CN1402782A - MAGE-A12 antigenic peptides and uses thereof - Google Patents

Abstract

The present invention provides antigenic peptides derived from MAGE-A12 polypeptide presented by HLA molecules. The invention also provides therapeutic and diagnostic methods related to the polypeptide.

Description

MAGE-A12 antigenic peptide and its application

Field of the invention

The present invention relates to encoding nucleic acid molecules and polypeptides preferentially expressed in tumors (including malignant melanoma, bladder cancer, kidney cancer, lung cancer, esophageal cancer, etc.). Use of nucleic acid molecules and encoded polypeptides in therapy and diagnosis.

Background of the invention

The phenotypic changes that distinguish tumor cells from normal cells are often the result of one or more changes in the cell's genome. Genes that are expressed in tumor cells but not in normal cells may be referred to as "tumor-associated" genes. These tumor-associated genes are markers of the tumor phenotype. The expression of tumor-associated genes can also be an important event in the tumorigenesis process.

Typically, the host recognizes tumor-associated genes that are not expressed in normal non-tumor cells as foreign. In this way, the expression of tumor-associated genes can stimulate the host's immune response against tumor cells. Tumor-associated genes can also be expressed in normal cells in certain tissues and do not stimulate an immune response. In such tissues, because the immune system cannot "see" the cells within these immunologically specialized tissues, ordinary immunologically recognizable fragments of protein products on the cell surface are unlikely to stimulate an immune response. Examples of immunologically specific tissues include brain and testis.

Discovery of tumor-associated expression of genes provides a means of identifying cells as tumor cells. Diagnostic compounds can be based on tumor-associated genes and can be used to determine the presence and location of tumor cells. Also, when a tumor-associated gene is associated with an aspect of the tumor phenotype (e.g., unregulated growth or metastasis), the tumor-associated gene can be used to provide therapeutics, such as antisense nucleic acids that reduce or substantially eliminate the expression of such genes, thereby reducing Or substantially eliminate aspects of the phenotype that depend on the expression of specific tumor-associated genes.

The process by which the mammalian immune system recognizes and reacts to foreign or foreign substances is complex. An important aspect of the system is the T cell response. This response requires T cells to recognize and react with complexes of cell surface molecules, either human leukocyte antigens ("HLA"), or the major histocompatibility complex ("MHC"), and peptides. Peptides are derived from cell-processed macromolecules that also present HLA/MHC molecules. See Chapters 6-10 of Male et al. Advanced Immunology (J.P. Lipincott Company, 1987) in this regard. The interaction between T cells and HLA/peptide complexes is restrictive, requiring T cells to be specific for a particular combination of HLA molecules and peptides. If the specific peptide is absent, there will be no T cell response even if its group pair complex is present. Likewise, if the specific complex is absent but the T cells are present, there will be no response. This mechanism is related to the response of the immune system to foreign substances, to autoimmune pathology, and to abnormal responses to cells. Much work has focused on the mechanisms by which proteins are processed into HLA-binding peptides. See Barinaga, Science 257:880, 1992; Fremont et al., Science 257:919, 1992; Matsumura et al., Science 257:927, 1992; Latron et al., Science 257:964,1992 in this respect.

The mechanism by which T cells recognize cellular abnormalities has also been implicated in cancer. For example, in PCT application PCT/US92/04354, filed May 22, 1992, published November 26, 1992 (herein incorporated by reference), a family of genes is disclosed which are processed into peptides and then expressed in On the cell surface, it can cause tumor cell lysis through specific CTL. These genes are said to encode the "Tumor Rejection Antigen Precursor" or "TRAP" molecule from which the peptide derived is called "Tumor Rejection Antigen" or "TRA". For more information on this family of genes, see Traversari et al., J. Exp. Med. 176:1453-1457, 1992; van der Bruggen et al., Science 254:1643, 1991; De Plaen et al., Immun Genetics 40:360-369, 1994 and US Patent No. 5,342,774.

No. 5,405,940, the disclosure of which is incorporated herein by reference, teaches nonapeptides presented by HLA-A1 molecules. The references teach that given the specificity of a particular peptide for a particular HLA molecule, it should be predictable that a particular peptide binds to one HLA molecule but not others. This is important because different individuals have different HLA phenotypes. Thus, when specific peptides are identified as having diagnostic and therapeutic derivatives as counterparts to specific HLA molecules, this is only relevant to the specific HLA phenotype for the individual. This requires further work in this area, as cellular abnormalities are not limited to one specific HLA phenotype, and targeted therapy requires knowledge of some of the abnormal cells to be treated.

In US Patent No. 5,629,166 (hereby incorporated by reference), the fact that MAGE-1 expression products are processed into a second TRA is disclosed. This second TRA is presented by the HLA-Cwl6 molecule, also known as HLA-C*1601. The disclosure shows that a given TRAP can generate multiple TRAs.

In US Patent No. 5,487,974 (herein incorporated by reference), tyrosinase is described as a tumor rejection antigen precursor. This reference discloses the processing of molecules produced by some normal cells, such as melanocytes, into tumor cells to generate tumor rejection antigens presented by HLA-A2 molecules.

In US Patent No. 5,620,886 (hereby incorporated by reference in its entirety), it is disclosed that a second TRA not derived from tyrosinase is presented by the HLA-A2 molecule. TRA is derived from TRAP but is encoded by the known MAGE gene. This disclosure shows that specific HLA molecules can present TRAs derived from different sources.

Other TRAPs are disclosed in US Patent Nos. 5,571,711, 5,610,013, 5,587,289, and 5,589,334, and PCT Application WO96/10577. TRAP is processed into tumor rejection antigens, which are presented by various HLA molecules.

There are many patients who would benefit from treatment that includes other antigenic peptides because the patient's tumor does not express a previously known antigenic peptide, or because the patient does not express the appropriate HLA molecules. Therefore, there is a need to identify other tumor-associated antigens that are presented by MHC class I molecules and recognized by CD8* lymphocytes.

Summary of the invention

It has been found that the human MAGE-A12 gene (SEQ ID NO: 1) encodes a tumor rejection antigen presented by HLA-Cw*07. A peptide derived from the MAGE-A12 polypeptide (SEQ ID NO: 1) potently induces the activation and proliferation of CD8+ cytotoxic T lymphocytes when presented by antigen-presenting cells containing HLA class I molecules.

According to one aspect of the present invention, an isolated MAGE-A12 HLA class I binding peptide is provided. Peptides include the amino acid sequence of SEQ ID NO: 6 or functional variants thereof that bind HLA class I molecules. Functional variants include one or more amino acid additions, substitutions or deletions. In certain embodiments, the isolated MAGE-A12 HLA class I binding peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, functional fragments thereof, and functional variants thereof. In a preferred embodiment, the isolated peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, functional fragments thereof, and functional variants thereof. The isolated MAGE-A12 HLA class I binding peptide is preferably not a full length MAGE-A12 polypeptide sequence.

According to another aspect of the present invention, there is provided an isolated MAGE-A12 HLA class I binding peptide, which comprises an amino acid sequence fragment of SEQ ID NO: 2 that binds HLA Cw ^* 07, or a functional variant thereof. Functional variants include one or more amino acid additions, substitutions or deletions. The functional variant binds HLA Cw*07. Preferred embodiments include SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

In some embodiments, the aforementioned isolated MAGE-A12 HLA class I binding peptide is non-hydrolyzable. Preferably, the non-hydrolyzable peptide is selected from the group consisting of D-amino acid-containing peptides, -psi[ _CH2NH ]-reduced amide peptide bond-containing peptides, -psi[ _COCH2 ]-ketomethylene Peptides containing -psi[CH(CN)NH]-(cyanomethylene)aminopeptide bonds, peptides containing -psi[ _CH2CH (OH)]-hydroxyethylene peptide bonds, Peptides containing -psi[ _CH2O ]-peptide bonds, peptides containing -psi[ _CH2S ]-thiomethylene peptide bonds.

According to one aspect of the present invention there is provided a composition comprising the aforementioned isolated MAGE-A12 HLA class I binding peptide and one or more isolated HLA class I or II binding peptides of a non-MAGE-A12 tumor antigen. Preferably, the MAGE-A12 HLA class I binding peptide and the non-MAGE-A12 HLA binding peptide are combined as a multi-epitopic polypeptide.

According to another aspect of the present invention, there is provided an isolated nucleic acid encoding the aforementioned peptide. The nucleic acid did not encode full-length MAGE-A12. In certain embodiments, the nucleic acid comprises a fragment of the nucleotide sequence of SEQ ID NO:1. Expression vectors are also provided according to the invention. Expression vectors include the isolated aforementioned nucleic acids operably associated with a promoter. In certain embodiments, the expression vector also includes a nucleic acid encoding HLA-Cw ^* 07. In another aspect of the present invention, host cells transfected or transformed with the aforementioned nucleic acids or expression vectors are provided. In certain embodiments, the host cell also expresses the HLA-Cw ^* 07 molecule.

According to another aspect of the present invention, there is provided a method of selectively increasing a population of T lymphocytes comprising T lymphocytes specific for a MAGE-A12 HLA binding peptide. The method comprises combining a source of T lymphocytes comprising a population of T lymphocytes with a presenting MAGE-A12 HLA binding peptide and an HLA molecule in an amount sufficient to selectively increase the population of T lymphocytes comprising T lymphocytes specific for the MAGE-A12 HLA binding peptide Reagent contact of the complex. In certain embodiments, the agent is an antigen presenting cell contacted with a MAGE-A12 protein or an HLA binding fragment thereof. In other embodiments, the MAGE-A12 HLA binding peptide is selected from the group consisting of (i) a peptide comprising a fragment of the amino acid sequence of SEQ ID NO: 2; (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 6 and (iii) functional variants of the (i) and (ii) peptides.

According to another aspect of the present invention, there is provided a method of diagnosing a lesion characterized by the expression of a MAGE-A12 HLA binding peptide. The method includes contacting a biological sample isolated from a subject with an agent that binds the complex, and determining the binding between the complex and the agent to determine the lesion. In certain embodiments, the MAGE-A12 HLA binding peptide is selected from the group consisting of (i) peptides comprising fragments of the amino acid sequence of SEQ ID NO: 2; (ii) peptides comprising the amino acid sequence of SEQ ID NO: 6 peptides; and (iii) functional variants of (i) and (ii) peptides.

According to another aspect of the invention, there is provided a method of treating a subject suffering from a condition characterized by the expression of MAGE-A12. The method comprises administering to a subject a MAGE-A12 HLA binding peptide in an amount sufficient to reduce the lesion. In certain embodiments, the MAGE-A12 HLA binding peptide is selected from the group consisting of (i) peptides comprising fragments of the amino acid sequence of SEQ ID NO: 2; (ii) peptides comprising the amino acid sequence of SEQ ID NO: 6 peptides; and (iii) functional variants of (i) and (ii) peptides.

According to another aspect of the invention, there is provided a method of treating a subject suffering from a condition characterized by the expression of MAGE-A12. The method comprises administering to a subject an amount sufficient to alleviate the lesion of a composition comprising an isolated MAGE-A12 HLA class I binding peptide and an isolated non-MAGE-A12 tumor antigen HLA class I or II binding peptide.

According to another aspect of the invention, there is provided a method of treating a subject suffering from a condition characterized by the expression of MAGE-A12. The method comprises administering to the subject an amount of an agent that selectively increases the presence of a complex of the HLA molecule and the MAGE-A12 HLA binding peptide in the subject in an amount sufficient to alleviate the disorder. In certain embodiments, the reagent comprises a MAGE-A12 HLA binding peptide. In a preferred embodiment, the MAGE-A12 HLA binding peptide is selected from the group consisting of (i) peptides comprising fragments of the amino acid sequence of SEQ ID NO: 2; (ii) peptides comprising the amino acid sequence of SEQ ID NO: 6; and (iii) functional variants of (i) and (ii) peptides.

According to another aspect of the invention, there is provided a method of treating a subject suffering from a condition characterized by the expression of MAGE-A12. The method comprises administering to the subject an amount of autologous T lymphocytes specific for a complex of an HLA molecule and a MAGE-A12 binding peptide sufficient to alleviate the lesion. In certain embodiments, the MAGE-A12 HLA binding peptide is selected from the group consisting of (i) a peptide comprising a fragment of the amino acid sequence of SEQ ID NO: 2; (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 6 and (iii) functional variants of the (i) and (ii) peptides.

According to another aspect of the present invention, there is also provided a method of identifying functional variants of the MAGE-A12 HLA binding peptide. The method includes selecting MAGE-A12 HLA-binding peptides, HLA-binding molecules that bind to MAGE-A12 HLA class I-binding peptides, and T cells stimulated by MAGE-A12 HLA-binding peptides presented by HLA-binding molecules; mutagenesis of MAGE-A12 HLA-binding peptides The first amino acid residue of the peptide to prepare a variant peptide; determine the binding of the variant peptide to the HLA-binding molecule and the stimulation of T cells where the variant peptide binds to the HLA-binding molecule and the T-cells are subjected to the variant presented by the HLA-binding molecule Peptide stimulated indicates that the variant peptide is a functional variant. In certain embodiments, the MAGE-A12 HLA binding peptide is selected from the group consisting of (i) peptides comprising fragments of the amino acid sequence of SEQ ID NO: 2; (ii) peptides comprising the amino acid sequence of SEQ ID NO: 6 peptides; and (iii) functional variants of (i) and (ii) peptides. In other embodiments, the method comprises the step of comparing the stimulation of T cells by the MAGE-A12 HLA binding peptide to the stimulation of T cells by the functional variant to determine the effectiveness of the stimulation of the T cells by the functional variant. Also provided are functional variants of the isolated MAGE-A12 HLA binding peptides identified by the method.

According to another aspect of the present invention, there is provided an isolated polypeptide that selectively binds to the aforementioned MAGE-A12 HLA-binding peptide, so long as the isolated polypeptide is not an HLA molecule. In certain embodiments, the isolated polypeptide antibodies, preferably monoclonal antibodies. In other embodiments, the isolated polypeptide is an antibody fragment selected from the group consisting of a Fab fragment, a F(ab) ₂ fragment or a fragment comprising a CDR3 region selective for the MAGE-A12 HLA binding peptide.

According to another aspect of the present invention, there are provided isolated T lymphocytes which selectively bind a complex of an HLA molecule and a MAGE-A12 HLA binding peptide. In certain embodiments, the MAGE-A12 HLA binding peptide is selected from the group consisting of (i) peptides comprising fragments of the amino acid sequence of SEQ ID NO: 2; (ii) peptides comprising the amino acid sequence of SEQ ID NO: 6 peptides; and (iii) functional variants of (i) and (ii) peptides.

According to another aspect of the present invention, there is provided an isolated antigen presenting cell comprising a complex of an HLA molecule and a MAGE-A12 HLA binding peptide. In certain embodiments, the MAGE-A12 HLA binding peptide is selected from the group consisting of (i) peptides comprising fragments of the amino acid sequence of SEQ ID NO: 2; (ii) peptides comprising the amino acid sequence of SEQ ID NO: 6 peptides; and (iii) functional variants of (i) and (ii) peptides.

According to another aspect of the invention, there is provided a method of identifying candidate analogs of a MAGE-A12 HLA binding peptide. The method comprises providing an HLA molecule that binds a MAGE-A12 HLA-binding peptide, contacting the HLA molecule with a test molecule, determining binding of the test molecule to the HLA molecule, wherein the test molecule that binds the HLA molecule is a candidate analog of the MAGE-A12 HLA-binding peptide . In certain embodiments, the method comprises forming a complex of the HLA molecule and the candidate analog, contacting the complex with a T cell that binds the complex of the HLA molecule and the MAGE-A12 HLA binding peptide, and testing the activation of the T cell . In some of these methods, T cell activation is indicated by a property selected from the group consisting of T cell proliferation, T cell production of interferon-γ, T cell production of tumor necrosis factor, T cell response to Cytolysis of target cells.

According to one aspect of the present invention, vaccine compositions are provided. The vaccine composition includes the aforementioned MAGE-A12 HLA-binding peptide, the aforementioned T lymphocytes, the aforementioned antigen-presenting cells, and/or the aforementioned isolated nucleic acid molecules. In certain embodiments, the foregoing vaccine composition includes an adjuvant and/or a pharmaceutically acceptable carrier.

In another aspect of the invention, protein microarrays comprising one or more isolated MAGE-A12 HLA class I binding peptides, or functional variants thereof that bind HLA class I molecules are provided. Functional variants include one or more amino acid additions, substitutions or deletions. In certain embodiments, the isolated MAGE-A12 HLA class I binding peptide comprises the amino acid sequence of SEQ ID NO:6. In other embodiments, the isolated MAGE-A12 HLA class I binding peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and functional variants thereof. The present invention also provides the application of the microarray in diagnosis, especially in the diagnosis of cancer. The diagnostic method includes contacting the protein microarray with a biological sample obtained from a subject suspected of having a lesion, and determining the composition of the biological sample in association with the isolated MAGE-A12 HLA class I binding peptide. In certain embodiments, the composition of the biological sample is selected from the group consisting of antibodies, T lymphocytes, and HLA molecules. A preferred lesion is cancer.

The invention also provides pharmaceutical preparations comprising any one or more of the compositions described herein. Such pharmaceutical preparations may include a pharmaceutically acceptable diluent carrier or excipient. The present invention also provides the use of such a composition for the preparation of medicines, especially for the preparation of medicines for treating cancer.

In the aforementioned methods and compositions, the HLA molecule is preferably HLA Cw ^* 07, more preferably HLA Cw ^* 0701. Lesions, as used herein, are cancers such as bladder cancer, malignant melanoma, esophageal cancer, lung cancer, head and neck cancer, breast cancer, colon cancer, myeloma, brain tumor, malignant sarcoma, prostate cancer, and kidney cancer.

These and other objects of the invention are described in more detail in connection with the detailed description of the invention.

Brief description of the diagram of the invention

Figure 1 demonstrates CTL501 D/19 recognition of self and allogeneic HLA-Cw*07 positive tumor cell lines: (A) lysis, (B) TNF release.

Figure 2 demonstrates that CTL 501D/19 recognizes an antigen encoded by MAGE-12 presented by HLA-Cw7.

Figure 3 demonstrates the determination of the MAGE-A12 region encoding the antigenic peptide recognized by CTL 501 D/19.

Figure 4 demonstrates lysis of the autologous cell line LB-831-EBV pulsed with the MAGE-12 peptides VRIGHLYIL (SEQ ID NO: 4) and RIGHLYIL (SEQ ID NO: 6) by CTL 501D/19.

Detailed description of the invention

The present invention provides isolated MAGE-A1 peptides, some of which are presented by HLA class I molecules, such peptides stimulate the proliferation and activation of CD8+ T lymphocytes. Such peptides are referred to herein as "MAGE-A12 immunogenic polypeptides" and "MAGE-A12 HLA class I binding peptides" and "MAGE-A12 HLA peptides", among others. Accordingly, one aspect of the invention is an isolated peptide comprising the amino acid sequence of SEQ ID NO:6.

The example below shows the isolation of a peptide that is a MAGE-A12 HLA binding peptide. These exemplary peptides are the processed translation products of the SEQ ID NO: 1 nucleic acid. Thus, those of ordinary skill in the art will appreciate that the translation product from which the MAGE-A12 immunogenic polypeptide is processed into the final form for presentation may be of any length or sequence so long as it includes the HLA binding peptide. In certain embodiments, the HLA binding peptide comprises a MAGE-A12 HLA binding peptide comprising the amino acid sequence of SEQ ID NO: 4, 5 or 6. As demonstrated in the Examples below, peptides or proteins as small as 8 amino acids are amenable to processing, are presented by HLA class I molecules and can efficiently stimulate CD8+ T lymphocytes. Peptides of SEQ ID NO: 4, 5, or 6 can have one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids added in one end or both ends. The added amino acid may correspond to the MAGE-A12 polypeptide (SEQ ID NO: 2), or may be unrelated. As is well known in the art, the antigenic portion of this peptide is cleaved under physiological conditions and presented by HLA class I molecules.

Other HLA-binding peptides derived from the MAGE-A12 polypeptide can stimulate immune responses when presented by HLACw*07 molecules. The invention includes all such immunogenic fragments of MAGE-A12 polypeptides.

As used herein, a "functional variant" or "variant" of a MAGE-A12 HLA-binding peptide is one that contains one or more modifications to the basic amino acid sequence of the MAGE-A12 HLA-binding peptide and maintains those disclosed herein. Molecules with HLA class I binding properties and the ability to stimulate proliferation and/or activation of CD8+ T lymphocytes. Modifications to generate functional variants of MAGE-A12 immunogenic polypeptides can be made to 1) enhance properties of the MAGE-A12 HLA-binding peptide, such as peptide stability in expression systems or stability of protein-protein binding such as HLA-peptide binding 2) providing new activities or properties to the MAGE-A12 immunogenic polypeptide, such as adding antigenic epitopes or adding identifiable components; or 3) providing different amino acid sequences that produce the same or similar T cell stimulating properties. Modifications to the MAGE-A12 HLA-binding peptide can be made to the nucleic acid encoding the peptide, including deletions, point mutations, truncations, amino acid substitutions, and amino acid additions. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of linker molecules, addition of identifiable components such as biotin, addition of fatty acids, substitution of one amino acid for another, and the like. Modifications also include fusion proteins comprising all or part of the amino acid sequence of the MAGE-A12 immunogenic polypeptide.

The amino acid sequences of MAGE-A12 immunogenic polypeptides may be of natural or non-natural origin, i.e., they may be native MAGE-A12 immunogenic polypeptide molecules or may comprise modified sequences, as long as the amino acid sequences remain stimulatory when presented. The ability to cytolytic T cells and retain the property of binding to HLA class I molecules such as HLACw*07 molecules. For example, the MAGE-A12 immunogenic polypeptide herein may be a fusion protein of a MAGE-A12 HLA binding peptide and an unrelated amino acid sequence, a synthetic peptide of the amino acid sequences shown in SEQ ID NO: 4, 5 and 6, a tagged peptide , peptides isolated from patients with MAGE-A12 expressing cancers, peptides isolated from cultured cells expressing MAGE-A12, peptides conjugated to non-peptide molecules (as in certain drug delivery systems) and including Other molecules of SEQ ID NO:6 amino acid sequence.

Preferably, the MAGE-A12 HLA binding peptide is non-hydrolyzable. To provide such peptides, MAGE-A12 HLA binding peptides can be selected from a library of non-hydrolyzable peptides (such as peptides containing one or more D-amino acids or peptides containing one or more amino acids bound by non-hydrolyzable peptide bonds). Alternatively, one can select a peptide that is optimal for inducing CD8+ T lymphocytes, and then modify such a peptide as necessary to reduce the likelihood of being hydrolyzed by proteases. For example, to determine susceptibility to proteolytic cleavage, peptides can be labeled and incubated with cell extracts or purified proteases, and then isolated to determine which peptide bonds are susceptible to proteolytic cleavage, such as by sequencing the peptides and proteolytic fragments. Alternatively, potential susceptibility peptide bonds can be determined by comparing the amino acid sequence of the MAGE-A12 immunogenic polypeptide with the known cleavage site specificity of a test panel of proteases. Based on the results of this test, individual peptide bonds that are likely to be proteolyzed can be replaced by non-hydrolyzable peptide bonds by in vitro peptide synthesis.

A number of non-hydrolyzable peptide bonds and methods for synthesizing peptides containing such peptide bonds are well known in the art. Non-hydrolyzable peptide bonds include -psi[CH ₂ NH]-reduced amide peptide bond, -psi[COCH ₂ ]-ketomethylene peptide bond, -psi[CH(CN)NH]-(cyanomethylene)aminopeptide bond, -psi[ _CH2CH (OH)]-hydroxyethylene peptide bond, -psi[ _CH2O ]-peptide bond, -psi[ _CH2S ]-thiomethylene peptide bond.

Non-peptide analogs of peptides, eg, which provide a stable structure or reduced biodegradation, are also contemplated. Peptidomimetic analogs can be prepared by substituting one or more residues with non-peptidic constituents based on the selected MAGE-A12 HLA binding peptide. Preferably, the non-peptide composition allows the peptide to maintain its native conformation, or stabilize a preferred, eg biologically active, conformation. An example of a method for preparing non-peptidomimetic analogs from peptides is described in Nachman et al., Regul. Pept. 57:359-370 (1995). Peptide analogs can also be selected from synthetic compounds (eg combinatorial libraries of small organic molecules) or natural molecule libraries based on the HLA binding properties and/or T cell stimulating properties of such molecules. Assays, such as binding assays, to identify analogs of MAGE-A12 immunogenic polypeptides obtained from libraries are well known in the art. Peptide as used herein includes all of the foregoing.

Functional variants of the MAGE-A12 immunogenic polypeptide containing conservative amino acid substitutions are generally preferred if the variant comprises a change in the MAGE-A12 immunogenic polypeptide (SEQ ID NO: 4, 5 or 6), i.e., maintains Substitution of source amino acid properties such as charge, hydrophobicity, conformation, etc. Examples of amino acid conservative substitutions include substitutions made at amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Other methods of identifying functional variants of MAGE-A12 immunogenic polypeptides are disclosed in PCT application (US/96/03182) by Strominger and Wucherpfennig. These methods depend on the visualization of amino acid sequence motifs to which potential epitopes can be compared. Each motif describes a limited set of amino acid sequences in which the residues at each (relative) position may (a) be restricted to a single residue, (b) vary within the restricted set of residues, or (c) can vary among all possible residues. For example, a motif may specify that the residue at the first position may be any of the residues valine, leucine, isoleucine, methionine, or phenylalanine; it may specify that at the second position The residue must be histidine; the residue specified at the third position may be any amino acid residue; the residue specified at the fourth position may be the residues valine, leucine, isoleucine , any of methionine, phenylalanine, tyrosine, or tryptophan; the residue specified at the fifth position must be lysine.

Sequence motifs of functional variants of MAGE-A12 HLA-binding peptides can be determined by analyzing the binding domain or binding pocket of the major histocompatibility complex HLA Cw protein and/or T cell receptors of the MAGE-A12 immunogenic polypeptides described herein. Body (“TCR”) contact points are visualized. By providing a detailed structural analysis of the residues involved in forming the HLA class I binding pocket, one of ordinary skill in the art can make predictions for sequence motifs that bind any HLA class I protein.

Using these sequence motifs as study, assessment, or design criteria, one of ordinary skill in the art can identify peptide classes that have a reasonable likelihood of binding to a particular HLA molecule and interacting with T cells to induce a T cell response (herein Functional variants of the disclosed MAGE-A12 HLA-binding peptides). These peptides can be synthesized and tested for activity as described herein. Using these motifs, sequence homology with pure sequence homology (which excludes multiple peptides that are antigenically similar but quite different in sequence) or with unrestricted "conservative" substitutions (which includes Peptides with different points) represent, by contrast, a method by which one of ordinary skill in the art can assess the likelihood of a peptide being used in the treatment of a disease.

The Strominger and Wucherpfennig PCT application (references cited herein, incorporated herein by reference in their entirety), describes HLA class II and TCT binding pockets that contact residues of HLA class II peptides. Likewise, the functionality of the MAGE-A12 HLA-binding peptide that binds to HLA class I and T cell receptors is preserved by keeping the residues likely to bind in the HLA class I and/or TCR binding pocket consistent, or allowing only specific substitutions Variants can be prepared.

Localization of one or more antigenic peptides within a protein sequence can be obtained by following established principles of binding likelihood (e.g., Parker et al., J. Immunol. 152:163, 1994; Rammensee et al., Immunogenetics 41:178- 228, 1995) with the aid of HLA peptide binding predictions. HLA binding predictions are available via the Internet at the (US) National Institutes of Health (National Institutes of Health) website at the World Wide Web site at Uniform Resource Locator (URL) http://bimas.dcrt.nih.gov Easy to do.

Methods of identifying MAGE-A12 immunogenic polypeptides are provided. Typically, the method involves taking a MAGE-A12 HLA-binding peptide, an HLA class I binding molecule that binds the MAGE-A12 HLA-binding peptide, and T cells stimulated with the MAGE-A12 HLA-binding peptide presented by the HLA class I binding molecule. In a preferred embodiment, the MAGE-A12 immunogenic polypeptide comprises the amino acid sequence of SEQ ID NO:6. More preferably, the peptide comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. The first amino acid residue of the MAGE-A12 HLA-binding peptide was mutated to generate variant peptides. Amino acid residues can be mutagenized according to the HLA and T cell contact point principles described above. Any method for preparing variant peptides can be used, such as synthesis of variant peptides, recombination of vegetation variant peptides using mutant nucleic acid molecules, and the like.

Binding of the variant peptides to HLA class I binding molecules and/or stimulation of T cells is determined according to standard methods. For example, as exemplified below, the variant peptide can be contacted with an antigen presenting cell containing an HLA class I molecule bound to a MAGE-A12 HLA binding peptide, forming a complex of the variant peptide and the antigen presenting cell. This complex can then be contacted with T cells that recognize the MAGE-A12 HLA-binding peptide presented by the HLA class I binding molecule. T cells can be obtained from a patient suffering from a condition characterized by expression of MAGE-A12. Recognition of the variant peptide by T cells can be determined by measuring indicators of T cell stimulation, such as TNF or IFNy production.

Binding of the variant peptides to HLA class I binding molecules and/or stimulation of T cells by the variant peptides presented by the HLA class I binding molecules suggest that the variant peptides are functional variants. The method may also comprise the step of comparing the T cell stimulatory effect of the MAGE-A12 HLA binding peptide with the T cell stimulatory effect of the functional variant to determine the effectiveness of the functional variant for T cell stimulatory effect. By comparing MAGE-A12 HLA binding peptides with functional variants, peptides with increased T cell stimulatory properties can be prepared.

If desired, variants of the MAGE-A12 HLA binding peptides prepared by any of the aforementioned methods can be sequenced to determine the amino acid sequence and deduce the nucleotide sequence encoding the variant.

Accordingly, nucleic acid sequences encoding MAGE-A12 immunogenic polypeptides or variants thereof, including allelic variants, are also part of the invention. In screening for nucleic acid encoding MAGE-A12 immunogenic polypeptides, nucleic acid hybridization, such as Southern blotting or Northern blotting, can be performed using ³² P probes under stringent conditions. "Stringent conditions" as used herein refers to parameters well known in the art. Nucleic acid hybridization parameters can be found in literature compiling this method, such as Molecular Cloning: A Laboratory Manual, J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989, or Molecular Biology's Current Protocols, edited by FMAusubel et al., John Wiley & Sons Ltd., New York. Exemplary stringent conditions include hybridization at 65°C in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 25mM NaH ₂ PO ₄ (pH 7), 0.5% SDS, 2mM EDTA) . SSC is 0.15M sodium chloride/0.015M sodium citrate, pH 7; SDS is sodium dodecyl sulfate; EDTA is ethylenediaminetetraacetic acid. After hybridization, the membrane onto which the DNA is transferred can be washed, eg, with 2×SSC at room temperature, followed by 0.1-0.5×SSC/0.1×SDS at room temperature to 68°C. After washing the membrane to which the DNA encoding the MAGE-A12 immunogenic polypeptide was finally transferred, the membrane was exposed to X-ray film to measure the radioactive signal.

There are other conditions, reagents that can be used and can produce similar degrees of stringency. Such conditions are well known to the skilled person and therefore they are not given here. It will be appreciated, however, that the skilled artisan will be able to manipulate these conditions in such a way that homologues and alleles of nucleic acids encoding MAGE-A12 immunogenic polypeptides of the invention will be unambiguously identified. Methods of screening cells and libraries expressing such molecules and subsequently routinely isolating, followed by isolating and sequencing the relevant nucleic acid molecules are also well known to the skilled artisan.

The present invention also includes the use of nucleic acid sequences comprising variable codons encoding the same amino acid residues of the MAGE-A12 immunogenic polypeptide. For example, as disclosed herein, the peptide VRIGHLYIL (SEQ ID NO: 4) is a MAGE-A12 HLA binding peptide. Leucine residues can be encoded by the codons CUA, CUC, CUG, CUU, UUA and UUG. Each of the six codons is equivalent for encoding a leucine residue. Thus, it will be apparent to those of ordinary skill in the art that any leucine-encoding nucleotide triplet can be used to direct the protein synthesis apparatus to incorporate leucine in vivo or in vitro. Similarly, triplets of nucleotide sequences encoding other amino acid residues of the MGAE-A12 HLA binding peptide comprising SEQ ID NO: 4 include: GUA, GUC, GUG and GUU (codons for valine); GGU, GGA, GGG, GGC (glycine codons); UGC and UAU (tyrosine codons). Other amino acid residues can be similarly encoded by various nucleotide sequences. Accordingly, the present invention includes degenerate nucleic acids that differ in codon sequence from the native MAGE-A12 immunogenic polypeptide-encoding nucleic acid due to the degeneracy of the genetic code.

A preferred nucleic acid encoding a MAGE-A12 polypeptide is one that preferably expresses a MAGE-A12 immunogenic polypeptide, such as an HLA binding peptide as described herein. The MAGE-A12 nucleic acids of the invention do not encode all MAGE-A12 polypeptides, but do include nucleotide sequences encoding MAGE-A12 HLA binding peptides.

The invention also provides modified nucleic acid molecules comprising additions, substitutions and deletions of one or more nucleotides. In preferred embodiments, these modified nucleic acids and/or polypeptides encoded by them retain at least one activity or function of the unmodified nucleic acid molecules and/or polypeptides, such as antigenicity, enzymatic activity, receptor binding, MHC class I and Class II molecules bind peptides to form complexes, etc. In certain embodiments, the modified nucleic acid molecule encodes a modified polypeptide, preferably the polypeptide contains conservative amino acid substitutions as described elsewhere herein. The modified nucleic acid molecule is structurally related to the unmodified nucleic acid molecule, and in preferred embodiments, the modified nucleic acid molecule is sufficiently related in structure to the unmodified nucleic acid molecule such that stringent conditions well known to those skilled in the art The modified nucleic acid and the unmodified nucleic acid molecule hybridize.

For example, modified nucleic acid molecules can be prepared that encode polypeptides with single amino acid changes (eg, preferably amino acids other than those that are HLA binding contacts). Each of these nucleic acid molecules may have one, two or three nucleotide substitutions in addition to the degenerate nucleotide changes corresponding to the genetic codes described herein. Likewise, modified nucleic acid molecules encoding polypeptides containing two amino acid changes can be prepared, such nucleic acid molecules having, for example, 2-6 nucleotide changes. Those skilled in the art can readily envision numerous modified nucleic acids identical to these nucleic acids, including, for example, substitutions of nucleotides in the codons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6 ,etc. In the preceding examples, every combination of two amino acids is included in the group of modified nucleic acid molecules, as well as all nucleotide substitutions encoding amino acid substitutions. Other nucleic acids encoding polypeptides with other substitutions (i.e., 3 or more), additions or deletions (e.g., by introducing stop codons or splice sites) can be readily envisioned by one of ordinary skill in the art Molecules can also be prepared and are included in the invention. Any of the aforementioned nucleic acids or polypeptides can be assayed by routine experimentation for the maintenance of structural relevance and activity of the nucleic acids and/or polypeptides disclosed herein.

It is also understood that the present invention includes the use of sequences in expression vectors, and transfection of host cells and cell lines, such as prokaryotic cells (E. coli), or eukaryotic cells (e.g., dendritic cells, CHO cells, COS cells , yeast expression system and recombinant baculovirus expression in insect cells). Expression vectors require the relevant sequences, ie, those described above, to be operably associated with a promoter. In addition, it has been found that the human HLA-Cw ^* 07 molecule presents the MAGE-A12 HLA class I binding peptide, and the expression vector may also include a nucleic acid sequence encoding the HLA-Cw*07 molecule. (For other class I or class II binding peptides, different HLA molecules can be used.) Where the vector includes both coding sequences, the vector can be used to transfect cells that do not normally express either. When the host cell already expresses the HLA-Cw ^* 07 molecule, the MAGE-A12 HLA class I binding peptide coding sequence can be used alone. Of course, if desired, there is no limitation to the specific host cell that can be used as a vector containing two coding sequences that can be used in host cells that do not express HLA-Cw ^* 07 molecules, and encode MAGE-A12 HLA class I binding Peptide nucleic acids can also be used in antigen presenting cells expressing HLA-Cw ^* 07 molecules. As used herein, "HLA-CW ^* 07 molecule" includes subtypes HLA-Cw ^* 0701 (07011, 07012), 0702, 0703, 0704, 0705, 0706, 0707, 0708, 0709, 0710, 0711, 0712, 0713 and 0714. The HLA-Cw ^* 07 molecule also includes subtypes that can be found in Bodmer et al., Tissue Antigens 49:297,1996. A list of currently identified HLA-Cw ^* 07 subtypes can be found in the IMGT/HLA database at the Internet URL http://www.ebi.ac.uk/imgt/hla/.

It is also understood that the present invention includes the use of sequences in expression vectors, including recombinant plasmids, phagemids, viruses, etc., and the use of sequences to transfect host cells and cell germlines, such as prokaryotic cells (E.coli) , or eukaryotic cells (eg, dendritic cells, CHO cells, COS cells, yeast expression systems, and recombinant baculovirus expression in insect cells). . Expression vectors require the relevant sequences, ie, those described above, to be operably associated with a promoter. Delivery of expression vectors containing the MAGE-A12 sequence in vivo or in vitro can be by nucleic acid delivery systems known in the art (see, eg, Allsopp et al., European Journal of Immunology, 26(8):1951-1959, 1996). Recombinant vectors comprising viruses selected from the following virus groups may be used in such delivery systems, e.g. as vaccines, including adenoviruses, adeno-associated viruses, poxviruses (including vaccinia viruses and attenuated poxviruses such as NYVAC ), Semliki Forest virus, Venezuelan equine encephalitis virus, retrovirus, Sindbis virus, and Ty virus-like particles, plasmids (such as "naked" DNA), bacteria. Other viruses, expression vectors and the like used to prepare vaccines are known to those of ordinary skill in the art. The MAGE-A12 delivery system can be tested in a standard model system, such as the mouse, to determine the effectiveness of the delivery system. The system could also be tested in human clinical trials.

In addition, non-MAGE-A12 tumor-associated peptides can also be administered to increase immune responses via HLA class I and/or class II. It is well established that cancers can express more than one tumor-associated gene. It is within the scope of routine experimentation for one of ordinary skill in the art to determine whether a particular subject expresses other tumor-associated genes, and includes HLA I derived from expression products of such genes in MAGE-A12 compositions and vaccines. and/or HLA class II binding peptides.

Particularly preferred are nucleic acids encoding a series of antigenic epitopes (referred to as "polytopes"). Multiple antigenic epitopes can be arranged in a contiguous manner or in an overlapping manner (see, e.g., Thomoson et al., Proc. Natl. Acad, Sci. USA 92:5845-5849, 1995; Gilbert et al., Nature Biotechnol. , 1997), with or without natural flanking sequences, epitopes can be spaced by irrelevant linker sequences if desired. Multiple epitopes are processed into product individual epitopes that are recognized by the immune system to generate an immune response.

Thus, MAGE-A12 HLA-binding peptides presented by MHC molecules and recognized by CTLs (or T helper lymphocytes), such as SEQ ID NO: 4, 5, and 6, can be compared with other tumor rejection antigens (e.g., by preparing hybrid nucleic acid or polypeptides) combine to form "multi-epitopes". Exemplary tumor-associated peptides that can be administered to induce or enhance an immune response are derived from tumor-associated genes and encoded proteins, including MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE -A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7 , GAGE-8, GAGE-9, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE -B4), tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, NY-ESO-1, LAGE-1, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7. For example, tumor-specific antigenic peptides include those listed in Table I below.

Table I: Example Antigens Gene MHC peptide Location SEQ ID NO: MAGE-A1 HLA-A1 EADPTGHSY 161-169 9 HLA-Cw16 SAYGEPRKL 230-238 10 MAGE-A3 HLA-A1 EVDPIGHLY 168-176 11 HLA-A2 FLWGPRALV 271-279 12 HLA-B44 MEVDPIGHLY 167-176 13 BAGE HLA-Cw16 AARAVFLAL 2-10 14 GAGE-1,2 HLA-Cw16 YRPRPRRY 9-16 15 RAGE HLA-B7 SPSSNRIRNT 11-20 16 GnT-V HLA-A2 VLPDVFIRC(V) 2-10/11 17, 18 MUM-1 HAL-B44 EEKLIVVLF exon 2/intron 19 EEKLSVVLF (wild type) 20 CDK4 HLA-A2 ACDPHSGHFV 23-32 twenty one ARDPHSGHFV (wild type) twenty two β-catenin HLA-A24 SYLDSGIHF 29-37 twenty three Tyrosinase SYLDSGIHS (wild type) twenty four HLA-A2 MLLAVLYCL 1-9 25 HLA-A2 YMNGTMSQV 369-377 26 HLA-A2 YMDGTMSQV 369-377 42 HAL-A24 AFLPWHRLF 206-214 27 HLA-B44 SEIWRDIDF 192-200 28 HLA-B44 YEIWRDIDE 192-200 29 HLA-DR4 QNILLSNAPLGPQFP 56-70 30 HLA-DR4 DYSYLQDSDPDSFQD 448-462 31 Melan-A ^MART HLA-A2 (E) AAGIGILTV 26/27-35 32, 33 Gp100 ^Pmell17 HLA-A2 ILTVILGVL 32-40 34 HLA-A2 KTWGQYWQV 154-162 35 HLA-A2 ITDQVPFSV 209-217 36 HLA-A2 YLEPPGVTA 280-288 37 HLA-A2 LLDG TATLR 457-466 38 HLA-A2 VLYRYGSFSV 476-485 39 PRAME HLA-A24 LYVDSLFEL 301-309 40 MAGE-A6 HLA-Cw16 KISGGPRISYPL 292-303 41 NY-ESO-1 HLA-A2 SLLMWITQCFL 157-167 43 HLA-A2 SLLMWITQC 157-165 44 HLA-A2 QLSLLMWIT 155 45

Other examples of HLA class I HLA class II binding peptides are known to those of ordinary skill in the art (e.g., see Coulie, Stem Cell 13:393-403, 1995) and can be used in a manner similar to that described herein in In the present invention. Those of ordinary skill in the art can prepare polypeptides comprising one or more MAGE-A12 peptides and one or more of the aforementioned tumor-repelling peptides, or nucleic acids encoding such polypeptides, according to standard methods of molecular biology.

Thus, a polyepitope is a group of two or more potentially immunogenic or immune response stimulating peptides that can be combined in various arrangements (eg concatenated, overlapping). Polytopes (or nucleic acids encoding polytopes) can be administered using standard immunization protocols, such as to animals, to test the effectiveness of polytopes in stimulating, enhancing and/or promoting an immune response.

Peptides forming polyepitopes, which can be joined together directly or through the use of flanking sequences, and the use of polyepitopes as vaccines are well known in the art (see, e.g., Thomson et al., Proc. Natl. Acad. Sci. USA92(13):5845-5849; Gilbert et al., Nature Biotechnol.15(12):1280-1284, 1997; Thomson et al., Journal of Immunology, 157(2):822-826, 1996; Tem et al., J. Exp. Med 171(1):299-306, 1990). For example, Tam showed that polytopes including MHC class I and class II binding epitopes successfully elicit antibodies and protective immunity in a murine model. Tam also demonstrated that polyepitope processing involving epitope "strings" generates individual antigenic epitopes that are presented by MHC molecules and recognized by CTLs. Thus, polytopes containing different numbers and combinations of antigenic epitopes can be prepared and tested for CTL recognition and effectiveness in increasing the immune response.

It is well known that tumors express a panel of tumor antigens, of which only certain subsets may be expressed in any given patient's tumor. Polytopes can be prepared corresponding to different combinations of antigenic epitopes representing subsets of tumor rejection antigens expressed in a particular patient. Polyepitopes can also be prepared to reflect a broad spectrum of tumor rejection antigens known to be expressed by a tumor type. Polyepitopes can be introduced into patients in need of such therapeutic e.g. polypeptide constructs, such as by using nucleic acid delivery systems known in the art (see, e.g., Allsopp et al., European Journal of Immunology, 26(8):1951-1959 , 1996). Adenoviruses, poxviruses, Ty-virus-like particles, adeno-associated viruses, plasmids, bacteria, etc. can be used in such delivery systems. Multi-epitope delivery systems can be tested in murine models to determine the effectiveness of the delivery system. The system could also be tested in human clinical trials.

As it has been found that human HLA-Cw ^* 07 molecules present MAGE-A12 immunogenic polypeptides, the expression vector may also include nucleic acid sequences encoding HLA-Cw ^* 07 molecules. Nucleic acids encoding single-chain soluble HLA/peptide complexes comprising MAGE-A12 immunogenic peptides fused to HLA-Cw ^* 07 can be prepared as described by lone et al. (J. Immunother. 21:283-294, 1998) .

In cases where the vector includes both coding sequences, the vector can be used to transfect cells that do not normally express either. When the host cell already expresses the HLA-Cw ^* 07 molecule, the MAGE-A12 HLA class I binding peptide coding sequence can be used alone. Of course, if desired, there is no limitation to the specific host cell that can be used as a vector containing two coding sequences that can be used in host cells that do not express HLA-Cw ^* 07 molecules, and encode MAGE-A12 HLA class I binding Peptide nucleic acids can also be used in antigen presenting cells expressing HLA-Cw ^* 07 molecules.

As used herein, a "vector" may be any number of nucleic acids into which desired sequences may be inserted in restriction linkage for transport between different genetic environments or for expression in a host cell. Vectors typically comprise DNA, although RNA vectors can also be used. Vectors include, but are not limited to, plasmids, phagemids, bacterial and viral genomes as described herein, such as adenovirus, poxvirus and BCG. A cloning vector is a vector that can replicate in a host cell or a vector that replicates after it is integrated into the host cell genome, and is further characterized by having one or more endonuclease restriction sites on which the vector can be It can be cleaved in a defined manner and the desired DNA sequence can be ligated into it so that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence can occur multiple times as the number of copies of the plasmid in the host bacterium increases, or once per host before the host reproduces by mitosis. In the case of phages, replication can occur actively during the lytic phase or passively during the lysogenic phase. An expression vector is one into which a desired DNA sequence can be inserted by restriction linkage so that it can be operably associated with regulatory sequences and expressed as an RNA transcript. The vector may further include one or more marker sequences suitable for identifying cells that have or have not been transfected or transformed with the vector. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activity can be detected by standard assays known in the art (e.g., beta-form milk glycosidase, luciferase or alkaline phosphatase), and genes that significantly affect the phenotype of transformed or transfected cells, hosts, colonies or phage (eg, green fluorescent protein). Preferred vectors are those capable of replicating themselves and expressing the structural gene products presented in the DNA segment with which they are operably associated.

As used herein, a coding sequence and a regulatory sequence are "operably" associated when they are covalently associated in such a manner that the expression or transcription of the coding sequence is placed under the control and influence of the regulatory sequence . If translation of the coding sequence into a functional protein is required, if induction of the promoter in the 5' regulatory sequence results in transcription of the coding sequence, and if the absence of (1) the linkage between the two DNA sequences results in the introduction of a frameshift mutation, ( 2) Interfering with the ability of the promoter region to direct the transcription of the coding sequence, (3) interfering with the ability of the corresponding RNA transcript to be translated into protein, the two DNA sequences can be said to be operably combined. Thus, a promoter region is operably associated with a coding sequence if the promoter region affects the transcription of the DNA sequence such that the resulting transcript may be translated into the desired protein or polypeptide. As noted above, certain preferred nucleic acids express only fragments of MAGE-A12 polypeptides that include the HLA binding peptides described herein.

The precise nature of the regulatory sequences required for gene expression may vary between species or cell types, but should generally include, 5'-untranslated and 5'-untranslated sequences involved in transcription and translation, respectively, such as the TATA box, cap sequence, CAAT sequence and more. In particular, 5'-non-transcriptional regulatory sequences will include the promoter region containing the promoter sequence operably associated with the transcriptional control of the gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the present invention may optionally include a 5' leader or signal sequence. The selection and design of suitable vectors is within the purview of those of ordinary skill in the art.

Expression vectors containing all the necessary factors for expression are commercially available and are well known to those skilled in the art. See, for example, Sambrook et al., "Molecular Cloning" A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are induced by introducing heterologous DNA (RNA) encoding MAGE-A12 immunogenic polypeptides into cells. Genetic engineering is performed. The heterologous DNA (RNA) is placed under the operable control of transcription factors to allow expression of the heterologous DNA in the host cell.

A preferred system for mRNA expression in mammalian cells is e.g. pcDNA3.1 (obtained from Invitrogen, Carlsbad, CA), which contains selectable markers such as genes with G418 resistance (facilitating selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequence. Therefore, suitable for expression in primates or canines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr virus (EBV) origin of replication, which facilitates maintenance of the plasmid as a multi-copy extrachromosomal genetic element. Another expression vector is the pEF-BOS plasmid containing the polypeptide elongation factor 1 alpha promoter, which efficiently stimulates transcription in vitro. This plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection assays is disclosed by, for example, Demoulin (Molecular Cell Biology, 16:4710-4716, 1996). Other preferred expression vectors are adenoviruses described by Stratford-Perricaudet, which lack the El and E3 proteins (J. Clin. Invest. 90:626-630, 1992). Immunization using an adenovirus-expressed protein was described by Warnier et al., using subcutaneous injection in murines for anti-PIA immunization (Int. J. Cancer, 67:303-310, 1996).

The present invention also includes so-called expression kits, which enable the skilled person to prepare the desired expression vector or vectors. Such expression kits comprise at least two of the previously discussed substances in isolation. Other components can be added as needed.

As described herein, the invention has a variety of uses, some of which are described herein. First, it enables the skilled person to diagnose lesions characterized by the expression of MAGE-A12 immunogenic polypeptides. These methods include determining the expression of a MAGE-A12 HLA-binding peptide, or determining the expression of a complex of a MAGE-A12 HLA-binding peptide and an HLA class I molecule, in a biological sample. Expression of the peptide or expression of the peptide and HLA class I complex can be determined by testing with binding pairs of the peptide or complex, such as antibodies. Expression of MAGE-A12 in biological samples (eg, tumor biosections) can also be determined by standard PCR amplification using MAGE-A12 primers. Examples of tumor expression are provided herein, and further exemplary conditions and primers for MAGE-A12 amplification can be found in US Ser. No. 09/018,422.

Preferably, the diagnostic method comprises contacting a biological sample isolated from a subject with a reagent specific for a MAGE-A12 HLA-binding peptide to detect the presence of the MAGE-A12 HLA-binding peptide in the biological sample. As used herein, "contacting" means placing the biological sample in sufficient proximity to the reagent under appropriate conditions (such as concentration, temperature, time, ionic strength) so that the reagent binds to the MAGE-A12 HLA present in the biological sample interactions between peptides. Generally, the conditions of contacting the reagent with the biological sample are those known to those skilled in the art to facilitate the molecules and their associated substances (such as proteins and their receptor receptors, antibodies and their protein antigens, nucleic acids) in the biological samples. and its complementary sequence associates). Exemplary conditions that facilitate specific interactions between a molecule and its cognate are described in US Patent No. 5,108,921 to Low et al.

Biological samples can be fixed in vivo or in vitro. For example, the biological sample can be a tissue in vivo, and reagents specific for MAGE-A12 immunogenic polypeptides can be used to detect the presence of such molecules in the tissue. Alternatively, biological samples can be fixed in vitro (eg, blood samples, tumor sections, tissue extracts). In a particularly preferred embodiment, the biological sample is a cell-containing sample, more preferably a tumor cell-containing sample.

The invention further includes nucleic acid or protein microarrays comprising MAGE-A12 HLA binding peptides or nucleic acids encoding such peptides. In this aspect of the invention, standard techniques of microarray technology can be used to assess the expression of MAGE-A12 HLA-binding peptides and/or to identify biological constituents that bind such peptides. The composition of biological samples includes antibodies, HLA molecules, lymphocytes (especially T lymphocytes), and the like. Microarray technology, which is also known by other names including protein chip technology and solid phase protein array technology, is well known to those of ordinary skill in the art based on, but not limited to, obtaining An array of identified peptides or proteins binds a target molecule or biological component to the peptides, and such binding is assessed. See, eg, G. MacBeath and S.L. Schreiber, "Printing Proteins as Microarrays for High-Throughput Function Determination," Science, 289(5485):1760-1763, 2000. Nucleic acid alignment, especially the alignment similarity rule for binding MAGE-A12 HLA-binding peptides, can also be used for diagnosis. For example, to identify subjects with a condition characterized by expression of the MAGE-A12 HLA binding peptide.

Microarray substrates include, but are not limited to, glass, silicon, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, various clays, nitrocellulose, or nylon. Microarray substrates can be coated with compounds to enhance the synthesis of probes (peptides or nucleic acids) on the substrate. A coupling reagent or set of reagents on a substrate can be used to covalently bind a first nucleotide or amino acid to the substrate. Various coupling reagents or sets of reagents are well known to those skilled in the art. Peptide or nucleic acid probes can be synthesized directly onto a substrate in a predetermined support network. Alternatively, the peptide or nucleic acid can be spotted on the substrate, in which case the substrate can be coated with a compound to enhance the binding of the probe to the substrate. In these embodiments, pre-synthesized probes can be applied to the substrate in precise, pre-determined amounts and in a grid pattern, preferably using a computer-controlled robot to print the probes in a contact-printed manner or in a non-contact manner. (e.g. inkjet or piezo-electronic delivery) applied to the substrate. Probes can be covalently bound to a substrate.

In some embodiments, one or more control peptides or nucleic acid molecules are attached to the substrate. Preferably, control nucleic acid molecules allow determination of factors such as binding properties, reagent quality and availability, hybridization success, and analytical limits and success rates.

The present invention also enables the skilled artisan to treat subjects suffering from lesions characterized by the expression of a MAGE-A12 immunogenic polypeptide. Treatment includes administration of agents that increase the complex of MAGE-A12 HLA-binding peptides and HLA class I molecules in the subject, and administration of CD8+ T lymphocytes specific for this complex. Agents used in the aforementioned treatments include MAGE-A12 immunogenic polypeptides and functional variants thereof, complexes of such peptides and HLA class I binding molecules (such as HLA Cw*07), bearing MAGE-A12 immunogenic polypeptides Antigen-presenting cells in complexes with HLA class I binding molecules, soluble single-chain fusions of HLA and MAGE-A12 polypeptides, etc. The present invention also enables the skilled person to selectively increase the population of T lymphocytes, CD8+ T lymphocytes specific for the MAGE-A12 HLA binding peptide.

The isolation of the MAGE-A12 HLA-binding peptide also enables the isolation or design of nucleic acids encoding the MAGE-A12 HLA-binding peptide. Nucleic acids can be used to produce MAGE-A12 HLA binding peptides or proteins containing such peptides in vitro or in prokaryotic host cells or in eukaryotic host cells. Various methods well known to those skilled in the art can be used to obtain isolated MAGE-A12 HLA binding peptides. For example, expression vectors can be introduced into cells to produce peptides. In another approach, mRNA transcripts can be microinjected or otherwise introduced into cells to produce encoded peptides. Translation of mRNA in cell-free extracts such as in a reticulocyte lysis system can also be used to generate peptides. Peptides including the MAGE-A12 HLA binding peptides of the present invention can also be synthesized in vitro. To obtain isolated MAGE-A12 HLA-binding peptides, those skilled in the art can also readily use known methods for isolating peptides. These methods include, but are not limited to, immunochromatography, HPLC, size exclusion chromatography, ion exchange chromatography and immunoaffinity chromatography.

These isolated MAGE-A12 HLA-binding peptides, or complexes of peptides and HLA class I molecules such as HLA-Cw ^* 07 molecules, can be combined with, for example, auxiliary substances to prepare therapeutic applications characterized by expressing MAGE-A12 immunogenicity Vaccines in Peptide Diseases. In addition, vaccines can be prepared from cells presenting the MAGE-A12 HLA-binding peptide/HLA complex on their surface, such as transfected dendritic cells, transfected B cells, non-proliferative transfectants, and the like. In all cases where the cells are used as vaccines, these cells may be cells transfected with the coding sequence with one or both of the components required to stimulate CD8+ lymphocytes, or may already express both molecules without the need for transfection. stained cells. Vaccines can also include expression vectors and naked DNA or RNA encoding MAGE-A12 HLA binding peptides, their precursors, or fusion proteins thereof, and vaccines can be prepared in vitro and administered by injection, particle bombardment, nasal inhalation, and other methods. Vaccines of the "naked nucleic acid" type have been shown to stimulate an immunogenic response, including the generation of CTLs specific for the peptide encoded by the naked nucleic acid (Science 259:1745-1748, 1993).

MAGE-A12 HLA-binding peptides, and complexes of MAGE-A12 HLA-binding peptides and HLA molecules, can also be used to prepare antibodies using standard techniques in the art. Standard literature works setting out the general principles of antibody production include Catty, D., Antibodies, A Practical Approach, Vol. 1, IRL Press, Washington DC (1988); Klein, J., Immunology; The Science of Cell-Non-Cell Discrimination (Immunology: The Science of Cell-Non-Cell Discrimination) , John Wiley and Sons, New York (1982); Kennett, R., et al. Monoclonal Antibodies, Hybridoma, A New Dimension In Biological Analyzes (monoclonal antibodies, hybridomas, directions in biological analysis ), Plenum Press, New York (1980); Campbell, A., Monoclonal Antibody Technology, in Laboratory Techniques and Biochemistry and Molecular Biology (laboratory techniques and Monoclonal Antibody Technology in Bioactivity and Molecular Biology ) , Volume 13 (Burdon, R. et al. EDS.), Elsevier Amsterdam (1984); and Eisen, HN, Microbiology (Microbiology), 3rd Edition, Davis , BD et al. EDS (Harper & Rowe, Philadelphia (1980)).

Accordingly, the antibodies of the present invention can be prepared by various methods, including administering to animals proteins, protein fragments, cells expressing proteins or fragments thereof, and appropriate HLA class I molecules to induce polyclonal antibodies. Monoclonal antibodies can be prepared according to techniques well known in the art.

It is worth noting that, as is well known in the art, only a small fraction of the antibody molecule (the paratope) is involved in the binding of the antibody to its antigenic epitope (see, Clark, WR (1986) The Experimental Foundations of Modern Immunology (now Immunological Experiments Essential) Wiley & Sons Ltd., New York; Roitt, I. (1991) Essential Immunology , Seventh Edition, Blackwell Scientific Publications. Oxford). For example, pFc' and Fc regions are effectors of complementary cascades but are not involved in antigen binding. From antibodies in which the pFc' region has been enzymatically cleaved, or produced without the pFc' region, designated F(ab') ₂ fragments, both antigen-binding sites of the intact antibody are maintained. Similarly, from antibodies in which the Fc region has been enzymatically cleaved, or made without the Fc region, designated Fab fragments, retain an antigen-binding site of the intact antibody. Also, a Fab fragment comprising a covalently bound antibody light chain and a portion of an antibody heavy chain is called Fd. The Fd fragment is the main determinant of antibody specificity (a single Fd fragment can be associated with up to 10 different light chains without altering antibody specificity), and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see , such as Clark, 1986; Roitt, 1991). In the heavy chain Fd fragment and light chain IgG immunoglobulins, there are four framework regions (FR1 to FR4), separated by three complementarity determining regions (CDR1 to CDR3), respectively. The CDRs, especially the CDR3 region, and more specifically the heavy chain CDR3 play a large role in antibody specificity.

It is now well established in the art that non-CDR regions of mammalian antibodies can be replaced with similar regions of co-specific or different specific antibodies while maintaining the epitope specificity of the source antibody. This is most clearly demonstrated in the development and use of "humanized" antibodies in which non-human CDRs are covalently attached to human FR and/or Fc/pFc' regions to produce functional antibodies. See, eg, US Patent Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.

Thus, for example, PCT International Application Serial No. WO92/04381 teaches the preparation and use of humanized murine RSV antibodies in which at least part of the murine FR regions have been replaced by human FR regions. Such antibodies, including fragments of intact antibodies that have the ability to bind antigen, are often referred to as "chimeric" antibodies.

Therefore, it is obvious to those of ordinary skill in the art that the present invention also provides F(ab) ₂ , Fab, Fv and Fd fragments; wherein Fc and/or FR and/or CDR1 and/or CDR2 and/or or chimeric antibodies in which the light chain CDR3 region has been replaced by homologous human or non-human sequences; chimeric antibodies in which FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences F(ab) ₂ fragment antibodies; chimeric Fab fragment antibodies wherein FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; wherein FR and/or CDR1 and/or Or a chimeric Fd fragment antibody in which the CDR2 region has been replaced by a homologous human or non-human sequence. The invention also includes so-called single chain antibodies and human monoclonal antibodies, such as antibodies produced by murine animals with functional human immunoglobulin loci.

Such antibodies can be used to identify tissues expressing the protein or to purify the protein. For therapeutic purposes antibodies may also be conjugated to specific labeling reagents for imaging or to antineoplastic agents including, but not limited to, methotrexate, radioiodinated compounds, toxins such as ricin, other cytostatic drugs or cytolytics drugs, etc. Antibodies prepared according to the invention are also preferably specific for the peptide/HLA complexes described herein.

When "disorder" or "condition" is used herein, it refers to any pathological condition in which a MAGE-A12 immunogenic polypeptide is expressed. Such lesions include cancers including bladder cancer, malignant melanoma, esophageal cancer, lung cancer, head and neck cancer, breast cancer, colon cancer, myeloma, brain tumor, malignant sarcoma, prostate cancer and kidney cancer. Some methods of treatment according to the disclosure are predicated on inducing a subject's immune system response to MAGE-A12 immunogenic polypeptide presenting cells. One such approach is to administer autologous CD8+ T cells specific for a complex of a MAGE-A12 HLA-binding peptide and an HLA class I molecule to a subject suspected of having an abnormal cellular phenotype. It is within the skill of the skilled person to develop such CD8+ T cells in vitro. Typically, sample cells obtained from a subject, such as blood cells, are contacted with cells that present the complex and are capable of stimulating proliferation of CD8+ T lymphocytes. Target cells can be transfectants, such as transfected COS cells, or transfected antigen-presenting cells with HLA class I molecules, such as dendritic cells or B cells. These transfectants transfect the desired complexes on their surface and, when bound to CD8+ T lymphocytes of interest, stimulate their proliferation. COS cells are other suitable host cells that are widely used. The subjects are then administered clonally expanded autologous CD8+ T lymphocytes. CD8+ T lymphocytes stimulate the subject's immune response, so as to achieve the desired therapeutic purpose.

Another method for selecting antigen-specific CTL clones has been described (Altman et al., Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416, 1998), in which MHC I Fluorophore tetramers of molecule/peptide complexes to detect specific CTL clones. Briefly, soluble MHC class I molecules are folded in vitro in β2-microglobulin and class I-bound peptide antigens. After purification, the MHC/peptide complexes are purified and labeled with biotin. Tetramers are prepared by mixing biotinylated peptide-MHC complexes with labeled avidin (such as phycoerythrin) at a molar ratio of 4:1. The tetramers are then contacted with a CTL source such as peripheral blood or lymph nodes. Tetramers bind CTLs that recognize peptide antigen/MHC class I complexes. Cells bound by tetramers can be sorted by fluorescence activated cell sorter to isolate active CTL. The isolated CTL can then be expanded in vitro for use as described above.

To describe the method of treatment in detail, the method called adoptive transfer (Grenberg, J. Immunol. 136(5): 1917, 1986; Riddel et al., Science 257: 238, 1992; Lynch et al., European Journal of Immunology, 21: 1403-1410, 1991; Kast et al., Cell 59:603-614, 1989), cells presenting the desired complex are contacted with CTLs, resulting in the proliferation of CTLs specific for them. The expanded CTLs are then administered to a subject suffering from a cellular abnormality characterized by certain abnormalities in cells presenting a particular complex. CTLs lyse abnormal cells, thereby achieving the desired therapeutic purpose.

The aforementioned treatments assume that at least some of the subject's abnormal cells present the associated HLA/TRA complex. This is readily ascertainable, since the art is very familiar with methods for identifying cells presenting a particular HLA molecule, and how to identify cells expressing DNA presenting the sequence of interest, in this case a tumor-associated gene sequence. Once cells presenting the relevant complexes have been identified by the aforementioned screening techniques, these cells can be combined with a sample obtained from a patient, where the sample contains CTLs. If complex-presenting cells are lysed by pooled CTL samples, it can be assumed that tumor-associated gene-derived TRAs are present and the subject is a suitable candidate for the aforementioned therapeutic approach.

Adoptive transfer is not the only therapy for which the present invention can be used. CD8+ T lymphocytes can also be stimulated in vivo using a variety of methods. One approach is to use non-proliferating cells to express the complex. The cells used in this method may be cells that normally express the complex, they may be dendritic cells or cells transfected with one or both of the genes required for presentation of the complex. This approach is exemplified by Chen et al. (Proc. Natl. Acad. Sci. USA 88: 110-114, 1991), showing the use of transfected cells expressing HPV-E7 peptides in therapy. A variety of cell types can be used. Similarly, vectors with one or two genes of interest can also be used. Viral or bacterial vectors are particularly preferred. For example, a nucleic acid encoding a MAGE-A12 HLA-binding peptide can be operably associated with promoter and enhancer sequences that direct the expression of the MAGE-A12 HLA-binding peptide in certain tissues or cell types. Nucleic acids can be incorporated into expression vectors. The expression vector can be an unmodified extrachromosomal nucleic acid, a plasmid or a viral genome constructed or modified to allow insertion of foreign nucleic acid (such as a nucleic acid encoding a MAGE-A12 HLA-binding peptide). Nucleic acids encoding MAGE-A12 HLA-binding peptides can also be inserted into the retroviral genome, which facilitates integration of the nucleic acids into the genome of the target tissue or cell type. In these systems, the ones of interest are carried by microorganisms, such as vaccinia viruses, retroviruses or bacterial BCG, and substances that actually "infect" the host cell. Cells presenting the complex of interest are recognized by autologous CD8+ T cells and subsequently proliferate.

A similar effect can be obtained by conjugating the MAGE-A12 HLA-binding peptide with an adjuvant to facilitate its incorporation into HLA class I presenting cells in vivo. If the MAGE-A12 HLA-binding peptide is larger than the HLA class I binding portion, the MAGE-A12 HLA-binding peptide can be processed if desired to generate a peptide set of HLA molecules that do not require further processing when TRA is presented. Typically, a subject is injected subcutaneously with an effective amount of a MAGE-A12 immunogenic polypeptide. The initial dose may be followed by booster doses according to standard immunization protocols in the art.

As part of certain immunization compositions, one or more cancer-associated antigens or stimulatory fragments thereof may be administered with one or more adjuvants to induce or augment an immune response. An adjuvant is a substance that is incorporated into or administered with an antigen that boosts an immune response. Adjuvants can enhance immunogenic responses by providing antigen depots (eg, extracellularly or within macrophages), activating macrophages, and stimulating specific groups of lymphocytes. A wide variety of adjuvants are well known in the art. Specific examples of adjuvants include Monophosphoryl Lipid A (MPL, SmithKline Beecham), congeners obtained after acid hydrolysis and purification of Salmonella minnesota Re595 lipopolysaccharide; saponins, including QS21 (SmithKline Beecham), obtained from Quillaja saponaria extract Pure QA-21 saponins purified in ®; DQS21 (SmithKlineBeecham), QS-7, QS-17, QS-18, and QS-L1 described in PCT application WO96/33739 (So et al., Molecular Cell 7: 178-186, 1997); Incomplete Freund's adjuvant; Complete Freund's adjuvant; montanide; nucleotides); various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or vitamin E. Preferably, the peptide is administered in admixture with DQS21/MPL, the ratio of DQS21 to MPL is generally about 1:10 to 10:1, preferably about 1:5 to 5:1, more preferably about 1:1. Typically for human administration, DQS21 and MPL are present in the vaccine formulation in the range of about 1 ug to about 10 ug. Other adjuvants are also known in the art and can be used in the present invention (see, eg, Goding, Monoclonal Antibodies: Principles and Practice, Second Edition 1986). Methods of preparing mixtures or emulsions of peptides and adjuvants are well known to those of ordinary skill in the vaccine arts.

Other agents that stimulate an immune response in a subject can also be administered to the subject. For example, other cytokines may also be used in vaccine regimens due to their lymphocyte modulating properties. Many other cytokines for this purpose are well known to those of ordinary skill in the art, including interleukin-12 (IL-12), which has been shown to enhance the protective effect of vaccines (see, e.g., Science 268: 1432-1434, 1995), GM-CSF and IL-18. Thus, cytokines can be administered in combination with antigens and adjuvants to increase the immune response to the antigen.

There are many other immune response enhancing compounds that can be used in vaccine regimens. These compounds include co-stimulatory molecules provided in polymorphic or nucleic acid form. Such co-stimulatory molecules include B7-1 and B7-2 (CD80 and CD86, respectively), which are expressed on dendritic cells (DCs) and interact with the molecule CD28 expressed on T cells. This interaction provides co-stimulation (signal 2) of antigen/MHC/TCR-stimulated (signal 1) T cells, increasing T cell proliferation and effector function. B7 also interacts with CTLA4 (CD152) on T cells, and studies on CTLA4 and B7 ligands have shown that the interaction between B7-CTLA4 can enhance anti-tumor immunity and CTL proliferation (Zheng, et al., Proc. Natl. Acad. Sci. USA 95:6284-6289, 1998).

B7 is not normally expressed on tumor cells, so they are not effective antigen presenting cells (APCs) for T cells. Induction of B7 expression will more effectively stimulate CTL proliferation and effector function. The combination of B7/IL-6/IL-12 co-stimulation has been shown to induce the distribution of IFN-γ and Th1 cytokines in the T cell population, resulting in further enhancement of T cell activity (Gajewski et al., Journal of Immunology, 154: 5637-5648, 1995). Transfection of tumor cells with B7 has been discussed in in vitro CTL expansion for adoptive transfer by Wang et al. (J. Immnother. 19:1-8, 1996). Other delivery systems for the B7 molecule would include nucleic acid (naked DNA) immunization (Kim et al., Nature Biotechnol. 15:7:641-646, 1997) and recombinant viruses such as adenoviruses and poxviruses (Kim et al., Gene Ther .4:726-735, 1997). These systems are also suitable for the application and construction of expression cassettes for the co-expression of B7 with other selected molecules, such as the antigens or antigen fragments (including polyepitopes) or cytokines discussed herein. These delivery systems can be used to induce appropriate molecules in vitro as well as for use in in vivo vaccination situations. Direct stimulation of T cells using anti-CD28 antibodies in vivo and in vitro is also contemplated. Similarly, the inducible co-stimulatory molecule ICOS, which induces T cell responses to exogenous antigens, can also be modulated, for example by using anti-ICOS antibodies (Hutloff et al., Nature 397:263-266, 199).

Lymphocyte function-associated antigen-3 (LFA-3) is expressed on APCs and some tumor cells and interacts with CD2 expressed on T cells. This interaction induces the production of IL-2 and IFN-γ in T cells, and thus can complement or replace, B7/CD28 co-stimulatory interaction (Parra et al., Journal of Immunology, 158:637-642, 1997; Fenton et al. People, J. Immunother. 21:95-108, 1998).

Lymphocyte function-associated antigen-1 (LFA-1 ) is expressed on leukocytes and interacts with ICAM-1 expressed on APCs and some tumor cells. This interaction induces T cell IL-2 and IFN-γ production, and is thus able to complement the single-displacement, B7/CD28 co-stimulatory interaction (Fenton et al., 1998). Thus, LFA-1 is another example of a co-stimulatory molecule that can be provided in various ways in the B7 immunization regimen discussed above.

Full CTL activation and effector function requires Th cell help through interactions between Th cell CD40L (CD40 ligand) molecules and CD40 molecules expressed by DCs (Ridge et al., Nature 393:474, 1998; Bennett et al. , Nature 393:478, 1998; Schoenberger et al., Nature 393:480, 1998). The mechanism of this co-stimulatory signal may include B7 upregulation and associated IL-6/IL-12 production by DCs (APCs). Thus, the CD-40-CD40L interaction complements the Signal 1 (antigen/MGC-TCR) and Signal 2 (B7-CD28) interactions.

Direct stimulation of CD cells using anti-CD40 antibodies is expected to enhance responses to tumor-associated antigens, which are often encountered outside the inflammatory environment or presented by non-professional APCs (tumor cells). In these cases, Th helper and B7 co-stimulatory signals were not provided. This mechanism could be used in antigen-pulsed DC-based therapy, or in cases where Th epitopes have not been restricted to known tumor-associated antigen precursors.

When administered, the medical compositions of the present invention are administered as pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, supplemental immune enhancing agents such as adjuvants and cytokines, and any other therapeutic agents. "Pharmaceutically acceptable" means a non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient. The characteristics of the carrier will depend on the route of administration.

The therapeutics of the present invention may be administered by any conventional route, including injection or by infusion over a period of time. The administration may be oral, intravenous, intraperitoneal, intramuscular, intranasal, intracavity, subcutaneous, intradermal or transdermal.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as palm oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, ethanol/aqueous solutions, emulsions or suspensions. Includes saline and buffered media. Injectable vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases, among others.

The present invention also contemplates gene therapy. Methods of performing gene therapy in vitro are described in US Patent No. 5,399,346 and in the presentations filed during the filing of that patent, all of which are publicly available documents. Typically, gene therapy involves introducing a functional copy of a gene in vitro into cells of a subject that contain a defective copy of the gene, and then returning the genetically engineered cells to the subject. A functional copy of a gene is under the operable control of regulatory factors that cause the gene to be expressed in a genetically engineered cell. Many transfection and transduction techniques, as well as appropriate expression vectors, are well known to those of ordinary skill in the art, some such techniques are described in PCT application WO95/00654. In vivo gene therapy using vectors such as adenovirus is also contemplated according to the invention.

The preparations of the invention are administered in an effective amount. An effective amount refers to the amount of a pharmaceutical preparation which, alone or in combination with further doses, stimulates the desired response. In the case of treating cancer, the desired response is to inhibit the development of the cancer. This may involve only temporarily slowing the progression of the disease, although more preferably, involves permanently halting the progression of the disease. In the case of induction of an immune response, the desired response is an increase in antibodies or T lymphocytes specific to the MAGE-A12 immunogen used. These desired responses can be monitored by conventional methods, or can be monitored according to the diagnostic methods of the invention described herein.

Where it is desired to stimulate an immune response using the medicinal composition of the invention, this may also include stimulation of a humoral antibody response leading to increased antibody titers in serum, clonal expression of cytotoxic lymphocytes, or some other desired immunogenic response. It is believed that doses of immunogen ranging from 1 ng/kg to 100 mg/kg, depending on the mode of administration, will be effective. A preferred range is believed to be between 500 ng and 500 mg/kg. The absolute amount will depend on a variety of factors, including the substance chosen to be administered, whether it is administered in single or multiple doses, and individual patient parameters including age, physical condition, size, weight, and stage of the disease. These factors are well known to those of ordinary skill in the art and can be obtained by routine experimentation.

Example

materials and methods

cell line

Bladder cancer cell line LB831-BLC, LB831 (HLA-A ^* 2403, -A3, -B ^* 4403, -B ^* 4901, -Cw) derived from a primary invasive bladder tumor (pT3, G3) in a 65-year-old Caucasian patient ^* 0401, -Cw ^* 07). The cell line chromatin pattern exhibited at passage 7 showed changes in chromosome number from 56 to 144, confirming that the LB831 cell line was a tumor line. MI13443-MEL is a malignant melanoma cell line, and LE9211-RCC is a renal carcinoma cell line. Both germlines were derived from HLA-Cw7-negative patients. LB373-MEL is a malignant melanoma line derived from HLA-Cw7-negative patients. Tumor cells were cultured in Iscove medium (Life Technologies, Gaithersburg, MD) containing 10% human serum or 10% FCS (Life Technologies) in an 8% carbon dioxide incubator. Supernatants from B95-8 cells transfected with EBV at 1 ug/ml of Cyclosporine A (Sandoz, Basel, Switzerland) and 20% (v/v) were derived from PBL of patient LB831 using standard techniques. Lymphoblastoid cell line LB-831-EBV. This cell line was grown in RPMI-1640 medium (Life Technologies) containing 10% FCS in a 5% carbon dioxide incubator. Prepare PBL-PHA cells by stimulating PBL with 1.0% (volume ratio) PHA (Difco) and 100 U/ml IL-2 (Eurocetus, Amsterdam, Netherlands), and place PBL-PHA cells in an incubator containing 8% carbon dioxide at 10 Cultured in Iscove's medium with % human serum. All media were supplemented with L-arginine (116ug/ml), L-asparagine (36ug/ml), L-glutamine (216ug/ml), streptomycin (0.1mg/ml) and penicillin ( 200U/ml).

Anti-tumor CTL clones

Blood mononuclear cells from patient LB831 were isolated using Lymphoprep (Nycomed, Oslo, Norway) density gradient centrifugation and stored at -80°C. Irradiated B7-1 transfected LB831-BLC cells as stimuli and CD8+ T lymphocytes as responders were mixed as previously described (Gueguen et al., Journal of Immunology 160:6188-6194, 1998). To implement self-mixed lymphocyte-tumor cell culture. CD8+ T lymphocytes were sorted using magnetic beads covalently bound to anti-CD8 antibody (MACS, Miltenyi Biotec GmbH). Irradiated non-CD8+ cells were added to the mixed cultures in the first stimulation. On the third day, IL-2 (25 U/ml) was added. After one week, 5×10 ⁵ lymphocytes were restimulated with irradiated B7-1 transfected tumor cells and 25 U/ml IL-2. On day 28, lymphocytes were obtained from culture by limiting dilution in Iscove medium supplemented with IL-2 (50 U/ml). Long-term culture of CTL clones was performed as previously described (Herin et al., Int. J. Cancer 39:390-396, 1987).

Cytotoxicity test

CTLs were tested for lytic activity in a chromium release assay as previously described (Boon et al., J. Exp. Med. 152:1184-1193, 1980). Briefly, 1000 chromium-labeled cells in 100 ul were incubated in 96-dish microplates at different effector-to-target ratios with equal volumes of CTLs. Chromium release was measured after four hours of incubation. For peptide testing, labeled LB831-EBV cells were incubated with different concentrations of peptides for 30 min at 37 °C. CTL was then added and chromium release was determined as described above/

CTL stimulation assay

A total of 3000 CTLs were added to micro dishes containing 10000 stimulator cells in Iscove's medium supplemented with 10% human serum and 25 U/ml of IL-2. After 24 hours, the supernatant was collected and assayed for cytotoxicity against 13 cells of the WEHI-164 clone in the MTT colony count assay (Hansen et al., J. Immunol. Meth. 119:203-210, 1989). The TNF content of the supernatant was determined (Espevik and Nissen-Meyer, J. Immunol. Methods 95:99-105, 1986). By adding 1/20-1/30 diluted ascitic fluid to the test, with mAbsW6/32 (anti-HLA class I), Bi.23.3 (anti-HLA-B and -C), B9.4.1 (anti-CD8) , 1B8.2 (anti-CD4, donated by D. Olive, INSERM U119, Marseille, France), GAPA-3 (anti-HLA-A3), C7709A2.6 (anti-HLA-A24) perform inhibition.

transient transfection

Transient transfection was performed with LipofectAMINE ^™ reagent (Life Technologies). Briefly, 5 × ¹⁰⁴ 293-EBNA cells (293 cells expressing EBV nuclear antigen EBNA-1) were plated in flat-bottomed 96-dish plates with 100 ng of DNA from MAGE-A12 cDNA or cloned into pcDNA3 (Invitrogen, Carlsbad, CA) 50ng of the plasmid pcDNA3 containing HLA-Cw ^* 0701, and 1.5ul LipofectAMINE ^TM transfection. LB373-MEL cells (10,000) were transfected with 150 ng of HLA-Cw7 construct and 1 ul of LipofectAMINE ^™ . After 24 hours, transfected cells were assayed in a CTL stimulation assay.

Cloning of HLA-Cw7 cDNA from LB831-BLC was performed as previously described (Gueguen et al., 1998). Its sequence is identical to allelic subtype Cw ^* 07011, except at position 1087 of the coding sequence, where G is substituted for A. This nucleotide change results in the substitution of alanine for threonine in the intracytoplasmic region of the molecule. The same difference between the Cw ^* 0704 and Cw ^* 0711 alleles has been described (Baurain and Coulie, Tissue Antigen 53:510-512, 1999).

Stable transfection of tumor cell germlines

Bladder cancer cells LB831-BLC were transfected by calcium phosphate precipitation as previously described (Traversari et al., Immunogenetics 35: 145-152). 1 × ¹⁰ cells were transfected with 20 ug of plasmid pEF-BOS purine-PL3 containing cDNA B7-1. Briefly, using cDNA obtained from the cell line LB23-EBV as a template, the sense primer 5'-GGGTCCAAATTGTTGGCTTTCACT (SEQ ID NO:7) and the antisense primer 5'-GAAGAATGCCTCATGATCCCCA (SEQ ID NO:8) were used in a PCT reaction. ) amplifies B7-1. PCR conditions were described by Gueguen et al. 1998. The B7-1 insert was then cloned into the plasmid pEF-BOS purine-PL3, which was obtained from pEF-BOS by insertion of the puromycin resistance gene and polylinker (Mishizuma and Nagata, Nucleic Acids Res. 18: 5322, 1990) derived. Puromycin resistant LB831-BLC cells were picked in 0.8ug/ml puromycin (Sigma, St. Louis, MO) and then cloned by limiting dilution.

Cloning of Subgene Fragment of Gene MAGE-A12

The MAGE-A12 cDNA containing the complete open reading frame (ORF) of 945 base pairs was used as a template for PCR amplification (DePlaen et al., Immunogenetics 40:360-369, 1994). Eight fragments containing the first 195, 342, 525, 540, 591, 651, 683 and 816 nucleotides of the MAGE-A12 ORP were amplified using the following primers, respectively:

5'-CCTACCTGCTGCCCTGACCA-3' (LHE7; SEQ ID NO: 46) and reverse primer

5'-CCAACTAAGCCATCTTCCTA-3' (LHE2; SEQ ID NO: 47)

5'-CCAACTAAGCCATCTTCCTA-3' (LHE3; SEQ ID NO: 48)

5'-GTGACAAGGATCTACAAGTG-3' (LHE4; SEQ ID NO: 49)

5'-CCAGTCAGGTGACAAGGATG-3' (LHE10; SEQ ID NO: 50)

5'-CCTGTCTAGGGCACGATCTG-3' (LHE8; SEQ ID NO: 51)

5'-CTCCTAAGGGGCACAGTCGC-3' (LHE8; SEQ ID NO: 52)

5'-TCAGATGCCTACAACACT-3' (LHE5; SEQ ID NO: 53)

5'-GGACCCTACAGGAACTCGTRA-3' (LHE6; SEQ ID NO: 54). Taq DNA polymerase (TaKaRa, Taq) was used for PCR amplification. The first denaturation step was performed at 94°C for 5 minutes, followed by 25 cycles of amplification consisting of: 1 minute at 94°C for all primers, 2 minutes at 62°C for LHE3, LHE4 and LHE5 primers or 64°C for LHE2, LHE6, LHE8, LHE9 and LHE10 primers for 2 min, and all primers at 72 °C for 3 min. The cycle ends with a final extension step of 10 min at 72 °C. The PCR product was cloned into the pcDNA3 vector using the BidirectionalEukaryotic TOPO TA cloning kit (Invitrogen).

PCR test of MAGE-A12 expression

The expression of MAGE-A12 in tumor tissues was detected by RT-PCR. Total RNA purification and cDNA synthesis were performed as previously described (Weynants et al., Int. J. Cancer 56:826-829, 1994). One fortieth of cDNA generated from 2 ug of total RNA was synthesized using sense primer 5'-CGTTGGAGGTCAGAAACAG-3' (SEQ ID NO:55) and antisense primer 5'-GCCTCCCACTGATCTTTAGCAA-3' (SEQ ID NO:56) Amplify. For PCR, the first denaturation step was performed at 94°C for 4 minutes, followed by 32 cycles of amplification including: 1 minute at 94°C, 2 minutes at 62°C, and 3 minutes at 72°C. The cycle ends with a final extension step of 15 min at 72°C.

Example 1: Derived from the gene MAGE-A12 and derived from the HLA-

A Novel Antigenic Peptide Recognized by Cw ^* 0701 Restricted CTL

LB831-BLC is a bladder cancer cell line expressing at least three antigens recognized by self CTLs. One of them has been described (Gueguen et al., Immunology 160:618806194, 1998). Self-mixed lymphocyte-tumor cell cultures were performed by mixing irradiated B7-1 transfected LB831-BLC cells cultured in serum as stimulators and CD8+ T lymphocytes as responders. CD8+ T lymphocytes were sorted using magnetic beads covalently bound to anti-CD8 antibody (Magnetic Activated Cell Sorter, MACS, Miltenyi Biotec, Gergisch-Gladbach, Germany). Irradiated non-CD8+ cells were added to the mixed cultures in the first stimulation. On the third day, IL-2 (50 U/ml) was added. After one week, 5×10 ⁵ lymphocytes were restimulated with irradiated B7-1 transfected tumor cells and 25 U/ml IL-2. On day 28, lymphocytes were obtained from culture by limiting dilution in Iscove medium supplemented with IL-2 (50 U/ml). A panel of BL831-specific CTL-clones was obtained in which CTL 501D/19 recognized autologous tumor germlines but not autologous EBV-transformed B-cells (i.e., it recognized an antigen other than the first three, called LB831- D).

To determine the MHC restriction on these CTL clones, the effect of anti-HLA mAb on clone stimulation was studied. When stimulated with LB831-cells, CTL clone 501D/19 produced TNF in the presence of anti-HLA class I mAb W6/32 or in the presence of a monoclonal antibody directed against the common determinant of HLA-B and -C molecules (mAb B1 .23.2), this output is completely blocked. Since the HLA classes of LB831 are HLA-A ^* 2403, -A ^* 3, -B ^* 4901, -Cw ^* 0401, and -Cw ^* 07, this result indicates that the target gene consists of HLA-B ^* 44, B ^* 49, CW ^* 04 Presentation or by CW ^* 04 Presentation.

Additional target cells are recognized by CTL501D/19 in a standard 4 hr chromium release assay. As shown in Figure 1A, CTL 501D/19 also lysed two allogeneic tumor lines, malignant melanoma MI13443-MEL and renal carcinoma cell line LE9211-RCC. Targets are LB831-BLC (autologous bladder cancer cell line); LB831-EBV (autologous EBV-transformed B cells); LE9211-RCC (allogeneic HLA-Cw4 and HLA-Cw7-positive kidney cancer cell line); MI13443 - MEL (allogeneic HLA-Cw4 and HLA-Cw7-positive malignant melanoma cell lines); and K562 (natural killer target). Allogeneic and autologous tumor cell lines were pretreated with IFN[gamma] for 1 or 5 days, respectively, before use as targets, and chromium release was measured after 4 hours.

All aforementioned tumor cells were negative for B ^* 44 and B ^* 49, but positive for Cw ^* 04 and Cw ^* 07. To confirm which HLA presents tumor antigens, malignant melanomas were transiently transfected with Cw ^* 04 or Cw ^* 07 and tested for recognition by CTL501D/19. Only Cw ^* 07 transfectants were recognized by CTL. As shown in Figure 1B, LB373-MEL (a malignant melanoma cell line derived from an HLA-Cw7-negative patient) was transiently transfected with the HLA-Cw7 plasmid construct. Three thousand CTLs were added to 10000 stimulator cells, and TNF production by CTLs was measured 24 hours later. Since CTL501D/19 also recognizes the HLA-Cw ^* 07-positive malignant melanoma cell line NaMe16, it can be concluded that the antigen LB831-D is presented by HLA-Cw ^* 07 molecules. The allelic subtype of Cw ^* 07 of LB831-BLC was determined and found to be HLA-Cw ^* 07011.

To determine whether the LB831-D antigen recognized by CTL501D/19 is encoded by a known gene, 293-EBNA cells were treated with an expression vector containing the HLA-Cw ^* 0701 cDNA and including MAGE, which was found to be abundantly expressed in bladder tumor samples from patient LB831. , BAGE, GAGE, RAGE, LAGE ^NY-ESO cDNA (and others) were co-transfected with a series of genes. The ability of transfectants to stimulate CTL501D/19 to produce TNF was determined. 24 hours after transfection, cells were incubated with 3000 cells of CTL501D/19. After 24 hours, TNF in the supernatant was determined by measuring the cytotoxicity of TNF on WEHI-164-13 cells. LB831-BLC cells were used as a positive control. CTL produced TNF only when stimulated with 293-EBNA cells transfected with HLA-Cw ^* 07 and the gene MAGE-A12 (Fig. 2). No stimulation was observed in 293-EBNA cells transfected with HLA-Cw7 alone or with HLA-Cw7 and any other gene combination.

By RT-PCR, it could be determined that all tumor cell lines recognized by CTL501D/19 expressed MAGE-A12, confirming that the antigen is encoded by this gene. Other HLA-Cw7-restricted CTL clones generated in MLTC were also detected. Six other CTL clones were found to recognize 293-EBNA cells transfected with MAGE-A12 and HLA-Cw7.

To identify the MAGE-A12 sequence encoding the antigenic peptide, MAGE-A12 fragments of different lengths were generated by PCR. These subgene fragments were cloned into pcDNA3 and transfected into 293-EBNA cells together with the HLA-Cw ^* 0701 construct. The CTL stimulation test was carried out with the transfectants (Fig. 3). Transfected cells were incubated with CTL501D/19 for 24 hours and TNF production in the supernatant was determined by measuring its toxicity to WEHI-164.13 cells. The numbering of the PCR fragments corresponds to the nucleotides of the coding region.

Cells transfected with 540 base pairs or more stimulated CTL501D/19, whereas cells transfected with a shorter fragment failed to stimulate CTL501D/19. This indicated that the end of the sequence encoding the antigenic peptide was located between nucleotides 525 and 540 of the MAGE-A12 ORF. In the amino acid sequence corresponding to nucleotides 525-540, two overlapping nonapeptides were found, EVVRIGHLY (codons 168-176 of MAGE-A12; SEQ ID NO: 3) and VRIGHLYIL (codons 170-178 of MAGE-A12 ; SEQ ID NO: 4), which is consistent with the HLA-Cw7 peptide binding motif, ie, tyrosine or leucine at the C-terminus (Rammensee et al., Immunogenetics 41: 178, 1995). The decapeptide VVRIGHLYIL (SEQ ID NO:5), the nonapeptide VRIGHLYIL (SEQ ID NO:4) and the octapeptide RIGHLYIL (SEQ ID NO:6) sensitize the autolymphoblast lineage LB-831-EBV to cleavage by CTL501D/19 (Figure 4). Autologous cell lines were loaded with MAGE-A12 peptide VRIGHLYIL (SEQ ID NO: 4), VVRIGHLYIL (SEQ ID NO: 5), or RIGHLYIL (SEQ ID NO: 6) and contacted with CTL501D/19. Chromium-labeled cells were pulsed with indicated concentrations of peptide (VRIGHLYIL (SEQ ID NO: 4) or RIGHLYIL (SEQ ID NO: 6)) for 30 min. CTL501D/19 was added at an effector to target ratio of 10. Chromium release was measured after 4 hours. Peptides lacking a C-terminal leucine (eg, EVVRIGHLY, SEQ ID NO: 3) were also assayed, but not recognized. Half-maximal cleavage, 100 pM, was obtained using a very low concentration of the nonapeptide VRIGHLYIL (SEQ ID NO: 4), indicating that CTL501D/19 recognizes this peptide very efficiently.

Example 2: Expression of MAGE-A12 in tumor samples

Expression of MAGE-A12 in tumor samples and/or cell lines of various tumors was determined by RT-PCR. PCR amplification was performed using MAGE-A12 specific primers and conditions as described above. The results of the MAGE-A12 RT-PCR amplification are provided in Table I.

Expression of MAGE-A12 detected by RT-PCR Sample type Number of test samples MAGE-A12 positive samples N% Malignant melanoma of the skin, primary 83 28 34 Transferred 243 151 62 326 179 55 Esophageal squamous cell carcinoma 19 5 26 Adenocarcinoma 5 2 40 twenty four 7 29 squamous cell carcinoma of the lung 93 26 28 Adenocarcinoma 43 14 33 136 40 29 head and neck squamous cell carcinoma 85 twenty three 27 Bladder cancer superficial (<T2) 70 7 10 Infiltrated (≥T2) 53 18 34 123 25 20 breast cancer 50 8 16 rectal cancer 46 5 11 Myeloma stage I-II 11 0 0 Phase III 27 4 15 38 4 11 brain tumor 11 1 9 sarcoma 13 1 8 prostate gland gland twenty two 1 5 cancer kidney cancer 6 0 0 Uterine tumor 5 0 0 Thyroid tumor 4 0 0 Pleural mesothelioma 4 0 0 leukemia 112 0 0 equivalent

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are covered by the following claims. All references disclosed in this article are incorporated in this article in full.

Sequence Listing

<110> Ludwig Cancer Institute

<120> MAGE-A12 antigenic peptide and its application

<130>L0461/7075WO

<140>PCT/US00/28852

<141>2000-10-19

<150>US 60/160,374

<151>1999-10-19

<150>US 60/179,570

<151>2000-02-01

<160>56

<170> FastSEQ version 3.0 for Windows

<210>1

<211>4523

<212>DNA

<213>Homo sapiens

<220>

<221> CDS

<222>(2960)...(3904)

<400>1tggcctggga cccgcagcca ttctctacaa ggggtgcagc tgtgcaaatg cacagacgtt     60acagaaacag agtatctcct gccaatcact tcatccaaca gccaggagtg aggaagagga    120ccctcttgag tgaggactga gggtccaccc tcccccacgt agtgaccaca gaatccagct    180cagtccctct tgtcagccct gctaaactta ggcaataatg tcaccccgac cgcacccctc    240ccccagtgcc acttcagggg gactcagagt cagagacttg gtctgagggg agcagacaca    300atcggcagag gatggcggtc caggctcagc ctggcatcca agtcaggacc ttgagggatg    360accaaaggcc cctcccaccc ccaactcccc caaccccacc aggatctaca gcctcatgat    420ccccgtccct atccctaccc ctacccccaa caccatcttc atcgttacct ccacctccat    480ctggatcccc atccaggaag aatccagttc cacccctgct gtgaacccag ggaagtcacg    540gggccggatg tgacgccact gacttgcgcg ttggaggtca gagaacagcg agattctcgc    600cctgagcaac ggcctgacgt cggcggaggg aagcaggcgc aggctccgtg aggaggcaag    660gtaagatgcc gagggaggac tgaggcgggc ctcaccccag acagagggcc cccaataatc    720cagcgctgcc tctgctgcca ggcctggacc accctgcagg ggaagacttc tcaggctcag    780tcgccaccac ctcaccccgc caccccccgc cgctttaacc gcagggaact ctggtgtaag     840agctttgtgt gaccagggca gggctggtta gaagtgctca gggcccagac tcagccagga     900atcaaggtca ggaccccaag aggggactga gggtaacccc cccgcacccc caccaccatt     960cccatccccc aacaccaacc ccacccccat cccccaacac caaacccacc accatcgctc    1020aaacatcaac ggcaccccca aaccccgatt cccatcccca cccatcctgg cagaatcgga    1080gctttgcccc tgcaatcaac ccacggaagc tccgggaatg gcggccaagc acgcggatcc    1140tgacgttcac atctgtggct cagggaggga agggggtcgg tatcgtgagt acggcctttg    1200ggaagcagag gatgggccca agcccctcct ggaagataat ggagtccgga gggctcccag    1260catgccagga caggggccca aagtacccct gtctcaaact gagccacctt ttcattcggc    1320cgcgggaatc ctagggatac agacccactt cagcagggag ttggagccca gccctgcgag    1380gagtcaaggg gaggaagaag agggaggact gaggggacct tggagtccag atcagtggca    1440accttgggct gggggatcct gggcacagtg gcctaatgtg ccccatgctc attgcgactt    1500cagggtgaca gatttgcggg ctgtggtctg aggagtggca cttcaggtca gcagagggag    1560gaatcccagg atctgccgga cccaaggtgt gcccccttta tgaggactgg ggataccccc    1620ggcccagaaa gaagggatgc cacagagtct ggctgtccct tattcttagc tctaagggaa    1680ccggatcaga gatagctcca attggcaatc tcatttgtac cacaggcagg aggttgggga    1740accctcaggg agataaggtg ttggtgtaaa gaggagctgt ctgctcattt cagggggttg    1800ggggttgagg aagggcagtc cccggcagga gtaaagatga gtaacccaca ggaggccatc    1860agaagcctca ccctagaacc aaaggggtca gccctggaca acctacctgg gagtgacagg    1920atgtggctcc tcctcacttc tgtttccaga tctcagggag ttgaggtcct tttcttcaga    1980gggtgactca ggtcaacaca ggggccccca tgtagtcgac agacacagtg gtcctaagat    2040ctaccaagca tccaggtgag aagcctgagg taggattgag ggtacccctg ggccagaacg    2100ctgacagagg gccccacaga aatctgccct gcccctgcta ttccctcaga gagcctgggg    2160caaggctacc tgctgaggtc cctccattat cctgggatct ttgatgtcag ggaaagggag    2220gccttggtct gaaggggctg cactcaggtc actagacgga ggttctcagg ccctagcagg    2280agtagtggtg aggaccaagc aggctcgtca cccaggacac ctggactcca atgaatttgg    2340acatctctca ttgtcctttg tgggaggatc tggttatgta tggccagatg ttggtcccct    2400catatccttc tgtaccgtat cagggatgtg aattcttgcc atgagagttt ctttggccag    2460caaaagggcg gtattaggcc ctgcaaggag aaaggtgagg gccctgagtg agcacagaag    2520gaccctccac cccagtagag tggggacctc acagagtctg gccgaccctc ctgacaattt    2580tgggaatctg tggctgtact tgcagtctgc accctgaggc ccatggattc ctctcctagg    2640aatcaggagt tccaagaaca aggcagtgag gccttggtct gaggcagtgt cctgaggtca    2700cagagcagag ggggtgcaga cagtgccaac actgaaggtt tgccttgaat gcacaccaag    2760cgcaccggcc ccagaacaca tggactccag agggcctggc ctcaccctcc ctactgtcat    2820tccttcagcc tcagcatgtg ctggccggct gtaccctgag gcgccctctc acttgttcct    2880tcaggttctg aggagacagg ccccggagca gcactagctc ctgcccacac tcctacctgc    2940tgccctgacc agagtcatc atg cca ctt gag cag agg agt cag cac tgc aag 2992

                                   Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys

1 5 10CCT GAG GAA GGC CTT GCC CAA GGA GCC CTG GGC TTG GGT GGT 3040pro Glu GLU GLY Leu Glu Gln Glu Ala Leu Val Gly Gly Gly Gly Gly Gly Gly Gly Gly

15 20 25GCG CAG GCT GCT GCT GAG GAG GAG GAG ACT GCC TCC TCC TCC TCT 3088Ala Gln Ala THR GLU Glu Gln Gln Gln Gl Ala Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Serial

30 35 40ACT CTA GAA GAA GTC ACC CTG CTG GAG GAG GCC GCC GCC GAG TCA CCA 3136thr Leu Val THR Leu ARG Glu Val Pro Ala Ala Glu Ser Pro

45                  50                  55agt cct ccc cac agt cct cag gga gcc tcc acc ctc ccc act acc atc      3184Ser Pro Pro His Ser Pro Gln Gly Ala Ser Thr Leu Pro Thr Thr Ile60                  65                  70                  75aac tat act ctc tgg agt caa tcc gat gag ggc tcc agc aac gaa gaa 3232Asn Tyr Thr Leu Trp Ser Gln Ser Asp Glu Gly Ser Ser Asn Glu Glu

80 85 90CAG GAA GGG CCA AGC ACC TTT CCT GAC CTG GAG GAG AGC TTC CAA GTA 3280GLN GLY Pro Serte Pro ASP Leu Glu Gln Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val

95 1005GCA CTC AGT AGG AAG AAG AAG GCT GAG GAG GTT CAT TTT CTC CTC CTC 3328ALA Leu Serg Lys Met Ala Glu Val His PHE Leu Leu LYS LYS

110 115 120TATA GCC Agg GAG GAG CCA TTC AAG GAA GAA ATG CTG GGG AGT GTC 3376tyr ARG GLU PRO PHR LYS ALA GLU MET Leu Gly Ser Val Val Val Val Val Val Val Val Val Val Val

125                 130                 135atc aga aat ttc cag gac ttc ttt cct gtg atc ttc agc aaa gcc tcc      3424Ile Arg Asn Phe Gln Asp Phe Phe Pro Val Ile Phe Ser Lys Ala Ser140                 145                150                  155gag tac ttg cag ctg gtc ttt ggc atc gag gtg gtg gaa gtg gtc cgc 3472Glu Tyr Leu Gln Leu Val Phe Gly Ile Glu Val Val Glu Val Val Arg

160 165 170ATC GGC CAC TTG TAC ATC CTT GTC ACC CTG GGC CTC CTC TAC GCT 3520ile GLY HIS Leu Tyr Ile Leu Val Leu Gly Leu Serial Tyr Ala

175 180 185GGC CTG CTG GGC GAC AAT CAG ATC GTG CCC AAG AAG AAG AAG CTC CTC ATA 3568Gly Leu Leu GLY ASN GLN Ile Val Leu Leu Ile

190 195 200ATC GTC CTG GCC ATC GCA AAA GAG GGC GAC GCC CCC CCT GAG GAG 3616ile Val Leu Ala Ile Ala Lys GLU GLY ALA PRO Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu

205                 210                 215aaa atc tgg gag gag ctg agt gtg ttg gag gca tct gat ggg agg gag      3664Lys Ile Trp Glu Glu Leu Ser Val Leu Glu Ala Ser Asp Gly Arg Glu220                 225                 230                 235gac agt gtc ttt gcg cat ccc agg aag ctg ctc acc caa gat ttg gtg 3712Asp Ser Val Phe Ala His Pro Arg Lys Leu Leu Thr Gln Asp Leu Val

240 245 250CAG GAC TAC CTG GAG GAG GAG GAG CGG CAG CCC CCC GGC AGT GCA 3760GLN GLU Asn Tyr Leu Tyr ARG GLN Val Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

255 260 265TGC Tac GAG TTC CTG TGG GGT CCA AGG GCC CTC GAA ACC AGC TAT 3808CYS Tyr Glu TRP GLY PRE ARA Leu Val Glu THRU

270 275 280GTG AA GTC CTG CTG CAC CAT TTG CTA AAG AAG AGT GGA GGG CCT CAC ATT 3856val Lys Val Leu His Leu Leu Lys Ile Serle Serle Serp

285                 290                 295ccc tac cca ccc ctg cat gaa tgg gct ttt aga gag ggg gaa gag tga      3904Pro Tyr Pro Pro Leu His Glu Trp Ala Phe Arg Glu Gly Glu Glu  *300                 305                 310gtctgagcac gagttgcagc cagggccagt gggagggagt ctgggccagt gcaccttcca    3964aggccctatc cattagtttc cactgcctcg tgtgacatga ggcccattct tcactctttg    4024aagagagcag tcagtattgt tagtagtgag tttctgttct attggatgac tttgagattt    4084atctttgttt cctgttggaa ttgttcaaat gttcctttta acggatggtt gaatgaactt    4144cagcatccaa gtttatgaat gacagtagtc acacatagtg ctgtttatat agtttaggag    4204taagagtgtt gttttttatt cagatttggg aaatccattc cattttgtga attgtgacaa    4264ataacagcag tggaaaaagt atgtgcttag aattgtgaaa gaattagcag taaaatacat    4324gagataaaga cctcaagaag ttaaaagata cttaattctt gccttatacc tcacttcatt    4384ctgtaaattt gaaaaaaaag cgtggatacc tggatatcct tggcttcttt gagaatttaa    4444gagaaattaa atctgaataa ataattcttc ctgttcactg gctcatttat tttccattca    4504ctcagcatct gctctgtgg                                                    1

<210>2

<211>314

<212>PRT

<213>Homo sapiens

<400> 2MET Pro Leu Gln ARG Serg Serg Serg Serg Serg Serg Serg Serg Serg Serg Serg Serg Serg Serg Serg

20 25 30Thr Glu Glu Gln Glu Thr Ala Ser Ser Ser Ser Thr Leu Val Glu Val

35 40 45Thr Leu Arg Glu Val Pro Ala Ala Glu Ser Pro Ser Pro Pro His Ser

50 55 60pro Gln Gln Gln Gln Gl Ala Seru Pro Thr THR ILE Asn Tyr THR Leu TRP65 75 80SER GLN Ser ASN GLU GLU GLU GLU GLU GLU GLY Pro Ser

85 90 95Thr Phe Pro Asp Leu Glu Thr Ser Phe Gln Val Ala Leu Ser Arg Lys

100 105 110 Met Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu

115 120 125Pro Phe Thr Lys Ala Glu Met Leu Gly Ser Val Ile Arg Asn Phe Gln

130 135 140ASP PHE PRO VAL ILE PHE PHE Ser Lys Ala Ser Glu Gln Leu145 150 155 160 Val Ile Glu Val Val Val Val His Leu Tyr

165 170 175Ile Leu Val Thr Cys Leu Gly Leu Ser Tyr Ala Gly Leu Leu Gly Asp

180 185 190Asn Gln Ile Val Pro Lys Thr Gly Leu Leu Ile Ile Val Leu Ala Ile

195 200 205Ile Ala Lys Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu

210 215 220leu Ser Val Leu Glu Ala Serg GLY ARG GLU ASP Ser Val PHE ALA2225 230 235 240HIS PRO ARS Leu Leu ThRn ASP Leu Val Gln Tyr Leu ASN Tyr Leuuu

245 250 255Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu

260 265 270Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His

275 280 285His Leu Leu Lys Ile Ser Gly Gly Pro His Ile Pro Tyr Pro Pro Leu

290 295 300His Glu Trp Ala Phe Arg Glu Gly Glu Glu305 310

<210>3

<211>9

<212>PRT

<213>Homo sapiens

<400>3Glu Val Val Arg Ile Gly His Leu Tyr1 5

<210>4

<211>9

<212>PRT

<213>Homo sapiens

<400>4Val Arg Ile Gly His Leu Tyr Ile Leu1 5

<210>5

<211>10

<212>PRT

<213>Homo sapiens

<400>5Val Val Arg Ile Gly His Leu Tyr Ile Leu1 5 10

<210>6

<211>8

<212>PRT

<213>Homo sapiens

<400>6Arg Ile Gly His Leu Tyr Ile Leu1 5

<210>7

<211>24

<212>DNA

<213>Homo sapiens

<400>7gggtccaaat tgttggcttt cact 24

<210>8

<211>22

<212>DNA

<213>Homo sapiens

<400>8gaagaatgcc tcatgatccc ca 22

<210>9

<211>9

<212>PRT

<213>Homo sapiens

<400>9Glu Ala Asp Pro Thr Gly His Ser Tyr1 5

<210>10

<211>9

<212>PRT

<213>Homo sapiens

<400>10Ser Ala Tyr Gly Glu Pro Arg Lys Leu1 5

<210>11

<211>9

<212>PRT

<213>Homo sapiens

<400>11Glu Val Asp Pro Ile Gly His Leu Tyr1 5

<210>12

<211>9

<212>PRT

<213>Homo sapiens

<400>12Phe Leu Trp Gly Pro Arg Ala Leu Val1 5

<210>13

<211>10

<212>PRT

<213>Homo sapiens

<400>13Met Glu Val Asp Pro Ile Gly His Leu Tyr1 5 10

<210>14

<211>9

<212>PRT

<213>Homo sapiens

<400>14Ala Ala Arg Ala Val Phe Leu Ala Leu1 5

<210>15

<211>8

<212>PRT

<213>Homo sapiens

<400>15Tyr Arg Pro Arg Pro Arg Arg Tyr1 5

<210>16

<211>10

<212>PRT

<213>Homo sapiens

<400>16Ser Pro Ser Ser Asn Arg Ile Arg Asn Thr1 5 10

<210>17

<211>9

<212>PRT

<213>Homo sapiens

<400>17Val Leu Pro Asp Val Phe Ile Arg Cys1 5

<210>18

<211>10

<212>PRT

<213>Homo sapiens

<400>18Val Leu Pro Asp Val Phe Ile Arg Cys Val1 5 10

<210>19

<211>9

<212>PRT

<213>Homo sapiens

<400>19Glu Glu Lys Leu Ile Val Val Leu Phe1 5

<210>20

<211>9

<212>PRT

<213>Homo sapiens

<400>20Glu Glu Lys Leu Ser Val Val Leu Phe1 5

<210>21

<211>10

<212>PRT

<213>Homo sapiens

<400>21Ala Cys Asp Pro His Ser Gly His Phe Val1 5 10

<210>22

<211>10

<212>PRT

<213>Homo sapiens

<400>22Ala Arg Asp Pro His Ser Gly His Phe Val1 5 10

<210>23

<211>9

<212>PRT

<213>Homo sapiens

<400>23Ser Tyr Leu Asp Ser Gly Ile His Phe1 5

<210>24

<211>9

<212>PRT

<213>Homo sapiens

<400>24Ser Tyr Leu Asp Ser Gly Ile His Ser1 5

<210>25

<211>9

<212>PRT

<213>Homo sapiens

<400>25 Met Leu Leu Ala Val Leu Tyr Cys Leu1 5

<210>26

<211>9

<212>PRT

<213>Homo sapiens

<400>26Tyr Met Asn Gly Thr Met Ser Gln Val1 5

<210>27

<211>9

<212>PRT

<213>Homo sapiens

<400>27Ala Phe Leu Pro Trp His Arg Leu Phe1 5

<210>28

<211>9

<212>PRT

<213>Homo sapiens

<400>28Ser Glu Ile Trp Arg Asp Ile Asp Phe1 5

<210>29

<211>9

<212>PRT

<213>Homo sapiens

<400>29Tyr Glu Ile Trp Arg Asp Ile Asp Phe1 5

<210>30

<211>15

<212>PRT

<213>Homo sapiens

<400>30Gln Asn Ile Leu Leu Ser Asn Ala Pro Leu Gly Pro Gln Phe Pro1 5 10 15

<210>31

<211>15

<212>PRT

<213>Homo sapiens

<400>31Asp Tyr Ser Tyr Leu Gln Asp Ser Asp Pro Asp Ser Phe Gln Asp1 5 10 15

<210>32

<211>10

<212>PRT

<213>Homo sapiens

<400>32Glu Ala Ala Gly Ile Gly Ile Leu Thr Val1 5 10

<210>33

<211>9

<212>PRT

<213>Homo sapiens

<400>33Ala Ala Gly Ile Gly Ile Leu Thr Val1 5

<210>34

<211>9

<212>PRT

<213>Homo sapiens

<400>34Ile Leu Thr Val Ile Leu Gly Val Leu1 5

<210>35

<211>9

<212>PRT

<213>Homo sapiens

<400>35Lys Thr Trp Gly Gln Tyr Trp Gln Val1 5

<210>36

<211>9

<212>PRT

<213>Homo sapiens

<400>36Ile Thr Asp Gln Val Pro Phe Ser Val1 5

<210>37

<211>9

<212>PRT

<213>Homo sapiens

<400>37Tyr Leu Glu Pro Gly Pro Val Thr Ala1 5

<210>38

<211>10

<212>PRT

<213>Homo sapiens

<400>38Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu1 5 10

<210>39

<211>10

<212>PRT

<213>Homo sapiens

<400>39Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val1 5 10

<210>40

<211>9

<212>PRT

<213>Homo sapiens

<400>40Leu Tyr Val Asp Ser Leu Phe Phe Leu1 5

<210>41

<211>12

<212>PRT

<213>Homo sapiens

<400>41Lys Ile Ser Gly Gly Pro Arg Ile Ser Tyr Pro Leu1 5 10

<210>42

<211>9

<212>PRT

<213>Homo sapiens

<400>42Tyr Met Asp Gly Thr Met Ser Gln Val1 5

<210>43

<211>11

<212>PRT

<213>Homo sapiens

<400>43Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu1 5 10

<210>44

<211>9

<212>PRT

<213>Homo sapiens

<400>44Ser Leu Leu Met Trp Ile Thr Gln Cys1 5

<210>45

<211>9

<212>PRT

<213>Homo sapiens

<400>45Gln Leu Ser Leu Leu Met Trp Ile Thr1 5

<210>46

<211>20

<212>DNA

<213>Homo sapiens

<400>46cctacctgct gccctgacca 20

<210>47

<211>20

<212>DNA

<213>Homo sapiens

<400>47cctaaggact gtggggagga 20

<210>48

<211>20

<212>DNA

<213>Homo sapiens

<400>48ccaactaagc catcttccta 20

<210>49

<211>20

<212>DNA

<213>Homo sapiens

<400>49gtgacaagga tctacaagtg 20

<210>50

<211>20

<212>DNA

<213>Homo sapiens

<400>50ccagtcaggt gacaaggatg 20

<210>51

<211>20

<212>DNA

<213>Homo sapiens

<400>51cctgtctagg gcacgatctg 20

<210>52

<211>20

<212>DNA

<213>Homo sapiens

<400>52ctcctaaggg gcacagtcgc 20

<210>53

<211>20

<212>DNA

<213>Homo sapiens

<400>53tcagatgcct acaacacact 20

<210>54

<211>20

<212>DNA

<213>Homo sapiens

<400>54ggaccctaca ggaactcgta 20

<210>55

<211>20

<212>DNA

<213>Homo sapiens

<400>55cgttggaggt cagagaacag 20

<210>56

<211>22

<212>DNA

<213>Homo sapiens

<400>56gccctccact gatctttagc aa 22

Claims (57)

1. isolated M AGE-A12HLA I class binding peptide comprise the aminoacid sequence of SEQ ID NO:6, or it is in conjunction with one or more aminoacid addition that comprise of HLA I quasi-molecule, replaces or the functional variant of disappearance.

2. isolated M AGE-A12 HLA I class binding peptide according to claim 1, wherein isolating peptide comprises the aminoacid sequence of choosing from following group, comprise SEQID NO:4, SEQ ID NO:5, their fragment and their functional variant.

3. isolated M AGE-A12 HLA I class binding peptide according to claim 1, wherein isolating peptide comprises the aminoacid sequence of choosing from following group, comprise SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, their fragment and their functional variant.

4. an isolated M AGE-A12 HLA I class binding peptide comprises the fragment in conjunction with the SEQ ID NO:2 aminoacid sequence of HLACw*07, or comprises one or more aminoacid addition, its functional variant of replacing or lacking, and wherein functional variant is in conjunction with HLA Cw ^*07.

5. according to claim 1 or 4 described isolated M AGE-A12 HLA I class binding peptides, wherein isolating peptide is a non-hydrolysable.

6. isolated M AGE-A12 HLA I class binding peptide according to claim 5, wherein peptide is chosen from following group, comprises containing the amino acid whose peptide of D-, contains-psi[CH ₂NH]-peptide of reducing amide peptide bond, contain-psi[COCH ₂The peptide of]-ketone methylene radical peptide bond contains-psi[CH (CN) NH]-peptide of (cyanogen methylene radical) amino peptide bond, contain-psi[CH ₂CH (OH)]-peptide of hydroxy ethylene peptide bond, contain-psi[CH ₂O]-peptide of peptide bond, contain-psi[CH ₂S]-peptide of thio-methylene peptide bond.

7. one kind comprises the isolating HLA I class of the described isolated M AGR-A12 HLA of claim 1 I class binding peptide and non--MAGE-A12 tumour antigen or the composition of II class binding peptide.

8. one kind comprises the isolating HLA I class of the described isolated M AGR-A12 HLA of claim 4 I class binding peptide and non--MAGE-A12 tumour antigen or the composition of II class binding peptide.

9. according to claim 7 or 8 described compositions, wherein the HLA I class of MAGE-A12 HLA I class binding peptide and non--MAGE-A12 tumour antigen or II class binding peptide are combined into the multi-epitope polypeptide.

10. the isolating nucleic acid of the peptide of choosing in the group of a coding any one peptide from comprise claim 1-4, its amplifying nucleic acid total length MAGE-A12 that do not encode.

11. isolating nucleic acid according to claim 10, its amplifying nucleic acid comprise SEQ IDNO:1 nucleotide sequence fragment.

12. an expression vector comprises with promotor and can operate the described isolating nucleic acid of bonded claim 11.

13. expression vector according to claim 12 further comprises coding HLA-Cw ^*The nucleic acid of 07 molecule.

14. one kind with expression vector transfection or the transformed host cells chosen from the group of the expression vector of the expression vector that comprises claim 12 and claim 13.

15. expression vector transfection or transformed host cells with a claim 12, wherein host cell expression HLA Cw ^*07 molecule.

16. a selectivity increase contains the method to the special T lymphocyte populations of MAGE-A12 HLA binding peptide, comprising: the T lymphocyte source that will contain the T lymphocyte populations be enough to optionally increase the reagent of presenting the MAGE-A12HLA binding peptide and the mixture of HLA molecule that contains the amount of the special lymphocytic T lymphocyte populations of T of MAGE-A12 HLA binding peptide and contact.

17. method according to claim 16, wherein reagent is with MAGE-A12 protein or the contacted antigen presenting cell of its HLA binding fragment.

18. method according to claim 16, wherein MAGE-A12 HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQ ID NO:2 aminoacid sequence; The peptide that (ii) comprises SEQ ID NO:6 aminoacid sequence; And (iii) (i) and the (ii) functional variant of peptide.

19. method of diagnosing characteristics to be to express the pathology of MAGE-A12 HLA binding peptide, comprise and to determine that from being tried the isolating biological sample of body and the special reagent of MAGE-A12 HLA binding peptide being contacted pathology is determined in the combination between reagent and the MAGE-A12 HLA binding peptide.

20. method according to claim 19, wherein MAGE-A12 HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQ ID NO:2 aminoacid sequence; The peptide that (ii) comprises SEQ ID NO:6 aminoacid sequence; And (iii) (i) and the (ii) functional variant of peptide.

21. a diagnosis suffers from the method for being tried body that characteristics are to express the pathology of MAGE-A12, comprises being contacted with reagent in conjunction with mixture from trying the isolating biological sample of body, determines that pathology is determined in the combination between reagent and the mixture.

22. method according to claim 21, wherein MAGE-A12 HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQ ID NO:2 aminoacid sequence; The peptide that (ii) comprises SEQ ID NO:6 aminoacid sequence; And (iii) (i) and the (ii) functional variant of peptide.

23. a treatment suffers from the method for being tried body that characteristics are to express the pathology of MAGE-A12, comprises to being tried body taking the MAGE-A12HLA binding peptide of the amount that is enough to alleviate pathology.

24. method according to claim 23, wherein MAGE-A12 HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQ ID NO:2 aminoacid sequence; The peptide that (ii) comprises SEQ ID NO:6 aminoacid sequence; And (iii) (i) and the (ii) functional variant of peptide.

25. a treatment suffers from the method for being tried body that characteristics are to express the pathology of MAGE-A12, comprises to being tried body taking the claim 7 of the amount that is enough to alleviate pathology or the composition of claim 8,

26. a treatment suffers from the method for being tried body that characteristics are to express the pathology of MAGE-A12, comprises to being tried body taking the reagent that optionally increasing of the amount that is enough to alleviate pathology tried the amount of the mixture of HLA molecule and MAGE-A12 HLA binding peptide in the body.

27. method according to claim 26, wherein reagent comprises MAGE-A12

The HLA binding peptide.

28. according to claim 26 or 27 described methods, wherein MAGE-A12 HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQ ID NO:2 aminoacid sequence; The peptide that (ii) comprises SEQ ID NO:6 aminoacid sequence; And (iii) (i) and the (ii) functional variant of peptide.

29. a treatment suffers from the method for being tried body that characteristics are to express the pathology of MAGE-A12, comprises that wherein the T lymphocyte is special to the mixture of HLA molecule and MAGE-A12 binding peptide to being tried amount self the T lymphocyte that body is taken is enough to alleviate pathology.

30. method according to claim 29, wherein MAGE-A12 HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQ ID NO:2 aminoacid sequence; The peptide that (ii) comprises SEQ ID NO:6 aminoacid sequence; And (iii) (i) and the (ii) functional variant of peptide.

31. method of differentiating the functional variant of MAGE-A12 HLA binding peptide, comprise and choose MAGE-A12 HLA binding peptide, in conjunction with the HLA binding molecule of MAGE-A12 HLA I class binding peptide, and be subjected to the T cell that the MAGE-A12 HLA binding peptide presented by the HLA binding molecule stimulates; First amino-acid residue of mutagenesis MAGE-A12HLA binding peptide prepares variant peptides; Determine combining and the hormesis of T cell of variant peptides and HLA binding molecule, wherein this variant peptides of expression of stimulating of the variant peptides that variant peptides combines with the HLA binding molecule and the T cell is subjected to being presented by the HLA binding molecule is a functional variant.

32. method according to claim 31, wherein MAGE-A12 HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQ ID NO:2 aminoacid sequence; The peptide that (ii) comprises SEQ ID NO:6 aminoacid sequence; And (iii) (i) and the (ii) functional variant of peptide.

33. method according to claim 31 comprises that further comparison MAGE-A12HLA binding peptide determines the step of functional variant to the validity of the hormesis of T cell to the hormesis and the functional variant of T cell to the step of the hormesis of T cell.

34. an isolated polypeptide, it is optionally in conjunction with any one polypeptide among the claim 1-4, as long as isolated polypeptide is not the HLA molecule.

35. isolated polypeptide according to claim 34, the wherein antibody of isolated polypeptide.

36. isolated polypeptide according to claim 35, wherein antibody is monoclonal antibody.

37. isolated polypeptide according to claim 34, wherein isolated polypeptide is the antibody fragment of choosing from following group, comprises the Fab fragment, F (ab) ₂Fragment or or comprise that MAGE-A12 HLA binding peptide is had the optionally fragment in CDR3 zone.

38. one kind optionally in conjunction with the isolating T lymphocyte of the mixture of HLA molecule and MAGE-A12 HLA binding peptide.

39. according to the described isolating T lymphocyte of claim 38, wherein MAGE-A12 HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQ IDNO:2 aminoacid sequence; The peptide that (ii) comprises SEQ ID NO:6 aminoacid sequence; And (iii) (i) and the (ii) functional variant of peptide.

40. isolating antigen presenting cell that comprises the mixture of HLA molecule and MAGE-A12 HLA binding peptide.

41. according to the described antigen presenting cell of claim 40, wherein the MAGE-A12HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQ ID NO:2 aminoacid sequence; The peptide that (ii) comprises SEQ ID NO:6 aminoacid sequence; And (iii) (i) and the (ii) functional variant of peptide.

42. vaccine composition that comprises any one polypeptide and pharmaceutically acceptable carrier among the claim 1-4.

43., further comprise assistant agent according to the described vaccine composition of claim 42.

44. one kind comprises from the T lymphocyte that comprises claim 38 and 39 and the antigen presenting cell of claim 40 and 41, and the vaccine composition of pharmaceutically acceptable carrier.

45., further comprise assistant agent according to the described vaccine composition of claim 44.

46. one kind comprises the isolated nucleic acid molecule of claim 10 and the vaccine composition of pharmaceutically acceptable carrier.

47., further comprise assistant agent according to the described vaccine composition of claim 46.

48. functional variant by the isolated M AGE-A12 HLA binding peptide of the method discriminating of claim 31.

49. method of differentiating the candidate analogue of MAGE-A12 HLA binding peptide, the HLA molecule that provides in conjunction with MAGE-A12 HLA binding peptide is provided, the HLA molecule is contacted with test molecule, determine combining of test molecule and HLA molecule, wherein the test molecule in conjunction with the HLA molecule is the candidate analogue of MAGE-A12 HLA binding peptide.

50. according to the described method of claim 49, further comprise the mixture of making HLA molecule and candidate analogue, with the T cells contacting of mixture with the mixture that combines HLA molecule and MAGE-A12 HLA binding peptide, the activation of test T cell.

51. according to the described method of claim 50, wherein the activation of T cell is indicated by the character of choosing from following group, comprises the propagation of T cell, the interferon-that the T cell produces, the tumour necrosis factor that the T cell produces, the T cell is to the cytolysis of target cell.

52. one kind comprises isolated M AGE-A12 HLAI class binding peptide, or it is in conjunction with one or more aminoacid addition that comprises of HLAI quasi-molecule, the little arrangement of protein (microarrays) of the functional variant of replacing or lacking.

53. according to the little arrangement of the described protein of claim 52, wherein isolated M AGE-A12 HLA I class binding peptide comprises SEQ ID NO:6 aminoacid sequence.

54. according to the little arrangement of the protein of claim 52, wherein isolated M AGE-A12HLA I class binding peptide comprises the aminoacid sequence of choosing from following group, comprises SEQ ID NO:4, SEQ ID NO:5, and their functional variant.

55. a diagnosis suffers from the method for being tried body that characteristics are to express the pathology of MAGE-A12, comprise the little arrangement of protein is contacted with the biological sample that is tried the body acquisition of suffering from pathology from suspection, determine the composition of biological sample and combining of isolated M AGE-A12 HLAI class binding peptide.

56. according to the described method of claim 55, wherein the composition of biological sample is chosen from following group, comprises antibody, T lymphocyte and HLA molecule.

57. according to the described method of claim 55, wherein pathology is a cancer.

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