EP1232178A2 - Compositions and methods for regulating tumor-associated antigen expression - Google Patents

Compositions and methods for regulating tumor-associated antigen expression

Info

Publication number
EP1232178A2
EP1232178A2 EP00983714A EP00983714A EP1232178A2 EP 1232178 A2 EP1232178 A2 EP 1232178A2 EP 00983714 A EP00983714 A EP 00983714A EP 00983714 A EP00983714 A EP 00983714A EP 1232178 A2 EP1232178 A2 EP 1232178A2
Authority
EP
European Patent Office
Prior art keywords
tumor
cells
antigen
agent
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00983714A
Other languages
German (de)
English (en)
French (fr)
Inventor
James T. Kurnick
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
General Hospital Corp
Original Assignee
General Hospital Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by General Hospital Corp filed Critical General Hospital Corp
Publication of EP1232178A2 publication Critical patent/EP1232178A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464491—Melan-A/MART
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to methods and compositions for the treatment of tumors.
  • the invention relates to methods and agents for the treatment of tumors expressing a tumor-antigen [also known as a tumor-associated antigen (TAA). lineage specific, differentiation, or self -antigen], and more specifically to methods and agents for the treatment of melanomas expressing the Melan- A/M ART- 1 antigen.
  • TAA tumor-associated antigen
  • the invention also relates to methods for identifying agents that regulate expression of tumor -antigens.
  • TIL tumor-infiltrating lymphocytes
  • TAA tumor-associated antigens
  • TILs show strong in vitro lytic activity which is often directed against targets expressing HLA-A2 and the immunodominant peptide of Melan- A/MART-1 (AAGIGILTV. SEQ ID NO:l) (Kawakami Y. and Rosenberg SA. Int Rev Immunol. 1997. 14: 173-92; Zhai. Y..
  • 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- associated antigens in tumor cells.
  • the invention therefore, is particularly useful, inter alia, for treating subjects with autologous. solid tumors having cells that express, or that can be induced to express, tumor-associated antigens.
  • One category of materials according to the invention is tumor-antigen expression down-regulating agents. These agents include tumor cell isolates and isolated or substantially purified materials. Another category of materials according to the invention is inhibitors of such agents that down-regulate tumor-antigen expression.
  • a malignant melanoma cell isolate 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.
  • the malignant melanoma cells are low-Melan- A/MART- 1 antigen-expressing cells.
  • the cell isolate comprises a polypeptide.
  • the cell isolate is a substantially pure polypeptide.
  • the substantially pure polypeptide is a polypeptide selected from the group consisting of Oncostatin M.
  • the cell isolate is a supernatant, or fraction thereof, of Melan- A/MART- 1 antigen-expressing malignant melanoma cells.
  • the cell isolate comprises at least one polypeptide selected from the group consisting of Oncostatin M. Stanniocalcin, and Tissue Factor Pathway
  • the malignant melanoma cells express low levels of
  • the malignant melanoma cells express high levels of Melan- A/MART- 1 antigen.
  • a substantially pure organic agent 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.
  • the culture conditions include a ratio of 5 x 10 6 cells/ml medium, and the agent is heat sensitive at 80°C, proteinase K sensitive, and binds to and elutes-off blue- and/or red- Sepharose ® (Pharmacia Biotech, Inc., Piscataway, NJ).
  • the organic agent is a polypeptide.
  • the polypeptide is selected from the group consisting of Oncostatin M. Stanniocalcin, and Tissue Factor Pathway Inhibitor-2.
  • 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.
  • an isolated organic agent 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.
  • the malignant melanoma cell isolate comprises at least one polypeptide.
  • the malignant melanoma cell isolate is a substantially pure polypeptide.
  • the substantially pure polypeptide is a polypeptide selected from the group consisting of Oncostatin M, Stanniocalcin. and Tissue Factor Pathway Inhibitor-2.
  • the isolated binding organic agent is a polypeptide.
  • the polypeptide when the isolated binding organic agent is a polypeptide, can be an antibody or an antibody fragment selected from the group consisting of a Fab fragment, a F(ab) fragment, or a fragment including a CDR3 region selective for the polypeptide.
  • the antibody or fragment thereof may be chimeric or humanized.
  • the antibody can be selected from the group consisting of an anti-Oncostatin-M antibody, an anti-Stanniocalcin antibody. and an anti-Tissue Factor Pathway Inhibitor-2 antibody.
  • 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, and inhibits down-regulation of
  • the substantially pure organic agent comprises at least one polypeptide having Melan- A/MART- 1 expression down-regulating properties in malignant melanoma cells.
  • the substantially pure organic agent is a substantially pure polypeptide.
  • the substantially pure polypeptide is a polypeptide selected from the group consisting of Oncostatin M, Stanniocalcin. and Tissue Factor Pathway Inhibitor-2.
  • 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) fragment, or a fragment including a CDR3 region selective for the polypeptide.
  • the antibody or fragment thereof may be chimeric or humanized.
  • the antibody can be selected from the group consisting of an anti-Oncostatin-M antibody, an anti- Stanniocalcin antibody, and an anti-Tissue Factor Pathway Inhibitor-2 antibody.
  • a pharmaceutical composition includes an isolated binding organic agent which binds selectively to a substantially pure organic agent that down-regulates Melan- A/M ART- 1 expression in malignant melanoma cells.
  • the isolated binding organic agent that binds selectively the substantially pure organic agent inhibits such down-regulation, and is present in an effective amount to inhibit such down-regulation.
  • the composition also includes a pharmaceutically acceptable carrier.
  • a pharmaceutical composition includes an isolated binding organic agent which binds selectively a malignant melanoma cell isolate that down-regulates Melan- A/MART- 1 expression in malignant melanoma cells.
  • the isolated binding organic agent that binds selectively the malignant melanoma cell isolate inhibits such down-regulation, and is present in an effective amount to inhibit such down-regulation.
  • the composition also includes a pharmaceutically acceptable carrier.
  • the invention in another aspect provides a method for isolating a tumor cell-derived tumor-antigen expression down-regulating agent.
  • the method involves (a) preparing a culture 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 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.
  • the origin of the tumor cells may be of: biliary tract cancer; brain cancer, including glioblastomas and medulloblastomas; breast cancer; cervical carcinoma; choriocarcinoma: colon cancer; endometrial cancer: esophageal cancer; gastric cancer; 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 Page s disease; liver cancer: lung cancer; lymphomas.
  • neuroblastomas oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells: pancreas cancer; prostate cancer; rectal cancer: sarcomas, including leiomyosarcoma, rhabdomyosarcoma. liposarcoma, fibrosarcoma and osteosarcoma; skin cancer, including melanoma. Kaposi ' s sarcoma, basocellular cancer and squamous cell cancer: testicular cancer, including germinal tumors (seminoma.
  • 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 and its immunogenic epitopes CAP-1 and CAP-2, etv6, amll.
  • PSA Prostate Specific Antigen
  • PSMA prostate-specific membrane antigen
  • 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. RCAS1 , ⁇ -fetoprotein. E-cadherin.
  • the fraction from the plurality of fractions can be undiluted or concentrated.
  • cancers or tumors escaping immune recognition and tumor-antigens associated with such tumors include acute lymphoblastic leukemia (etv6; amll; cyclophilin b), glioma (E-cadherin: ⁇ -catenin: ⁇ -catenin; ⁇ -catenin; pl20ctn), bladder cancer
  • p21ras billiary cancer
  • breast cancer MUC family; HER2/neu: c-erbB-2
  • cervical carcinoma p53; p21ras
  • colon carcinoma p21ras: HER2/neu: c-erbB-2; MUC family
  • colorectal cancer Colorectal associated antigen (CRC)— C017-1A/GA733; APC
  • CEA choriocarcinoma
  • CEA epithelial cell-cancer
  • gastric cancer HER2/neu; c- erbB-2; ga733 glycoprotein
  • hepatocellular cancer ⁇ -fetoprotein
  • hodgkins lymphoma hepatocellular cancer
  • lymphoid cell-derived leukemia 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 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).
  • HER2/neu c-erbB-2
  • testicular cancer NY-ESO-1.
  • T cell leukemia HTLV-1 epitopes
  • melanoma Melan- A/MART- 1 ; cdc27; MAGE-3: p21ras: gpl00 Pmel 117 ).
  • the invention in a further aspect provides a method of screening for tumor-antigen expression modulating agents.
  • the method involves (a) contacting an agent suspected of being a tumor antigen expression modulating agent with a tumor-antigen expressing tumor cell, (b) measuring tumor-antigen expression of the tumor cell, and (c) determining whether tumor-antigen expression on the tumor cell is modulated as a result of such contacting, for example, by comparison to a control. Both up-modulating and down-modulating agents can be identified. Tumor cells and tumor-antigens expressed by the tumor cells are as described above.
  • 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.
  • a method for isolating an agent that up-regulates tumor-antigen expression.
  • the method includes (a) providing a tumor- antigen expression down-regulating agent which may be isolated according to any of the foregoing methods of the invention, (b) preparing a culture of tumor cells, wherein the tumor cells may be identical to those used in the isolation of the tumor-antigen expression down- regulating agent of (a), (c) contacting the isolated tumor-antigen expression down-regulating agent of (a) and a putative inhibitory agent of the isolated tumor-antigen expression down- regulating agent of (a) with the culture cells of (b), (d) determining tumor-antigen expression in the culture cells, and (e) comparing the tumor-antigen expression determined in (d) with a control.
  • Tumor cells and tumor-antigens expressed by the tumor cells are as described above.
  • the tumor cells are melanoma cells and the tumor-antigen is Melan- A/MART-1.
  • 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).
  • a method is provided for enhancing a melanoma-specific immune response in a subject with melanoma.
  • the method involves administering to a subject in need of such treatment an isolated binding organic agent which binds selectively to: (i) a substantially pure organic agent, or (ii) a malignant melanoma cell isolate, either of which down-regulates Melan-A/MART-1 expression in malignant melanoma cells when contacted with malignant melanoma cells, in an amount effective to inhibit down- regulation of Melan-A/MART-1 expression in malignant melanoma cells and enhance a melanoma-specific immune response in the subject.
  • the isolated binding organic agent is at least one polypeptide.
  • the polypeptide is selected from the group consisting of Oncostatin M. Stanniocalcin. and Tissue Factor Pathway Inhibitor-2.
  • the method further comprises co-administering to the subject an anti-tumor agent other than the agents of the invention.
  • methods for preparing medicaments useful in enhancing a melanoma-specific immune response in a subject with melanoma are also provided.
  • Figure 1 shows graphs depicting Melan-A/MART-1 expression on different melanoma tumor lines and the differences culture density has on such expression: Fig. IA on MU-tumor; Fig. IB on MA-tumor; Fig.lC on MO-tumor: Fig. ID control, HLA-I expression on MU-tumor; Fig. IE control. HLA-A2 expression on MU-tumor.
  • Figure 2 shows graphs depicting the effect of MART- 1 /Melan- A expression on target cell recognition and lysis by TIL;
  • Fig. 2A shows MU-TIL lysis of autologous MU-tumor;
  • Fig. 2B shows MU-TIL lysis of Melan- A/MART- 1 -negative targets;
  • Fig. 2C shows anti-HLA- A2-specif ⁇ c lysis of targets.
  • SEQ ID NO:l is the immunodominant amino acid sequence of the Melan-A/MART-1 peptide.
  • SEQ ID NO:2 is a melanocyte lineage-derived peptide for Tyrosinase.
  • SEQ ID NO:3 is a melanocyte lineage-derived peptide for Tyrosinase.
  • SEQ ID NO:4 is a melanocyte lineage-derived peptide for MAGE-3.
  • agents and pharmaceutical compositions containing such agents that modulate expression of tumor-associated antigens in tumor cells 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.
  • TAA down-modulation is mediated through an agent secreted by the tumor cells (i.e.. autocrine secretion/down-modulation).
  • autocrine secretion/down-modulation an agent secreted by the tumor cells
  • Down-modulating (down-regulating), 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 directly determined by detecting a decrease in the level of mRNA for the gene encoding the antigen, or the level of peptide expression of the tumor-antigen, using any suitable means known to the art, such as nucleic acid hybridization or, preferably, antibody detection methods, respectively (see Examples).
  • Inhibition of tumor-antigen expression can also be determined indirectly, for example, by detecting a change in tumor-cell lysis ability by TILs that specifically recognize the tumor-antigen.
  • the present invention relates in one aspect to a malignant melanoma cell isolate with Melan-A/MART-1 expression down-regulating activity. Therefore, in one important 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 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.
  • isolated means separated from the environment of the cells, such as a supernatant, a lysate. a fraction thereof, etc.
  • isolated means separated from its native environment and present in sufficient quantity to permit its identification or use according to the invention. "Isolated” , when referring to a protein or polypeptide. means, for example: (i) selectively produced by expression cloning, or
  • 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.
  • 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.
  • the agent is heat sensitive at 80°C. proteinase K sensitive, and binds to and elutes-off blue- and/or Red Sepharose®.
  • the substantially pure organic agent is at least one polypeptide.
  • the at least one polypeptide with Melan-A/MART-1 expression down-regulating properties in malignant melanoma cells has a molecular weight between about 20kD and about 30 kD, and preferably at about 25kD.
  • the agent that down-regulates Melan-A/MART-1 expression in malignant melanoma cells when contacted with malignant melanoma cells is at least one. at least two. or at least three polypeptides. each polypeptide selected from the group consisting of Oncostatin M, Stanniocalcin, and Tissue Factor Pathway Inhibitor-2.
  • Oncostatin M is the subject of United States patent 5.618.715 to Shoyab et al., which is expressly incorporated herein by reference.
  • Stanniocalcin is the subject of United States patents 5.994,301, 5,877,290, and 5,837,498, and of WO 952441 1. all of which are expressly incorporated herein by reference.
  • Tissue Factor Pathway Inhibitor-2 is the subject of United States patents 5,849.875, 5,773,251, 5.576.294. 5.466.783. 5.106.833. and 4.966.852. all of which are expressly inco ⁇ orated herein by reference.
  • the malignant melanoma cell isolate can be obtained from a non-homogenous proteinaceous solution such as a cell culture supernatant or cell homogenate.
  • Malignant melanoma cells can be isolated from a subject using a tumor biopsy, by disaggregating the biopsy sample, and forming cell suspensions.
  • These malignant melanoma cell suspensions can be cultured according to standard cell culture techniques. In small scale, the cultures can be contained in culture plates, flasks, and dishes. In important embodiments, under standard culture conditions (e.g., on typical culture plates, flasks, and dishes that are 'static' -i.e. nonperfused), the culture conditions include a ratio of 5 x 10 6 cells/ml of medium. In larger
  • 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.
  • the malignant melanoma cell isolate can be obtained from the supernatants of the above-described cell cultures, although the entire culture can be in 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.
  • 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.
  • the collected can be obtained from the supernatants of the above-described cell cultures, although the entire culture can be in 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.
  • the supernatant is removed by aspiration or by centrifugation of
  • 15 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.
  • standard chromatographic procedures include, but are not limited to, size-exclusion chromatography, FPLC. HPLC, gel filtration chromatography, ion-exchange 0 chromatography, hydrophobic chromatography, immune-affinity chromatography, electrophoresis. etc.
  • 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 5 activity of the fraction can be measured as described above, or according to the assays described in greater detail in the Examples. Other suitable methods will be known to one of ordinary skill in the art and can be employed using routine experimentation.
  • the fractions which are positive for the malignant melanoma cell isolate can be subjected to additional rounds of screening using the foregoing methodology.
  • the purity of o the fraction can be assessed after each round of culture stimulation by subjecting an aliquot of the fraction to SDS-PAGE or other analytical methods for visualizing the mixture of constituents in the fraction.
  • the nature of the malignant melanoma cell isolate as a protein, nucleic acid, lipid, carbohydrate etc.. can be confirmed at any time by treating an aliquot of a positive fraction with non-specific degradative enzymes for the foregoing classes of molecules and testing the treated fraction in the same assays detailed above.
  • the malignant melanoma cell isolate can then be further purified for the active down- regulating agent, if desired, using immunological and molecular biological methods (see, e.g. Molecular Cloning: A Laboratory 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).
  • a fraction positive for one active agent e.g., the agent that is heat sensitive at 80°C, proteinase K sensitive, binds to and elutes-off blue-Sepharose®.
  • 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.
  • a membrane such as polyvinylidene fluoride by electroblotting, and N-terminal amino sequence determined by Edman degradation, mass spectroscopy, etc.
  • Any sequence information can be used to screen databases for homology to existing proteins and also to generate degenerate nucleic acids useful for screening a cDNA library by standard methods such as colony hybridization or polymerase chain reaction.
  • 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.
  • any cDNA libraries, expression libraries, etc.. are preferably created from malignant melanoma cells.
  • the malignant melanoma cell isolate comprises at least one, at least two. or at least three polypeptides. each polypeptide selected from the group consisting of Oncostatin M, Stanniocalcin. and Tissue Factor Pathway Inhibitor-2.
  • the invention also makes it possible to isolate agents which bind to the malignant melanoma cell isolate as disclosed herein, including antibodies and cellular binding partners of the malignant melanoma cell isolate, such as receptors or ligands.
  • 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 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 their respective binding partners, and in purification protocols to isolate the active agent in the malignant melanoma cell isolate.
  • the binding organic agents also can be used to block the effects of the agent down-regulating TAA expression in malignant melanoma cells.
  • the invention therefore, embraces organic binding agents which, for example, can be antibodies or fragments of antibodies having the ability to selectively bind the active agent(s) in malignant melanoma cell isolates and inhibit the Melan-A/MART-1 expression down- modulating properties of the agent(s) in the isolate.
  • the organic binding agents are peptides.
  • 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 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.
  • the organic binding peptides are antibodies that bind to one or more polypeptides. each polypeptide selected from the group consisting of Oncostatin M, Stanniocalcin. and Tissue Factor Pathway Inhibitor-2.
  • Anti-Oncostatin-M antibodies are known in the art. and include Mouse and Goat anti- human Oncostatin M from Research Diagnostics. Flanders. NJ.
  • Anti-stanniocalcin antibodies and methods of preparing such antibodies are known in the art, some of which are described in detail in United States patent 5.877.290 to Olsen.
  • Anti-Tissue Factor Pathway Inhibitor antibodies are also known in the art. and include sheep anti-human Tissue Factor Pathway Inhibitor antibodies from Enzyme Research Laboratories. South Bend, IN.
  • an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc * region designated an F(ab') fragment
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • 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 fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDRl through CDR3 complementarity determining regions
  • the present invention also provides for F(ab') 2 , Fab, Fv and Fd fragments: chimeric antibodies in which the Fc and/or FR 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 single chain antibodies.
  • 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.
  • polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries.
  • Combinatorial libraries also can be synthesized of peptides containing one or
  • Libraries further can be synthesized of peptides and non-peptide synthetic moieties.
  • antibodies and other binding molecules may be used for example to identify tissues expressing protein or to purify protein.
  • Antibodies also may be coupled to specific diagnostic labeling agents for imaging of cells and tissues that express the
  • the agent which is a tumor-antigen expression down-regulating agent is an agent present in a tumor cell culture supernatant, tumor cell eluate. and/or tumor cell lysate.
  • the tumor cell may be of a cancer or tumor type thought to escape immune recognition.
  • cancers or tumors may be of the folowing origin: biliary tract cancer; brain cancer, including
  • glioblastomas and medulloblastomas breast cancer; cervical carcinoma: choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer: hematological neoplasms, including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS associated leukemias and adult T-cell leukemia lymphoma: intraepithelial neoplasms, including Bowen's disease and Paget ' s disease; liver cancer; lung cancer: lymphomas,
  • 2o 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 leiomvosarcoma, rhabdomyosarcoma. liposarcoma. fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma, basocellular cancer
  • tumor-antigen can be Melan- A/MART-1.
  • Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein in (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)-C017-1A/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.
  • PSMA prostate-specific membrane antigen
  • T-cell receptor/CD3-zeta chain MAGE- family of tumor antigens. GAGE- 1.2. BAGE, RAGE. GnT-N, MUM-1. CDK4.
  • the fraction from the plurality of fractions can be undiluted or concentrated.
  • cancers or tumors escaping immune recognition and tumor- antigens associated with such tumors include acute lymphoblastic leukemia (etv6; amll ; cyclophilin b), glioma (E-cadherin; ⁇ -catenin; ⁇ -catenin; ⁇ -catenin: pl20ctn), bladder cancer (p21ras), billiary cancer (p21ras), breast cancer (MUC family;
  • HER2/neu c-erbB-2
  • testicular cancer NY-ESO-1
  • T cell leukemia HTLV- 1 epitopes
  • melanoma Melan-A/MART-1 ; cdc27; MAGE-3; p21ras; gpl00 Pmel 1 17 .
  • a subject is a human, non-human primate, cow. horse, pig. sheep, goat, dog, cat or rodent. In all embodiments, human subjects are preferred.
  • the isolated Melan-A/MART- 1 expression down- modulation inhibitors of the invention are administered in therapeutically effective amounts.
  • a therapeutically effective amount means that amount necessary to delay the onset
  • a therapeutically effective amount will vary with the subject ' s age. condition, and sex. as well as the nature and extent of the disease in the subject, all of which can be determined by one of ordinary skill in the art.
  • the dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication.
  • a therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg. preferably from about
  • the therapeutically effective amount of the isolated Melan-A/MART-1 expression down-modulation inhibitors of the invention is that amount effective to inhibit Melan- A/MART-1 antigen expression down-modulation, and can be determined using, for example, standard tests known in the art.
  • 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 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.
  • the isolated Melan-A/MART-1 expression down-modulation inhibitors of the invention may be co-administered with an anti-cancer agent other than an agent of the invention (e.g., other than an isolated Melan-A/MART-1 expression down-modulation inhibitor), that can act cooperatively, additively or synergistically with an agent of the invention, for treating or preventing cancers that express (and. it is believed, through autocrine secretions down-modulate) tumor-antigens.
  • co-administered means administered substantially simultaneously with another agent (e.g., in different or same compositions/formulations).
  • a Melan- A/MART-1 expression down-modulation inhibitor of the invention is administered to the subject close enough in time with the administration of the other agent (e.g., an anti-cancer agent) whereby the two agents may exert an additive or even synergistic effect to upregulate Melan-A/MART-1 expression and inhibit growth and/or proliferation of the cancer.
  • the other agent e.g., an anti-cancer agent
  • Anti-cancer agents other than agents of the invention include, but are not limited to: Acivicin: Aclarubicin: Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin: Altretamine; Ambomycin; Ametantrone Acetate: Aminoglutethimide: Amsacrine: Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine: Azetepa: Azotomycin: Batimastat; Benzodepa: Bicalutamide; Bisantrene Hydrochloride: Bisnafide Dimesylate: Bizelesin; Bleomycin Sulfate: Brequinar Sodium: Bropirimine; Busulfan: Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin: Carmustine: Carubicin Hydrochloride; Carzelesin; Cedefmgol: Chlorambucil; Cirolemycin
  • Enpromate Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;
  • Etoprine Fadrozole Hydrochloride
  • Fazarabine Fenretinide: Floxuridine
  • 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: Melengestrol Acetate: Melphalan: Menogaril: Mercaptopurine; Methotrexate: Methotrexate Sodium; Metoprine: Meturedepa; Mitindomide; Mitocarcin; Mitocromin: Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride: Mycophenolic Acid; Nocodazole
  • the above-described drug therapies are well known to those of ordinary skill in the art and are administered by modes known to those of skill in the art.
  • the drug therapies are administered in amounts which are effective to achieve the physiological goals in combination with the isolated Melan-A/MART-1 expression down-modulation inhibitors of the invention.
  • An isolated Melan-A/MART-1 expression down-modulation inhibitor tumor-antigen
  • TAA TAA expression upregulating molecule
  • a pharmaceutical composition may include the isolated Melan-A/MART-1 expression down- modulation inhibitor in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.
  • the compositions can be sterile and contain a therapeutically effective amount of the isolated Melan-A/MART-1 expression down-modulation inhibitor in a unit of weight or volume suitable for administration to a patient.
  • 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.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • Pharmaceutically acceptable further means a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • the characteristics of the carrier will depend on the route of administration.
  • Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, 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 may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • modes of administration include oral, rectal, topical, nasal, intradermal. or parenteral routes.
  • 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 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 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.
  • the acceptable vehicles and solvents that may be employed are water. Ringer ' s solution, and isotonic sodium chloride solution. In addition.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • 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.
  • 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 Melan-A/MART-1 expression down-modulation inhibitor into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the isolated Melan- A/MART- 1 expression down-modulation inhibitor.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the isolated Melan- A/MART-1 expression down-modulation inhibitors described above, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include the above-described polymeric systems, as well as polymer base systems such as poly(lactide-glycolide). copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in. 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 tri-glycerides: hydrogel release systems; sylastic systems: peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants: and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • hydrogel release systems sylastic systems: peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants: and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which the isolated Melan-A/MART-1 expression down-modulation inhibitor is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • Long-term sustained release means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • the invention also embraces methods for isolating tumor cell-derived tumor-antigen expression down-regulating agents.
  • a method according to this aspect of the invention typically involves: (a) preparing a culture of tumor cells that have down-regulated tumor- 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.
  • the tumor-antigen expressing cells contacted in step (d) with the supernatants (cell isolates, or fractions thereof) of the cultures of tumor cells that have down-regulated tumor- antigen expression cells of step (a), are of the same origin (i.e.. patient/tissue/cell line source) with the tumor cells that have down-regulated tumor-antigen expression cells of step (a), the only difference being that the tumor-antigen expressing cells still express the tumor-antigen.
  • the origin of the tumor cells may be of: biliary tract cancer; brain cancer, including glioblastomas and medulloblastomas: breast cancer; cervical carcinoma; choriocarcinoma: colon cancer; endometrial cancer: esophageal cancer; gastric cancer; hematological neoplasms, including acute lymphocytic and myelogenous leukemia: multiple myeloma; AIDS associated leukemias and adult T-cell leukemia lymphoma: intraepithelial neoplasms, including Bowen ' s disease and Paget ' s disease: liver cancer: lung cancer: lymphomas.
  • 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 leiomvosarcoma. rhabdomyosarcoma. liposarcoma. fibrosarcoma and osteosarcoma
  • skin cancer including melanoma, Kaposi ' s sarcoma, basocellular cancer and squamous cell cancer
  • testicular cancer including germinal tumors (seminoma.
  • 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.
  • DPPIV Dipeptidyl peptidase IV
  • ADAbp adenosine deaminase- binding protein
  • CRC Colorectal associated antigen
  • PSA Prostate Specific Antigen
  • PSMA prostate-specific membrane antigen
  • 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, RCASl, ⁇ -fetoprotein. E-cadherin. ⁇ - catenin, ⁇ -catenin and ⁇ -catenin, pl20ctn. gpl00 Pmel 117 , PRAME, NY-ESO-1, cdc27.
  • the fraction from the plurality of fractions can be undiluted or concentrated.
  • cancers or tumors escaping immune recognition and tumor- antigens associated with such tumors include acute lymphoblastic leukemia (etv ⁇ : amll: cyclophilin b), glioma (E-cadherin; ⁇ -catenin; ⁇ -catenin: ⁇ -catenin; pl20ctn).
  • bladder cancer p21ras
  • billiary cancer p21ras
  • breast cancer MUC family: HER2/neu: c-erbB-2).
  • cervical carcinoma p53; p21ras).
  • colon carcinoma p21ras: HER2/neu: c-erbB-2; MUC family
  • colorectal cancer Colorectal associated antigen (CRC)— CO 17- 1A/GA733; APC
  • CEA choriocarcinoma
  • CEA epithelial cell-cancer
  • gastric cancer HER2/neu; c-erbB-2; ga733 glycoprotein
  • ⁇ -fetoprotein hepatocellular cancer
  • 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 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.
  • 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).
  • the fraction from the plurality of fractions can be undiluted or concentrated.
  • the tumor cell isolate can be obtained from a non-homogenous proteinaceous solution such as a cell culture supernatant or a cell homogenate.
  • Tumor cells can be isolated from a subject using a tumor biopsy, by disaggregating the biopsy sample, and forming cell suspensions. These tumor cell suspensions can be cultured according to standard cell culture techniques. In small scale, the cultures can be contained in culture plates, flasks, and dishes.
  • a ratio of 5 x 10 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 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).
  • the tumor cell isolate can be obtained from the supernatants of the above-described cell cultures [i.e.. of the cultured cells of step (a)], although the entire culture can be homogenized and subjected to the steps described below for isolation of a tumor cell isolate that down-regulates tumor-antigen expression when contacted with tumor cells that express a tumor antigen.
  • 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.
  • the collected supernatant is (in its entirety) the tumor cell isolate.
  • the tumor cell supernatant can be fractionated according to standard chromatographic procedures to facilitate further isolation of the desired agent.
  • the fractions of tumor cell isolate-containing supernatant then are used to down-regulate tumor-antigen expression in tumor cells when contacted with the tumor cells.
  • the down-regulating activity of the fraction can be measured according to assays described elsewhere herein. Other suitable methods will be known to one of ordinary skill in the art and can be employed using routine experimentation.
  • Typical controls include identically isolated and cultured cells, with the exception that the supernatant medium in the control cultures is removed at regular intervals during the culture period (e.g. every 2-6 hours), being replaced with fresh culture medium. This media change effectively eliminates any down-regulatory effects a control-tumor cell isolate may exert on the control-tumor cell tumor-antigen expression.
  • the fractions which are positive for the tumor cell isolate can be subjected to additional rounds of screening using the foregoing methodology.
  • the purity of the fraction can be assessed after each round of culture stimulation by subjecting an aliquot of the fraction to SDS-PAGE or other analytical methods for visualizing the mixture of constituents in the fraction.
  • the nature of the tumor cell isolate as a protein, nucleic acid, lipid. carbohydrate etc. can be confirmed at any time by treating an aliquot of a positive fraction with nonspecific 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 immunological and molecular biological methods (.see, e.g.
  • the invention in a further aspect provides a method of screening for tumor-antigen expression modulating agents.
  • the method involves (a) contacting an agent suspected of being a tumor antigen expression modulating agent with a tumor-antigen expressing tumor cell, (b) measuring tumor-antigen expression of the tumor cell, and (c) determining whether tumor-antigen expression on the tumor cell is modulated as a result of such contacting, for example, by comparison to a control. Both up-modulating and down-modulating agents can be identified using such methods.
  • Tumor cells and tumor-antigens expressed by the tumor cells are as described above.
  • 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.
  • a method for isolating an agent that up-regulates tumor-antigen expression.
  • the method involves (a) providing a tumor- antigen expression down-regulating agent which may be isolated according to any of the foregoing methods of the invention, (b) preparing a culture of tumor cells, wherein the tumor cells may be identical (as to the patient/tissue/cell line source) to those used in the isolation of the tumor-antigen expression down-regulating agent which may be isolated according to any of the foregoing methods of the invention, (c) contacting the isolated tumor-antigen expression down-regulating agent and its putative inhibitory agent with the culture cells of step (b), (d) determining tumor-antigen expression in the culture cells, and (e) comparing the tumor- antigen expression determined in (d) with a control.
  • Tumor cells and tumor-antigens expressed by the tumor cells are as described above.
  • the tumor cells are melanoma cells and the tumor-antigen is Melan-A/MART-1.
  • 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). Controls typically include cultures similar to the foregoing control cultures described earlier.
  • 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.
  • a plurality of cultures are run in parallel, each culture containing different concentrations of the putative inhibitory agent in order to obtain a different level of tumor- antigen expression.
  • one of these concentrations serves as a negative control, i.e.. at zero concentration of agent or at a concentration of agent below the limits of assay detection.
  • Putative inhibitory agents encompass numerous chemical classes, although typically they are organic compounds.
  • 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 comprise functional chemical groups necessary for structural interactions with polypeptides and/or nucleic acids, and typically include at least an amine. carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups.
  • the putative inhibitory agents can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Putative inhibitory agents also can be biomolecules such as peptides.
  • the putative inhibitory agents are polypeptides that bind the isolated tumor-antigen expression down-regulating agent (e.g.. antibodies).
  • 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 libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation. alkylation. esterification. amidification. etc. to produce structural analogs of the agents.
  • reagents also can be included in the culture media. These include reagents such as neutral proteins (e.g., albumin), etc., which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce 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.
  • neutral proteins e.g., albumin
  • 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.
  • incubation temperature typically is between 4°C and 40°C.
  • Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours.
  • 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 expression is detected by any convenient method available to the user, supra.
  • TIL Tumor and Tumor Infiltrating Lymphocytes
  • TIL tumor infiltrating lymphocytes
  • MU, MO, MA and EW were obtained from cutaneous metastatic melanoma deposits and some (MU, MO and MA), were previously described (Pandolfi F, et al., Clin. Exp. Immunol 1994, 95:141-7; Pandolfi F, et al., Cancer Res, 1991, 51:3164-3170).
  • MU, MO and MA cutaneous metastatic melanoma deposits and some (MU, MO and MA)
  • Pandolfi F et al., Clin. Exp. Immunol 1994, 95:141-7
  • Pandolfi F et al., Cancer Res, 1991, 51:3164-3170.
  • 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- A/MART-1 specific TIL.
  • Conditioned medium from melanoma tumor lines were generated by culturing cells at
  • TIL were assayed for the ability to lyse melanoma target cells in 4 hour Cr-release assays as previously described (Hishii M, et al., Proc. Natl. Acad Sci USA. 1997, 94:1378- 1383).
  • 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.
  • EBV- transformed B lymphocyte targets EBV-3 (HLA-A1. B8. DR3). EBV-19 (HLA-A2. B18. 51
  • TIL Tumor Infiltrating T Lymphocytes
  • the IL-2 responding TIL propagated from patient MU were over 98% CD3+, CD8+, (CD4-) T cells expressing the ⁇ TCR. As previously reported (Pandolfi F. et al., Clin. Exp.
  • Tumour target lysis is shown a follows: ⁇ 10% specific lysis: -; 10-20%: + : 20-40%: ++ ;
  • 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- 1 15, indicating that MU-9 was also likely to be Melan-A/MART-1 peptide specific. As noted, clones MU-45, MU-63 and MU-79 were identical to one another as determined by TCR gene is sequencing.
  • the diminution of expression of the Melan-A/MART-1 antigen 5 correlated with reduced susceptibility of the target cells to lysis by HLA-A2-restricted, Melan- A/MART-1 -specific CTL.
  • the level of Melan-A/MART-1 antigen expression by tumor cells diminished, while levels of HLA-A2 cell surface expression did not decrease, the T cell recognition of these targets decreased.
  • the target cells were pulsed with the Melan-A/MART-1 peptide (AAGIGILTV. SEQ ID NO:l) (Chen Y, et al.. Proc Natl Acad 0 Sci USA, 1996. 93:5915-5919; Mattei. S.. et al..
  • the Melan-A/MART-1 silencing activity could be heat inactivated by treatment of the supernatants at 80°C for 60 minutes. A similar treatment at 60°C did not influence the activity. o To define this silencing activity in more detail, an assessment of cytokine and chemokine production by tumor cells which lacked Melan-A/MART-1 protein expression was performed. These Melan-A/MART-1 silencing supernatants were found to contain several known cytokines. but they lacked TNF ⁇ . which has been demonstrated to have a partial down- regulatory activity directed at the Melan-A/MART-1 promoter (Butterfield. L. H., et al.. Gene, 5 1997. 191:129-34).
  • Material purified by binding and elution from Red Sepharose, followed by binding and elution to Con-A-Sepharose, can be size fractionated on G-50 Sephadex. with the activity centering at approximately 25kD.
  • the "activity” parallels the appearance of a "band” on polyacrylamide gel electrophoresis of material fractionated on G-50 Sephadex.
  • tryptic digest sequence analysis (performed at Harvard MicroChemistry, Cambridge, MA) showed the presence of sequences corresponding to at least 7 different human proteins. Some of these proteins include Oncostatin-M (OSM), stanniocalcin (STC), and tissue factor pathway inhibitor-2 (TFPI-2).
  • OSM Oncostatin-M
  • STC stanniocalcin
  • TFPI-2 tissue factor pathway inhibitor-2
  • Recombinant Oncostatin-M shows "antigen-silencing" activity when tested on the human melanoma cell line, "MU-89" (James T. Kurnick. Massachusetts General Hospital).
  • OSM Oncostatin-M
  • MU-89 human melanoma cell line
  • recombinant OSM causes down-modulation of Melan-A/MART-1 expression comparable to that of "active" supernatants isolated from human melanoma cell lines, such as the EW tumor cell line (James T. Kurnick. Massachusetts General Hospital).
  • the down modulation of Melan-A/MART-1 on the MU-89 cell line minimal to undetectable.
  • a commercially available polyclonal antibody (Research Diagnostics, Flanders, NJ) to OSM can block the activity of the recombinant OSM.
  • the same polyclonal antibody can block the activity of the most purified fraction of EW-supernatant which had been purified by Red Sepharose binding and elution. followed by binding to and elution from Con-A— Sepharose, and fractionation on G-50 Sephadex. to isolate material of approximately 25kD.
  • the same polyclonal anti-OSM antibody which blocks recombinant OSM and "purified" fractions from EW-supernatant can only partially (approximately 50%) block the unfractionated EW- supernatant.
  • OSM-depleted EW-supernatant fluid (produced by passage over an anti-OSM immuno-affmity column ) is still active in down-modulating
  • EW-supernatants (as determined by quantitative ELISA) is approximately 1 ng/ml. yet the antigen-silencing activity of this material is equivalent of recombinant OSM at a dose of
  • Oncostatin-M a known cytokine, with well-characterized structure, and several known functions, is capable of mediating antigen-silencing in the melanoma system we have investigated, however, an additional molecule or molecules produced by melanoma cells are also able to manifest antigen silencing.
  • Antibodies to the receptor for Oncostatin-M reported to neutralize the OSM activity in other assay systems, do not block the antigen silencing activity of either purified, recombinant OSM or of the EW-supernatant.
  • the gpl 30 molecule is reported to be a shared chain in the OSM receptor, the
  • Oncostatin-M. and other molecules capable of manifesting antigen-silencing activity may act through additional receptors. including, but not limited to. oncostatin-M binding receptor molecules.
  • Oncostatin-M is produced by at least one melanoma cell line (EW), (detectable protein was demonstrated by ELISA), and mRNA transcripts can be isolated from several melanoma cell lines. Melanomas and melanocytes have nto been previously known to express oncostatin-
  • Antigen-silencing activity can be demonstrated from many different melanoma cell lines, including Melan-A/MART-1 negative cell lines, and from PMA-stimulated U937, a pro-monocytic leukemic cell line which can be shown to produce Oncostatin-M.
  • Oncostatin-M can be used at several fold the dose needed to manifest antigen-silencing (use up to lOOng/ml) without causing significant cellular toxicity, but EW supernatants contain significant toxic activities. even in partially purified fractions, indicating that additional factors are influencing the cellular viability and phenotype.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Urology & Nephrology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Physics & Mathematics (AREA)
EP00983714A 1999-11-16 2000-11-15 Compositions and methods for regulating tumor-associated antigen expression Withdrawn EP1232178A2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US16580699P 1999-11-16 1999-11-16
US165806P 1999-11-16
PCT/US2000/031511 WO2001036472A2 (en) 1999-11-16 2000-11-15 Compositions and methods for regulating tumor-associated antigen expression

Publications (1)

Publication Number Publication Date
EP1232178A2 true EP1232178A2 (en) 2002-08-21

Family

ID=22600560

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00983714A Withdrawn EP1232178A2 (en) 1999-11-16 2000-11-15 Compositions and methods for regulating tumor-associated antigen expression

Country Status (5)

Country Link
EP (1) EP1232178A2 (ja)
JP (1) JP2003515535A (ja)
AU (3) AU2236201A (ja)
CA (1) CA2388517A1 (ja)
WO (3) WO2001036472A2 (ja)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE261126T1 (de) * 2002-08-01 2004-03-15 Mtm Lab Ag Verfahren für lösung-basierte diagnose
DE602006015957D1 (de) * 2005-06-23 2010-09-16 Siemens Healthcare Diagnostics Quantitative assays für ras p21 in körperflüssigkeiten
JP5397692B2 (ja) * 2007-11-28 2014-01-22 国立大学法人名古屋大学 悪性黒色腫抗原の発現上昇剤及びその用途
JP5739816B2 (ja) 2008-12-19 2015-06-24 バクスター・インターナショナル・インコーポレイテッドＢａｘｔｅｒ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｉｎｃｏｒｐ０Ｒａｔｅｄ Ｔｆｐｉインヒビターおよび使用法
WO2011115712A2 (en) 2010-03-19 2011-09-22 Baxter International Inc Tfpi inhibitors and methods of use
WO2013141965A1 (en) 2012-03-21 2013-09-26 Baxter International Inc. Tfpi inhibitors and methods of use
EP2971045B1 (en) 2013-03-13 2019-06-19 Health Research, Inc. Compositions and methods for use of recombinant t cell receptors for direct recognition of tumor antigen
US9550828B2 (en) * 2013-09-05 2017-01-24 Boise State University Oncostatin M (OSM) antagonists for preventing cancer metastasis and IL-6 related disorders
US11633457B2 (en) 2019-04-11 2023-04-25 Boise State University Pharmaceutical compositions comprising oncostatin m (OSM) antagonist derivatives and methods of use

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5618715A (en) * 1985-12-20 1997-04-08 Oncogen Limited Partnership Oncostatin M and novel compositions having anti-neoplastic activity
ZA912137B (en) * 1990-03-29 1992-11-25 Oncogen Monocional antibodies that inhibit growth of kaposi's sarcoma
NZ237533A (en) * 1990-03-29 1992-12-23 Bristol Myers Squibb Co Monoclonal antibodies which bind to oncostatin m, cell lines producing them
US6020478A (en) * 1997-02-28 2000-02-01 Incyte Pharmaceuticals, Inc. Human tumor-associated antigen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0136472A2 *

Also Published As

Publication number Publication date
JP2003515535A (ja) 2003-05-07
AU2043201A (en) 2001-05-30
AU1615701A (en) 2001-05-30
WO2001036472A3 (en) 2002-01-10
AU2236201A (en) 2001-05-30
WO2001036472A2 (en) 2001-05-25
CA2388517A1 (en) 2001-05-25
WO2001035903A2 (en) 2001-05-25
WO2001036461A3 (en) 2002-01-17
WO2001036461A2 (en) 2001-05-25

Similar Documents

Publication Publication Date Title
CN110545883B (zh) 针对cd19的人源化抗原结合结构域及其使用方法
KR102012113B1 (ko) 항-pd-1 항체 및 그의 사용 방법
CN107206025A (zh) 用于过继细胞治疗的方法和组合物
EP0625051A1 (en) Hepatic growth factor receptor
US11925662B2 (en) Compositions and methods of enhancing anti-tumor response using hybrid neutrophils
March et al. Identification and functional characterization of the hepatic stellate cell CD38 cell surface molecule
US20230172980A1 (en) Chimeric Antigen Receptor, Construction Method Therefor and Application Thereof
RU2598719C2 (ru) Средства для лечения заболевания
EP3263599A1 (en) Anti-cancer agent and antimetastatic agent using fstl1, and concomitant drug for same
Chen et al. Programmed cell death protein-1/programmed death-ligand 1 blockade enhances the antitumor efficacy of adoptive cell therapy against non-small cell lung cancer
JPH07507202A (ja) ヒトｍｄｒ１マルチドラッグ耐性遺伝子産物に対するモノクローナル抗体およびその使用
EP1232178A2 (en) Compositions and methods for regulating tumor-associated antigen expression
CN111040036B (zh) 抗gpc3单克隆抗体、由其修饰的免疫效应细胞及其应用
EP4137519A1 (en) Fusion protein comprising ifn-gamma and anti-erbb antibody for the treatment of cancers
EP1712563A1 (en) Method of isolating monocytes
AU2003222221A1 (en) Map-kinase inhibitors as regulators of tumour-associated antigen expression
Kotani et al. Expression of functional Fas antigen on adult T-cell leukemia
CN115925945A (zh) 抗tigit人源化抗体或其抗原结合片段及其应用
EP4314253A2 (en) Methods and compositions for t-cell coculture potency assays and use with cell therapy products
JP2023519644A (ja) Ｓｓｅａ－４結合メンバー
US20220106402A1 (en) Antibody
WO2024120484A1 (en) Antibodies against edil3 and methods of use thereof
Shukla et al. Targeting of palpable B16-F10 melanoma tumors with polyclonal antibodies on white blood cells
AU684806B2 (en) Human cell adhesion protein AAMP-1 and uses thereof
CN117120091A (zh) 靶向pd-1和tim-3的双特异性抗体

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020614

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20031205

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20050909