testicular cancer cells, T cell leukemia cells, in and melanoma cells, and the tumor-cell antigen is selected from the group consisting of

Melan-A/MART-1, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)— C017- 1 A/GA733, Carcinoembryoπic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amll, Prostate Specific Antigen (PSA) and its immunogenic epitopes is 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, p53, MUC family, HER2/neu, p21ras, RCAS 1 , α- fetoprotein, E-cadherin, α-cateπiπ, β-catenin and γ-catenin, pl20ctn, gpl00^{pmeU ,7} _I PRAME, NY-ESO- 1, cdc27, adeno atous polyposis coli protein (APC), fodrin, Smad 0 family of tumor antigens, lmp-1 , EBV-encoded nuclear antigen (EBNA)- l , or c-erbB-

2.

27. The method of claim 25, wherein the agent suspected of being a tumor-antigen expression modulating agent is an agent present in a tumor cell-culture supernatant, S tumor cell eluate, or tumor cell lysate.

28. A method for isolating an agent that up-regulates tumor-antigen expression, comprising:

(a) isolating a tumor-antigen expression down-regulating agent according to in Claim 21,

(b) preparing a culture of tumor cells, wherein the tumor cells are 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 control tumor-antigen expression.

29. The method of claim 28, wherein 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). in

30. The method of claim 28, wherein the tumor cells used in the isolation of the tumor- antigen expression down-regulating agent of (a) are melanoma cells and the tumor- antigen is Melan-A/MART-1.

is 31. A method of enhancing a melanoma-specific immune response in a subject with melanoma, comprising: administering to a subject in need of such treatment the isolated binding organic agent of claim 9, in an amount effective to inhibit down-regulation of Melan-A/MART- 1 expression in malignant melanoma cells and enhance a melanoma-specific immune n response in the subject.

32. The method of claim 31, wherein the isolated binding organic agent of claim 9 is a polypeptide.

5 33. The method of claim 31, further comprising co-administering an anti-tumor agent other than the isolated binding organic agent of claim 9.

34. A method of enhancing a melanoma-specific immune response in a subject with melanoma, comprising: in administering to a subject in need of such treatment the isolated binding organic agent of claim 14, 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.

35. The method of claim 34, wherein the isolated binding organic agent of claim 14 is a polypeptide.

36. The method of claim 34, further comprising co-administering an anti-tumor agent other than the isolated binding organic agent of claim 14.