WO1998055133A1 - Antigene associe a un melanome, epitopes des cellules t et leurs procedes d'utilisation - Google Patents

Antigene associe a un melanome, epitopes des cellules t et leurs procedes d'utilisation Download PDF

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WO1998055133A1
WO1998055133A1 PCT/US1998/011533 US9811533W WO9855133A1 WO 1998055133 A1 WO1998055133 A1 WO 1998055133A1 US 9811533 W US9811533 W US 9811533W WO 9855133 A1 WO9855133 A1 WO 9855133A1
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seq
cell epitope
leu
cell
ala
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PCT/US1998/011533
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English (en)
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Malcolm S. Mitchell
Robert J. Deans
Boris R. Minev
June Kan-Mitchell
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The Regents Of The University Of California
University Of Southern California
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Priority to AU77243/98A priority Critical patent/AU7724398A/en
Publication of WO1998055133A1 publication Critical patent/WO1998055133A1/fr

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • 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
    • 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
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • 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

Definitions

  • the present invention relates generally to tumor biology and cancer therapy and more specifically to a melanoma associated antigen and T cell epitopes of the antigen, as well as to methods of using such compositions to stimulate an immune response against melanoma cells.
  • melanoma is treated using surgical, chemotherapeutic or, in some cases, radiotherapeutic methods, or combinations of these methods.
  • Surgical methods can be curative only when the melanoma is detected early and has not metastasized.
  • radiotherapy when used, generally only is effective when the tumor is localized. In the majority of cases, however, the melanoma has metastasized by the time it has been diagnosed and, therefore, chemotherapy is indicated, sometimes in combination with surgery or radiotherapy.
  • chemotherapy suffers from the disadvantage that it generally is not specific for the melanoma cells, but also kills rapidly dividing normal cells. In fact, toxicity to normal cells generally limits the dose of chemotherapy that a patient can tolerate.
  • the cancer cells can become resistant to the chemotherapeutic agent and, therefore, refractory to the treatment. Thus, the duration of response to chemotherapy, radiotherapy or surgery can be too brief.
  • immunotherapy holds great promise for treating a cancer such as melanoma, particularly because it can be effective against disseminated disease and because it is expected to be specific only for the cancer cells.
  • Efforts at immunotherapy of melanoma have been attempted using crude vaccines being composed, for example, of "killed" melanoma cells isolated either from the patient to be treated or from another patient, lysates of such cells or cell extracts.
  • a potential therapeutic melanoma vaccine designated MELACINE, has been formulated from lysates of melanoma cells obtained from two different patients and has produced some positive results when used to treat patients having substantial disease or minimal residual disease.
  • the present invention provides a substantially purified polypeptide portion of a melanoma associated antigen, MG50, comprising the amino acid sequence shown as SEQ ID NO: 2, and provides substantially purified T cell epitopes, comprising a contiguous amino acid sequence of SEQ ID NO: 2, particularly a contiguous sequence within the sequence shown as amino acids 1187 to 1447 of SEQ ID NO: 2.
  • the invention provides cytotoxic T cell epitopes, comprising 8 to 11 contiguous amino acids of SEQ ID NO: 2, such as the cytotoxic T cell epitopes RPRPEQEPLP (SEQ ID NO: 4), DVTSGNTVY (SEQ ID NO : 5) and VLFCAWGTL (SEQ ID NO : 6), and provides helper T cell epitope comprising 12 to 25 contiguous amino acids of SEQ ID NO: 2.
  • the invention provides MG50 cytotoxic T cell epitopes fused to a signal peptide or a functional portion thereof, which facilitates presentation of the epitope as a complex with an MHC molecule at the surface of an antigen presenting cell.
  • the invention provides MG50 cytotoxic T cell epitopes fused to a signal peptide or a functional portion thereof, which facilitates presentation of the epitope as a complex with an MHC molecule at the surface of an antigen presenting cell.
  • MRYMILGLLALAAVCSARPRPEQEPLP (SEQ ID NO : 21) , which is a cytotoxic T cell epitope fused to a signal peptide.
  • the invention provides a chimeric polypeptide, comprising an MG50 polypeptide encoded by SEQ ID NO: 1 or an MG50 T cell epitope encoded by SEQ ID NO: 1, fused to a second polypeptide, which is not MG50 or an MG50 T cell epitope, wherein the second polypeptide can facilitate detection of the MG50 component, for example, or can render an MG50 T cell epitope immunogenic .
  • the invention also provides antibodies, or antigen binding fragments thereof, that specifically bind the MG50 melanoma associated antigen (SEQ ID NO : 2) or a peptide encoded by SEQ ID NO: 1, for example, an MG50 T cell epitope.
  • an antibody of the invention can specifically bind an MG50 T cell epitope that is fused to a signal peptide or a functional portion thereof or can specifically bind a chimeric polypeptide of the invention.
  • Such antibodies which can be monoclonal antibodies, are useful, for example, to prepare an anti-idiotypic antibody, which specifically binds to the antibody of the invention and provides a mimic of the MG50 antigen used to raise the antibody.
  • the invention also provides a substantially purified nucleic acid molecule having the nucleotide sequence shown as SEQ ID NO: 1 (nucleotides 1 to 6848), including subsequences shown as nucleotides 1 to 5509, nucleotides 1 to 3555, nucleotides 1 to 4336, nucleotides 3555 to 4336, nucleotides 3555 to 5509 and nucleotides 3555 to 6848.
  • the invention further provides a substantially purified nucleic acid molecule encoding an MG50 polypeptide comprising SEQ ID NO: 2.
  • the invention provides nucleic acid molecules encoding MG50 T cell epitopes, such nucleic acid molecules comprising a portion of SEQ ID NO: 1, particularly of nucleotides 1 to 5509 or nucleotides 3555 to 4336 of SEQ ID NO: 1, or comprising a nucleotide sequence encoding a portion of SEQ ID NO: 2, particular amino acids 1187 to 1447 of SEQ ID NO: 2.
  • the invention further provides vectors containing a nucleic acid molecule of the invention, for example, expression vectors or viral vectors, and provides cells containing such vectors.
  • antigen presenting cells which contain and express a nucleic acid molecule of the invention, are provided, such cells which can present an MG50 T cell epitope complexed with an MHC molecule at its surface .
  • the present invention also provides methods of identifying the presence of an MG50 melanoma associated antigen in an individual, for example, by contacting a biological sample obtained from the individual with an antibody that specifically binds an MG50 antigen, wherein specific binding of the antibody to a component of the sample identifies the presence of the MG50 melanoma associated antigen in the individual.
  • the invention provides methods of identifying the presence in an immune response against an MG50 melanoma associated antigen in an individual, by contacting a biological sample obtained from the subject with a peptide encoded by SEQ ID NO: 1 and detecting an immunoeffector function of the sample due to contact with the peptide, thereby identifying the presence of an immune response against an MG50 melanoma associated antigen in the individual.
  • a biological sample obtained from the subject with a peptide encoded by SEQ ID NO: 1 and detecting an immunoeffector function of the sample due to contact with the peptide, thereby identifying the presence of an immune response against an MG50 melanoma associated antigen in the individual.
  • the ability of an MG50 peptide comprising an MG50 T cell epitope to stimulate the proliferation of T cells which are a component of the biological sample, identifies the presence of an immune response in the individual from whom the sample was obtained.
  • the invention also provides methods for producing a population of antigen presenting cells that express an MG50 T cell epitope complexed with an MHC molecule on their surfaces.
  • the antigen presenting cells can be contacted with an MG50 melanoma associated antigen comprising the polypeptide encoded by SEQ ID NO: 1 or an MG50 T cell epitope encoded by SEQ ID NO: 1, which can be fused to a signal peptide or a functional portion thereof, if desired.
  • the antigen presenting cells can be contacted with a nucleic acid molecule encoding an MG50 polypeptide, for example, SEQ ID NO: 1 or a nucleic acid molecule encoding SEQ ID NO: 2, or with a nucleic acid molecule encoding an MG50 T cell epitope, which can be fused to a signal peptide.
  • the invention further provides populations of antigen presenting cells produced by such a method of the invention and provides methods for stimulating T lymphocytes to react specifically against cancer cells expressing an MG50 melanoma associated antigen by contacting the T cells with such antigen presenting cells .
  • the invention also provides methods for treating an individual having a cancer in which the cancer cells express an MG50 melanoma associated antigen.
  • an individual can be treated by administration of antigen presenting cells that express an MG50 T cell epitope complexed with an MHC molecule on their surfaces or can be treated by administration of T lymphocytes that have been stimulated in vi tro to react with cancer cells expressing an MG50 melanoma associated antigen.
  • an individual can be treated by administration of a composition comprising an MG50 melanoma associated antigen, or a nucleic acid molecule encoding such an antigen, for example, an MG50 polypeptide encoded by SEQ ID NO: 1 or an MG50 T cell epitope encoded by SEQ ID NO: 1.
  • the composition can contain an immunostimulatory agent such as an adjuvant, for example, DETOX, or a cytokine, for example, interleukin-2 or interferon- ⁇ , or the immunostimulatory agent can be administered separately.
  • the invention also provides methods of preventing the formation of a cancer due to cancer cells expressing an MG50 melanoma associated antigen.
  • Figures 1A to IE show the nucleotide sequence
  • SEQ ID NO: 1 encoding a portion of the MG50 melanoma associated antigen.
  • the open reading frame is underlined.
  • One potential polyadenylation signal is double underlined and a second is indicated in bold and underlined.
  • Figure 2 shows the amino acid sequence (SEQ ID NO: 2) of the MG50 melanoma associated antigen encoded by the open reading frame shown in Figure 1 (SEQ ID NO: 1) .
  • the present invention provides a substantially purified polypeptide portion of a melanoma associated antigen, MG50, comprising the amino acid sequence shown as SEQ ID NO: 2.
  • the invention provides substantially purified T cell epitopes, comprising a contiguous amino acid sequence of SEQ ID NO: 2 or an amino acid sequence encoded by SEQ ID NO: 1, particularly an amino acid sequence encoded by nucleotides 1 to 5509 of SEQ ID NO: 1, preferably by nucleotides 3555 to 4336 of SEQ ID NO: 1.
  • substantially purified when used in reference to an MG50 polypeptide, means that the polypeptide is relatively free from contaminating lipids, proteins, nucleic acids or other cellular material normally associated with an MG50 polypeptide in a cell. Methods for obtaining a substantially purified MG50 polypeptide of the invention are provided, below.
  • MG50 or a T cell epitope of MG50 is useful for stimulating specific reactivity of immunoeffector cells against cells expressing the MG50 melanoma associated antigen.
  • MG50 is considered a melanoma associated antigen because it is expressed on melanoma cells obtained from different individuals.
  • MG50 is considered a shared tumor antigen because it is expressed on different types of cancer cells, including, for example, melanoma cells, lung cancer cells and rhabdomyosarcoma cells.
  • Various melanoma associated antigens which are shared among melanoma cells in different patients, have been identified.
  • MAGE-1 The product of the MAGE-1 gene, the MZ2-E antigen (hereinafter "MAGE-1"), was the first shared melanoma antigen identified (Treversari et al . , Immunogenetics 35:145- (1991); van der Bruggen et al . , Science 254:1643- (1991)). MAGE-1 was determined to stimulate cytotoxic T cell ("Tc cell”) activity and is HLA-A1 restricted (Traversi et al . , J. Exp . Med. 176:1453-1457 (1992)). Additional related melanoma antigens subsequently were identified and named MAGE-2, MAGE-3 and MAGE-4 based on their homology to MAGE-1. The MAGE antigens do not appear to be expressed in normal tissues .
  • MART-1 is another example of a shared human melanoma antigen (Kawakami et al . , Proc. Natl. Acad. Sci .. USA 91:3515-3519 (1994)). MART-1 is expressed in melanoma cells and, to a lesser extent, melanocytes and retina, and, therefore, appears restricted to cells of melanocyte lineage. Unlike the MAGE antigens, which are HLA-A1 restricted, MART-1 is presented in association with HLA-A2 molecules, which are expressed in about 50% of the population. In comparison, only about 10% of the population express HLA-A1 molecules.
  • MG50 also is a melanoma associated antigen and was cloned from the Ml melanoma cell line by subtractive hybridization; the gene encoding MG50 is present on chromosome 2 (Hutchins et al . , Cancer Res . 51:1418-1425 (1991) ; Weiler, "Molecular Characterization of a Novel Human Melanoma Associated Gene (MG50) , Dissertation submitted to the University of Southern California, December, 1993; Weiler et al . , Genomics 22:243-244 (1994) ; Genome Data Bank, Accession No. : locus D2S448 (G00-252-144) , each of which is incorporated herein by reference) .
  • the MG50 gene which encodes the polypeptide shown as SEQ ID NO: 2, contains cryptic coding sequences located downstream of a polyadenylation signal.
  • a nucleic acid molecule encoding MG50 was cloned and, early in the sequencing of the cDNA, a peptide, CSEQPFPEHTASVQHAD (SEQ ID NO : 3) was prepared based on the MG50 cDNA sequence (see Weiler, supra , 1993; referred to a "pep-50") .
  • This peptide which was suspected of being encoded by a nucleic acid sequence located downstream of the MG50 coding sequence, stimulated proliferation of melanoma specific T cells (Weiler, supra , 1993) .
  • CSEQPFPEHTASVQHAD SEQ ID NO : 3
  • CSEQPFPEHTASVQHAD (SEQ ID NO : 3) is encoded by a cryptic MG50 coding sequence, as is the peptide RPRPEQEPLP (SEQ ID NO: 4), which also stimulates proliferation of melanoma specific T cells.
  • RPRPEQEPLP (SEQ ID NO: 4)
  • CSEQPFPEHTASVQHAD (SEQ ID NO : 3) are not encoded by the same reading frame of SEQ ID NO: 1.
  • RPRPEQEPLP SEQ ID NO: 4
  • CSEQPFPEHTASVQHAD SEQ ID NO: 3
  • T cell epitopes of MG50 can be identified in cryptic coding regions of SEQ ID NO : 1 and can comprise an amino acid sequence encoding by various reading frames of the cryptic coding sequence.
  • RPRPEQEPLP (SEQ ID NO: 4) is a T cell epitope that is recognized by cytotoxic T cells in the context of the Class I MHC molecule HLA-B7. Additional MG50 peptide sequences having the characteristics of T cell epitopes that are recognized by HLA-Al MHC molecules (SEQ ID NO: 5) or by HLA-A2 MHC molecules (SEQ ID NOS : 6-17) also have been identified (see Table 1) . Such MG50 epitopes were identified by homology to consensus HLA-Al or HLA-A2 epitopes (see Kaat et al . , J. Immunol. 152:3904-3912 (1994); Falk and Rotzschko, Sem.
  • the invention provides cytotoxic T cell epitopes, comprising 8 to 11 contiguous amino acids encoded by SEQ ID NO: 1, for example, the cytotoxic T cell epitopes RPRPEQEPLP (SEQ ID NO: 4), DVTSGNTVY (SEQ ID NO: 5) and VLFCAWGTL (SEQ ID NO: 6) .
  • cytotoxic T cell epitope means a T cell epitope that is recognized by and stimulates cytotoxic T cells.
  • helper T cell epitopes comprising 12 to 25 contiguous amino acids encoded by SEQ ID NO: 1.
  • helper T cell epitope means a T cell epitope that is recognized by and stimulates helper T cells.
  • T cell epitope means a peptide that is complexed with an MHC molecule and, when complexed with the MHC molecule, can be bound to a T cell receptor.
  • T cell epitopic fragment is used herein to mean a peptide portion of a protein, which can be formed due to proteolysis of the protein, that has the characteristics of a T cell epitope as defined above. In view of these definitions, it should be recognized that the terms “T cell epitope” and “T cell epitopic fragment” often can be used synonymously.
  • a "T cell epitopic fragment” specifically comprises a peptide sequence that is present in a protein
  • a "T cell epitope” can comprise a peptide containing an amino acid sequence that is the same as or different from the corresponding sequence present in the protein from which the epitope was derived.
  • the term “T cell epitope” broadly encompasses a T cell epitopic fragment.
  • the term “peptide” or “peptide portion, " when used in reference to MG50, means an amino acid sequence of at least two contiguous amino acids of SEQ ID NO: 2 (amino acids 1 to 1497) , particularly of amino acids 1187 to 1447 of SEQ ID NO: 2, and that are unique to MG50.
  • a T cell epitope varies in size based on the MHC molecule that binds the epitope.
  • class I MHC molecules bind peptides containing about 8 to 11 amino acids, generally peptides containing 8 to 10 amino acids and, most often, 9 or 10 amino acids.
  • class II MHC molecules bind peptides containing about 12 to 25 amino acids, generally peptides containing 13 to 18 amino acids.
  • Class I MHC molecules are, or can be, expressed by all nucleated cells, including antigen presenting cells (see below) , and present T cell epitopes to Tc cells.
  • the epitopes presented by class I MHC molecules often are produced by proteolysis of endogenously expressed proteins, including proteins expressed in virally infected cells and in tumor cells. The epitope likely associates with the class I molecule in the endoplasmic reticulum, then the complex is transported to the cell surface.
  • Tc cells which express the CD8 surface antigen ("CD8 + ") and a T cell receptor, then bind the epitope associated with the class I molecule, thereby activating the effector function of the Tc cells (see, generally, Kuby, "Immunology" 3d ed. (W.H. Freeman and Co. , 1997) ) .
  • class II MHC molecules In comparison to class I molecules, which are expressed on by nucleated cells, class II MHC molecules only are expressed by antigen presenting cells (APC's), including B lymphocytes (“B cells”), dendritic cells, mononuclear phagocytic cells, macrophages, including Langerhans cells and, in humans, venular endothelial cells, and present a T cell epitope to helper T cells ("Th cells”), stimulating the Th cells, such stimulation being effective in immunity to tumors (see, generally, Abbas et al . , “Cellular and Molecular Immunology,” 2d ed. (W.B. Saunders Co. 1995); Jones and Mitchell, Trends Biotechnol. 14:349-355 (1996), each of which is incorporated herein by reference; see, also, Kuby, supra,
  • APC's express both class I and class II MHC molecules and, therefore, can activate Tc cells and Th cells .
  • the epitopes that are bound by class II molecules generally are derived by proteolysis of exogenous proteins, which are internalized in the APC by phagocytosis or endocytosis.
  • APC's such as macrophages, can express co-stimulatory B7 molecules, B7-1 (CD80) and B7-2 (CD86) , which are recognized by a cell surface molecule (CD28) that is expressed by certain T cells, including naive T cells, and is involved in activation of the T cells.
  • Th cells Binding of a T cell epitope and B7 molecule by Th cells stimulates activation of two subsets of Th cells, Thl cells, which express interleukin-2 (IL-2), interferon- ⁇ , tumor necrosis factor- ⁇ and tumor necrosis factor- ⁇ and are involved in the cell-mediated immune functions, including activation of Tc cells; and Th2 cells, which secrete IL-4, IL-5, IL-6 and IL-10 and are involved in the activation of B cells (Quan and Mitchell, in "Current Research and
  • MG50 cytotoxic T cell epitopes fused to a signal peptide or a functional portion thereof are provided. Fusion of a signal peptide, for example, to the N-terminus of a cytotoxic MG50 T cell epitope, can facilitate presentation of the epitope as a complex with an MHC molecule at the surface of an antigen presenting cell (see Minev et al . , Cancer Res.
  • the invention provides MRYMILGLLALAAVCSARPRPEOEPLP (SEQ ID NO: 21) and MTNKCLLOIALLLCFSTTALSRPRPEOEPLP (SEQ ID NO: 22), which contain the cytotoxic T cell epitope, RPRPEQEPLP (SEQ ID NO: 4), fused to two different signal peptides (signal peptide is underlined) .
  • Signal peptides are well known in the art and consist generally of three functional portions: a basic N-terminal region of about 1 to 3 positively charged amino acids; a central hydrophobic region of about 8 to 12 hydrophobic amino acids; and a polar C-terminal region of about 5 to 7 amino acids with higher average polarity than the central hydrophobic region.
  • Recognition of a signal peptide by a signal peptidase which is located within the endoplasmic reticulum of a eukaryotic cell, results in cleavage of the signal peptide from the remainder of the molecule, for example, the MG50 T cell epitope .
  • a signal peptide or a functional portion thereof can be based on any naturally occurring signal sequence or can be a non-naturally occurring sequence having the general characteristics of a signal peptide or of a functional portion of the signal peptide.
  • the term "functional portion,” when used in reference to a signal peptide means the basic N-terminal region of about 1 to 3 positively charged amino acids; the central hydrophobic region of about 8 to 12 hydrophobic amino acids; or polar C-terminal region of about 5 to 7 amino acids with higher average polarity than the central hydrophobic region.
  • the substitution, for example, of the central hydrophobic 8 to 12 amino acids of a signal peptide with an MG50 T cell epitope having the appropriate hydrophobicity provides a unique type of T cell epitope having characteristics of an MG50 T cell epitope fused to a signal sequence.
  • Contact of an APC with such an MG50 T cell epitope results in efficient transport of the epitope complexed with an MHC molecule to the surface of an APC.
  • the invention provides a chimeric MG50 polypeptide, comprising an MG50 polypeptide, comprising SEQ ID NO: 2, or a peptide portion of MG50 such as an MG50 T cell epitope encoded by SEQ ID NO: 1, fused to a second peptide or polypeptide, which is not MG50 or a peptide portion of MG50.
  • a chimeric MG50 polypeptide provides certain advantages. For example, where the second polypeptide is an enzyme, such as alkaline phosphatase, horseradish peroxidase or luciferase, detection of the MG50 component of the fusion is facilitated by detecting the presence of the enzyme activity.
  • detectable markers for example, a FLAG epitope
  • a detectable marker for example, a FLAG epitope
  • an MG50 T cell epitope or, if desired, to an MG50 polypeptide for the purpose of detecting the presence of the MG50 or MG50 T cell epitope.
  • Such a detectably labeled chimeric polypeptide is useful, for example, in an immunoassay to identify the presence of anti-MG50 antibodies or of MG50 reactive immunoeffector cells in a biological sample obtained from a subject.
  • a chimeric polypeptide of the invention also can be MG50 or an MG50 T cell epitope fused to a second polypeptide such as glutathione-S-transferase (GST) or the His-6 peptide.
  • GST glutathione-S-transferase
  • Such a chimeric polypeptide can be particularly useful for purifying the MG50 or MG50 T cell epitope.
  • GST readily binds to glutathione, which can be attached to an insoluble matrix, thereby providing a simple affinity chromatography method of purifying a GST-MG50 chimeric polypeptide.
  • the His-6 sequence readily binds to a cation such as nickel ion, thus allowing for purification of a His-6-MG50 chimeric polypeptide.
  • Such chimeric polypeptides also can be used to purify antibodies that specifically bind to an MG50 component of the chimeric polypeptide .
  • a chimeric polypeptide of the invention also can be a peptide portion of the MG50 polypeptide, for example, an MG50 T cell epitope, fused to a carrier protein such as bovine serum albumin, bovine gamma-globulin, human gamma-globulin, keyhole limpet hemocyanin, or ovalbumin.
  • a carrier protein such as bovine serum albumin, bovine gamma-globulin, human gamma-globulin, keyhole limpet hemocyanin, or ovalbumin.
  • Such a chimeric polypeptide can render a haptenic MG50 peptide immunogenic and, therefore, can be useful for inducing an anti-MG50 antibody response, thus providing a means for obtaining anti-MG50 antibodies (see Harlow and Lane, “Antibodies: A laboratory manual” (Cold Spring Harbor Laboratory Press 1988), which is incorporated herein by reference; see, also, Kuby, supra, 1997) .
  • MG50, an MG50 T cell epitope, an MG50 T cell epitope fused to a signal sequence, or a chimeric MG50 polypeptide can be produced by a variety of routine methods, including, as appropriate, biochemical purification, recombinant DNA methods or chemical synthesis.
  • An MG50 polypeptide or MG50 T cell epitope, for example, can be produced by recombinant DNA methods (see, generally, Sambrook et al . , Sambrook et al . , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989), which is incorporated herein by reference) .
  • an MG50 T cell epitope can be produced by cloning a nucleic acid encoding the epitope into an expression vector such as a baculovirus vector, then isolating the expressed epitope from an appropriate insect host cell.
  • an MG50 polypeptide also can be expressed in a mammalian cell, where it can be post-translationally modified in a manner expected for a native MG50 protein. Appropriate expression vectors and host cells are well known in the art (see Kriegler, Gene Transfer and Expression. A
  • An MG50 T cell epitope for example, can be synthesized using well known chemical methods, including, for example, automated solid phase methods. Chemical synthesis of an MG50 T cell epitope can be particularly desirable because the method allows for the introduction of amino acid analogs such a (D) -amino acids into the peptide, if desired. The incorporation of a (D) -amino acid, for example, can increase the stability of the T cell epitope, which can be particularly useful for preparing a vaccine or for preparing a diagnostic kit.
  • Native MG50 protein can be purified from a melanoma cell lysate using, for example, an antibody of the invention.
  • MG50 T cell epitopes can be purified from antigen presenting cells obtained from an individual suffering from a cancer that expresses MG50.
  • Methods for obtaining such T cell epitopes are well known in the art and include immunoaffinity chromatography, gel filtration chromatography or gel electrophoresis (see, for example, Hunt et al . , Science 256:1817-1820 (1992); Chicz et al., Nature 358:764-768 (1992); see, also, Guide to Protein Purification (Academic Press, Inc. 1990) , each of which is incorporated herein by reference .
  • the invention also provides antibodies, or antigen binding fragments thereof, that specifically bind to an MG50 melanoma associated antigen comprising SEQ ID NO: 2, particularly to amino acids 1187 to 1447 of SEQ ID NO: 2, or to a peptide encoded by SEQ ID NO: 1, for example, an MG50 T cell epitope.
  • an antibody of the invention can specifically bind an MG50 T cell epitope that is fused to a signal peptide or a functional portion thereof or can specifically bind a chimeric polypeptide of the invention.
  • Such antibodies which can be monoclonal antibodies, are useful, for example, to prepare an anti-idiotypic antibody, which specifically binds to the antibody of the invention and provides a mimic of the MG50 antigen used to raise the antibody.
  • the antibody also can be labeled with a detectable label, for example, a radionuclide, biotin, or an enzyme, using known methods (see, Harlow and Lane, supra , 1988; Hermanson, "Bioconjugate Techniques” (Academic Press 1996) , which is incorporated herein by reference) .
  • an antibody is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies.
  • An antibody of the invention, or an antigen binding fragment thereof is characterized in that it specifically can bind with an MG50 epitope with an affinity of at least about 1 x 10 5 M "1 and, generally, at least about 1 x 10 6 M "1 .
  • antigen binding fragments of an antibody include, for example, Fab, F(ab') 2 , Fd and Fv fragments that retain specific binding activity for an MG50 epitope.
  • An antibody of the invention can be a either naturally occurring or non-naturally occurring, and can include, for example, a single chain antibody, a chimeric, bifunctional and humanized antibody, as well as an antigen-binding fragment thereof.
  • a non-naturally occurring antibody can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (see Huse et al . , Science 246:1275-1281 (1989), which is incorporated herein by reference) .
  • spleen cells from a mouse immunized with MG50 or a peptide portion thereof can be fused to an appropriate myeloma cell line such as SP/02 myeloma cells to produce hybridoma cells.
  • Cloned hybridoma cell lines can be screened using labeled antigen to identify clones that secrete the desired monoclonal antibodies.
  • Hybridomas expressing, for example, anti-MG50 monoclonal antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibodies, which are useful, for example, for preparing standardized kits containing the antibody.
  • a recombinant phage that expresses, for example, a single chain anti-MG50 antibody provides a monoclonal antibody that can used for preparing a kit.
  • a monoclonal antibody specific for MG50 or a peptide portion of MG50 can be used to prepare anti-idiotypic antibodies, which present an epitope that mimics the epitope recognized by the monoclonal antibody used to prepare the anti-idiotypic antibodies.
  • the anti-idiotypic antibody can be useful for detecting the presence of MG50 reactive T cells in a biological sample obtained from an individual.
  • vaccines containing anti-idiotypic antibodies can have antitumor prophylactic effects and induce the involvement of Th cells (Mitchell, Brit. Med. Bull. 51:631-646 (1995); Quan et al . , J. Clin. Oncol. 15:2103-2110 (1997), each of which is incorporated herein by reference) .
  • the invention also provides a substantially purified nucleic acid molecule having the nucleotide sequence shown as SEQ ID NO: 1 (see, also, Weiler, supra , 1993) .
  • the invention provides subsequences of SEQ ID NO: 1, including nucleotides 1 to 5509, nucleotides 1 to 3555, nucleotides 1 to 4336, nucleotides 3555 to 4336, nucleotides 3555 to 5509 and nucleotides 3555 to 6848.
  • the portion of SEQ ID NO: 1 shown as nucleotides 1 to 5510 also is available at GenBank Accession No. D86983 (submitted by N. Nomura; August 2, 1996).
  • substantially purified when used in reference to a nucleic acid molecule of the invention, means that the nucleic acid molecule is relatively free from contaminating lipids, proteins, nucleic acids or other cellular material normally associated with a nucleic acid molecule in a cell.
  • a substantially purified nucleic acid molecule of the invention can be obtained by chemical synthesis of the nucleotide sequence shown as SEQ ID NO: 1 or by cloning the molecule using, for example, a method of the polymerase chain reaction (PCR) , wherein appropriate primers are selected based on SEQ ID NO: 1.
  • nucleic acid molecules of the invention Due to the degeneracy of the genetic code and in view of the disclosed MG50 amino acid sequence shown in SEQ ID NO: 2, particularly of amino acids 1187 to 1447 of SEQ ID NO : 2, additional nucleic acid molecules of the invention would be well known to those skilled in the art. Such nucleic acid molecules have a nucleotide sequence that is different from the sequence shown as nucleotides 1 to 4488, particularly of nucleotides 3555 to 4336, of SEQ ID NO: 1 but, nevertheless, encode the amino acid sequence shown as amino acids 1 to 1497, particularly amino acids 1187 to 1447, respectively, of SEQ ID NO: 2.
  • the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an MG50 polypeptide comprising SEQ ID NO: 2.
  • the invention provides nucleic acid molecules encoding MG50 T cell epitopes, such nucleic acid molecules comprising a portion of SEQ ID NO: 1 or comprising a nucleotide sequence encoding a portion of SEQ ID NO: 2.
  • a nucleic acid molecule encoding when used in reference to MG50 or to a peptide portion of MG50, including an MG50 T cell epitope, indicates 1) the polynucleotide sequence of one strand of a double stranded DNA molecule comprising the nucleotide sequence that codes for MG50 or a peptide portion of MG50 and can be transcribed into an RNA that encodes MG50 or the peptide, or 2) an RNA molecule, which can be translated into MG50 or a peptide portion thereof.
  • a double stranded DNA molecule also comprises a second polynucleotide strand that is complementary to the coding strand and that the disclosure of a polynucleotide sequence comprising a coding sequence necessarily discloses the complementary polynucleotide sequence.
  • the invention provides polynucleotide sequences, including, for example, polydeoxyribonucleotide or polyribonucleotide sequences that are complementary to the nucleotide sequence shown as SEQ ID NO: 1 or to a nucleic acid molecule encoding MG50, comprising the amino acid sequence shown as SEQ ID NO: 2, or to a peptide portion of SEQ ID NO: 2.
  • polynucleotide is used in its broadest sense to mean two or more nucleotides or nucleotide analogs linked by a covalent bond.
  • the invention also provides nucleotide sequences of SEQ ID NO: 1, particularly of nucleotides 3555 to 4336 of SEQ ID NO: 1, which specifically hybridize to a nucleic acid molecule encoding MG50. It is recognized, for example, that SEQ ID NO: 1 shares regions of homology with nucleic acid molecules encoding mammalian peroxidases and Drosophila peroxidasin.
  • nucleotide sequence of SEQ ID NO: 1 hybridizes under stringent hybridization conditions to a nucleotide sequence encoding MG50, but not to a nucleic acid molecule encoding a mammalian peroxidase, such as those disclosed as GenBank Accession Numbers X15313, X15378, M29907, X14346, L77979, or the like, or to a nucleotide sequence encoding Drosophila peroxidasin, such as that disclosed as GenBank Accession No. U11052.
  • a nucleotide sequence of the invention is useful, for example, as a probe, which can hybridize to a nucleic acid molecule encoding MG50 and allow the identification of the nucleic acid molecule in a sample.
  • a nucleotide sequence of the invention is characterized, in part, in that it is at least nine nucleotides in length, such sequences being particularly useful as primers for PCR, and can be at least fourteen nucleotides in length or, if desired, at least seventeen nucleotides in length, such nucleotide sequences being particularly useful as hybridization probes, although such sequences also can be used for PCR.
  • a nucleotide sequence of the invention comprises at least six nucleotides, preferably at least nine nucleotides, 5' to nucleotide 5509 of SEQ ID NO: 1, where SEQ ID NO: 1 is shown in the conventional manner from the 5 '-terminus ( Figure 1A; upper left) to the 3 '-terminus, most preferably at least nine contained within nucleotides 3555 to 4336 of SEQ ID NO: 1.
  • the invention also provides vectors comprising a nucleic acid molecule of the invention and host cells, which are appropriate for maintaining such vectors.
  • Vectors which can be cloning vectors or expression vectors, are well known in the art and commercially available.
  • An expression vector comprising a nucleic acid molecule of the invention, which can encode, for example, MG50 or a T cell epitope thereof, can be used to express the nucleic acid molecule in a cell.
  • an expression vector contains the elements necessary to achieve, for example, sustained transcription of the nucleic acid molecule, although such elements also can be inherent to the nucleic acid molecule cloned into the vector.
  • an expression vector contains or encodes a promoter sequence, which can provide constitutive or, if desired, inducible expression of a cloned nucleic acid sequence, a poly-A recognition sequence, and a ribosome recognition site, and can contain other regulatory elements such as an enhancer, which can be tissue specific.
  • the vector also contains elements required for replication in a procaryotic or eukaryotic host system or both, as desired.
  • Such vectors which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (Promega, Madison WI ; Stratagene, La Jolla CA; GIBCO/BRL, Gaithersburg MD) or can be constructed by one skilled in the art (see, for example, Meth. Enzymol .. Vol. 185, D.V. Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Cane. Gene Ther. 1:51-64 (1994); Flotte, J. Bioenerg. Biomemb. 25:37-42 (1993); Kirshenbaum et al . , J. Clin. Invest 92:381-387 (1993), which is incorporated herein by reference) .
  • viral vectors such as bacteri
  • a nucleic acid molecule can be introduced into a cell by any of a variety of methods known in the art (see Ausubel et al . , Current Protocols in Molecular Biology. John Wiley and Sons, Baltimore, MD (1994) ; Chang, Somatic Gene Therapy, Chap. 11 (CRC Press, Inc., 1995), each of which is incorporated herein by reference; see, also, Sambrook et al . , supra, 1989) . Such methods include, for example, transfection, lipofection, microinjection, electroporation and infection with recombinant viral vectors or the use of liposomes .
  • nucleic acid molecule by infection with a viral vector is particularly advantageous in that it can efficiently introduce the nucleic acid molecule into a cell ex vivo or in vivo (see, for example, U.S. Patent No. 5,399,346, issued March 21, 1995, which is incorporated herein by reference) .
  • the invention also provides methods for producing a population of antigen presenting cells (APC's), which can express an MG50 T cell epitope complexed with an MHC molecule on their surfaces.
  • APC's are well known in the art and include dendritic cells, mononuclear phagocytic cells, B lymphocytes, Langerhans cells or human venular cells.
  • APC's that contain and express a nucleic acid molecule of the invention are provided. Such a nucleic acid molecule can be introduced into an APC using methods as discussed above.
  • the APC's are contacted with an MG50 melanoma associated antigen encoded by SEQ ID NO: 1 or a peptide portion thereof, particularly a peptide portion encoded by a nucleotide sequence contained within nucleotides 1 to 5509 or within nucleotides 3555 to 4226 of SEQ ID NO: 1, or are contacted with an MG50 T cell epitope encoded by SEQ ID NO: 1, particularly by a sequence within nucleotides 1 to 5509 or within nucleotides 3555 to 4336 of SEQ ID NO: 1, which can be fused to a signal peptide or a functional portion thereof, if desired.
  • the invention also provides populations of APC's that are produced by a method of the invention and express on their cell surfaces an MG50 T cell epitope complexed with an MHC molecule .
  • APC's can be contacted, for example, with an MG50 T cell epitope fused to a signal peptide to produce a population of APC's encompassed within the claimed invention.
  • the MG50 T cell epitope is loaded into the cytosol of T cells using osmotic lysis of pinocytic vesicles. T cells exposed to hypertonic medium take-up the fusion peptides due to the formation of pinocytic vesicles in the medium. The pinocytic vesicles break in the cytosol when the cells are placed in hypotonic culture medium, due to the increased internal osmotic pressure.
  • the signal sequence then facilitates translocation of the MG50 T cell epitope from the cytosol into the endoplasmic reticulum, thereby increasing the efficiency with which the epitope is presented at the cell surface complexed with an MHC molecule.
  • APC's produced by a method of the invention can present an MG50 T cell epitope with a class II molecule or a co-stimulatory B7 molecule to a T cell to activate the T cell.
  • the invention further provides methods for stimulating T lymphocytes to react specifically against cancer cells expressing an MG50 melanoma associated antigen by contacting the T cells with an APC that presents an MG50 T cell epitope complexed with an MHC molecule on its surface. Although such a stimulation can occur in vivo, for example, by administration of the
  • APC's of the invention to an individual, such stimulation of T cells also can be performed in vi tro .
  • the invention provides an isolated population of T cells, which are specifically reactive with cancer cells that express an MG50 melanoma associated antigen.
  • a population of specifically reactive T cells can be obtained by contacting naive APC's and T cells in vi tro with MG50 or an MG50 epitope, then isolating the T cells from the APC's.
  • Such in vi tro methods of producing APC's that express an MG50 T cell epitope complexed with an MHC molecule on its cell surface or of producing T cells specifically reactive with a cell expressing an MG50 melanoma associated antigen are particularly useful because the respected populations of the cells can be expanded such that a large number of the cells can be isolated.
  • the skilled artisan will recognize that the APC's and the T cells can be autologous with respect to each other or can be allogeneic (see, for example, Mitchell, supra, 1995) .
  • the invention also provides methods for treating an individual having a cancer containing cancer cells that express an MG50 melanoma associated antigen. It is recognized that the methods of the invention can be curative in some cases. However, a method of the invention also can be useful where it is palliative and, therefore, increases the quality of life of an individual. In particular, the artisan skilled in cancer therapy will recognize that a method of the invention can be particularly useful in combination with conventional cancer therapeutic modalities, including surgery, radiotherapy and chemotherapy.
  • An individual having a cancer containing cancer cells that express MG50 can be treated, for example, by administration of APC's that express an MG50 T cell epitope complexed with an MHC molecule on their surfaces. Administration of such APC's can stimulate an active immune response in the subject by presenting MG50 T cell epitopes to the individual's T lymphocytes.
  • the individual can be treated by administration of
  • T lymphocytes that have been stimulated in vi tro to react with cancer cells expressing an MG50 melanoma associated antigen, thus providing a means of passive immunization of the individual .
  • the present invention also provides methods of treating an individual having a cancer containing cancer cells expressing an MG50 melanoma associated antigen by administering an MG50 vaccine to the individual.
  • the term "vaccine,” when used in reference to the present invention means a formulation that is suitable for administration to a mammal, particularly a human, and contains an MG50 component selected from 1) an MG50 polypeptide, comprising SEQ ID NO: 2; 2) an MG50 T cell epitope encoded by SEQ ID NO : 1, which can be fused to a signal peptide, if desired; 3) an anti-idiotypic antibody of the invention, which is a mimic of an MG50 epitope; or 4) a nucleic acid molecule encoding an MG50 polypeptide or MG50 T cell epitope.
  • a vaccine of the invention generally contains a pharmaceutically acceptable carrier, for example, an aqueous solution such as physiologically buffered saline or other solvent or vehicle such as a glycol, glycerol, oil such as olive oil or injectable organic ester.
  • a pharmaceutically acceptable carrier also can include a physiologically acceptable compound that acts, for example, to stabilize the MG50 component of the formulation or to increase the absorption of the MG50 component.
  • Physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • a vaccine of the invention can contain an immunostimulatory agent such as an adjuvant, for example, DETOX (Ribi Immunochem) , alum or Freund's complete or incomplete adjuvant.
  • a vaccine can contain an immunostimulatory agent such as a cytokine, for example, interleukin-2 (IL-2), IL-4, IL-7, IL-12, IL-15, interferon- (Ifn- ⁇ ), Ifn- ⁇ , granulocyte- macrophage colony stimulating factor (R&D Systems, Inc.; Minneapolis MN) , an accessory molecule such as ICAM-1 or B7, or an agent from plants such as QS-21 (see Mitchell, supra, 1995; Jones & Mitchell, supra, 1996), although such agents also can be added separately from the vaccine, if desired.
  • a vaccine that includes about 10 ⁇ g to about 10 mg of MG50 or an MG50 T cell epitope can be administered in conjunction with about 2.5 million units/meter 2 to about 20 million units/meter 2 of interferon- ⁇ .
  • a combined modality can be administered about three to five times per week and can be continued for up to about two years.
  • An MG50 vaccine can be administered for preventive purposes or for therapeutic purposes.
  • the vaccine can be administered for preventive purposes, for example, to minimize the likelihood that a cancer such as melanoma, which expresses MG50, will occur in those individuals that are at high risk for the disease.
  • Such individual include, for example, those suffering from familial dysplastic nevus syndrome or from atypical Spitz nevi , those individuals having a large number of moles or having an irregularly shaped mole, or those individuals living in high incidence regions such as Australia, Hawaii or the southeastern United States.
  • an MG50 vaccine can be administered for therapeutic purposes to an individual suffering from a cancer that contains cancer cells expressing MG50 and can prevent the further growth or spread of the cancer or induce regression of the cancer.
  • Such cancers can be, for example, melanoma, lung cancer or rhabdomyosarcoma and other cancers expressing MG50 can be identified using methods as disclosed herein.
  • An MG50 vaccine can be administered in a manner similar to other vaccines, for example, subcutaneously, orally, intradermally, intramuscularly or intravenously.
  • it can be advantageous to administer one or more booster vaccinations.
  • the need to administer a booster vaccination and the timing of such vaccinations can be determined experimentally by measuring, for example, the presence or proliferation of MG50 reactive Tc cells in the individual .
  • the MG50 component of a vaccine is administered to the individual in an amount that is sufficient to stimulate an immune response, particularly a cellular immune response.
  • an amount will vary, for example, depending on whether the MG50 component is an MG50 polypeptide or an MG50 T cell epitope or a nucleic acid molecule encoding the MG50 component.
  • the amount will vary, for example, depending on whether stimulation of the immune response is in vivo or in vi tro; whether the administration is a first administration or a booster administration; whether an immunostimulatory agent such as an adjuvant is administered; and, when administered in vivo, on the route of administration.
  • an MG50 polypeptide or MG50 T cell epitope is administered per immunization.
  • Methods for determining a sufficient amount of an MG50 component is required to stimulate an immune response are well within the means of the skilled artisan and generally are determined in Phase I and Phase II clinical trials (see, for example, Powell and Newman, Vaccine Design: The subunit and adjuvant approach (Plenum Publ . Corp.; 1994), which is incorporated herein by reference) .
  • the nucleic acid molecule can be contained in a vector (see Goeddel, supra , 1990) .
  • a nucleic acid molecule can be inserted into such a vector using known methods (see, Sambrook, supra , 1989) .
  • vectors, including expression vectors are available and contain, for example, a promoter such as the cytomegalovirus or SV40 promoter, which can direct expression of MG50 or an MG50 T cell epitope in a cell (see, Gacesa and Ramji, Vectors, Essential Data.
  • Viral vectors based, for example, on a retrovirus, an adenovirus, an adeno-associated virus, a vaccinia virus, or the like, are particularly useful (see, for example, Anderson et al., U.S. Patent No. 5,399,347, issued March 21, 1995; Lee et al . , U.S. Patent No. 5,532,220, issued July 2, 1996; Collins et al . , U.S. Patent No. 5,240,846, issued August 31, 1993; and Ram et al . , Cancer Res. 53:83-88 (1993); Karlsson et al .
  • a nucleic acid molecule of the invention can be admininstered using a liposome.
  • Methods of making a liposome containing a nucleic acid molecule are known in the art (see, Nabel et al., Proc. Natl. Acad . Sci. USA. 90:11307-11311 (1993), which is incorporated herein by reference; and Nabel et al . , supra, 1990) .
  • Such liposomes can be made target specific by incorporating, for example, lipid-conjugated antibodies into the structure of the liposome (see, Holmberg et al . , J. Liposome Res. , 1:393-406 (1990), which is incorporated herein by reference) or by incorporating a ligand or a receptor, that is bound by a corresponding receptor or ligand, respectively, that is expressed on the target cell.
  • the present invention also provides methods of identifying the presence of an MG50 melanoma associated antigen in an individual .
  • a method can be performed, for example, by contacting a biological sample obtained from the individual with an antibody that specifically binds an MG50 epitope, wherein specific binding of the antibody to a component of the sample identifies the presence of the MG50 melanoma associated antigen in the individual.
  • a biological sample can be, for example, a tissue or tumor sample, which can be obtained by a biopsy procedure from an individual suspected of having a cancer in which the cancer cells express an MG50 melanoma associated antigen.
  • the invention provides methods of identifying the presence in an immune response against an MG50 melanoma associated antigen in an individual.
  • a method can be performed, for example, by contacting a biological sample obtained from the subject with a peptide comprising at least six contiguous amino acids, generally at least 8 contiguous amino acids, encoded by SEQ ID NO: 1 and detecting an immunoeffector function of the sample due to contact with the peptide, thereby identifying the presence of an immune response against an MG50 melanoma associated antigen in the individual.
  • An immunoeffector function can be, for example, the presence of anti-MG50 antibodies in the biological sample or the presence of MG50 reactive T cells in the sample.
  • a biological sample can be, for example, a blood sample or a lymph tissue sample.
  • the peptide can be an MG50 T cell epitope and the ability of epitope to stimulate the proliferation of T cells, which are a component of the biological sample, identifies the presence of MG50 reactive T cells in the sample and, therefore, the presence of an immune response against MG50 in the individual from whom the sample was obtained.
  • This example describes methods for obtaining a nucleic acid molecule encoding the MG50 melanoma associated antigen.
  • Subtractive hybridization of cDNA obtained from melanoma cell line MSM M-l ("Ml") against an excess of mRNA from a squamous lung carcinoma cell line Lu-1 was used to clone cDNA sequences differentially expressed in the Ml melanoma cells (Hutchins et al . , Cancer Research 51:1418-1425 (1991), which is incorporated herein by reference) . Twelve candidate differentially expressed clones were obtained, six of which were considered novel based on a lack of sequence homology to sequence in the GenBank database (Hutchins et al . , supra , 1991) . One of these six clones, designated "melanoma gene-50" (“MG50”) was selected for further characterization.
  • MSM M-l MSM M-l
  • Lu-1 squamous lung carcinoma cell line
  • MG50 is encoded by an mRNA of about 8.1 kilobases (kb) .
  • MG50 mRNA was detected in melanoma cells, lung carcinoma cells, rhabdomyosarcoma cells, fetal brain, fetal heart and human placenta.
  • Figure 1 shows a 6848 nucleotide portion of the cDNA encoding MG50 (SEQ ID NO: 1) .
  • Nucleotides 5510 to 6848 of SEQ ID NO: 1) were reported previously (Weiler, supra , 1993) However, efforts to continue sequencing the cDNA in the 5' direction largely were unsuccessful.
  • primers were made based on the 5' end of the portion of the sequence described by Weiler ( supra, 1993) and used to obtain more 5' sequences, which then were sequenced and used to search the Merck EST database.
  • MG50 Based on the 8.1 kb mRNA for MG50, it is estimated that approximately 1300 nucleotides remain to be sequenced to obtain the full length MG50 cDNA sequence. At least some of the 1300 nucleotides are expected to be 5' to the sequence shown in SEQ ID NO: 1, since an ATG initiation codon has not yet been identified.
  • the remaining MG50 cDNA sequences can be obtained, for example, using a PCR method such as a RACE method.
  • a deduced amino acid sequence encoded by nucleotides 1 to 4488 of the MG50 cDNA is shown in Figure 2 (SEQ ID NO: 2) .
  • the 1496 amino acid polypeptide shares homology to Drosophila peroxidasin and to products of the human peroxidase gene family.
  • the 1496 amino acid MG50 polypeptide (SEQ ID NO: 2) is shown because the first stop codon encoded by SEQ ID NO: 1 occurs at nucleotides 4489 to 4491. However, if additional amino acids are deduced beyond this stop codon, cryptic coding sequences are revealed.
  • the sequence is considered to be a cryptic coding region because the peptide RPRPEQEPLP (SEQ ID NO: 4), which is encoded by nucleotides 5410 to 5439 of SEQ ID NO : 1 has the characteristics of an MG50 T cell epitope. Specifically, the peptide of SEQ ID NO: 4 binds more strongly to HLA-B7 than any other epitope tested in a competitive binding assay and stimulates proliferation of CD8 + T cells that were specifically reactive with melanoma cells expressing MG50 (see Example II) .
  • the coding sequence of SEQ ID NO: 4 is downstream of nine stop codons, including the stop codon at nucleotides 4489 to 4491. While the presence of this T cell epitope (SEQ ID NO: 4) in the cryptic region of SEQ ID NO: 1 may be fortuitous, another possibility is that the cryptic coding region, or a portion of this region, is translated in melanoma cells.
  • This example provides peptides that are encoded by SEQ ID NO: 1 and have characteristics of MHC class I restricted T cell epitopes.
  • the peptide RPRPEQEPLP (SEQ ID NO: 4) , which is encoded by nucleotides 5410 to 5439 of SEQ ID NO : 1, in the cryptic region, stimulated proliferation of CD8 + T cells that were specifically reactive with melanoma cells expressing MG50 (see Example II) .
  • T2 cells were transduced to express HLA-B7, then incubated with the peptide and CD8 + T cells (Tc cells) , which were obtained from a patient having a melanoma that expressed MG50.
  • Tc cells CD8 + T cells
  • CD8 T cells were generated against the RPRPEQEPLP (SEQ ID NO : 4) peptide in vi tro, then reacted against RPRPEQEPLP (SEQ ID NO : 4) pulsed Cos- 7 cells, which were transduced to express HLA-B7.
  • the Cos-7 cells were lysed, demonstrating that the Tc cells recognized the MG50 T cell epitope in the context of HLA-B7 and were specifically reactive for the peptide.
  • T cell epitopes encoded within the open reading frame of SEQ ID NO: 1 were identified by homology to consensus HLA-Al and HLA-A2 epitope sequences (see Kaat et al . , supra , 1994; Falk and Rotzschko, supra , 1993) .
  • SEQ ID NO: 5 a peptide having that characteristics expected of an HLA-Al epitope
  • SEQ ID NOS : 6-17 12 peptides having characteristics of an HLA-A2 epitope
  • Cos-7 cells can be cotransduced with a cDNA encoding HLA-Al or HLA-A2 , as appropriate, and with a nucleic acid molecule encoding a potential T cell epitope.
  • the cotransduced Cos-7 cells then can be incubated with Tc cells that are specifically reactive with melanoma cells expressing MG50 and MG50 T cell epitopes can be identified by detecting lysis of the Cos-7 cells.
  • NAME Campbell, Cathryn A.
  • AAA ACC TAC GCG GAG TCG GGG AAC GCG CAG GCA GCG GCC ATC TGT GAA 720
  • CAC CGC GCC AAC GAG CAG CTG GGC CTG ACC AGC ATG CAC ACG CTG TGG 3024
  • CAAGGAATTG TGTTGTCTAA NCCAAAGGGG GAAGCTGACT GTGTATTACC AAAAAAAATT 6549 CTGTAATNCA AACCNAAATG TCTGCGGAAT CACCAGTTTG ATACTCTCTG TAATCAGAGC 6609

Abstract

La présente invention décrit une partie d'un polypeptide sensiblement purifiée appartenant à un antigène associé à un mélanome, le MG50 (SEQ ID NO: 2) ainsi que des épitopes de cellules T sensiblement purifiés de MG50. L'invention décrit un épitope de cellule T cytotoxique dont la séquence d'acides aminés est: RPRPEQEPLP (SEQ ID NO: 4) et un épitope de cellule T auxiliaire dont la séquence d'acides aminés est: CSEQPFPEHTASVQHAD (SEQ ID NO: 3). L'invention décrit également des anticorps qui se lient spécifiquement au MG50 ou à un épitope de cellule T de MG50 ainsi que des fragments de ces anticorps se liant aux antigènes, une molécule d'acide nucléique sensiblement purifiée (SEQ ID NO: 1) codant une partie d'un antigène associé à un mélanome, le MG50, des molécules d'acide nucléique codant les épitopes de cellules T de MG50, des vecteurs comprenant de telles molécules d'acide nucléique et des cellules comprenant de tels vecteurs, par exemple, des cellules représentant l'antigène et renfermant une molécule d'acide nucléique de l'invention. Par ailleurs, cette invention décrit des procédés permettant d'identifier un antigène associé à un mélanome de MG50 chez un sujet et des procédés permettant d'identifier une réaction immunitaire contre l'antigène associé au mélanome chez le sujet en question. De plus, l'invention décrit des procédés de stimulation des lymphocytes T réactifs contre les cellules cancéreuses exprimant un antigène de MG50 associé à un mélanome, ainsi que des procédés de traitement d'un sujet ayant des cellules cancéreuses exprimant un antigène de MG50 associé à un mélanome.
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CN114920822A (zh) * 2021-02-02 2022-08-19 复旦大学附属中山医院 一种识别C7orf50突变体抗原短肽的T细胞受体及其应用
CN114920822B (zh) * 2021-02-02 2023-06-30 复旦大学附属中山医院 一种识别C7orf50突变体抗原短肽的T细胞受体及其应用

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