WO2001035810A2 - Immunotherapie et diagnostic du cancer utilisant du cytochrome p450 1b1 - Google Patents

Immunotherapie et diagnostic du cancer utilisant du cytochrome p450 1b1 Download PDF

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WO2001035810A2
WO2001035810A2 PCT/US2000/031513 US0031513W WO0135810A2 WO 2001035810 A2 WO2001035810 A2 WO 2001035810A2 US 0031513 W US0031513 W US 0031513W WO 0135810 A2 WO0135810 A2 WO 0135810A2
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Prior art keywords
peptide
patient
ibl
cytochrome
cell
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PCT/US2000/031513
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English (en)
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WO2001035810A3 (fr
Inventor
Joachim L. Schultze
Robert H. Vonderheide
David Sherr
Lee M. Nadler
Britta Maecker
Michael Von Bergwelt-Baildon
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Dana-Farber Cancer Institute, Inc.
Trustees Of Boston University
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Application filed by Dana-Farber Cancer Institute, Inc., Trustees Of Boston University filed Critical Dana-Farber Cancer Institute, Inc.
Priority to EP00980436A priority Critical patent/EP1241945A4/fr
Priority to CA002390882A priority patent/CA2390882A1/fr
Priority to US10/130,413 priority patent/US7385023B1/en
Publication of WO2001035810A2 publication Critical patent/WO2001035810A2/fr
Publication of WO2001035810A3 publication Critical patent/WO2001035810A3/fr

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    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001157—Telomerase or TERT [telomerase reverse transcriptase]
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • 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
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4612—B-cells
    • 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
    • A61K39/4615—Dendritic cells
    • 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
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • 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
    • A61K39/464454—Enzymes
    • 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
    • A61K39/464454—Enzymes
    • A61K39/464457—Telomerase or [telomerase reverse transcriptase [TERT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • TAAs tumor-specific cytotoxic T lymphocytes
  • T cell-defined TAAs such as the MAGE genes
  • SEREX T cell-defined TAAs
  • TAA-derived peptides from viral peptides that are almost exclusively of high binding affinity and high MHC/peptide complex stability (Feltkamp et al., Mol., Immunol.
  • the invention provides a method of treating a patient that has or is at risk of having a cell that expresses cytochrome P450 IBl (CYP1B1).
  • This method involves administering to the patient a cytotoxic T lymphocyte (CTL)(autologous or allogeneic) that leads to death of (from here on said as kill) the cell in a CYP IBl -specific, major histocompatibility complex-restricted fashion.
  • CTL cytotoxic T lymphocyte
  • the CTL can be generated, for example, by activation with an antigen presenting cell that has been pulsed with CYP IBl, or a peptide of CYP IBl that binds to a major histocompatibility complex molecule.
  • the invention also includes a second method of treating a patient that has or is at risk of having a cell that expresses CYP IBl.
  • This method involves administering to the patient an antigen presenting cell (APC) that activates in the patient a cytotoxic T lymphocyte that kills the cell in a CYP IBl -specific, major histocompatibility complex-restricted fashion.
  • the APC can be pulsed with CYPIBI or a peptide of CYPIBI that binds to a major histocompatibility complex molecule.
  • Another method included in the invention is a third method of treating a patient that has or is at risk of having a cell that expresses CYPIBI.
  • This method involves administering to the patient CYPIBI or a peptide of CYPIBI that binds to a major histocompatibility complex molecule, which is processed by an antigen presenting cell in the patient, which, in turn, activates a cytotoxic T lymphocyte in the patient to induce cell death of the cell that expresses CYPIBI in a CYP IB 1- specific, major histocompatibility complex-restricted fashion.
  • the CYPIBI polypeptide or peptide of CYPIBI used in this method can be administered to the patient in association with an adjuvant.
  • the invention also includes a fourth method of treating a patient that has or is at risk of having a cell that expresses CYPIBI.
  • This method involves administering to the patient a nucleic acid molecule encoding CYPIBI or a peptide of CYPIBI that binds to a major histocompatibility complex molecule.
  • the nucleic acid molecule is expressed in the patient so that it can be processed by an antigen presenting cell in the patient, which activates a cytotoxic T lymphocyte in the patient to induce cell death of the cell that expresses CYPIBI, in a CYP IBl -specific, major histocompatibility complex-restricted fashion.
  • the nucleic acid molecule encoding CYPIBI or a peptide of CYPIBI can be present in an expression vector.
  • Each of the methods described above can also include treatment based around a second (or more) tumor associated antigen, e.g., telomerase (hTERT, PCT/US99/25438), or a peptide thereof that binds to MHC (e.g., the 1540 peptide).
  • a second (or more) tumor associated antigen e.g., telomerase (hTERT, PCT/US99/25438)
  • a peptide thereof that binds to MHC e.g., the 1540 peptide.
  • the patient can have a tumor containing cells that express CYPIBI.
  • APCs used in these methods can be, for example, a dendritic cell or a CD40-activated B cell.
  • the peptide of CYPIBI in these methods can bind to a class I or a class II major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • the molecule can be, for example, an HLA-A2 molecule
  • the peptide of CYPIB 1 can include the amino acid sequence of CYP239 (SEQ ID NO:l; SLNDVMPWL), CYP246 (SEQ ID ⁇ O:2; WLQYFPNPI), CYP190 (SEQ ID NO:3; FLDPRPLTV), or CYP528 (SEQ ID NO:4; LLDSAVQNL).
  • Examples of other CYPIBI sequences that can be used in these methods are set forth in the Sequence Appendix and in Tables 3- 10.
  • the invention also includes a method of assessing the level of immunity of a patient to CYPIBI or a peptide of CYPIBI that binds to a major histocompatibility complex molecule.
  • the level of cytotoxic T lymphocytes specific for CYPIBI or a peptide of CYPIBI is measured in a sample from a patient.
  • the sample can be obtained from the patient before, during, or after a cancer treatment is administered to the patient.
  • a sample can also be obtained, for example, before and after treatment.
  • the invention also includes CYPIBI peptides that bind to major histocompatibility complex molecules, for example, a peptide that consists essentially of the amino acid sequence set forth in SEQ ID NO: 1 (CYP239), SEQ
  • CYP246 SEQ ID NO:3 (CYP190), or SEQ ID NO:4 (CYP528).
  • an ex vivo generated cytotoxic T lymphocyte that specifically kills a cell expressing CYPIBI in a specific, major histocompatibility complex-restricted fashion
  • an ex vivo generated antigen presenting cell e.g., a dendritic cell or a CD40-activated B cell
  • a dendritic cell or a CD40-activated B cell that presents a peptide of CYPIBI in the context of a major histocompatibility complex molecule.
  • polypeptide is a chain of amino acids linked to one another by peptide bonds.
  • a “protein” can be made up of one or more polypeptides, while a “peptide” is generally understood to be (or include) a fragment of a polypeptide, and to consist of a chain of peptide bond-linked amino acids that is shorter in length than a full length polypeptide from which it may be derived.
  • tumor associated antigen such as CYPIBI
  • CYPIBI is an immunogenic molecule, such as a protein, that is, generally, expressed at a higher level in tumor cells than in non-tumor cells, in which, preferably, it may not be expressed at all, or only at low levels.
  • a tumor associated antigen, or TAA is said to be
  • cytochrome P450 IBl polypeptide or a "CYPIBI polypeptide” is a full length, non- fragmented polypeptide of CYPIBI, while a "cytochrome P450
  • IBl peptide or a "CYPIBI peptide,” is (or includes) a fragment of such a
  • CYPIBI polypeptides can be of any length, up to just under the full length of a CYPIBI polypeptide. However, preferably, for use in the invention, CYPIBI peptides are of a relatively short length, such as, for example, eight, nine, ten, eleven, or twelve amino acids.
  • a CYPIBI peptide may include sequences that are not present in a corresponding CYPIBI polypeptide, provided that the CYPIBI peptide also includes a stretch of at least, for example, eight, nine, ten, eleven, or twelve consecutive amino acids that have a sequence that is identical to a sequence of eight, nine, ten, eleven, or twelve consecutive amino acids in a CYPIBI polypeptide. Peptides including amino acid substitutions can also be considered as
  • a CYPIBI peptide can include a region of at least nine amino acids, of which any six or more are identical to the amino acids within a nine amino acid stretch in CYPIBI.
  • at least seven, more preferably, at least eight, and, most preferably, all nine of the amino acids in a CYPIBI peptide nine amino acid region are identical to a nine amino acid region in the CYPIBI.
  • a CYPIBI polypeptide corresponding to CYPIBI includes 533 amino acids that are substantially identical (see below) to the amino acid sequence of CYPIBI (Sutter et al, J. Biol. Chem. 269:13092-13099, 1994; Tang et al, J. Biol. Chem. 271:28324-28330, 1996; Genbank Accession No. U56438), or such a polypeptide can include the amino acid sequence of CYPIBI, as well as additional sequences.
  • CYPIBI polypeptides of the invention include regions that bind to major histocompatibility complex (MHC) antigens.
  • MHC major histocompatibility complex
  • Preferred examples of CYPIBI peptides that are included in the invention are CYP239 (SEQ ID NO:l), CYP246 (SEQ ID NO:2), CYP190 (SEQ ID NO:3), and CYP528 (SEQ ID NO:4). Additional CYPIBI peptides are listed in the Sequence Appendix, as well in Tables 3-10, and still more CYPIBI peptides can be identified using methods described below (also see PCT/US99/25438).
  • a CYPIBI peptide or polypeptide can be fused to amino acid sequences that do not naturally occur in CYPIBI.
  • a CYPIBI peptide or polypeptide can be attached to the surface of a cell or to a molecule or a macromolecule (e.g., a histocompatibility antigen), or a CYPIBI peptide or polypeptide can be conjugated to immunogens or adjuvants that are known to those of skill in this art, for example, keyhole limpet hemocyanin (KLH), for the purpose of eliciting a CYP IBl -specific immune response.
  • KLH keyhole limpet hemocyanin
  • CYPIBI peptides are CYP239 (SEQ ID NO:l), CYP246 (SEQ ID NO:2), CYP190 (SEQ ID NO:3), and CYP528 (SEQ ID NO:4).
  • CYPIBI nucleic acid molecule is meant a DNA or RNA (e.g., mRNA) molecule that encodes a CYPIBI polypeptide or CYPIBI peptide, as are defined above.
  • CYP IBl -expressing tumor cell is meant a tumor cell that expresses CYPIBI.
  • a CYP IBl -expressing tumor cell can express a level of CYPIBI that is equal to, or, preferably, greater than the level of CYPIBI expressed by the normal cell type from which the CYP IBl -expressing tumor cell has originated, or other non-tumor cells.
  • the tumor cell expresses at least 10% more CYPIBI, more preferably, at least 25% more, still more preferably at least 50% more, and most preferably at least 150% more CYPIBI than the normal cell type from which the CYP IBl -expressing tumor cell has originated, or another non- tumor cell.
  • CYPIBI expression levels in a CYP IB 1- expressing tumor cell can be increased by, for example, increased transcription of the CYPIBI gene, increased CYPIBI mRNA stability or translation, increased CYPIBI polypeptide stability, or increased CYPIBI enzymatic activity. Increasing such CYPIBI expression levels may be useful in the invention to increase the likelihood that a tumor cell will be recognized as a target of the immunotherapeutic methods described herein (see below).
  • histocompatibility antigen is meant a molecule, such as a major histocompatibility complex (MHC) class I, MHC class II, or minor histocompatibility antigen, that mediates interactions of cells of the immune system with each other and with other cell types.
  • MHC major histocompatibility complex
  • histocompatibility antigens examples include MHC class I antigens, such as HLA-A (e.g., Al, A2, A3, Al l, A24, A31, A33, and A38), HLA-B, and HLA-C, MHC class II antigens, such as HLA-DR, HLA-DQ, HLA-DX, HLA-DO, HLA-DZ, and HLA-DP, and minor histocompatibility antigens, such as HA-1.
  • HLA-A e.g., Al, A2, A3, Al l, A24, A31, A33, and A38
  • HLA-B e.g., Al, A2, A3, Al l, A24, A31, A33, and A38
  • HLA-B e.g., Al, A2, A3, Al l, A24, A31, A33, and A38
  • HLA-B e.g., Al, A2, A3, Al l, A24, A31
  • CTLs By “generating CTLs” is meant an in vivo, in vitro, or ex vivo process by which CTLs (e.g., CYP IBl -specific CTLs) are activated (e.g., stimulated to grow and divide) and/or selected.
  • CTLs e.g., CYP IBl -specific CTLs
  • activated e.g., stimulated to grow and divide
  • a peptide of CYPIBI is said to "specifically bind" to an MHC antigen if the peptide adheres to a histocompatibility antigen under physiological conditions.
  • binding can be similar to that of a peptide antigen that is naturally processed and presented in the context of MHC in an antigen presenting cell.
  • a cytotoxic T lymphocyte (CTL) or antibody is said to "specifically recognize” a CYPIBI polypeptide or a CYPIBI peptide if it binds to the polypeptide or peptide, but does not substantially bind to other, unrelated polypeptides or peptides.
  • a CTL is said to "specifically kill” a cell if it specifically recognizes and lyses a cell that expresses an antigen (e.g., CYPIBI) to which it has been activated, but does not substantially recognize or lyse cells not expressing the antigen.
  • an antigen e.g., CYPIBI
  • CYPIBI CYP IB 1- specific CTL
  • CYP IBl -specific antibody an antibody that can specifically recognize and bind to a CYPIBI peptide or polypeptide, and that does not substantially recognize and bind to other, unrelated molecules.
  • a CYPIBI polypeptide is "presented” if a peptide of CYPIBI is displayed on the extracellular surface of a cell (e.g., an antigen presenting cell), such that it can result in the in vivo, ex vivo, or in vitro generation of CYP IBl - specific CTLs or the lysis of a tumor cell by a CYP IBl -specific CTL.
  • a cell e.g., an antigen presenting cell
  • the displayed CYPIBI peptide is bound to a histocompatibility antigen.
  • CYPIBI vaccination administration of an immunogenic preparation including one or more CYPIBI peptides, CYPIBI polypeptides, CYPIBI nucleic acid molecules, fragments of any of these molecules, CYP IB 1- presenting cells (e.g.
  • CYP IBl -specific immune cells such as CTLs
  • Such vaccination stimulates a CYPIB 1 -specific immune response within the subject.
  • the vaccination can result in partial or complete inhibition of tumor growth, or partial or complete tumor regression, provided that the patient's tumor expresses CYPIBI.
  • vaccination can provide prophylaxis against the development of new CYPIBI -expressing tumors.
  • a “vaccine,” as used herein, is an immunogenic composition that can be administered in the vaccination method described above.
  • a vaccine includes, for example, one or more CYPIBI peptides, CYPIBI polypeptides, CYPIBI nucleic acid molecules, fragments of any of these molecules, CYP IB 1- presenting cells (e.g., dendritic cells or CD40-activated B cells), or mixtures thereof.
  • a vaccine composition can also include an adjuvant, which is a molecule that stimulates an immune response to a co-administered vaccine antigen. Examples of adjuvants that can be used in the invention are provided below.
  • a vaccine composition can also include other tumor associated antigens (e.g., hTERT) or peptides thereof (PCT/US99/25438).
  • immune cell any cell that plays a role in cell-mediated or humoral immunity, including CTLs and antigen-presenting cells, e.g., B cells, T helper cells, and dendritic cells.
  • sample is meant a tumor or tissue biopsy, a lymph node biopsy, bone marrow, cells, blood, serum, urine, stool, sputum, saliva, or other specimen obtained from a patient.
  • a sample can be analyzed to determine the level of CYP IBl -specific CTLs, the level of CYP IBl -specific antibodies, or the level of any other immune response indicator (e.g., a cytokine) in the patient from whom it was taken by methods that are known in the art.
  • ELISA can be used to measure levels of CYPIBI -specific antibodies
  • ELISPOT can be used to measure cytokine levels.
  • Cr 51 release (T cell cytotoxicity) assays and assays that test the binding of CTLs to tetrameric CYPIBI peptide/MHC complexes, as described herein, can be used to measure levels of CYPIB 1- specific CTLs.
  • reference sample is meant a sample in which the level of CYP IBl -specific CTLs or the level of CYP IBl -specific antibodies have been measured, and to which the level of CYPIBI -specific CTLs or the level of CYP IBl -specific antibodies in a test subject's sample are compared.
  • Reference levels can be higher, lower, or the same as patient sample levels. Comparison of a test sample to a reference sample provides an assessment of the CYPIBI -specific immune response in the test subject. In addition, comparison of a patient's sample levels to reference sample levels can allow a diagnosis of cancer and/or a prognosis of a cancer in a patient having a tumor that includes CYP 1 B 1 - expressing cells.
  • cancer treatment any therapy (e.g., chemotherapy, radiation therapy, administration of a tumor associated antigen (e.g., CYPIBI)- specific CTLs, administration of an APC presenting a peptide of a TAA (e.g., CYPIBI), or vaccination with a TAA (e.g., CYPIBI), a nucleic acid molecule encoding a TAA (e.g., CYPIBI), or a fragment thereof, to enhance an anti-tumor immune response) administered either alone or in combination with other therapies, that alleviates disease in at least some patients to which the treatment is administered.
  • a cancer treatment can reduce or inhibit tumor growth, or can induce partial or complete tumor regression.
  • a cancer treatment can be prophylactic, in that it inhibits or prevents the development of new tumors in healthy individuals, in patients that are in remission from cancer, have metastatic cancer, or have a high risk of developing cancer.
  • a protective therapy such as CYPIB 1 -specific CTLs, CYPIBI peptide presenting APCs, or a vaccine including, for example, one or more CYPIBI peptides, CYPIBI polypeptides, or CYPIBI nucleic acid molecules, or a combination thereof
  • a protective therapy such as CYPIB 1 -specific CTLs, CYPIBI peptide presenting APCs, or a vaccine including, for example, one or more CYPIBI peptides, CYPIBI polypeptides, or CYPIBI nucleic acid molecules, or a combination thereof
  • Subjects with a relatively high risk of developing a tumor include those having a family history of cancer, those having one or more genetic mutations that are associated with a high risk for cancer (e.g., a mutation that inactivates a tumor suppressor gene), those having relatively high levels of CYPlBl-specific CTLs or CYP IBl -specific antibodies, those who have cancer or are in remission from cancer, and those who have been exposed to agents known or suspected to cause cancer.
  • pharmaceutically acceptable carrier is meant a carrier that is physiologically acceptable to a treated patient, while retaining the therapeutic properties of the compound with which it is administered.
  • One exemplary pharmaceutically acceptable carrier is physiological saline.
  • Other physiologically acceptable carriers and their formulations are known to those skilled in the art, and are described, for example, in Remington 's Pharmaceutical Sciences (18 th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA.
  • substantially identical is used herein to describe a polypeptide or nucleic acid molecule exhibiting at least 50%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% identity to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences is at least 8 amino acids, preferably at least 16 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids.
  • the length of comparison sequences is at least 24 nucleotides, preferably at least 50 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 110 nucleotides.
  • Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705).
  • the CYPIBI polypeptides, peptides, and nucleic acid molecules of the invention can be identical or substantially identical to naturally occurring molecules, and thus may or may not include non- wild type sequences.
  • substantially pure peptide or “substantially pure polypeptide” is meant a peptide, polypeptide, or a fragment thereof, which has been separated from the components that naturally accompany it.
  • the peptide or polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the peptide or polypeptide is a CYPIBI peptide or polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure.
  • a substantially pure CYPIBI peptide or polypeptide can be obtained, for example, by extraction from a natural source (e.g., a tumor cell), by expression of a recombinant nucleic acid molecule encoding a CYPIBI peptide or polypeptide, or by chemically synthesizing the peptide or polypeptide. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • a protein is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state.
  • a protein that is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates is substantially free from its naturally associated components.
  • substantially pure peptides and polypeptides not only include those derived from eukaryotic organisms, but also those synthesized in E. coli or other prokaryotes.
  • substantially pure DNA or “isolated DNA” is meant DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA is derived, flank the gene.
  • the term thus includes, for example, a recombinant DNA that is incorporated into a vector; an autonomously replicating plasmid or virus; or the genomic DNA of a prokaryote or eukaryote; or DNA that exists as a separate molecule (e.g., a cDNA, or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
  • transformation means any method for introducing foreign molecules into a cell.
  • Lipofection, DEAE- dextran-mediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, transduction (e.g., bacteriophage, adenoviral refroviral, or other viral delivery), electroporation, and biolistic fransformation are just a few of the methods known to those skilled in the art that can be used in the invention.
  • transformed cell By “transformed cell,” “transfected cell,” or “transduced cell,” is meant a cell (or a descendent of a cell) into which a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a polypeptide of the invention has been introduced by means of recombinant DNA techniques.
  • a nucleic acid molecule e.g., a DNA or RNA molecule
  • promoter is meant a minimal sequence sufficient to direct franscription. Promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific, temporal- specific, or inducible by external signals or agents can also be used in the invention; such elements can be located in the 5' or 3' or infron sequence regions of the native gene.
  • operably linked is meant that a gene and one or more regulatory- sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
  • expression vector is meant a genetically engineered plasmid or virus, derived from, for example, a bacteriophage, adenovirus, refrovirus, poxvirus, herpesvirus, or artificial chromosome, that is used to transfer a peptide or polypeptide coding sequence (e.g., a CYPIBI peptide coding sequence), operably linked to a promoter, into a host cell, such that the encoded peptide or polypeptide is expressed within the host cell.
  • a peptide or polypeptide coding sequence e.g., a CYPIBI peptide coding sequence
  • Fig. 1 is a graph showing the level of peptide binding of MAGE-3
  • CYP239, and CYP246 to TAP-deficient T2 cells.
  • Figs. 2A-2D are graphs showing that CTL derived from healthy donors recognize CYP239 and CYP246 peptides.
  • A CTL raised against the CYP239 peptide specifically lyse CYP239 pulsed ( ⁇ ), but not impulsed T2 cells (D), or T2 cells pulsed with an irrelevant peptide (O; F271 from MAGE-3).
  • B CTL generated against the CYP246 peptide recognize only T2 cells pulsed with CYP246 ( ⁇ ), but not control T2 cells (D, unpulsed; O, pulsed with F271).
  • the diagrams display representative experiments for 11/13 healthy donors positive for CYP239-specific CTL induction and 4/10 healthy donors positive for CYP246- specific CTL induction.
  • C CYP239-specific CTL recognize autologous CD40- B cells pulsed with CYP239 peptide ( ⁇ ), but not unpulsed autologous CD40-B ( ) or allogeneic HLA-A2 mismatched CD40-B unpulsed (O) or pulsed with CYP239 peptide (•).
  • D Analogous results were obtained for CYP246-specific CTL using the same target cells unpulsed or pulsed with the CYP246 peptide.
  • 2E is a series of graphs showing a representative teframer analysis of CYP239- and CYP246-specific CTL after 4 weeks in culture.
  • the A2/TAX teframer served as a negative confrol.
  • Percent teframer + CD8 + T cells is shown.
  • Positive teframer staining correlated with specific cytotoxicity in 51 Cr assays.
  • Fig. 2F is a graph showing the cytotoxicity of expanded CYP239-specific tetramer sorted CTL against T2 cells either unpulsed (D), pulsed with CYP239 ( ⁇ ) or RT-pol476 (O).
  • Fig. 3 is a graph showing the level of specific lysis of CD40-activated B cells that were titrated with increasing concentrations of peptide before exposure to peptide-specific CTLs.
  • Figs. 4A-4H are graphs showing that CYP239 and CYP246-s ⁇ ecific CTL are cytotoxic for HLA-A2 + melanoma, multiple myeloma and ovarian carcinoma cell lines. Expression of CYPIBI in all tumor cell lines was confirmed by Western blot analysis. HLA-A2 + cell lines are shown by solid symbols; HLA-A2 " cell lines by open symbols.
  • Targets for CYP239-specific CTL were (A, B) melanoma cell lines K029 (•) and SK-MEL-2 (O), (C, D) multiple myeloma cell lines U266 (•), IM-9 ( ⁇ ) and HS-Sultan (O), and (E, F) ovarian carcinoma cell lines 36M ( ⁇ ) and SK-OV-3 (D).
  • HLA-A2 + fibroblast cell line GM847 ( ⁇ ) and primary monocytes from 3 HLA-A2 + (O, O, V) and one HLA-A2 " healthy donors (D) were not lysed by either (G) CYP239-specific or (H) CYP246-specific CTL. Results of one representative experiment are shown. Similar results were obtained for each of 2 to 6 CTL tested per target.
  • Fig. 5 is a graph showing the specific lysis of tumor cells pulsed with CYP239 by CYP239-specific CTLs.
  • Figs. 6A-6C are graphs showing lysis of CYPIB 1 + HLA-A2 + primary lymphoma and acute myeloid leukemia (AML).
  • Two HLA-A2 + CYPIB 1 + follicular lymphoma samples ( ⁇ and •) from lymph node biopsies were lysed by (A) CYP239-specific and (B) CYP246-specific CTL, while no cytotoxicity occurred against an HLA- A2 " CYPIB 1 + FL (D). Experiments were performed from two different normal donors with similar results.
  • C CYP239-specific CTL lysed primary HLA-A2 + ( ⁇ ), but not HLA-A2 " (O) AML cells.
  • Figs. 7 A and 7B are each a series of graphs showing the generation of CYP239- and CYP246-s ⁇ ecific CTL from cancer patients.
  • Fig. 8 is a graph showing that the efficacy of a combination of CYPIBI and hTERT-specific CTL in a chromium release assay.
  • CYP239- and 1540- specific CTL were used individually or in combination against a mixture of T2 cells pulsed with either CYP239 or 1540 peptide.
  • Target cells were mixed at a 1 : 1 ratio using a final number of 5000 cells/well. Numbers shown reflect the number of effector cells added to each well.
  • Fig. 9 is a graph showing the specific lysis of target cells with CTLs specific for heteroclitic peptides CYP239-19 and CYP239-139.
  • Fig. 10 is a graph showing the stability of HLA-A2/peptide complexes including the indicated peptides, as determined by TAP-deficient T2 cell assays.
  • Fig. 11 is a graph showing the stability of HLA-A2/peptide complexes including CYP 190 and CYP528, as determined by TAP-deficient T2 cell assays.
  • Figs. 12A-12C are graphs showing that CYP190-specific CTL lyse peptide-pulsed T2 cells (A), HLA-A2 + myeloma cell lines, and HLA-A2 + primary ALL cells (C).
  • Figs. 13A and 13B are graphs showing that CYP190-specific CTL can be generated from cancer patients, such as a (A) prostate cancer patient (HLA- A2 + ), and (B) a multiple myeloma patient (HLA-A2 + ).
  • cancer patients such as a (A) prostate cancer patient (HLA- A2 + ), and (B) a multiple myeloma patient (HLA-A2 + ).
  • Fig. 14 is a graph showing the generation and verification of CYPIB 1- specific tetramers including CYP239, CYP246, or a control, Tax 11.
  • Fig. 15 is a schematic representation of a system to detect CYPIBI T cells by HLA-A2/peptide teframeric complexes.
  • Fig. 16 is a set of graphs showing the detection of CYP IBl -specific CTL in normal HLA-A2 + donors.
  • Fig. 17 is a set of graphs showing the detection of CYP IBl -specific
  • cytochrome P450 IBl includes peptides that bind to HLA molecules.
  • Antigen presenting cells APCs that present such peptides on their surfaces, in complexes with HLA, can activate cytotoxic T lymphocytes (CTLs) to specifically lyse cells expressing CYPIBI, in an MHC-restricted fashion.
  • CTLs cytotoxic T lymphocytes
  • CYPIBI is a mediator of dioxin-related effects on tumorigenesis, in combination with searches of public literature databases, such as PubMed, we identified CYPIBI as a potential universal tumor antigen. It is overexpressed in nearly 100% of human tumors (Murray et al, Cancer Res. 57:3026-3031, 1997), whereas the expression in normal tissue is low and limited to steroidogenic and steroid-responsive tissue (Buters et al. , Proc. Natl. Acad. Sci. USA 96:1977-1982, 1999). CYPIBI is a member of the superfamily of monooxygenases responsible for the metabolic activation of environmental carcinogens. Mice lacking CYPIBI have a much lower incidence of lymphoma than wild type mice after challenge with polycyclic aromatic hydrocarbons, further implicating that CYPIBI plays a role in oncogenesis.
  • TAAs tumor associated antigens
  • TAAs have been subsequently characterized in several other malignancies (Van Pel et al, Immunological Reviews 145:229-250, 1995; Rosenberg, Immunol. Today 18:175-182, 1997; Van den Eynde et al, Curr. Opin. Immunol. 9:684-693, 1997), raising the hypothesis that most, if not all, tumors express antigens that CTL can potentially attack.
  • the demonstration that TAA-specific immune responses can lead to tumor regression has been borne out extensively in animal models (Rosenberg, Immunity 10:281-287, 1999).
  • T cell-defined TAAs such as the MAGE genes
  • SEREX T cell-defined TAAs
  • TAA-derived peptides This quality distinguishes TAA-derived peptides from viral peptides that are almost exclusively of high binding affinity and high MHC/peptide complex stability (Feltkamp et al, Mol. Immunol. 31 : 1391-1401, 1994; Sette et al, J. Immunol. 153:5586-5592, 1994).
  • the low binding affinity of TAA-derived peptides is likely to be one of the reasons why natural CTL responses against such peptides are not successful for tumor eradication. This is in agreement with the finding that large numbers of TAA-specific CTLs co-exist with metastatic tumors in melanoma patients (Romero et al, J. Exp. Med. 188: 1641-1650, 1998).
  • a recent study has even demonstrated that despite expansion, such CTLs were hyporesponsive, showing reduced cytotoxic and cytokine responses (Lee et al, Nat. Med. 5:677-685, 1999).
  • TAAs To overcome these limitations of currently known TAAs, we have developed methods to identify more universal TAAs, and, in particular, those containing T cell epitopes with high MHC binding affinity and high MHC/peptide complex stability. Such TAAs and MHC-binding peptides thereof can trigger sufficient CTL responses against a broad range of tumor types. Rather than analyzing tumor-derived T cell clones or tumor-specific antibodies derived from patients, an alternative strategy was used, in which TAA and their CTL epitopes are deduced from genes known to be selectively expressed in tumors.
  • cytochrome P450 IBl cytochrome P450 IBl
  • This TAA contains at least two peptide epitopes that (1) bind to HLA-A*0201 with high affinity and high MHC/peptide complex stability, (2) are naturally processed and presented by HLA-A*0201 molecules on the cell surface of a panel of tumor cell lines, (3) elicit peptide-specific HLA-resfricted CTL responses, and (4) are recognized by such CTL on a wide variety of different tumor histologies.
  • TAAs tumor associated antigens
  • CD8 + CTL responses depends upon the binding affinity of the target peptide to MHC, the peptide-MHC complex stability, and the avidity of T cell receptor (TCR) binding for the peptide complex (Sette et al, J. Immunol.
  • HLA-A*0201 -binding peptides determines the contributions for each of the 20 amino acids at each of the positions of a peptide using a linear programming algorithm.
  • LPpep When tested on a data set of over 1000 peptides having known binding affinities, LPpep has a higher sensitivity (>0.75) and specificity (> 0.9) than four other available methods.
  • Antigen-specific T cells in vitro is a classical immunological technique.
  • Antigen-specific T cells can be generated relatively easily if the peptides used to make such cells are: (1) immunodominant, (2) of viral or other non-self origin, (3) expressed at a reasonably high copy number on the cell surface (Porgador et al, Immunity 6:715-726, 1997), and (4) of high affinity for, and of low dissociation rate (high MHC/peptide complex stability) from, MHC, and if the T cell pool under study has been exposed to the antigen in vivo prior to ex vivo analysis (recall response).
  • the frequency analysis of peptide-specific T cells by teframer technology revealed a significantly higher frequency than earlier assays based on in vitro expansion had suggested.
  • T cells are primarily stimulated with peptide-pulsed DC, and repeatedly stimulated with peptide-pulsed CD40-B cells. Peptide-specificity and HLA-restriction is analyzed after a total of 2-5 stimulations, depending on the antigen under study.
  • This system is not only very powerful in amplifying rare T cells against TAA-derived peptides, but has several other advantages: (1) it is relatively cheap compared to transgenic mice, (2) a single blood draw is sufficient to generate all cellular components necessary, and (3) the use of professional APCs for restimulation is superior to PBMC.
  • cytotoxicity assays using radioactive chromium.
  • cytotoxicity analysis is an important component of the characterization of a novel TAA, since tumor cell lysis is the ultimate goal of any TAA-directed immunotherapeutic intervention.
  • assays are not suitable to determine the frequency of peptide-specific CTLs.
  • the sensitivity of cytotoxicity assays to identify very small numbers of specific CTLs is insufficient.
  • two new technologies namely the teframer technology (Altman et al, Science 274:94-96, 1996) and cytokine ELISPOT analysis (Herr et al, J.
  • TAA TAA.
  • a list of peptides predicted to bind to all HLA alleles available are listed in the Sequence Appendix. The prediction was carried out using three different algorithms that are freely available on the Internet: http://engpub 1.bu.edu/LPpep-cgi/peptide2.cgi http://www.uni-tuebingen.de/uni/kxi http://bimas.dcrt.nih.gov/molbio/hla_bind/ Analysis of the CYPIBI sequence by two independent prediction algorithms 5 (BIMAS and LPpep, see Experimental Methods, below) revealed two peptides (CYP239 and CYP246) predicted to bind to HLA-A*0201, the most common HLA allele (Table 1).
  • CYPIBI peptides are unique in the public gene databases and in particular are not found within any other member of the cytochrome P450 family. (Also see below for additional CYPIBI peptides (e.g., l o CYP 190 and CYP528) identified according to the invention.)
  • Both peptides stabilized HLA-A2 molecules on the surface of T2 cells to a similar extent as a positive control peptide (F271) derived from the tumor antigen MAGE-3 (Nijman et al, Eur. J. Immunol. 23:1215-1219,1993), which is known to bind HLA-A2 with high affinity.
  • F271 a positive control peptide derived from the tumor antigen MAGE-3
  • T2 cells were incubated with peptide in serum- free medium for up to 18 hours, harvested, 0 washed, and subsequently stained with FITC-labeled anti-HLA-A2 mAb BB7.2 (maximum peptide binding). Increase in fluorescence intensity was determined as a function of peptide binding.
  • T2 cells were cultured in serum-free media for an additional 2, 4, 6, or 24 hours, and subsequently analyzed for HLA-A2 expression by flow cytometry.
  • the MAGE-3 -derived peptide which induces CTL responses in the majority of all normal donors, demonstrated high binding affinity and complex stability.
  • CYP IBl -derived peptides bound to HLA-A2
  • CYP246 showed a significantly lower complex stability than CYP239 and MAGE-3.
  • attempts to induce CTL responses were successful in 13/15 donors 0 against CYP239, but only 4/9 donors for CYP246.
  • Our data further show that complex stability might be a more important factor than binding affinity for the likelihood to generate peptide-specific CTL responses.
  • CTL lines were generated ex vivo by repetitive stimulation with peptide pulsed autologous APC.
  • CTL specific for CYP239 were induced from peripheral blood mononuclear cells (PBMC) in 11 of 13 healthy HLA-A2 + donors (Fig. 2A). These CTL specifically lysed T2 cells pulsed with CYP239 peptide, while no cytotoxicity occurred against unpulsed T2 cells or T2 cells pulsed with the F271 peptide from MAGE-3.
  • CYP246 specific CTL were generated in 4 of 10 healthy HLA-A*0201 + donors (Fig. 2B).
  • HLA-A2 restriction was demonsfrated using autologous and HLA-A2 mismatched CD40-activated B cells (CD40-B) as targets (Figs. 2C and 2D).
  • CD40-B autologous and HLA-A2 mismatched CD40-activated B cells
  • CYP239-specific CTL lysed autologous CD40-B pulsed with CYP239, but not allogeneic HLA-A2- CD40-B pulsed with CYP239 (Fig. 2C). Similar results were obtained for CYP246- specific CTL (Fig. 2D).
  • Experiments titrating the concentration of peptide onto the CD40-activated B cells before the cytotoxicity assay further support the peptide-specificity of the CTL generated against the CYP239 peptide. Comparing the data with published data in the literature the cell line tested in this experiment 5 is of intermediate avidity. Alternatively, the cell line contains both high and low avidity CTL and the curve represents the sum
  • CYP239 and CYP246 CTL specificity was further demonsfrated using peptide/MHC teframers (Fig. 2E).
  • Frequency analysis using CYP239 l o teframers demonstrated that 1.4-2.4% of all CD8 + T cells recognized the CYP239 peptide, a percentage comparable to previously published data for gplOO specific (Yee et al, J. Immunol. 162:2227-2234, 1999) or proteinase-3 specific CTL lines (Molldrem et al, Cancer Res. 59:2675-268.1,1999).
  • CYP246-specific CTLs were detected with CYP246 teframer, but the frequency of specific CTL was lower
  • CYP239 teframer- positive CTL were sorted and expanded using phytohemagglutinin (PHA), IL-7, IL-2, and irradiated allogeneic PBMC. These CTL lysed T2 cells pulsed with CYP239 at extremely low E:T ratios, but not unpulsed T2 cells or T2 cells pulsed with an irrelevant HLA-A2 binding peptide (Fig. 2F).
  • PHA phytohemagglutinin
  • IL-7 interleukin-7
  • IL-2 interleukin-2
  • CYPIBI specific CTL lyse CYPIBI expressing tumors in an HLA-A2 restricted fashion 5
  • peptide-specificity of CTL is demonsfrated by lysis of peptide- pulsed target cells, it is important to show that tumor cells themselves process and present the peptide in the groove of their MHC molecules (Yee et al, J. Immunol. 162:2227-2234, 1999).
  • CYP239- and 0 CYP246-specific CTL from healthy donors were then screened for cytotoxicity (Figs. 4A-4H). CYP239 CTL (Fig.
  • CYPIBI expression has been reported in fibroblasts (Eltom et al, Carcinogenesis 19:1437-1444, 1998) and monocytes (Baron et al, Biochem. Pharmacol. 56:1105-1110, 1998)
  • HLA-A2 + fibroblast cell line GM847
  • primary peripheral blood derived monocytes from four healthy donors as targets for CYPIBI -specific CTL.
  • Western blot analysis showed that of these normal cells express low or absent levels of CYPIBI.
  • Figs. 4G and 4H CYP239 and CYP246-specific CTL failed to lyse these normal targets.
  • CD40-activated B cells strongly express CYPIBI protein (detected by Western blot), but these normal cells were not lysed by CYP239- or CYP246-specific CTL (Figs. 2C and 2D), suggesting that there is a differential expression of CYPIB peptides on tumor cells.
  • CYP239 peptide is most likely expressed at low levels on tumor cell MHC.
  • tumor cells could be more resistant to CTL-mediated lysis.
  • tumor cells were pulsed with the specific peptide before they were used in chromium release assays.
  • peptide-pulsing of tumor cells significantly increased killing of the target cells, suggesting that the level of naturally expressed CYP239 peptide is low on the tumor cells, however, that these cells can be readily killed once the level of peptide is increased.
  • any methodology to increase the expression of CYPIB 1 -derived peptides on the cell surface will make the tumor cell a susceptible target for CYP IBl -specific CTLs (Fig. 5).
  • CYPIBI " ' mice demonstrate a significantly reduced incidence in carcinogen- induced lymphomas (Buters et al, Proc. Natl. Acad. Sci. U.S.A. 96:1977-1982, 1999), we chose to study human primary follicular lymphoma (FL) as a model tumor target for CYPIBI specific CTL. Tumor cells from two HLA-A2 + FL samples and one HLA-A2 " FL sample were found to be CYPIB 1 + as assessed by Western blot analysis.
  • Generation of all cellular components of our ex vivo system i.e., dendritic cells, CD40-B, and CTL
  • expansion of CTL to CYP239 and CYP246 were similar to results obtained for healthy donors.
  • CYP239-specific CTL was Using peptide- pulsed T2 cells as targets, we demonsfrated CYP239-specific CTL in all four patients (Fig. 7 A). Due to lower numbers of PBMC available, CYP246-specific CTL cultures were only initiated in patients 3 and 4. CYP246-specific CTLs were detected in both patients These patient-derived lines showed tumor-specific lysis of HLA-A2 + myeloma cell lines U266 and IM-9, but not the HLA-A2 " myeloma cell line HS-Sultan (Fig. 7B) Because autologous tumor cells were not available from these patients, we tested the same FL samples described above as primary 5 tumor targets. CYP239-spec ⁇ f ⁇ c CTL from patient 1 lysed both HLA-A2 + FL samples but not the HLA- A2 (18% 0% at an E.T ration of 30.1).
  • the T2 assay described above was used to determine binding and dissociation rate of heteroclitic peptides engineered for optimal binding to HLA molecules.
  • Two heteroclitic peptides to CYP239 were tested and shown to have higher peptide/MHC-complex stabilities, as is shown in Table 4. While the confrol peptide MAGE-3 and the CYP239 peptide showed no significant binding at 24 hours (0.14 resp. 0.12), both heteroclitic peptides still bound to HLA-A*0201 (0.72 resp. 0.73).
  • Tables 5 and 6 show predicted mutations to improve HLA-A2 binding of CYPIBI 239 and CYPIBI 246.
  • CYP 190 and CYP528 show the longest half-life on the cell surface. Additional experiments were carried out to characterize these peptides, in particular, CYP 190. As is shown in Fig. 11, further binding studies using TAP- deficient T2 cells showed that CYP190/A2 complexes can be detected as long as 24 hours after peptide withdrawal. Moreover, as is shown in Figs. 12A-12C, CYP190-specific CTL can be generated from normal HLA-A2 + donors, and these CTL can lyse peptide-pulsed T2 cells (Fig.
  • CYP190-specific CTL can be generated from HLA-A2 + cancer patients (Fig. 13 A, prostate cancer patient, and Fig. 13B, multiple myeloma patient), and show specific lysis.
  • HLA- A3 binding epitopes from CYPIBI.
  • BIMAS server for example, we identified the peptides shown in Table 8, in which the positive confrol is a peptide derived from influenza A.
  • T cells from HLA-A2+ healthy donors were stained with CYP239 and CYP246 teframers directly ex vivo and 10 days after in vitro restimulation with CYP239 or CYP246 peptides.
  • the level of detection on day 10 is at 0.05% as determined from background staining of HLA-A2 " donors.
  • No expansion of CYP239-specific T cells was detected in healthy donors on day 10 (mean 0.022% ⁇ 0.018%).
  • CYP246-specific T cells were detected in 2 healthy donors with one rising to 0.5% (mean 0.032% ⁇ 0.022%). As is shown in Fig.
  • T cells from HLA-A2+ multiple myeloma patients were stained with CYP239 and CYP246 teframers directly ex vivo and 10 days after in vitro restimulation with CYP239 or CYP246 peptides.
  • the level of detection on day 10 is at 0.05% as determined from background staining of HLA- A2- donors. 4 patients showed T cells reactive against CYP239 >0.05% on day 10 (mean 0.068% ⁇ 0.055%), whereas 5 patients showed reactivity against CYP246 (mean 0.098% ⁇ 0.080%).
  • Table 10 shows the sequence of CYPIBI and the sequences of CYPIBI peptides that were identified by LPEP analysis as having binding affinity for HLA-A2.
  • Peripheral blood from healthy blood donors and cancer patients was obtained by leukapheresis and peripheral blood mononuclear cells (PBMC) were purified by Ficoll-density centrifugation (Schultze et al. , J. Clin. Invest. 100:2757-2765, 1997).
  • Primary NHL and AML samples were obtained from discarded specimens.
  • Leukapheresis products and tumor tissue were obtained following informal consent and approval by our institute's Review Board.
  • the melanoma cell line K029 was a kind gift of Dr. G. Dranoff (Dana- Farber Cancer Institute, Boston).
  • the fibroblast cell line GM847 was a kind gift of Dr. W. Hahn (Whitehead Institute of Biomedical Research, Cambridge).
  • the 36M ovarian carcinoma cell line was a kind gift of Dr. S. Cannisfra (Beth Israel Deaconess Hospital, Boston).
  • TAP-deficient T2 cell line The TAP-deficient T2 cell line; the multiple myeloma cell lines U266, IM9, and HS-Sultan; the melanoma cell line SK-MEL- 2; and the ovarian carcinoma cell line SK-OV-3 were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Peptides
  • Peptide Prediction Binding of peptides to HLA molecules can be predicted for the most common HLA alleles by computational methods (Parker et al, J. Immunol. 152:163-75, 1994; Gulukota et al, J. Mol. Biol. 267:1258-67, 1997).
  • To increase specificity of peptide prediction we used two independent algorithms: a matrix algorithm available on the BIMAS (Bioinformatics & Molecular Analysis Section at the NIH) web site (Parker et al, J. Immunol. 152:163-75, 1994) and a linear programming algorithm (LPpep) at Boston University (Z. Weng).
  • BIMAS predicts for the half-life of peptides bound to class I molecules, while LPpep predicts an arbitrary half inhibitory concentration (IC 50 ) in competition with a labeled reference peptide.
  • IC 50 half inhibitory concentration
  • TAP-deficient T2 cells were pulsed with 40 ⁇ g/ml of peptide and 3 ⁇ g/ml of ⁇ 2 -microglobulin (Sigma, St. Louis, MO) for 18 hours in serum-free IMDM (Life Technologies, Rockville, MD) at 37°C. Cells were washed three times in serum-free IMDM and HLA-A*0201 expression was measured by flow cytometry using FITC-conjugated mAb BB7.2 (ATCC).
  • FI (MFI pept j de pUlsed T2 / MFI unpU i sed T2 ) - 1).
  • CYPIBI expression was determined in microsomal cell fractions.
  • Microsomal protein was isolated by differential speed centrifugation. Cells were harvested, washed, and resuspended in hypotonic buffer. After mechanical homogenization high-density particles were pelleted by centrifugation for 20 minutes at 15,000g. The supernatant was collected and centrifuged for 1 hour at 180,000g. The pellet was resuspended in TEDG buffer, and 100 ⁇ g of microsomal protein was separated by SDS-PAGE and transferred to nifrocellulose membrane.
  • Western blot for CYPIBI was performed according to the manufacturer's recommendations (Gentest, Woburn, MA). Bands were visualized by enhanced chemiluminescent detection (NEN Life Science Products, Boston, MA).
  • CD8 + T cells >80% CD8 + , >95% CD3 + , ⁇ 2.0% CD4 + , and ⁇ 5% CD56 + ) were isolated from PBMC by negative selection using magnetic beads. B cells were activated via CD40, and DC were prepared from peripheral blood monocytes with IL-4 and GM-CSF (Schultze et al, J. Clin. Invest. 100:2757-2765, 1997).
  • DC were harvested after 7 days, pulsed withpeptide (40 ⁇ g/ml) and ⁇ 2-microglobulin (3 ⁇ g/ml) for 2 hr at 37°C, irradiated (33 Gy), and added to autologous CD8 + T cells at a T:DC ratio of 20:1 in RPMI media supplemented with 10% human AB serum, 2 mM glutamine, 15 ⁇ g/ml gentamicin, 20 mM HEPES, and 15 ng/ml IL-7 (Endogen, Woburn, MA).
  • T cell cultures were harvested and restimulated with irradiated (33 Gy), peptide-pulsed (10 ⁇ g/ml) autologous CD40-activated B cells.
  • IL-2 50 U/ml; Chiron Corp, Emeryville, CA
  • Flow cytometry was performed as described (Schultze et al, J Clin. Invest. 100:2757-2765, 1997).
  • Assessment of cytotoxic effector function and tetramer analysis were performed with CTL cultures always >90% CD37CD8 + , ⁇ 5% CD4 + , and ⁇ 5% CD56 + . Cytotoxicity Assay
  • CTL lines were used after at least four antigenic stimulations in standard 51 Cr release assays as previously described (Vonderheide et al, Immunity 10:673-679, 1999). Percent specific lysis was calculated from cpm of (experimental result - spontaneous release)/(maximum release - spontaneous release) xl00%. Monocytes as targets were isolated from PBMC by RosetteSep ® (Stem Cell Technologies, Vancouver) following the manufacturer's recommendations.
  • ALEXA- 488 Molecular Probes, Eugene, OR
  • CTL lines cells were incubated with the tetramer and CD8-PE (Beckman Coulter, Fullerton, CA) for 30 minutes at room temperature. Teframers were also used to sort CYP239-specific CTL. Tetramer sorted CTL were expanded by mitogen stimulation as described (Nalmori et al, Cancer Res. 59:2167-2173, 1999).
  • the invention provides methods for preventing or treating conditions associated with excessive cell proliferation and expression of CYPIBI, such as cancer.
  • conditions that can be prevented or treated using the methods of the invention include, for example, all cancers, e.g., melanoma, lymphoma, carcinoma, sarcoma, multiple myeloma, leukemia, lung cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer, and brain cancer.
  • pre-cancerous and non-cancerous conditions characterized by excessive cell proliferation, and expression of a CYPIBI can be treated using the methods of the invention as well.
  • carcinomas in situ e.g., ductal carcinoma in situ, lobular carcinoma in situ, and cervical carcinoma in situ, as well as adenoma and benign polyps can be treated using the methods of the invention.
  • Patients that can be treated using the methods of the invention include those whose conditions are at early, intermediate, or advanced stages of development. Patients can receive treatment according to the invention before, during, or after other types of treatment, such as chemotherapy, radiation, or surgery, or can receive the treatment of the invention in the absence of any other type of treatment.
  • the methods of the invention can also be used as general prophylactic measures; to prevent conditions from arising in patients that are at risk, or have early signs, of developing a condition associated with excessive cellular proliferation, such as cancer; or to prevent recurrence of such a condition.
  • Additional persons that can be treated, in particular, using vaccination methods of the invention are those who are to donate cells, such as cytotoxic T lymphocytes, for use in the treatment of another (see below).
  • cytotoxic T lymphocytes Central to the prophylactic and therapeutic methods of the invention is the pathway of cell-mediated immunity involving cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • an antigen is taken up and processed by an antigen presenting cell, so that a peptide of the antigen is presented on the surface of the cell, in the context of MHC.
  • antigen presenting cells then activate cytotoxic T lymphocytes, in an MHC-restricted fashion, to proliferate and kill target cells that express the antigen.
  • a CYPIBI antigen is administered to a patient, in whom the antigen is taken up by antigen presenting cells, which in turn activate CTLs.
  • an antigen presenting cell is contacted with a CYPIBI antigen ex vivo, where it takes up, processes, and presents the antigen, in the context of MHC.
  • ex vivo stimulated APCs are then administered to a patient, in whom they specifically activate CTLs.
  • CTLs are activated ex vivo with APCs presenting CYPIBI peptides, and the activated CTLs are then administered to a patient.
  • CYPIBI peptides alone or, preferably, in combination with another (or more) tumor associated antigen polypeptides or peptides (e.g., telomerase).
  • the prophylactic and therapeutic methods of the invention include one in which CYPIBI, or a fragment thereof that binds to MHC, is administered to a patient, in whom the antigen or fragment is taken up by and processed within an antigen presenting cell, which in turn activates a cytotoxic T cell in the patient.
  • This vaccination method can be carried out using CYPIBI, one or more MHC-binding peptides of CYPIBI, and, in addition to these (or a combination thereof), one or more universal TAAs or one or more MHC-binding peptides of more than one universal TAA, or a combination thereof.
  • the antigen can be administered in combination with an adjuvant to enhance the anti-TAA immune response, or the antigen can be packaged into a delivery system (see below).
  • Any reagent including CYPIBI or a MHC-binding peptide thereof can be used for vaccination.
  • These include, without limitation, full length CYPIBI, MHC-binding fragments of CYPIBI, as well as fusion proteins including CYPIBI and MHC-binding fragments thereof.
  • Peptides or polypeptides including CYPIBI peptides and polypeptides can include 8, 9, 10, 11, 12, or more amino acid stretches having sequence identity with a region of CYPIBI.
  • the peptides can include nine amino acid stretches, in which seven, eight, or all nine of the amino acids in the CYPIBI peptide nine amino acid sequence are identical to a region of nine amino acids in CYPIBI.
  • a CYPIBI peptide or polypeptide can include up to 533 amino acids that are identical to an amino acid sequence found in CYPIBI, for example, 9-20, 20-40, 40-80, 80-200, or 200-533 amino acids that are identical to an amino acid sequence found in CYPIBI.
  • Polypeptides containing CYPIBI peptides can contain additional amino acid stretches that do not correspond to the amino acid sequence of CYPIBI.
  • CYPIBI protein or peptide To vaccinate a patient to elicit a CYP IBl -specific immune response in the patient, it is necessary to obtain large amounts of a CYPIBI protein or peptide, and this can be accomplished by numerous standard methods, for example, chemical synthesis (e.g., Fmoc methods (Sigma Genosys); see above) or expression in eukaryotic or prokaryotic cells.
  • Recombinant CYPIBI peptides can be overexpressed in vivo by introducing coding sequences of the peptides into various types of cells, or in vitro, using cell-free expression systems that are known in the art. The peptide products can then be purified for generating CYP IBl -specific CTLs ex vivo and for vaccine production. Purified CYPIBI peptides are also useful for diagnostic assays that measure the presence of CYP IBl -specific CTLs in a test sample.
  • CYPIBI peptides can be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, IL, or by other methods known to those skilled in the art of peptide synthesis).
  • CYPIBI peptides can be produced in prokaryotic hosts (e.g., E. coli) or in eukaryotic hosts (e.g., S. cerevisiae, insect cells, such as Sf9 cells, or mammalian cells, such as COS-1, NIH 3T3, or HeLa cells). These cells are commercially available from, for example, the American Type Culture Collection, Rockville, Maryland (also see, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998).
  • the method of fransformation and the choice of expression vehicle depends on the host system selected. Transformation and transfection methods are described, e.g., by Ausubel et al, supra, and expression vehicles can be chosen from the numerous examples that are known in this field.
  • a nucleic acid molecule encoding a CYPIBI peptide is introduced into a plasmid or other vector, which is then used to transform living cells.
  • Constructs in which a cDNA containing the entire CYPIBI coding sequence, a fragment of the CYPIBI coding sequence, amino acid variations of the CYPIBI coding sequence, or fusion proteins of the aforementioned, inserted in the correct orientation into an expression plasmid can be used for protein expression.
  • Prokaryotic and eukaryotic expression systems allow various immunogenic domains of CYPIBI peptides or polypeptides to be recovered as fusion proteins, and then used for the generation of CYPIBI -specific CTLs.
  • Typical expression vectors contain promoters that direct the synthesis of large amounts of mRNA corresponding to the inserted CYPIBI peptide-encoding nucleic acid molecule in the plasmid-bearing cells. They can also include eukaryotic or prokaryotic "origin of replication" sequences, which allow for their autonomous replication within the host organism, sequences that encode genetic traits that allow vector-containing cells to be selected in the presence of otherwise toxic drugs (such as antibiotics), and sequences that increase the efficiency with which the synthesized mRNA is translated. Stable, long-term vectors can be maintained as freely replicating entities within cells by using regulatory elements of, for example, viruses (e.g., the OriP sequences from the Epstein Barr Virus genome). Cell lines can also be produced that have the vector integrated into genomic DNA, and, in this manner, the gene product is produced on a continuous basis.
  • viruses e.g., the OriP sequences from the Epstein Barr Virus genome
  • Plasmid vectors in this category contain several elements required for propagation of the plasmid in bacteria and expression of inserted DNA of the plasmid by the plasmid-carrying bacteria. Propagation of only plasmid-bearing bacteria is achieved by introducing into the plasmid selectable marker-encoding sequences that allow plasmid-bearing bacteria to grow in the presence of otherwise toxic drugs (e.g., antibiotics).
  • the plasmid also includes a franscriptional promoter that capable of producing large amounts of mRNA from the cloned gene.
  • the plasmid also, preferably, contains a polylinker to simplify insertion of the gene in the correct orientation within the vector.
  • the expression vector plasmid contains a fragment of the E. coli chromosome containing the lac promoter and the neighboring lacZ gene.
  • RNA polymerase normally transcribes the lacZ gene, producing lacZ mRNA, which is translated into the encoded protein, ⁇ -galactosidase.
  • the lacZ gene can be cut out of the expression vector with restriction endonucleases and replaced by a CYPIBI peptide gene sequence, or a fragment, fusion, or mutant thereof.
  • a CYPIBI peptide gene sequence or a fragment, fusion, or mutant thereof.
  • the appropriate expression vector containing a CYPIBI gene is constructed, it is introduced into an appropriate host cell by fransformation, transfection, or transduction techniques that are known in the art, including calcium chloride transformation, calcium phosphate transfection, D ⁇ A ⁇ -dexfran transfection, electroporation, microinjection, protoplast fusion, and liposome- mediated transfection.
  • the host cells that are fransformed with the vectors of this invention can include (but are not limited to) E. coli or other bacteria, yeast, fungi, insect cells (using, for example, baculoviral vectors for expression), human, mouse, or other animal cells.
  • Mammalian cells can also be used to express CYPIBI peptides using a vaccinia virus expression system, as is described by Ausubel et al, supra.
  • a vaccinia virus expression system as is described by Ausubel et al, supra.
  • In vitro expression of CYPIBI peptides, proteins, fusions, polypeptide fragments, or mutated versions thereof encoded by cloned DNA is also possible using the T7 late promoter expression system.
  • Plasmid vectors containing late promoters and the corresponding RNA polymerases from related bacteriophages such as T3, T5, and SP6 can also be used for in vitro production of proteins from cloned DNA.
  • E. coli can also be used for expression using an M13 phage such as mGPI-2.
  • vectors that contain phage lambda regulatory sequences or vectors that direct the expression of fusion proteins, for example, a maltose- binding protein fusion protein or a glutathione-S-fransferase fusion protein, also can be used for expression in E. coli.
  • Eukaryotic expression systems permit appropriate post-translational modifications to expressed proteins.
  • Transient transfection of a eukaryotic expression plasmid allows the transient production of CYPIBI peptides by a transfected host cell.
  • CYPIBI peptides can also be produced by a stably- transfected mammalian cell line.
  • a number of vectors suitable for stable transfection of mammalian cells are available to the public (e.g., see Pouwels et al, Cloning Vectors: A Laboratory Manual, 1985, Supp. 1987), as are methods for constructing such cell lines (see, e.g., Ausubel et al, supra).
  • cDNA encoding a CYPIBI peptide, protein, fragment, mutant, or fusion protein is cloned into an expression vector that includes the dihydrofolate reductase (DHFR) gene.
  • DHFR dihydrofolate reductase
  • Integration of the plasmid and, therefore, integration of the CYPIBI peptide-encoding gene into the host cell chromosome is selected by inclusion of 0.01-300 ⁇ M methotrexate in the cell culture medium (as is described by Ausubel et al, supra). This dominant selection can be accomplished in most cell types.
  • Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described by Ausubel et al, supra.
  • DHFR-containing expression vectors are pCVSEII-DHFR and pAdD26SV(A) (described by Ausubel et al, supra).
  • the host cells described above or, preferably, a DHFR-deficient CHO cell line e.g., CHO DHFR- cells, ATCC Accession No. CRL 9096
  • a DHFR-deficient CHO cell line e.g., CHO DHFR- cells, ATCC Accession No. CRL 9096
  • Other drug markers can be analogously used.
  • proteins such as those containing CYPIBI peptides
  • expression of proteins, such as those containing CYPIBI peptides, in eukaryotic cells allows the production of large amounts of normal or mutant proteins for isolation and purification, and the use of cells expressing a CYPIBI peptide-containing protein provides a functional assay system for antibodies generated against a CYPIBI peptide of interest.
  • Another preferred eukaryotic expression system is the baculovirus system using, for example, the vector pBacPAK9, which is available from Clontech (Palo Alto, CA). If desired, this system can be used in conjunction with other protein expression techniques, for example, the myc tag approach described by Evan et al. (Mol. Cell Biol. 5:3610-3616, 1985).
  • a recombinant CYPIBI protein Once a recombinant CYPIBI protein is expressed, it can be isolated from the expressing cells by cell lysis followed by protein purification techniques, such as affinity chromatography.
  • an anti-CYPlBl peptide antibody which can be produced by methods that are well-known in the art, can be attached to a column and used to isolate recombinant CYPIBI peptide-containing proteins. Lysis and fractionation of CYPIBI pep tide-harboring cells prior to affinity chromatography can be performed by standard methods (see, e.g., Ausubel et al, supra).
  • the recombinant protein can, if desired, be purified further, e.g., by high performance liquid chromatography (HPLC; e.g., see Fisher, Laboratory Techniques in Biochemistry and Molecular Biology, Work and Burdon, Eds., Elsevier, 1980).
  • HPLC high performance liquid chromatography
  • CYPIBI or a MHC-binding peptide thereof is administered to a patient in association with an adjuvant.
  • a chemical antigen e.g., Freund's incomplete adjuvant; cytoxan; an aluminum compound, such as aluminum hydroxide, aluminum phosphate, or aluminum hydroxyphosphate; liposomes; ISCOMS; microspheres; protein chochleates; vesicles consisting of nonionic surfactants; cationic amphiphilic dispersions in water; oil/water emulsions; muramidyldipeptide (MDP) and its derivatives such as glucosyl muramidyldipeptide (GMDP), threonyl-MDP, murametide and murapalmitin; and QuilA and its subfractions; as well as various other compounds such as monophosphoryl-lipid A (MPLA); gamma-inulin; calcifriol; and loxoribine) can be used.
  • MPLA monophosphoryl-lipid A
  • a biological response modifier which is a soluble mediator that affects induction of an immune response
  • cytokines e.g., IL-2 and GM-CSF
  • chemokines e.g., IL-2 and GM-CSF
  • co-stimulatory molecules e.g., B7, ICAM, class I monoclonal antibodies, stem cell factor, and stimulated T cells
  • bacterial products such as toxins or, preferably, subunits or fragments thereof that have reduced (if any) toxicity, but maintained adjuvant activity.
  • biological modifiers of the death response e.g., apoptosis sensitizers
  • compounds or treatment that increases the susceptibility of the target cell to treatment such as radiation and chemotherapy.
  • increasing expression of CYPIBI in the cell can increase susceptibility of the cell to treatment according to the invention.
  • cellular adjuvants can be used in the immunization methods of the invention.
  • a CYPIBI peptide can be administered to a patient on the surface of an antigen presenting cell, in the context of MHC.
  • professional antigen presenting cells e.g., dendritic cells, CD40-activated B cells, irradiated tumor cells (e.g., in association with GM-CSF), alternative antigen presenting cells, synthetic antigen presenting cells (e.g., lipid mycels and artificial APC-like scaffolds), and fusions of any of the above-listed cells can be used.
  • nucleic acid molecules that encodes such a protein or peptide can be used for vaccination.
  • nucleic acid molecules can be administered as "naked" DNA molecules, present in a plasmid or viral vector, or packaged into a liposome or cell, such as eukaryotic cell, prior to administration.
  • the nucleic acid molecules can be administered to a patient in vivo, or can be used to treat a cell ex vivo (e.g., an antigen presenting cell, such as a dendritic cell or a CD40-activated B cell), which is then administered to the patient.
  • a cell ex vivo e.g., an antigen presenting cell, such as a dendritic cell or a CD40-activated B cell
  • RNA e.g., mRNA
  • Boczkowski et al J. Exp. Med. 184:465-472, 1996; J. Exp. Med. 186:1177-1182, 1997.
  • a gene that encodes a polypeptide that includes CYPIBI or an MHC-binding peptide thereof must be delivered to cells in a form that can be taken up by the cells, in which a sufficient level of protein is expressed to induce an effective immune response.
  • Refroviral, adenoviral, lentiviral, poxviral, and other viral vectors are suited as nucleic acid expression vectors for in vivo delivery, because they show efficient infection and/or integration and expression; see, e.g., Cayouette et al, Hum. Gene Therapy, 8:423-430, 1997; Kido et al, Curr. Eye Res. 15:833-844, 1996; Bloomer et al, J. Virol.
  • any DNA fragment that encodes a polypeptide that contains a CYPIBI peptide can be cloned into a refroviral vector and transcribed via its endogenous promoter, via an exogenous promoter, via a promoter specific for the target cell type of interest, or, in the case of refroviral vectors, via the refroviral long terminal repeat.
  • viral vectors that can be used include adenovirus, adeno- associated virus, poxviruses, such as vaccinia virus or bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus.
  • Gene transfer in vivo can also be achieved by non-viral means.
  • a plasmid vector that encodes a polypeptide that contains a CYPIBI peptide can be injected directly into skeletal muscle or cardiac muscle by previously described methods (e.g., Wolff et al, Science, 247:1465-1468, 1990).
  • Expression vectors injected into skeletal muscle in situ are taken up into muscle cell nuclei and used as templates for expression of their encoded proteins.
  • CYPIBI peptide-encoding genes that are engineered to contain a signal peptide are secreted from CYPIBI peptide-expressing muscle cells, after which they induce an immune response.
  • Refroviral vectors adenoviral vectors, adenovirus-associated viral vectors, or other viral vectors also can be used to deliver genes encoding CYPIBI peptides or polypeptides to cells ex vivo.
  • Numerous vectors useful for this purpose are generally known (see, e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al, BioTechniques 6:608-614, 1988; Tolstoshev et al, Curr. Opin. Biotech. 1 :55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et al, Nucl. Acid Res. and Mol. Biol.
  • Refroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al, N. Engl. J. Med 323:370, 1990; Anderson et al, U.S. Patent No. 5,399,346).
  • Gene fransfer into cells ex vivo can also be achieved by delivery of nonviral vectors, such as expression plasmids, using methods such as calcium phosphate or DEAE dextran transfection, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell.
  • Cells that are to be transduced or transfected ex vivo can be obtained from a patient (e.g., peripheral blood cells, such as B cells or dendritic cells, bone marrow stem cells, or cells from a tumor biopsy) prior to transfection, and re- introduced after transfection.
  • a patient e.g., peripheral blood cells, such as B cells or dendritic cells, bone marrow stem cells, or cells from a tumor biopsy
  • the cells also can be derived from a source other than the patient undergoing gene transfer.
  • CYPIBI peptide expression can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element.
  • CMV human cytomegalovirus
  • SV40 simian virus 40
  • metallothionein promoters e.g., metallothionein promoters
  • enhancers known to preferentially direct gene expression in skeletal muscle cells can be used to direct CYPIBI peptide expression for vaccination in situ.
  • the enhancers used can include, without limitation, those that are characterized as tissue- or cell- specific in their expression.
  • CYPIBI peptides, CYPIBI polypeptides, and CYPIBI nucleic acid molecules can be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Administration can begin before a patient is symptomatic.
  • administration can be parenteral, intravenous, infra-arterial, subcutaneous, intramuscular, infracranial, infraorbital, ophthalmic, intravenfricular, infracapsular, infraspinal, intracisternal, infraperitoneal, infranasal, aerosol, by suppositories, or oral administration.
  • Therapeutic formulations can be in the form of liquid solutions or suspensions; for oral administration, formulations can be in the form of tablets or capsules; and for infranasal formulations, in the form of powders, nasal drops, or aerosols.
  • An adjuvant e.g., as listed above, can be included with the formulation.
  • Methods well known in the art for making formulations are found, for example, in Remington 's Pharmaceutical Sciences, (18 th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA.
  • Formulations for parenteral administration can, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers can be used to confrol the release of the compounds.
  • Other potentially useful parenteral delivery systems for CYPIBI peptides, polypeptides, and CYPIBI nucleic acid molecules include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation can contain excipients, for example, lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or can be oily solutions for administration in the form of nasal drops, or as a gel.
  • excipients for example, lactose
  • aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate
  • any cell that expresses an endogenous or exogenously-infroduced major histocompatibility antigen-encoding gene can be used to present a CYPIBI peptide to generate CYPIBI- specific CTLs in vitro.
  • a peptide-presenting cell expresses an endogenously or exogenously-infroduced CYPIBI polypeptide-encoding gene.
  • CYPIBI endogenous CYPIBI in antigen-presenting cells
  • cytokines such as IL-2
  • IL-2 cytokines
  • the antigen presenting cells are pulsed with CYPIBI or MHC-binding peptide thereof, and the pulsed cells are then used to generate CTLs for administration to a patient.
  • the CTLs used in these methods are obtained from the patient to whom they are to ultimately be administered (i.e., the cells are autologous).
  • donor cells i.e., allogeneic cells
  • a patient may be treated with an ex vivo, CYP IBl -activated CTL and/or an ex vivo, CYP 1 B 1 -pulsed APC (e.g. , a DC or a CD40-activated B cell), and this freatment can be carried out before, during, or after a vaccination approach (see above).
  • each approach (or a combination thereof) can employ multiple peptides of CYPIBI, peptides of other TAAs, or a combination thereof.
  • Patients who have one or more tumors containing CYP IBl -expressing tumor cells and patients who are at risk for developing such tumors can be vaccinated with compositions containing one or more CYPIBI peptides, CYPIBI polypeptides, CYPIBI nucleic acid molecules, cells presenting a CYPIBI peptide, or mixtures thereof (other TAA (e.g., hTERT) polypeptides, peptides, nucleic acid molecules, or APCs can also be included).
  • Subjects to be used as donors of CYP IBl -specific CTLs for fransfer into patients can be similarly vaccinated.
  • Levels of CYPlBl-specific CTLs that result from CYPIBI -specific vaccination of patients or other subjects, or ex vivo generation of CYPIBI specific CTLs can be monitored using well-known methods.
  • the increase is by at least 50%, more preferably, at least 100%, still more preferably, at least 200%, and most preferably, at least 400%.
  • non-antigen-specific immunotherapies e.g., administration of IL-2 or interferon
  • the efficacy of non-antigen-specific immunotherapies against tumors containing CYP IB 1- expressing cells can be monitored using similar approaches.
  • Levels of CYP IBl -specific CTLs can also be assessed in naive subjects who have not received CYPIBI vaccinations or other freatment for the purpose of generating CYP IBl -specific CTLs. Since some types of tumors (e.g., malignant melanoma, renal cell carcinoma, and non-Hodgkin's lymphoma) themselves elicit immune responses in their hosts, an increase in the level of CYP IBl -specific CTLs cells in a patient sample, compared to the level in a reference sample from a normal subject who does not have a tumor, or in a reference sample that was previously obtained from the patient, can indicate the development of a tumor in a patient not known to have a tumor or an increase in tumor burden (e.g., increased tumor size, or the development or increase in metastatic tumors) in a patient known to have a tumor.
  • an increase in tumor burden e.g., increased tumor size, or the development or increase in metastatic tumors
  • CYP IBl -specific CTLs can be measured using standard cytotoxicity assays, such as the Cr 51 release assay (Schultze et al, J. Clin. Invest. 100:2757, 1997), which is described above.
  • Another approach for measuring the level of CYPIB 1 -specific CTLs involves measuring the binding of peptide-specific CTLs to a teframeric peptide/MHC complex in vitro, as is described by Altman et al. (Science 274:94-96, 1996).
  • a fusion protein containing an HLA heavy chain molecule such as HLA-A*0201, plus a peptide that is a subsfrate for biotinylation at the C-terminus of the HLA polypeptide, is produced.
  • the fusion protein is folded in vitro in the presence 2-microglobulin and a CYPIBI peptide ligand.
  • the purified MHC/CYPIBI peptide complexes are then biotinylated at the C-terminus of the HLA heavy chain, and teframers are produced by mixing the biotinylated MHC/CYPIBI peptide complexes with phycoerythrin-labeled deglycosylated avidin at a molar ratio of 4: 1.
  • Samples that contain CTLs are mixed with the CYPIBI peptide/MHC teframeric complexes and the relative amount of CYP IBl -specific CTLs that bind to the CYPIBI peptide/MHC teframeric complexes can be measured for each sample by flow cytometry, using methods described by Altman et al, supra, and by other methods known to those of skill in this art.
  • Another method that can be used is ELISPOT (Herr et al, J. Immunol. Methods 203: 141-152, 1997).
  • Hl-A-A*0201 Nonamers HLA-A*0201 Decamers HLA-A*0201 Octamers HLA-A*0202 Nonamers HLA-A*0202 Decamers
  • Hl-A-A*0203 Octamers HLA-A1 Nonamers HLA-A1 Decamers HLA-A26 Nonamers HLA-A26 Decamers
  • LVALLVRGS 474 ELSKMQLFL 249 YFPNPVRTV 126 VSGGRSMAF 176 ELVALLVRG 469 RCIGEELSK 233 RTVGAGSLV 123 FRVVSGGRS 135 GHYSEHWKV 463 FSVGKRRCI 232 GRTVGAGSL 115 ADRPAFASF 107 LVQQGSAFA 450 GLINKDLTS 213 RYSHDDPEF 112 SAFADRPAF 512 KPKSFKVNV 417 DTVVFVNQW 202 ANVMSAVCF 99 GERAIHQAL 503 MNFSYGLTI 412 YHIPKDTVV 200 AVANVMSAV 93 PIVVLNGER 502 KMNFSYGLT 406 NTSVLGYHI 193 PRPLTVVAV 75 ARLARRYGD 476 SKMQLFLFI 391 FSSFVPVTI 190 FLDPRPLTV 63 AAAVGQAAH 467 KRRCIGEEL 322 ATITDIFGA 181
  • VALLVRGSA 523 RESMELLDS 77 LARRYGDVF 402 ATTANTSVL 162 SRQVLEGHV 510 TIKPKSFKV 62 NAAAVGQAA 377 NLPYVLAFL 150 SMMRNFFTR 480 LFLFISILA 58 PLIGNAAAV 374 DQPNLPYVL 125 VVSGGRSMA 458 SRVMIFSVG 56 AWPLIGNAA 372 MGDQPNLPY 106 ALVQQGSAF 446 LDKDGLINK 32 TVHVGQRLL 367 DRLPCMGDQ 81 YGDVFQIRL 425 WSVNHDPLK 24 LLLLSVLAT 309 HGGGARLDL 63 AAAVGQAAH 407 TSVLGYHIP 530 DSAVQNLQA 263 QLNRNFSNF 28 SVLATVHVG 354 TRVQAELDQ 503 MNFSYGLTI 239 SLVDVMPWL 26 LLSVLATVH 339 LQWLLLLFT 4
  • ELDQVVGRD 6 SPNDPWPLN 149 HSMMRNFFT 30
  • LATVHVGQR 348 RYPDVQTRV 529 LDSAVQNLQ 147
  • AAHSMMRNF 522 LRESMELLD 346
  • FTRYPDVQT 524 ESMELLDSA 140 HWKVQRRAA 518
  • VNVTLRESM 324 ITDIFGASQ 511 IKPKSFKVN 139
  • EHWKVQRRA 503 MNFSYGLTI 303 KAAGDSHGG 507 YGLTIKPKS 111
  • GSAFADRPA 486 ILAHQCDFR 290
  • RDMMDAFIL 494 RANPNEPAK 107 LVQQGSAFA 478
  • AHQCDFRAN 94 IVVLNGERA 475 LSKMQLFLF 263
  • QLNRNFSNF 484 ISILAHQCD 76 RLARRYG
  • ILDKFLRHCE 108 VQQGSAFADR 502 KMNFSYGLTI 273 LDKFLRHC 255 RTVFREFEQL 12 PLNPLSIQQT 501 AKMNFSYGLT 266 RNFSNFIL 239 SLVDVMPWLQ 531 SAVQNLQAKE 494 RANPNEPAKM 262 EQLNRNFS 235 VGAGSLVDVM 530 DSAVQNLQAK 469 RCIGEELSKM 233 RTVGAGSL 231 FGRTVGAGSL 524 E SMELLDSAV 389 MRFSSFVPVT 229 EEFGRTVG
  • ELSKMQLFLF 370 PCMGDQPNLP 363 VVGRDRLPCM 176 ELVALLVR 459 RVMIFSVGKR 352 VQTRVQAELD 347 TRYPDVQTRV 171 LSEARELV 443 ARFLDKDGLI 334 TLSTALQWLL 345 LFTRYPDVQT 157 TRQPRSRQ 442 PARFLDKDGL 309 HGGGARLDLE 337 TALQWLLLLF 154 NFFTRQPR 423 NQWSVNHDPL 295 AFILSAEKKA 329 GASQDTLSTA 142 KVQRRAAH 421 FVNQWSVNHD 262' EQLNRNFSNF 319 NVPATITDI F 141 WKVQRRAA 411 GYHIPKDTVV 252 NPVRTVFREF 295 AFILSAEKKA 127 SGGRSMAF
  • SAPPGPFAWP 380 YVLAFLYEAM 142 KVQRRAAHSM 528 LLDSAVQN 46 QLRSAPPGPF 374 DQPNLPYVLA 116 DRPAFASFRV 509 LTIKPKSF
  • HGGGARLDLE 306 GDSHGGGARL 348 RYPDVQTRVQ 162 SRQVLEGH
  • AVANVMSAVC 180 LLVRGSADG 357 QAELDQVVG 57 WPLIGNAAA 119 AFASFRVVSG 143 VQRRAAHSM 347 TRYPDVQTR 40 LRQRRRQLR 113 AFADRPAFAS 119 AFASFRVVS 315 LDLENVPAT 36 GQRLLRQRR 530 DSAVQNLQAK 113 AFADRPAFA 297 ILSAEKKAA 29 VLATVHVGQ 499 EPAKMNFSYG 88 RLGSCPIVV 272 ILDKFLRHC 23 LLLLLSVLA 493 FRANPNEPAK 71 HLSFARLAR 228 NEEFGRTVG 6 SPNDPWPLN 486 ILAHQCDFRA 26 LLSVLATVH 227 HNEEFGRTV 4 SLSPNDPWP 441 DPARFLDKDG 5 LSPNDPWPL 176 ELVALLVRG 531 SAVQNLQAK 403 TTANTSVLGY 460 VMIFSVGK
  • VLAFLYEAM 33 VHVGQRLLR 365 GRDRLPCMG 108 VQQGSAFAD
  • VAVANVMSAV 337 TALQWLLLLF 194 RPLTWAVAN 314 RLDLENVPAT 192 DPRPLTWAV 259 REFEQLNRNF 181 LVRGSADGAF 252 NPVRTVFREF

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Abstract

L'invention concerne des procédés destinés à réaliser une immunothérapie et un diagnostic du cancer utilisant du cytochrome P450 1B1 et des fragments peptidiques de ce composé.
PCT/US2000/031513 1999-11-15 2000-11-15 Immunotherapie et diagnostic du cancer utilisant du cytochrome p450 1b1 WO2001035810A2 (fr)

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US7241742B2 (en) 2000-10-31 2007-07-10 Mgi Pharma Biologics, Inc. CYP1B1 nucleic acids and methods of use
EP2295578A3 (fr) * 2000-10-31 2011-07-06 Eisai Inc. Acides nucléiques Cyp1b1 et leurs procédés d'utilisation
US8158594B2 (en) * 2000-10-31 2012-04-17 Eisai Inc. CYP1B1 nucleic acids and methods of use
WO2004035769A1 (fr) * 2002-10-14 2004-04-29 Ml Laboratories Plc Immunotherapie amelioree
US20070026396A1 (en) * 2003-01-31 2007-02-01 Gerd Wallukat Peptides directed against antibodies, which cause cold-intolerance, and the use thereof
US8440609B2 (en) * 2003-01-31 2013-05-14 Gerd Wallukat Peptides against autoantibodies causing intolerance to cold and use thereof

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EP1241945A2 (fr) 2002-09-25
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CA2390882A1 (fr) 2001-05-25

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