BACKGROUND OF THE INVENTION FIELD OF THE INVENTION 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.

BACKGROUND INFORMATION The incidence of malignant melanoma has been increasing rapidly. Although ultraviolet radiation is believed to be the primary cause of melanoma, familial occurrence of the disease indicates that hereditary factors also may be involved. Unfortunately, methods for successfully treating melanoma have not kept pace with the increasing incidence.

In general, melanoma, like other cancers, is treated using surgical, chemotherapeutic or, in some cases, radiotherapeutic methods, or combinations of these methods. Surgical methods, however, can be curative only when the melanoma is detected early and has not metastasized. Similarly, 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. However, 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. In addition, 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.

In theory, 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. For example, 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. However, use of crude melanoma vaccines in immunotherapy is problematic, for example, because the precise antigenic composition of such vaccines is largely undefined. It is generally believed that more effective immunotherapy requires the identification and isolation of proteins that are expressed relatively specifically by melanoma cells, preferably on their surface, but are not expressed on normal cells. Thus, a need exists to identify melanoma associated antigens. The present invention satisfies this need and provides additional advantages.

SUMMARY OF THE INVENTION 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. For example, 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.

In one embodiment, 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.

For example, the invention provides MRYMILGLLALAAVCSARPRPEQEPLP (SEQ ID NO: 21), which is a cytotoxic T cell epitope fused to a signal peptide. In another embodiment, 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. If desired, 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. In addition, 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. In an embodiment of the invention, 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 MMC 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. Conversely, 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. For example, 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. For example, 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. In addition, 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.

Accordingly, 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.

For example, such 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 vitro to react with cancer cells expressing an MG50 melanoma associated antigen. In addition, 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. If desired, 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.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A to 1E 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).

DETAILED DESCRIPTION OF THE INVENTION 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. In addition, 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. As used herein, the term "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.

As disclosed herein, 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. In addition, 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. 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. Sxp. 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 M1 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). As disclosed herein, 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). As disclosed herein, 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. Remarkably, RPRPEQEPLP (SEQ ID NO: 4) and CSEQPFPEHTASVQHAD (SEQ ID NO: 3) are not encoded by the same reading frame of SEQ ID NO: 1.

Specifically, while RPRPEQEPLP (SEQ ID NO: 4) is encoded by the same reading frame as the remainder of the MG50 polypeptide, although it is downstream of a potential polyadenylation signal, CSEQPFPEHTASVQHAD (SEQ ID NO: 3) is out of frame with respect to the MG50 coding sequence.

Thus, 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-A1 MMC 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-A1 or HLA-A2

epitopes (see Kaat et al., J. Immunol. 152:3904-3912 (1994); Falk and Rotzschko, Sem. Immunol. 5:81-94 (1993), each of which is incorporated herein by reference). The availability of MG50 T cell epitopes that are presented in the context of HLA-A2 MHC molecules provides that advantage that HLA-A2 molecules are expressed by a relatively large number of individuals as compared to HLA-B7.

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). As used herein, the term "cytotoxic T cell epitope" means a T cell epitope that is recognized by and stimulates cytotoxic T cells. Also provided are helper T cell epitopes, comprising 12 to 25 contiguous amino acids encoded by SEQ ID NO: 1. As used herein, the term "helper T cell epitope" means a T cell epitope that is recognized by and stimulates helper T cells.

As used herein, the term "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. In addition, the term "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. However, while 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. Thus, the term "T cell epitope" broadly encompasses a T cell epitopic fragment. In addition, 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. Specifically, 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. In comparison, 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)).

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, 1997). Thus, 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. In addition, 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. 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-y, tumor necrosis factor- and tumor necrosis factor-a 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 Clinical Management of Melanoma" (Kluwer Academic Publ.

1993), which is incorporated herein by reference; see pages 257-277; see, also, Kuby, supra, 1997; chaps. 1, 10 and 12; Jones and Mitchell, supra, 1996).

In an embodiment of the invention, 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. 54:4155-4161 (1994), which is incorporated herein by reference). For example, the invention provides MRYMILGLLALAAVCSARPRPEQEPLP (SEQ ID NO: 21) and MTNKCLLOIALLLCFSTTALSRPRPEQEPLP (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.

For use in the present invention, 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. As used herein, 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. As disclosed herein, 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.

In another embodiment, 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. Other such detectable markers, for example, a FLAG epitope, also can be fused to 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. Such a chimeric polypeptide can be particularly useful for purifying the MG50 or MG50 T cell epitope. For example, 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. Similarly, 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. 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). For example, 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. In addition, 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 Laboratory Manual, W.H. Freeman and Co., 1991, which is incorporated herein by reference) and are commercially available.

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. In addition, 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, Deutscher, 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. If desired, 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).

As used herein, the term "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 <BR> <BR> <BR> affinity of at least about 1 x 105 M1 and, generally, at least about 1 x 106 M-1. Such antigen binding fragments of an antibody include, for example, Fab, F(ab')21 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). These and other methods of making, for example, a chimeric, humanized, CDR-grafted, single chain, or bifunctional antibody are well known to those skilled in the art (Hoogenboom et al., U.S. Patent No.

5,564,332, issued October 15, 1996; Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

Methods for raising polyclonal antibodies, for example, in a rabbit, goat, mouse or other mammal, are well known in the art. In addition, monoclonal antibodies can be obtained using methods that are well known and routine in the art (Harlow and Lane, supra, 1988). Essentially, 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.

Similarly, 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. Where the epitope to which the monoclonal antibody includes, for example, an MG50 T cell epitope, the anti-idiotypic antibody can be useful for detecting the presence of MG50 reactive T cells in a biological sample obtained from an individual. In addition, 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). In particular, 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). As used herein, the term "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.

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. Thus, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an MG50 polypeptide comprising SEQ ID NO: 2. In addition, 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.

As used herein, the term "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.

It is recognized that 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. Accordingly, 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. As used herein, the term "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. Thus, a nucleotide sequence of SEQ ID NO: 1, which is considered to be within the claimed invention, 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. In addition, 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 lA; 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.

In general, 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. In particular, 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, Canc. Gene Ther. 1:51-64 (1994); Flotte, J.

Bioenera. Biomemb. 25:37-42 (1993); Kirshenbaum et al., J. Clin. Invest 92:381-387 (1993), which is incorporated herein by reference).

A nucleic acid molecule, either alone or contained a vector, 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. Introduction of a 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. In one embodiment of the invention, 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. In another embodiment, 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. Accordingly, 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. Thus, 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 vitro. Accordingly, the invention provides an isolated population of T cells, which are specifically reactive with cancer cells that express an MG50 melanoma associated antigen. In addition, it should be recognized that such a population of specifically reactive T cells can be obtained by contacting naive APC's and T cells in vitro with MG50 or an MG50 epitope, then isolating the T cells from the APC's. Such in vitro 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. Furthermore, 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. In addition, the individual can be treated by administration of T lymphocytes that have been stimulated in vitro 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. As used herein, 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.

If desired, 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. In addition, 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-a (Ifn-a), Ifn-y, 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. Other such agents include vectors that are rendered nonpathogenic, for example, by attenuation, liposomes and cell-sized microspheres (see Jones and Mitchell, supra, 1996). The skilled artisan will know how to formulate a vaccine of the invention using methods routine in the art. For example, a vaccine that includes about 10 yg to about 10 mg of MG50 or an MG50 T cell epitope can be administered in conjunction with about 2.5 million units/meter2 to about 20 million units/meter2 of interferon-a. Such 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 southwestern United States. In addition, 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.

In addition, following a first administration of the vaccine, 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. Such 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. In addition, the amount will vary, for example, depending on whether stimulation of the immune response is in vivo or in vitro; 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. In general, about 10 Ug to about 10 mg of 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 Desian: The subunit and adjuvant approach (Plenum Publ. Corp.; 1994), which is incorporated herein by reference).

Where adminstration is of a nucleic acid molecule encoding MG50 or an MG50 T cell epitope, 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). A variety of 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, John Wiley and Sons, NY (1994), which is incorporated herein by reference). 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., EMBO J. 5:2377-2385 (1986); Kleinerman et al., Cancer Res. 55:2831-2836 (1995); Hamada et al., Gynecol. Oncol., 63:219-227 (1996); Nabel et al., Science, 249:1285-1288 (1990); and Berkner, BioTechniques 6:616-629 (1989), each of which is incorporated herein by reference).

In addition to a viral vector, 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. Such 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. Such 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.

In addition, the invention provides methods of identifying the presence in an immune response against an MG50 melanoma associated antigen in an individual. Such 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. For example, 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.

The following examples are provided to illustrate embodiments of the invention.

EXAMPLE I NUCLEIC ACID MOLECULE ENCODING MG50 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 ("M1") against an excess of mRNA from a squamous lung carcinoma cell line Lu-l was used to clone cDNA sequences differentially expressed in the M1 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.

Based on northern blot analysis using the MG50 cDNA as a probe, 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. As disclosed herein, 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. Overlapping EST sequences were identified and used to extent the sequence of MG50 to nucleotide 4336 of SEQ ID NO: 1 (i.e., nucleotides 43366 to 5509).

Additional 5' sequences were determined by anchored PCR, RACE and DNA sequencing to nucleotide 3555 of SEQ ID NO: 1. Recently, the portion SEQ ID NO: 1 shown as nucleotides 1 to 5510 was submitted to GenBank as Accession No. D86983 and, therefore, nucleotides 1 to

3554 were added to produce the sequence shown as SEQ ID NO: 1 (Figures 1A to 1E).

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. Although additional stop codons are interspersed throughout the cryptic coding region, 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).

Remarkably, 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.

EXAMPLE II POTENTIAL MG50 T CELL EPITOPES 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). In these experiments, 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. Proliferation of the T cells indicated that the RPRPEQEPLP (SEQ ID NO: 4) peptide acts as an HLA-B7 restricted Tc cell epitope.

In other experiments, CD8 T cells were generated against the RPRPEQEPLP (SEQ ID NO: 4) peptide in vitro, 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.

Potential T cell epitopes encoded within the open reading frame of SEQ ID NO: 1 (i.e., present within SEQ ID NO: 2) were identified by homology to consensus HLA-A1 and HLA-A2 epitope sequences (see Kaat et al.,

supra, 1994; Falk and Rotzschko, supra, 1993). As shown in Table 1, a peptide having that characteristics expected of an HLA-A1 epitope (SEQ ID NO: 5) was identified and 12 peptides having characteristics of an HLA-A2 epitope (SEQ ID NOS: 6-17) were identified.

TABLE 1 Amino Acid Sequence *Amino Acid Position SEO ID NO: CSEQPFPEHTASVQHAD ** 3 RPRPEQEPLP 1801-1810 4 DVTSGNTVY 273-281 5 VLFCAWGTL 34-42 6 CMHLLLEAV 66-74 7 LLLEAVPAV 69-77 8 TLHCDCEIL 210-218 9 VLSVNVPDV 625-633 10 DLDSTVVAL 845-853 11 WLPKILGEV 1051-1059 12 PLLRGLFGV 1133-1141 13 RLGPTLMCL 1244-1252 14 LLSTQFKRL 1252-1260 15 EMQKTITDL 1408-1416 16 DLRTQIKKL 1415-1423 17 * - amino acid position with respect to SEQ ID NO: 2.

** - not encoded by same reading frame as SEQ ID NO: 2.

The ability of the peptides shown in Table 1, or other potential T cell epitope encoded by SEQ ID NO: 1, to act as an MG50 T cell epitope can be determined using a T cell proliferation assay as described above.

In addition, Cos-7 cells can be cotransduced with a cDNA

encoding HLA-A1 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.

Although the invention has been described with reference to the example provided above, it should be understood that various changes can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: The Regents of the University of California The University of Southern California (ii) TITLE OF INVENTION: A Melanoma Associated Antigen, T Cell Epitopes Thereof and Methods of Using Same (iii) NUMBER OF SEQUENCES: 26 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Campbell & Flores LLP (B) STREET: 4370 La Jolla Village Drive, Suite 700 (C) CITY: San Diego (D) STATE: California (E) COUNTRY: United States (F) ZIP: 92122 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/870,941 (B) FILING DATE: 06-JUN-1997 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Campbell, Cathryn A.

(B) REGISTRATION NUMBER: 31,815 (C) REFERENCE/DOCKET NUMBER: FP-UD 3175 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (619) 535-9001 (B) TELEFAX: (619) 535-8949 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6847 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..4489 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AGC CGG CCG TGG TGG CTC CGT GCG TCC GAG CGT CCG TCC GCG CCG TCG 48 Ser Arg Pro Trp Trp Leu Arg Ala Ser Glu Arg Pro Ser Ala Pro Ser 1 5 10 15 GCC ATG GCC AAG CGC TCC AGG GGC CCC GGG CGC CGC TGC CTG TTG GCG 96 Ala Met Ala Lys Arg Ser Arg Gly Pro Gly Arg Arg Cys Leu Leu Ala 20 25 30 CTC GTG CTG TTC TGC GCC TGG GGG ACG CTG GCC GTG GTG GCC CAG AAG 144 Leu Val Leu Phe Cys Ala Trp Gly Thr Leu Ala Val Val Ala Gln Lys 35 40 45 CCG GGC GCA GGG TGT CCG AGC CGC TGC CTG TGC TTC CGC ACC ACC GTG 192 Pro Gly Ala Gly Cys Pro Ser Arg Cys Leu Cys Phe Arg Thr Thr Val 50 55 60 CGC TGC ATG CAT CTG CTG CTG GAG GCC GTG CCC GCC GTG GCG CCG GAG 240 Arg Cys Met His Leu Leu Leu Glu Ala Val Pro Ala Val Ala Pro Gln 65 70 75 80 ACC TCC ATC CTA GAT CTT CGC TTT AAC AGA ATC AGA GAG ATC CAA CTT 288 Thr Ser Ile Leu Asp Leu Arg Phe Asn Arg Ile Arg Glu Ile Gln Pro 85 90 95 GGG GCA TTC AGG CGG CTG AGG AAC TTG AAC ACA TTG CTT CTC AAT AAT 336 Gly Ala Phe Arg Arg Leu Arg Asn Leu Asn Thr Leu Leu Leu Asn Asn 100 105 110 AAT GAG ATC AAG AGG ATA CTT AGT GGA GCA TTT GAA GAC TTG GAA AAT 384 Asn Gln Ile Lys Arg Ile Pro Ser Gly Ala Phe Glu Asp Leu Glu Asn 115 120 125 TTA AAA TAT CTC TAT CTG TAC AAG AAT GAG ATC GAG TCA ATT GAC AGG 432 Leu Lys Tyr Leu Tyr Leu Tyr Lys Asn Glu Ile Gln Ser Ile Asp Arg 130 135 140 CAA GCA TTT AAG GGA CTT GCC TCT CTA GAG CAA CTA TAC CTG CAC TTT 480 Gln Ala Phe Lys Gly Leu Ala Ser Leu Glu Gln Leu Tyr Leu His Phe 145 150 155 160 AAT GAG ATA GAA ACT TTG GAC CCA GAT TCG TTC GAG CAT CTC CCG AAG 528 Asn Gln Ile Glu Thr Leu Asp Pro Asp Ser Phe Gln His Leu Pro Lys 165 170 175 CTC GAG AGG CTA TTT TTG CAT AAC AAC CGG ATT ACA CAT TTA GTT CCA 576 Leu Glu Arg Leu Phe Leu His Asn Asn Arg Ile Thr His Leu Val Pro 180 185 190 GGG ACA TTT AAT GAG TTG GAA TCT ATG AAG AGA TTG CGA CTG GAC TCA 624 Gly Thr Phe Asn His Leu Glu Ser Met Lys Arg Leu Arg Leu Asp Ser 195 200 205 AAC ACA CTT GAG TGC GAC TGT GAA ATC CTG TGG TTG GCG GAT TTG CTG 672 Asn Thr Leu His Cys Asp Cys Glu Ile Leu Trp Leu Ala Asp Leu Leu 210 215 220 ARA ACC TAC GCG GAG TCG GGG AAC GCG GAG GCA GCG GCC ATC TGT GAA 720 Lys Thr Tyr Ala Glu Ser Gly Asn Ala Gln Ala Ala Ala Ile Cys Glu 225 230 235 240 TAT CCC AGA CGC ATC GAG GGA CGC TCA GTG GCA ACC ATC ACC CCG GAA 768 Tyr Pro Arg Arg Ile Gln Gly Arg Ser Val Ala Thr Ile Thr Pro Glu 245 250 255 GAG CTG AAC TGT GAA AGG CCC CGG ATC ACC TCC GAG CCC GAG GAC GCA 816 Glu Leu Asn Cys Glu Arg Pro Arg Ile Thr Ser Glu Pro Gln Asp Ala 260 265 270 GAT GTG ACC TCG GGG AAC ACC GTG TAC TTC ACC TGC AGA GCC GAA GGC 864 Asp Val Thr Ser Gly Asn Thr Val Tyr Phe Thr Cys Arg Ala Glu Gly 275 280 285 AAC CCC AAG CCT GAG ATC ATC TGG CTG CGA AAC AAT AAT GAG CTG AGC 912 Asn Pro Lys Pro Glu Ile Ile Trp Leu Arg Asn Asn Asn Glu Leu Ser 290 295 300 ATG AAG ACA GAT TCC CGC CTA AAC TTG CTG GAC GAT GGG ACC CTG ATG 960 Met Lys Thr Asp Ser Arg Leu Asn Leu Leu Asp Asp Gly Thr Leu Met 305 310 315 320 ATC CAG AAC ACA CAG GAG ACA GAC GAG GGT ATC TAC CAG TGC ATG GCA 1008 Ile Gln Asn Thr Gln Glu Thr Asp Gln Gly Ile Tyr Gln Cys Met Ala 325 330 335 AAG AAC GTG GCC GGA GAG GTG AAG ACG CAA GAG GTG ACC CTC AGG TAC 1056 Lys Asn Val Ala Gly Glu Val Lys Thr Gln Glu Val Thr Leu Arg Tyr 340 345 350 TTC GGG TCT CCA GCT CGA CCC ACT TTT GTA ATC CAG CCA CAG AAT ACA Phe Gly Ser Pro Ala Arg Pro Thr Phe Val Ile Gln Pro Gln Asn Thr 355 360 365 GAG GTG CTG GTT GGG GAG AGC GTC ACG CTG GAG TGC AGC GCC ACA GGC 1152 Glu Val Leu Val Gly Glu Ser Val Thr Leu Glu Cys Ser Ala Thr Gly 370 375 380 GAG CCC CCG CCG CGG ATC TCC TGG ACG AGA GGT GAC CGC ACA CCC TTG 1200 His Pro Pro Pro Arg Ile Ser Trp Thr Arg Gly Asp Arg Thr Pro Leu 385 390 395 400 CCA GTT GAC CCG CGG GTG AAC ATC ACG CCT TCT GGC GGG CTT TAC ATA Pro Val Asp Pro Arg Val Asn Ile Thr Pro Ser Gly Gly Leu Tyr Ile 405 410 415 GAG AAC GTC GTA GAG GGG GAC AGC GGA GAG TAT GCG TGC TCT GCG ACC 1296 Gln Asn Val Val Gln Gly Asp Ser Gly Glu Tyr Ala Cys Ser Ala Thr 420 425 430 AAC AAC ATT GAC AGC GTC CAT GCC ACC GCT TTC ATC ATC GTC GAG GCT 1344 Asn Asn Ile Asp Ser Val His Ala Thr Ala Phe Ile Ile Val Gln Ala 435 440 445 CTT CCT CAG TTC ACT GTG ACG CCT CAG GAC AGA GTC GTT ATT GAG GGC 1392 Leu Pro Gln Phe Thr Val Thr Pro Gln Asp Arg Val Val Ile Glu Gly 450 455 460 GAG ACC GTG GAT TTC CAG TGT GAA GCC AAG GGC AAC CCG CCG CCC GTC 1440 Gln Thr Val Asp Phe Gln Cys Glu Ala Lys Gly Asn Pro Pro Pro Val 465 470 475 480 ATC GCC TGG ACC AAG GGA GGG AGC CAG CTC TCC GTG GAC CGG CGG GAG 1488 Ile Ala Trp Thr Lys Gly Gly Ser Gln Leu Ser Val Asp Arg Arg His 485 490 495 CTG GTC CTG TCA TCG GGA ACA CTT AGA ATC TCT GGT GTT GCC CTC CAC 1536 Leu Val Leu Ser Ser Gly Thr Leu Arg Ile Ser Gly Val Ala Leu His 500 505 510 GAC GAG GGC GAG TAC GAA TGC GAG GCT GTC AAC ATC ATC GGC TCC CAG 1584 Asp Gln Gly Gln Tyr Glu Cys Gln Ala Val Asn Ile Ile Gly Ser Gln 515 520 525 AAG GTC GTG GCC CAC CTG ACT GTG GAG CCC AGA GTC ACC CCA GTG TTT 1632 Lys Val Val Ala His Leu Thr Val Gln Pro Arg Val Thr Pro Val Phe 530 535 540 GCC AGC ATT CCC AGC GAC ACA ACA GTG GAG GTG GGC GCC AAT GTG GAG 1680 Ala Ser Ile Pro Ser Asp Thr Thr Val Glu Val Gly Ala Asn Val Gln 545 550 555 560 CTC CCG TGC AGC TCC GAG GGC GAG CCC GAG CCA GCC ATC ACC TGG AAC 1728 Leu Pro Cys Ser Ser Gln Gly Glu Pro Glu Pro Ala Ile Thr Trp Asn 565 570 575 AAG GAT GGG GTT GAG GTG ACA GAA AGT GGA AAA TTT CAC ATC AGC CCT 1776 Lys Asp Gly Val Gln Val Thr Glu Ser Gly Lys Phe His Ile Ser Pro 580 585 590 GAA GGA TTC TTG ACC ATC AAT GAC GTT GGC CCT GCA GAC GCA GGT CGC 1824 Glu Gly Phe Leu Thr Ile Asn Asp Val Gly Pro Ala Asp Ala Gly Arg 595 600 605 TAT GAG TGT GTG GCC CGG AAC ACC ATT GGG TCG GCC TCG GTG AGC ATG 1872 Tyr Glu Cys Val Ala Arg Asn Thr Ile Gly Ser Ala Ser Val Ser Met 610 615 620 GTG CTC AGT GTG AAC GTT CCT GAC GTC AGT CGA AAT GGA GAT CCG TTT 1920 Val Leu Ser Val Asn Val Pro Asp Val Ser Arg Asn Gly Asp Pro Phe 625 630 635 640 GTA GCT ACC TCC ATC GTG GAA GCG ATT GCG ACT GTT GAC AGA GCT ATA 1968 Val Ala Thr Ser Ile Val Glu Ala Ile Ala Thr Val Asp Arg Ala Ile 645 650 655 AAC TCA ACC CGA ACA CAT TTG TTT GAC AGC CGT CCT CGT TCT CCA AAT 2016 Asn Ser Thr Arg Thr His Leu Phe Asp Ser Arg Pro Arg Ser Pro Asn 660 665 670 GAT TTG CTG GCC TTG TTC CGG TAT CCG AGG GAT CCT TAC ACA GTT GAA 2064 Asp Leu Leu Ala Leu Phe Arg Tyr Pro Arg Asp Pro Tyr Thr Val Glu 675 680 685 GAG GCA CGG GCG GGA GAA ATC TTT GAA CGG ACA TTG GAG CTC ATT CAG 2112 Gln Ala Arg Ala Gly Glu Ile Phe Glu Arg Thr Leu Gln Leu Ile Gln 690 695 700 GAG CAT GTA CAG CAT GGC TTG ATG GTC GAC CTC AAC GGA ACA AGT TAC 2160 Glu His Val Gln His Gly Leu Met Val Asp Leu Asn Gly Thr Ser Tyr 705 710 715 720 CAC TAC AAC GAC CTG GTG TCT CCA CAG TAC CTG AAC CTC ATC GCA AAC 2208 His Tyr Asn Asp Leu Val Ser Pro Gln Tyr Leu Asn Leu Ile Ala Asn 725 730 735 CTG TCG GGC TGT ACC GCC GAG CGG CGC GTG AAC AAC TGC TCG GAC ATG 2256 Leu Ser Gly Cys Thr Ala His Arg Arg Val Asn Asn Cys Ser Asp Met 740 745 750 TGC TTC CAC GAG AAG TAC CGG ACG CAC GAC GGC ACC TGT AAC AAC CTG 2304 Cys Phe His Gln Lys Tyr Arg Thr His Asp Gly Thr Cys Asn Asn Leu 755 760 765 GAG CAC CCC ATG TGG GGC GCC TCG CTG ACC GCC TTC GAG CGC CTG CTG 2352 Gln His Pro Met Trp Gly Ala Ser Leu Thr Ala Phe Glu Arg Leu Leu 770 775 780 AAA TCC GTG TAC GAG AAT GGC TTC AAC ACC CCT CGG GGC ATC AAC CCC 2400 Lys Ser Val Tyr Glu Asn Gly Phe Asn Thr Pro Arg Gly Ile Asn Pro 785 790 795 800 CAC CGA CTG TAC AAC GGG CAC GCC CTT CCC ATG CCG CGC CTG GTG TCC 2448 His Arg Leu Tyr Asn Gly His Ala Leu Pro Met Pro Arg Leu Val Ser 805 810 815 ACC ACC CTG ATC GGG ACG GAG ACC GTC ACA CCC GAC GAG GAG TTC ACC 2496 Thr Thr Leu Ile Gly Thr Glu Thr Val Thr Pro Asp Glu Gln Phe Thr 820 825 830 CAC ATG CTG ATG GAG TGG GGC CAG TTC CTG GAC CAC GAC CTC GAC TCC 2544 His Met Leu Met Gln Trp Gly Gln Phe Leu Asp His Asp Leu Asp Ser 835 840 845 ACG GTG GTG GCC CTG AGC CAG GCA CGC TTC TCC GAC GGA CAG CAC TGC 2592 Thr Val Val Ala Leu Ser Gln Ala Arg Phe Ser Asp Gly Gln His Cys 850 855 860 AGC AAC GTG TGC AGC AAC GAC CCC CCC TGC TTC TCT GTC ATG ATC CCC 2640 Ser Asn Val Cys Ser Asn Asp Pro Pro Cys Phe Ser Val Met Ile Pro 865 870 875 880 CCC AAT GAC TCC CGG GCC AGG AGC GGG GCC CGC TGC ATG TTC TTC GTG 2688 Pro Asn Asp Ser Arg Ala Arg Ser Gly Ala Arg Cys Met Phe Phe Val 885 890 895 CGC TCC AGC CCT GTG TGC GGC AGC GGC ATG ACT TCG CTG CTC ATG AAC 2736 Arg Ser Pro Val Cys Gly Ser Gly Met Thr SEr Leu Leu Met Asn 900 905 910 TCC GTG TAC CCG CGG GAG CAG ATC AAC GAG CTC ACC TCC TAC ATC GAC 2784 Ser Val Tyr Pro Arg Glu Gln Ile Asn Gln Leu Thr Ser Tyr Ile Asp 915 920 925 GCA TCC AAC GTG TAC GGG AGC ACG GAG CAT GAG GCC CGC AGC ATC CGC 2832 Ala Ser Asn Val Tyr Gly Ser Thr Glu His Glu Ala Arg Ser Ile Arg 930 935 940 GAC CTG GCC AGC GAG CGC GGC CTG CTG CGG GAG GGC ATC GTG GAG CGG 2880 Asp Leu Ala Ser His Arg Gly Leu Leu Arg Gln Gly Ile Val Gln Arg 945 950 955 960 TCC GGG AAG CCG CTG CTC CCC TTC GCC ACC GGG CCG CCC ACG GAG TGC 2928 Ser Gly Lys Pro Leu Leu Pro Phe Ala Thr Gly Pro Pro Thr Glu Cys 965 970 975 ATG CGG GAC GAG AAC GAG AGC CCC ATC CCC TGC TTC CTG GCC GGG GAC 2976 Met Arg Asp Glu Asn Glu Ser Pro Ile Pro Cys Phe Leu Ala Gly Asp 980 985 990 CAC CGC GCC AAC GAG CAG CTG GGC CTG ACC AGC ATG GAG ACG CTG TGG 3024 His Arg Ala Asn Glu Gln Leu Gly Leu Thr Ser Met His Thr Leu Trp 995 1000 1005 TTC CGC GAG CAC AAC CGC ATT GCC ACG GAG CTG CTC AAG CTG AAC CCG 3072 Phe Arg Glu His Asn Arg Ile Ala Thr Glu Leu Leu Lys Leu Asn Pro 1010 1015 1020 CAC TGG GAC GGC GAC ACC ATC TAC TAT GAG ACC AGG AAG ATC GTG GGT 3120 His Trp Asp Gly Asp Thr Ile Tyr Tyr Glu Thr Arg Lys Ile Val Gly 1025 1030 1035 1040 GCG GAG ATC CAG CAC ATC ACC TAC GAG CAC TGG CTC CCG AAG ATC CTG 3168 Ala Glu Ile Gln His Ile Thr Tyr Gln His Trp Leu Pro Lys Ile Leu 1045 1050 1055 GGG GAG GTG GGC ATG AGG ACG CTG GGA GAG TAC CAC GGC TAC GAC CCC 3216 Gly Glu Val Gly Met Arg Thr Leu Gly Glu Tyr His Gly Tyr Asp Pro 1060 1065 1070 GGC ATC AAT GCT GGC ATC TTC AAC GCC TTC GCC ACC GCG GCC TTC AGG 3264 Gly Ile Asn Ala Gly Ile Phe Asn Ala Phe Ala Thr Ala Ala Phe Arg 1075 1080 1085 TTT GGC CAC ACG CTT GTC AAC CCA CTG CTT TAC CGG CTG GAC GAG AAC 3312 Phe Gly His Thr Leu Val Asn Pro Leu Leu Tyr Arg Leu Asp Glu Asn 1090 1095 1100 TTC CAG CCC ATT GCA CAA GAT CAC CTC CCC CTT CAC AAA GCT TTC TTC 3360 Phe Gln Pro Ile Ala Gln Asp His Leu Pro Leu His Lys Ala Phe Phe 1105 1110 1115 1120 TCT CCC TTG CGG ATT GTG AAT GAG GGC GGC ATC GAT CCG CTT CTC AGG 3408 Ser Pro Phe Arg Ile Val Asn Glu Gly Gly Ile Asp Pro Leu Leu Arg 1125 1130 1135 GGG CTG TTC GGG GTG GCG GGG AAA ATG CGT GTG CCC TCG GAG CTG CTG 3456 Gly Leu Phe Gly Val Ala Gly Lys Met Arg Val Pro Ser Gln Leu Leu 1140 1145 1150 AAC ACG GAG CTC ACG GAG CGG CTG TTC TCC ATG GCA CAC ACG GTG GCT 3504 Asn Thr Glu Leu Thr Glu Arg Leu Phe Ser Met Ala His Thr Val Ala 1155 1160 1165 CTG GAC CTG GCG GCC ATC AAC ATC GAG CGG GGC CGG GAC CAC GGG ATC 3552 Leu Asp Leu Ala Ala Ile Asn Ile Gln Arg Gly Arg Asp His Gly Ile 1170 1175 1180 CCA CCC TAC CAC GAC TAC AGG GTC TAC TGC AAT CTA TCG GCG GCA CAC 3600 Pro Pro Tyr His Asp Tyr Arg Val Tyr Cys Asn Leu Ser Ala Ala His 1185 1190 1195 1200 ACG TTC GAG GAC CTG AAA AAT GAG ATT AAA AAC CCT GAG ATC CGG GAG 3648 Thr Phe Glu Asp Leu Lys Asn Glu Ile Lys Asn Pro Glu Ile Arg Glu 1205 1210 1215 AAA CTG AAA AGG TTG TAT GGC TCG ACA CTC AAC ATC GAC CTG TTT CCG 3696 Lys Leu Lys Arg Leu Tyr Gly Ser Thr Leu Asn Ile Asp Leu Phe Pro 1220 1225 1230 GCG CTC GTG GTG GAG GAC CTG GTG CCT GGC AGC CGG CTG GGC CCC ACC 3744 Ala Leu Val Val Glu Asp Leu Val Pro Gly Ser Arg Leu Gly Pro Thr 1235 1240 1245 CTG ATG TGT CTT CTC AGC ACA GAG TTC AAG CGC CTG CGA GAT GGG GAC 3792 Leu Met Cys Leu Leu Ser Thr Gln Phe Lys Arg Leu Arg Asp Gly Asp 1250 1255 1260 AGG TTG TGG TAT GAG AAC CCT GGG GTG TTC TCC CCG GCC CAG CTG ACT 3840 Arg Leu Trp Tyr Glu Asn Pro Gly Val Phe Ser Pro Ala Gln Leu Thr 1265 1270 1275 1280 GAG ATC AAG GAG ACG TCG CTG GCC AGG ATC CTA TGC GAC AAC GCG GAC 3888 Gln Ile Lys Gln Thr Ser Leu Ala Arg Ile Leu Cys Asp Asn Ala Asp 1285 1290 1295 AAC ATC ACC CGG GTG GAG AGC GAC GTG TTC AGG GTG GCG GAG TTC CCT 3936 Asn Ile Thr Arg Val Gln Ser Asp Val Phe Arg Val Ala Glu Phe Pro 1300 1305 1310 GAG GGC TAC GGC AGC TGT GAC GAG ATC CCC AGG GTG GAC CTC CGG GTG 3984 His Gly Tyr Gly Ser Cys Asp Glu Ile Pro Arg Val Asp Leu Arg Val 1315 1320 1325 TGG GAG GAC TGC TGT GAA GAC TGT AGG ACC AGG GGG GAG TTC AAT GCC 4032 Trp Gln Asp Cys Cys Glu Asp Cys Arg Thr Arg Gly Gln Phe Asn Ala 1330 1335 1340 TTT TCC TAT CAT TTC CGA GGC AGA CGG TCT CTT GAG TTC AGC TAC GAG 4080 Phe Ser Tyr His Phe Arg Gly Arg Arg Ser Leu Glu Phe Ser Tyr Gln 1345 1350 1355 1360 GAG GAC AAG CCG ACC AAG AAA ACA AGA CCA CGG AAA ATA CCC AGT GTT 4128 Glu Asp Lys Pro Thr Lys Lys Thr Arg Pro Arg Lys Ile Pro Ser Val 1365 1370 1375 GGG AGA GAG GGG GAA CAT CTC AGC AAC AGC ACC TCA GCC TTC AGC ACA 4176 Gly Arg Gln Gly Glu His Leu Ser Asn Ser Thr Ser Ala Phe Ser Thr 1380 1385 1390 CGC TCA GAT GCA TCT GGG ACA AAT GAC TTC AGA GAG TTT GTT CTG GAA 4224 Arg Ser Asp Ala Ser Gly Thr Asn Asp Phe Arg Glu Phe Val Leu Glu 1395 1400 1405 ATG GAG AAG ACC ATC ACA GAC CTC AGA ACA GAG ATA AAG AAA CTT GAA 4272 Met Gln Lys Thr Ile Thr Asp Leu Arg Thr Gln Ile Lys Lys Leu Glu 1410 1415 1420 TCA CGG CTC AGT ACC ACA GAG TGC GTG GAT GCC GGG GGC GAA TCT CAC 4320 Ser Arg Leu Ser Thr Thr Glu Cys Val Asp Ala Gly Gly Glu Ser His 1425 1430 1435 1440 GCC AAC AAC ACC AAG TGG AAA AAA GAT GCA TGC ACC ATT TGT GAA TGC 4368 Ala Asn Asn Thr Lys Trp Lys Lys Asp Ala Cys Thr Ile Cys Glu Cys 1445 1450 1455 AAA GAC GGG GAG GTC ACC TGC TTC GTG GAA GCT TGC CCC CCT GCC ACC 4416 Lys Asp Gly Gln Val Thr Cys Phe Val Glu Ala Cys Pro Pro Ala Thr 1460 1465 1470 TGT GCT GTC CCC GTG AAC ATC CCA GGG GCC TGC TGT CCA GTC TGC TTA 4464 Cys Ala Val Pro Val Asn Ile Pro Gly Ala Cys Cys Pro Val Cys Leu 1475 1480 1485 CAG AAG AGG GCG GAG GAA AAG CCC T AGGCTCCTGG GAGGCTCCTC 4509 Gln Lys Arg Ala Glu Glu Lys Pro 1490 1495 AGAGTTTGTC TGCTGTGCCA TCGTGAGATC GGGTGGCCGA TGGCAGGGAG CTGCGGACTG 4569 CAGACCAGGA AACACCCAGA ACTCGTGACA TTTCATGACA ACGTCCAGCT GGTGCTGTTA 4629 CAGAAGGCAG TGCAGGAGGC TTCCAACCAG AGCATCTGCG GAGAAGGAGG CACAGCAGGT 4689 GCCTGAAGGG AAGCAGGCAG GAGTCCTAGC TTCACGTTAG ACTTCTCAGG TTTTTATTTA 4749 ATTCTTTTAA AATGAAAAAT TGGTGCTACT ATTAAATTGC ACAGTTGAAT CATTTAGGCG 4809 CCTAAATTGG TTTTGCCTCC CAACACCATT TCTTTTTAAA TAAAGCAGGA TACCTCTATA 4869 TGTCAGCCTT GCCTTGTTCA GATGCCAGGA GCCGGCAGAC CTGTCACCCG CAGGTGGGGT 4929 GAGTCTCGGA GCTGCCAGAG GGGCTCACCG AAATCGGGGT TCCATCACAA GCTATGTTTA 4989 AAAAGAAAAT TGGTGTTTGG CAAACGGAAC AGAACCTTTG ATGAGAGCGT TCACAGGGAC 5049 ACTGTCTGGG GGTGCAGTGC AAGCCCCCGG CCTCTTCCCT GGGAACCTCT GAACTCCTCC 5109 TTCCTCTGGG CTCTCTGTAA CATTTCACCA CACGTCAGCA TCTAATCCCA AGACAAACAT 5169 TCCCGCTGCT CGAAGCAGCT GTATAGCCTG TGACTCTCCG TGTGTCAGCT CCTTCCACAC 5229 CTGATTAGAA CATTCATAAG CCACATTTAG AAACAGATTT GCTTTCAGCT GTCACTTGCA 5289 CACATACTGC CTAGTTGTGA ACCAAATGTG AAAAAACCTC CTTCATCCCA TTGTGTATCT 5349 GATACCTGCC GAGGGCCAAG GGTGTGTGTT GACAACGCCG CTCCCAGCCG GCCCTGGTTG 5409 CGTCCACGTC CTGAACAAGA GCCGCTTCCG GATGGCTCTT CCCAAGGGAG GAGGAGCTCA 5469 AGTGTCGGGA ACTGTCTAAC TTCAGGTTGT GTGAGTGCGT TAAAAAAAAA AAAAAAAAAA 5529 AGAATCCCTA TACCTCATTT GTATTTTTAA AATGCGTGAT GTTTTATGAA ATTGTGTCCA 5589 TTTTTTAGGT ATTAGATATG GCAGAAAAAC CATTTCCACT ATGCAAAGTT CTTTTAGACG 5649 TCAGTGAAAA TCAACTCTCA TACCTCATGG GTCTCTCTTT AATTGACCAA AACCTTCCAT 5709 TTTTCTCTTA AATACAAAGC GATCTGTGTT CTGAGCAACC TTTCCCCGAA CACACAGCTT 5769 CAGTGCAGCA CGCTGACCTG AGTATCCACC AGGTGCCAGG CACAGTTGCT GGGCNNACGG 5829 AGGCACCAAG GTCCGGGCCA CCTGCCCGCA GGCAAGGCCC AGCTGAGGTG GTGGGAGGGG 5889 AGCCCCTGAG GTCAGGGGCC GTTTCGGTTC AGGGTGGCAG GTGTCCAGCA CTGGGGTATG 5949 GCGTCGAGGC TTCCATGGGG TGGGGGAGGC CAGCTTCCTT CTGACAGGAT GGGCGCATAC 6009 AGTGCCTGGT GTGATTTGTG CACAACCCGT GTTCCAGGTG CACATCCTCC CAAGGAGACA 6069 CCCAGACCCT TCCAGCACGG GCCGGCCAAG TTGCTGCGGC GGAGGCAGCA TTTCAGCTGT 6129 GAGGAAGGTC ATTGGATTCA TGTGTTTTAT CTGTAAAAAT GGTTGTCTTA ACTTCTTAAC 6189 TCATATTGGT AAGTGATTGA TAAAAATTGG TTGGTGTTTT CATGACATGT GGACTTCTNT 6249 TGNATAGAAG TCAAATGTAG TGACAATTTG TGGAAGAGAT TCTTGTCAAA GTGAAATAGG 6309 AAATGTGTAA GTTCGTCTAA AAGCTGATGG TTATGTAAGT TGCTCAGGCA CTCAGATGAC 6369 AGCAGATTCT GGGTTCTGGG AGTGTTCTGT GCCTCTTACA TGCCCTGGAG GCCTCATGGT 6429 CTCAGTGCTG AGGCGGCACA CCTGTAGCAC ACCTGCGTAA TGTGCGGTCT GGGCCAGTCA 6489 CAAGGAATTG TGTTGTCTAA NCCAAAGGGG GAAGCTGACT GTGTATTACC AAAAAAAATT 6549 CTGTAATNCA AACCNAAATG TCTGCGGAAT CACCAGTTTG ATACTCTCTG TAATCAGAGC 6609 AGTNGNCTGA GGGCGGNCAG TNCCTGGGTG AACGTGTCTA GCAGCCACTG TGGGGGATCG 6669 CTGTAACAGG AGTGGAATGT ACATATTTAT TTACTTTTCT AACTGCTCCA ACAGCCAAAT 6729 GCCTTTTTTA TGACCATTGT ATTCAGTTCA TTACCAAAGA AATGTTTGCA CTTTGTAATG 6789 ATGCCTTTCA GTTCAAATAA ATGGGTCACA TTTTCAAATG GAAAAAAAAA AAAAAAAA 6847 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1496 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Ser Arg Pro Trp Trp Leu Arg Ala Ser Glu Arg Pro Ser Ala Pro Ser 1 5 10 15 Ala Met Ala Lys Arg Ser Arg Gly Pro Gly Arg Arg Cys Leu Leu Ala 20 25 30 Leu Val Leu Phe Cys Ala Trp Gly Thr Leu Ala Val Val Ala Gln Lys 35 40 45 Pro Gly Ala Gly Cys Pro Ser Arg Cys Leu Cys Phe Arg Thr Thr Val 50 55 60 Arg Cys Met His Leu Leu Leu Glu Ala Val Pro Ala Val Ala Pro Gln 65 70 75 80 Thr Ser Ile Leu Asp Leu Arg Phe Asn Arg Ile Arg Glu Ile Gln Pro 85 90 95 Gly Ala Phe Arg Arg Leu Arg Asn Leu Asn Thr Leu Leu Leu Asn Asn 100 105 110 Asn Gln Ile Lys Arg Ile Pro Ser Gly Ala Phe Glu Asp Leu Glu Asn 115 120 125 Leu Lys Tyr Leu Tyr Leu Tyr Lys Asn Glu Ile Gln Ser Ile Asp Arg 130 135 140 Gln Ala Phe Lys Gly Leu Ala Ser Leu Glu Gln Leu Tyr Leu His Phe 145 150 155 160 Asn Gln Ile Glu Thr Leu Asp Pro Asp Ser Phe Gln His Leu Pro Lys 165 170 175 Leu Glu Arg Leu Phe Leu His Asn Asn Arg Ile Thr His Leu Val Pro 180 185 190 Gly Thr Phe Asn His Leu Glu Ser Met Lys Arg Leu Arg Leu Asp Ser 195 200 205 Asn Thr Leu His Cys Asp Cys Glu Ile Leu Trp Leu Ala Asp Leu Leu 210 215 220 Lys Thr Tyr Ala Glu Ser Gly Asn Ala Gln Ala Ala Ala Ile Cys Glu 225 230 235 240 Tyr Pro Arg Arg Ile Gln Gly Arg Ser Val Ala Thr Ile Thr Pro Glu 245 250 255 Glu Leu Asn Cys Glu Arg Pro Arg Ile Thr Ser Glu Pro Gln Asp Ala 260 265 270 Asp Val Thr Ser Gly Asn Thr Val Tyr Phe Thr Cys Arg Ala Glu Gly 275 280 285 Asn Pro Lys Pro Glu Ile Ile Trp Leu Arg Asn Asn Asn Glu Leu Ser 290 295 300 Met Lys Thr Asp Ser Arg Leu Asn Leu Leu Asp Asp Gly Thr Leu Met 305 310 315 320 Ile Gln Asn Thr Gln Glu Thr Asp Gln Gly Ile Tyr Gln Gys Met Ala 325 330 335 Lys Asn Val Ala Gly Glu Val Lys Thr Gln Glu Val Thr Leu Arg Tyr 340 345 350 Phe Gly Ser Pro Ala Arg Pro Thr Phe Val Ile Gln Pro Gln Asn Thr 355 360 365 Glu Val Leu Val Gly Glu Ser Val Thr Leu Glu Gys Ser Ala Thr Gly 370 375 380 His Pro Pro Pro Arg Ile Ser Trp Thr Arg Gly Asp Arg Thr Pro Leu 385 390 395 400 Pro Val Asp Pro Arg Val Asn Ile Thr Pro Ser Gly Gly Leu Tyr Ile 405 410 415 Gln Asn Val Val Gln Gly Asp Ser Gly Glu Tyr Ala Gys Ser Ala Thr 420 425 430 Asn Asn Ile Asp Ser Val His Ala Thr Ala Phe Ile Ile Val Gln Ala 435 440 445 Leu Pro Gln Phe Thr Val Thr Pro Gln Asp Arg Val Val Ile Glu Gly 450 455 460 Gln Thr Val Asp Phe Gln Gys Glu Ala Lys Gly Asn Pro Pro Pro Val 465 470 475 480 Ile Ala Trp Thr Lys Gly Gly Ser Gln Leu Ser Val Asp Arg Arg His 485 490 495 Leu Val Leu Ser Ser Gly Thr Leu Arg Ile Ser Gly Val Ala Leu His 500 505 510 Asp Gln Gly Gln Tyr Glu Gys Gln Ala Val Asn Ile Ile Gly Ser Gln 515 520 525 Lys Val Val Ala His Leu Thr Val Gln Pro Arg Val Thr Pro Val Phe 530 535 540 Ala Ser Ile Pro Ser Asp Thr Thr Val Glu Val Gly Ala Asn Val Gln 545 550 555 560 Leu Pro Cys Ser Ser Gln Gly Glu Pro Glu Pro Ala Ile Thr Trp Asn 565 570 575 Lys Asp Gly Val Gln Val Thr Glu Ser Gly Lys Phe His Ile Ser Pro 580 585 590 Glu Gly Phe Leu Thr Ile Asn Asp Val Gly Pro Ala Asp Ala Gly Arg 595 600 605 Tyr Glu Cys Val Ala Arg Asn Thr Ile Gly Ser Ala Ser Val Ser Met 610 615 620 Val Leu Ser Val Asn Val Pro Asp Val Ser Arg Asn Gly Asp Pro Phe 625 630 635 640 Val Ala Thr Ser Ile Val Glu Ala Ile Ala Thr Val Asp Arg Ala Ile 645 650 655 Asn Ser Thr Arg Thr His Leu Phe Asp Ser Arg Pro Arg Ser Pro Asn 660 665 670 Asp Leu Leu Ala Leu Phe Arg Tyr Pro Arg Asp Pro Tyr Thr Val Glu 675 680 685 Gln Ala Arg Ala Gly Glu Ile Phe Glu Arg Thr Leu Gln Leu Ile Gln 690 695 700 Glu His Val Gln His Gly Leu Met Val Asp Leu Asn Gly Thr Ser Tyr 705 710 715 720 His Tyr Asn Asp Leu Val Ser Pro Gln Tyr Leu Asn Leu Ile Ala Asn 725 730 735 Leu Ser Gly Cys Thr Ala His Arg Arg Val Asn Asn Cys Ser Asp Met 740 745 750 Gys Phe His Gln Lys Tyr Arg Thr His Asp Gly Thr Cys Asn Asn Leu 755 760 765 Gln His Pro Met Trp Gly Ala Ser Leu Thr Ala Phe Glu Arg Leu Leu 770 775 780 Lys Ser Val Tyr Glu Asn Gly Phe Asn Thr Pro Arg Gly Ile Asn Pro 785 790 795 800 His Arg Leu Tyr Asn Gly His Ala Leu Pro Met Pro Arg Leu Val Ser 805 810 815 Thr Thr Leu Ile Gly Thr Glu Thr Val Thr Pro Asp Glu Gln Phe Thr 820 825 830 His Met Leu Met Gln Trp Gly Gln Phe Leu Asp His Asp Leu Asp Ser 835 840 845 Thr Val Val Ala Leu Ser Gln Ala Arg Phe Ser Asp Gly Gln His Gys 850 855 860 Ser Asn Val Gys Ser Asn Asp Pro Pro Gys Phe Ser Val Met Ile Pro 865 870 875 880 Pro Asn Asp Ser Arg Ala Arg Ser Gly Ala Arg Gys Met Phe Phe Val 885 890 895 Arg Ser Ser Pro Val Cys Gly Ser Gly Met Thr Ser Leu Leu Met Asn 900 905 910 Ser Val Tyr Pro Arg Glu Gln Ile Asn Gln Leu Thr Ser Tyr Ile Asp 915 920 925 Ala Ser Asn Val Tyr Gly Ser Thr Glu His Glu Ala Arg Ser Ile Arg 930 935 940 Asp Leu Ala Ser His Arg Gly Leu Leu Arg Gln Gly Ile Val Gln Arg 945 950 955 960 Ser Gly Lys Pro Leu Leu Pro Phe Ala Thr Gly Pro Pro Thr Glu Cys 965 970 975 Met Arg Asp Glu Asn Glu Ser Pro Ile Pro Gys Phe Leu Ala Gly Asp 980 985 990 His Arg Ala Asn Glu Gln Leu Gly Leu Thr Ser Met His Thr Leu Trp 995 1000 1005 Phe Arg Glu His Asn Arg Ile Ala Thr Glu Leu Leu Lys Leu Asn Pro 1010 1015 1020 His Trp Asp Gly Asp Thr Ile Tyr Tyr Glu Thr Arg Lys Ile Val Gly 1025 1030 1035 1040 Ala Glu Ile Gln His Ile Thr Tyr Gln His Trp Leu Pro Lys Ile Leu 1045 1050 1055 Gly Glu Val Gly Met Arg Thr Leu Gly Glu Tyr His Gly Tyr Asp Pro 1060 1065 1070 Gly Ile Asn Ala Gly Ile Phe Asn Ala Phe Ala Thr Ala Ala Phe Arg 1075 1080 1085 Phe Gly His Thr Leu Val Asn Pro Leu Leu Tyr Arg Leu Asp Glu Asn 1090 1095 1100 Phe Gln Pro Ile Ala Gln Asp His Leu Pro Leu His Lys Ala Phe Phe 1105 1110 1115 1120 Ser Pro Phe Arg Ile Val Asn Glu Gly Gly Ile Asp Pro Leu Leu Arg 1125 1130 1135 Gly Leu Phe Gly Val Ala Gly Lys Met Arg Val Pro Ser Gln Leu Leu 1140 1145 1150 Asn Thr Glu Leu Thr Glu Arg Leu Phe Ser Met Ala His Thr Val Ala 1155 1160 1165 Leu Asp Leu Ala Ala Ile Asn Ile Gln Arg Gly Arg Asp His Gly Ile 1170 1175 1180 Pro Pro Tyr His Asp Tyr Arg Val Tyr Gys Asn Leu Ser Ala Ala His 1185 1190 1195 1200 Thr Phe Glu Asp Leu Lys Asn Glu Ile Lys Asn Pro Glu Ile Arg Glu 1205 1210 1215 Lys Leu Lys Arg Leu Tyr Gly Ser Thr Leu Asn Ile Asp Leu Phe Pro 1220 1225 1230 Ala Leu Val Val Glu Asp Leu Val Pro Gly Ser Arg Leu Gly Pro Thr 1235 1240 1245 Leu Met Cys Leu Leu Ser Thr Gln Phe Lys Arg Leu Arg Asp Gly Asp 1250 1255 1260 Arg Leu Trp Tyr Glu Asn Pro Gly Val Phe Ser Pro Ala Gln Leu Thr 1265 1270 1275 1280 Gln Ile Lys Gln Thr Ser Leu Ala Arg Ile Leu Cys Asp Asn Ala Asp 1285 1290 1295 Asn Ile Thr Arg Val Gln Ser Asp Val Phe Arg Val Ala Glu Phe Pro 1300 1305 1310 His Gly Tyr Gly Ser Gys Asp Glu Ile Pro Arg Val Asp Leu Arg Val 1315 1320 1325 Trp Gln Asp Cys Cys Glu Asp Cys Arg Thr Arg Gly Gln Phe Asn Ala 1330 1335 1340 Phe Ser Tyr His Phe Arg Gly Arg Arg Ser Leu Glu Phe Ser Tyr Gln 1345 1350 1355 1360 Glu Asp Lys Pro Thr Lys Lys Thr Arg Pro Arg Lys Ile Pro Ser Val 1365 1370 1375 Gly Arg Gln Gly Glu His Leu Ser Asn Ser Thr Ser Ala Phe Ser Thr 1380 1385 1390 Arg Ser Asp Ala Ser Gly Thr Asn Asp Phe Arg Glu Phe Val Leu Glu 1395 1400 1405 Met Gln Lys Thr Ile Thr Asp Leu Arg Thr Gln Ile Lys Lys Leu Glu 1410 1415 1420 Ser Arg Leu Ser Thr Thr Glu Cys Val Asp Ala Gly Gly Glu Ser His 1425 1430 1435 1440 Ala Asn Asn Thr Lys Trp Lys Lys Asp Ala Cys Thr Ile Cys Glu Gys 1445 1450 1455 Lys Asp Gly Gln Val Thr Cys Phe Val Glu Ala Cys Pro Pro Ala Thr 1460 1465 1470 Gys Ala Val Pro Val Asn Ile Pro Gly Ala Gys Cys Pro Val Gys Leu 1475 1480 1485 Gln Lys Arg Ala Glu Glu Lys Pro 1490 1495 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Cys Ser Glu Gln Pro Phe Pro Glu His Thr Ala Ser Val Gln His Ala 1 5 10 15 Asp (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Arg Pro Arg Pro Glu Gln Glu Pro Leu Pro 1 5 10 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Asp Val Thr Ser Gly Asn Thr Val Tyr 1 5 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Val Leu Phe Gys Ala Trp Gly Thr Leu 1 5 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Cys Met His Leu Leu Leu Glu Ala Val 1 5 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Leu Leu Leu Glu Ala Val Pro Ala Val 1 5 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Thr Leu His Gys Asp Gys Glu Ile Leu 1 5 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: Val Leu Ser Val Asn Val Pro Asp Val 1 5 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Asp Leu Asp Ser Thr Val Val Ala Leu 1 5 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Trp Leu Pro Lys Ile Leu Gly Glu Val 1 5 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Pro Leu Leu Arg Gly Leu Phe Gly Val 1 5 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Arg Leu Gly Pro Thr Leu Met Gys Leu 1 5 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Leu Leu Ser Thr Gln Phe Lys Arg Leu 1 5 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Glu Met Gln Lys Thr Ile Thr Asp Leu 1 5 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Asp Leu Arg Thr Gln Ile Lys Lys Leu 1 5 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Arg Tyr Met Ile Leu Gly Leu Leu Ala Leu Ala Ala Val Gys Ser 1 5 10 15 Ala (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser 20 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: Arg Tyr Met Ile Leu Gly Leu Leu Ala Leu Ala Ala Val Cys Ser Ala 1 5 10 15 Met (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Met Arg Tyr Met Ile Leu Gly Leu Leu Ala Leu Ala Ala Val Gys Ser 1 5 10 15 Ala Arg Pro Arg Pro Glu Gln Glu Pro Leu Pro 20 25 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Gys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Arg Pro Arg Pro Glu Gln Glu Pro Leu Pro 20 25 30 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Met Arg Tyr Met Ile Leu Gly Leu Leu Ala Leu Ala Ala Val Cys Ser 1 5 10 15 Ala Ala Arg Pro Arg Pro Glu Gln Glu Pro Leu Pro 20 25 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Arg Tyr Met Ile Leu Gly Leu Leu Ala Leu Ala Ala Val Gys Ser Ala 1 5 10 15 Met Arg Pro Arg Pro Glu Gln Glu Pro Leu Pro 20 25 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: Met Arg Arg Pro Arg Pro Glu Gln Glu Pro Leu Pro Ala Ala Val Gys 1 5 10 15 Ser Ala (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Met Ala Arg Pro Arg Pro Glu Gln Glu Pro Leu Pro Ala Ala Ala Ala 1 5 10 15 Ala Gly