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

Abstract

The invention provides antigenic peptides derived from MAGE-A12 polypeptides and presented by HLA molecules. Methods for diagnosis and treatment which involve the polypeptides also are provided.

Description

MAGE-A12 antigen peptide and application thereof

The field of the invention

The present invention relates to preferentially be expressed in coding nucleic acid molecule and polypeptide in the tumour (comprising malignant melanoma, bladder cancer, kidney, lung cancer, esophagus cancer etc.).The application in treatment and diagnosis of nucleic acid molecule and coded polypeptide.

Background of the present invention

Distinguish between tumor cells and Normocellular phenotype change the result of the one or more variations that usually are cellular genome.The gene that is expressed in the tumour cell but is not expressed in the normal cell can be called " tumour is relevant " gene.These tumor-related genes are marks of tumor phenotypes.The expression of tumor-related gene also can be the critical event of tumour production process.

Generally, the host is identified as foreign gene with the tumor-related gene that not express in normal non-tumor cell.Like this, the expression of tumor-related gene can stimulation of host to the immunne response of tumour cell.Tumor-related gene also can be expressed in the normal cell in some tissue, and immune stimulatory is not replied.In this tissue, because immunity system can not " be seen " cell in these immunology teleocracies, the common immunology of the proteinaceous product on the cell surface can be discerned fragment and can not reply by immune stimulatory.The example of immunology teleocracy comprises brain and testis.

It is a kind of mode of tumour cell that the tumour correlated expression of discovery gene provides recognizing cells.Diagnostic compounds can be with tumor-related because foundation, and can be used to determine the existence and the position of tumour cell.And, when tumor-related gene is relevant with the tumor phenotypes aspect (as, non-adjusting growth or transfer), tumor-related gene can be used to provide therapeutant, as reducing or eliminate basically the antisense nucleic acid of this genetic expression, depend on the phenotype aspect that concrete tumor-related gene is expressed thereby reduce or eliminate basically.

Immune system identification and with the process of external source or foreign matter reaction be very complicated process.An importance of system is a t cell response.This reply need the T cell recognition and with the reaction of the mixture of cell surface molecule and peptide, cell surface molecule is meant human leucocyte antigen (" HLA "), or main histocompatibility complex (" MHC ").The macromole that peptide is handled from the cell of also presenting the HLA/MHC molecule is derived.Relevant this aspect is referring to people Advanced Immunology such as Male (J.P.Lipincott Company, 1987) 6-10 chapter.Interaction between T cell and the HLA/ peptide complex is restrictive, needs the special combination of T cell-specific in HLA molecule and peptide.If special peptide does not exist,, its group do not have t cell response even existing mixture yet.Similarly, the T cell exists if special mixture does not exist, and does not also have and replys.This mechanism is relevant to replying of allogenic material with immunity system, and is relevant with the autoimmunization pathology, relevant with the pair cell exception response.A lot of work concentrate on protein are worked on the mechanism in the HLA binding peptide.Relevant this aspect is referring to Barinaga, science, 257:880,1992; People such as Fremont, science 257:919,1992; People such as Matsumura, science 257:927,1992; People such as Latron, science 257:964,1992.

The mechanism of T cell recognition cellular abnormality is also relevant with cancer.For example, submit on May 22nd, 1992, among disclosed PCT application on November 26th, 1992 PCT/US92/04354 (with reference to this piece of writing of income), gene family is disclosed, wherein be machined in the peptide, then be expressed on the cell surface, it can cause the tumour cell cracking by special CTL.These genes are coding " tumor rejection antigen precursor " or " TRAP " molecule allegedly, and therefrom the deutero-peptide is called " Tumor rejection antigen " or " TRA ".Obtain the more information of this family gene, referring to people such as Traversari, J.Exp.Med.176:1453-1457,1992; People such as van der Bruggen, science 254:1643,1991; People such as De Plaen, immunogenetics 40:360-369,1994 and U.S. Patent No. 5,342,774.

U.S. Patent No. 5,405, in 940, its disclosure has wherein been taught the nonapeptide by the HLA-A1 molecular presentation with reference to this piece of writing of income.The reference professor should estimate that concrete peptide combines with a kind of HLA molecule under the specific situation of known concrete peptide to concrete HLA molecule, rather than other molecules.This point is very important, because different individualities has different HLA phenotypes.Like this, when determining that the group of concrete peptide as special HLA molecule has diagnosis and treatment derivative to thing, this is only relevant with concrete HLA phenotype for individual.This need be in the further work in this field, because cellular abnormality is not limited to a kind of concrete HLA phenotype, and goal treatment need be understood some paracytic situations to be treated.

In U.S. Patent No. 5,629, in 166 (with reference to these pieces of writing of income), disclose the MAGE-1 expression product and be worked into the fact among the 2nd TRA.This 2nd TRA has the HLA-Cw16 molecular presentation, is also referred to as HLA-C*1601.Disclosure shows that given TRAP can produce multiple TRA.

In U.S. Patent No. 5,487, in 974 (with reference to these pieces of writing of income), tyrosine oxidase is described to tumor rejection antigen precursor.This part reference discloses and will be worked into by the molecule that some normal cells (for example melanophore) produce in the tumour cell to produce the Tumor rejection antigen by the HLA-A2 molecular presentation.

In U.S. Patent No. 5,620, in 886 (it is in full with reference to these pieces of writing of income), disclosing is not by the HLA-A2 molecular presentation from the 2nd tyrosinase derived TRA.TRA derives from TRAP, but by known MAGE genes encoding.This part disclosure shows that concrete HLA molecule can be presented from difference source deutero-TRA.

Other TRAP are in U.S. Patent No. 5,571, and 711,5,610,013,5,587,289 and 5,589,334, and open among the PCT application WO96/10577.TRAP is processed into Tumor rejection antigen, and it is by multiple HLA molecular presentation.

There are a lot of patients from the treatment that comprises other antigen peptide, to be benefited, because patient tumors does not have to express the antigen peptide of knowing in advance, perhaps because the patient does not express suitable HLA molecule.Therefore, need determine to contain other tumor associated antigens of presenting by MHC I quasi-molecule by the identification of CD8* lymphocyte.

General introduction of the present invention

Have been found that the Tumor rejection antigen that human MAGE-A12 gene (SEQ ID NO:1) coding is presented by HLA-Cw*07 at present.From MAGE-A12 polypeptide (SEQID NO:1) deutero-peptide, when presenting, can induce the activation and the propagation of CD8+ cytotoxic T lymphocyte effectively by the antigen presenting cell that contains HLA I quasi-molecule.

According to one aspect of the present invention, provide isolated M AGE-A12 HLA I class binding peptide.Peptide comprises SEQ ID NO:6 aminoacid sequence or in conjunction with its functional variant of HLA I quasi-molecule.Functional variant comprises one or more aminoacid addition, replaces or disappearance.In certain embodiments, isolated M AGE-A12 HLA I class binding peptide comprises the aminoacid sequence of choosing from following group, comprise SEQ ID NO:4, SEQ ID NO:5, its function fragment and its functional variant.In preferred embodiments, isolating peptide comprises the aminoacid sequence of choosing from following group, comprises SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, its function fragment and its functional variant.Isolated M AGE-A12 HLA I class binding peptide preferably is not a total length MAGE-A12 peptide sequence.

According to another aspect of the present invention, isolated M AGE-A12 HLA I class binding peptide is provided, it comprises the Cw in conjunction with HLA ^*07 SEQ ID NO:2 aminoacid sequence fragment, or its functional variant.Functional variant comprises one or more aminoacid addition, replaces or disappearance.Functional variant is in conjunction with HLA Cw*07.Preferred embodiment comprises SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

In some embodiments, aforementioned isolated M AGE-A12 HLA I class binding peptide is a non-hydrolysable.Preferably, the peptide of non-hydrolysable 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.

One or more the isolating HLA I classes that comprise aforementioned isolated M AGE-A12HLA I class binding peptide and non--MAGE-A12 tumour antigen or the composition of II class binding peptide are provided according to one aspect of the present invention.Preferably, MAGE-A12 HLA I class binding peptide and non-MAGE-A12 HLA binding peptide are combined into the multi-epitope polypeptide.

According to another aspect of the present invention, provide the isolating nucleic acid of the aforementioned peptide of encoding.The nucleic acid total length MAGE-A12 that do not encode.In certain embodiments, nucleic acid comprises the fragment of SEQ ID NO:1 nucleotide sequence.Also provide according to expression vector of the present invention.Expression vector comprises operationally the isolating aforementioned nucleic acid in conjunction with promotor.In certain embodiments, expression vector also comprises coding HLA-Cw ^*07 nucleic acid.In another aspect of the present invention, provide with aforementioned nucleic acid or expression vector transfection or transformed host cells.In certain embodiments, host cell is also expressed HLA-Cw ^*07 molecule.

According to another aspect of the present invention, providing optionally increases the method that contains the special lymphocytic T lymphocyte populations of T of MAGE-A12 HLA binding peptide.Method comprises the T lymphocyte source that will contain the T lymphocyte populations and is enough to optionally increase the reagent of presenting the MAGE-A12 HLA 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 contacts.In certain embodiments, reagent is with MAGE-A12 protein or the contacted antigen presenting cell of its HLA binding fragment.In other embodiments, 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.

According to another aspect of the present invention, provide the diagnosis characteristics to be to express the method for the pathology of MAGE-A12 HLA binding peptide.Method comprises and will contact with reagent in conjunction with mixture from being tried the isolating biological sample of body, determines to combine to determine pathology between mixture and the reagent.In certain embodiments, 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.

According to another aspect of the present invention, provide treatment to suffer from the method for being tried body that characteristics are to express the pathology of MAGE-A12.Method comprises to being tried body takes the MAGE-A12 HLA binding peptide of the amount that is enough to alleviate pathology.In certain embodiments, 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.

According to another aspect of the present invention, provide treatment to suffer from the method for being tried body that characteristics are to express the pathology of MAGE-A12.Method comprises to being tried body takes the composition of the amount that is enough to alleviate pathology, composition comprise isolated M AGE-A12 HLA I class binding peptide and isolating non--the HLA I class or the II class binding peptide of MAGE-A12 tumour antigen.

According to another aspect of the present invention, provide treatment to suffer from the method for being tried body that characteristics are to express the pathology of MAGE-A12.Method comprises to being tried body takes 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.In certain embodiments, reagent comprises MAGE-A12 HLA binding peptide.In preferred embodiments, MAGE-A12 HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQID 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.

According to another aspect of the present invention, provide treatment to suffer from the method for being tried body that characteristics are to express the pathology of MAGE-A12.Method 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.In certain embodiments, MAGE-A12 HLA binding peptide is chosen from following group, comprises that (i) comprises the segmental peptide of SEQID 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.

According to another aspect of the present invention, also provide the method for the functional variant of differentiating MAGE-A12 HLA binding peptide.Method comprises chooses MAGE-A12 HLA binding peptide, in conjunction with the HLA binding molecule of MAGE-A12 HLA I class binding peptide, and is 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-A12 HLA 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.In certain embodiments, 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.In other embodiments, method comprises that comparison MAGE-A12 HLA binding peptide determines the validity of functional variant to the hormesis of T cell to the hormesis and the functional variant of T cell to the step of the hormesis of T cell.The functional variant of the isolated M AGE-A12 HLA binding peptide of differentiating by method is provided equally.

According to another aspect of the present invention, provide optionally isolated polypeptide, as long as isolated polypeptide is not the HLA molecule in conjunction with aforementioned MAGE-A12 HLA binding peptide.In certain embodiments, the antibody of isolated polypeptide, preferably monoclonal antibody.In other embodiments, 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.

According to another aspect of the present invention, provide optionally isolating T lymphocyte in conjunction with the mixture of HLA molecule and MAGE-A12 HLA binding peptide.In certain embodiments, 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.

According to another aspect of the present invention, provide the isolating antigen presenting cell of the mixture that comprises HLA molecule and MAGE-A12 HLA binding peptide.In certain embodiments, 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.

According to another aspect of the present invention, provide the method for the candidate analogue of differentiating MAGE-A12 HLA binding peptide.The HLA molecule that provides in conjunction with MAGE-A12 HLA binding peptide is provided method, and the HLA molecule is contacted with test molecule, determines combining of test molecule and HLA molecule, and wherein the test molecule in conjunction with the HLA molecule is the candidate analogue of MAGE-A12 HLA binding peptide.In certain embodiments, method comprises 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, and the activation of test T cell.In some method therein, 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, and the tumour necrosis factor that the T cell produces, the T cell is to the cytolysis of target cell.

According to one aspect of the present invention, provide vaccine composition.Vaccine composition comprises aforementioned MAGE-A12 HLA binding peptide, aforementioned T lymphocyte, aforementioned antigen presenting cell, and/or aforementioned isolated nucleic acid molecule.In certain embodiments, aforementioned vaccine composition comprises assistant agent and/or pharmaceutically acceptable carrier.

In another aspect of the present invention, provide to comprise one or more isolated M AGE-A12 HLA I class binding peptide, or it is in conjunction with the little arrangement of protein (microarrays) of the functional variant of HLA I quasi-molecule.Functional variant comprises one or more aminoacid addition, replaces or disappearance.In certain embodiments, isolated M AGE-A12HLA I class binding peptide comprises SEQ ID NO:6 aminoacid sequence.In other embodiments, isolated M AGE-A12 HLA I class binding peptide comprises the aminoacid sequence of choosing from following group, comprise SEQ ID NO:4, SEQ ID NO:5, and their functional variant.The present invention also provides this little application that is arranged in the diagnosis, the particularly application in diagnosing cancer.Diagnostic method comprises the little arrangement of protein is contacted with the biological sample that body obtains that tried of suffering from pathology from suspection, and determines the composition of biological sample and combining of isolated M AGE-A12 HLA I class binding peptide.In certain embodiments, the composition of biological sample is chosen from following group, comprises antibody, T lymphocyte and HLA molecule.Preferred pathology is a cancer.

The present invention also provides the medication preparation that contains any or multiple composition described herein product.Such medication preparation product can comprise pharmaceutically acceptable thinner carrier or vehicle.The present invention also provides and has used such preparation of compositions medicine, particularly the medicine of preparation treatment cancer.

In preceding method and composition, the HLA molecule is HLA Cw preferably ^*07, HLA Cw more preferably ^*0701.The employed pathology of this paper is a cancer, as bladder cancer, and malignant melanoma, esophagus cancer, lung cancer, head and neck cancer, mammary cancer, colorectal carcinoma, myelomatosis, cerebral tumor, pernicious sarcoma, prostate cancer and kidney.

These purposes of the present invention and other purposes are described in more detail in conjunction with detailed description of the present invention.

The summary of diagrammatic sketch of the present invention

Fig. 1 has demonstrated the recognition reaction that CTL501 D/19 to self and allotype HLA-Cw*07 positive tumor cell is: (A) cracking, (B) TNF discharges.

Fig. 2 has demonstrated the antigen of the MAGE-12 coding that CTL 501D/19 identification presented by HLA-Cw7.

Fig. 3 has demonstrated the MAGE-A12 zone of definite coding by the antigen peptide of CTL 501 D/19 identification.

Fig. 4 has demonstrated self clone LB-831-EBV with MAGE-12 peptide VRIGHLYIL (SEQ ID NO:4) and RIGHLYIL (SEQ ID NO:6) pulse by CTL 501D/19 cracking.

Detailed description of the present invention

The invention provides the MAGE-A1 peptide of separation, the some of them peptide is presented by HLA I quasi-molecule, and such peptide stimulates CD8+T Proliferation of lymphocytes and activation. Such peptide this paper is called " MAGE-A12 immunogenic polypeptide " and " MAGE-A12 HLA I class binding peptide " and " MAGE-A12 hla peptide ", etc. Therefore, one aspect of the present invention is the peptide that comprises the separation of SEQ ID NO:6 amino acid sequence.

Following example has shown the separation of the peptide that is MAGE-A12 HLA binding peptide. These example peptides are translation products of the processing of SEQ ID NO:1 nucleic acid. Like this, the those of ordinary skill in this field is appreciated that the translation product of the final form that MAGE-A12 immunogenic polypeptide therefrom is processed to present can be as long as it comprises any length or the sequence of HLA binding peptide. In certain embodiments, the HLA binding peptide comprises and contains SEQ ID NO:4, the MAGE-A12 HLA binding peptide of 5 or 6 amino acid sequence. As demonstrating among the following embodiment, littlely be suitable for processing to 8 amino acid whose peptides or protein, but presented and effective stimulus CD8+T lymphocyte by HLA I quasi-molecule. SEQ ID NO:4,5 or 6 peptide can have one, and two, three, four, five, six, seven, eight, nine, ten, or more amino acid is added on an end or two ends. The amino acid that adds can corresponding to MAGE-A12 polypeptide (SEQ ID NO:2), maybe can be incoherent. So well-known in the field, the cracking under physiological conditions of the antigen part of this peptide is presented by HLA I quasi-molecule.

Other can be replied by immune stimulatory when by HLA Cw*07 molecular presentation from the HLA binding peptide that the MAGE-A12 polypeptide is derived. The present invention includes all such immunogen fragments of MAGE-A12 polypeptide.

As used herein, " functional variety " of MAGE-A12 HLA binding peptide or " variant " are one or more molecules primary amino acid sequence modification and that keep HLA I class binding characteristic disclosed herein and stimulate the ability of CD8+T Proliferation of lymphocytes and/or activation to MAGE-A12 HLA binding peptide that contain. The modification that produces MAGE-A12 immunogenic polypeptide functional variety can be made with 1) strengthen the characteristic of MAGE-A12 HLA binding peptide, such as the stabilized peptide in expression system or protein-protein in conjunction with the stability such as the combination of HLA-peptide; 2) new activity or characteristic are provided for the MAGE-A12 immunogenic polypeptide, as adding epitope or adding identifiable composition; Or 3) provide the different aminoacids sequence that produces identical or similar T cytositimulation attribute. Modification to MAGE-A12 HLA binding peptide can be made the nucleic acid of encoded peptide, modifies to comprise disappearance, and point mutation, brachymemma, amino acid substitution and amino acid add. Replacedly, modification can directly be made polypeptide, as by cracking, adds linkers, and interpolation can be differentiated composition, such as biotin, adds aliphatic acid, another amino acid of amino acid substitution etc. Modify and also comprise the fused protein that contains all or part of MAGE-A12 immunogenic polypeptide amino acid sequence.

The amino acid sequence of MAGE-A12 immunogenic polypeptide can be natural or the non-natural source, namely, they can be the sequences that natural MAGE-A12 immunogenic polypeptide molecule maybe can comprise modification, as long as amino acid sequence keeps stimulating the ability of cytolytic T lymphocyte and the characteristic that maintenance is combined with HLA I quasi-molecule such as HLA Cw*07 molecule when presenting. For example, the MAGE-A12 immunogenic polypeptide can be the fused protein of MAGE-A12 HLA binding peptide and uncorrelated amino acid sequence in this article, at SEQ ID NO:4, the synthetic peptide of the amino acid sequence that shows in 5 and 6, the mark peptide, express the peptide that separates the patient of cancer from suffering from MAGE-A12, the peptide that separates from the cultured cell of expressing MAGE-A12 is with the peptide (as in the some drugs delivery system) of non-peptide molecule coupling and other molecules that comprise SEQ ID NO:6 amino acid sequence.

Preferably, MAGE-A12 HLA binding peptide is non-hydrolysable. For such peptide is provided, can choose MAGE-A12 HLA binding peptide in never hydrolyzable peptide (as contain the amino acid whose peptide of one or more D-or contain the amino acid whose peptide of the one or more non-hydrolysable peptide linkages) library. Replacedly, can choose the peptide of inducing CD8+T lymphocyte optimum, then modify on demand such peptide to reduce by the possibility of protease hydrolytic. For example, in order to determine the neurological susceptibility to proteolytic cleavage, can be peptide-labeled and with cell extract or purifying protein enzyme incubation, then separate to determine which kind of peptide bond to the proteolysis susceptible, as by peptide and proteolytic fragments are checked order. Replacedly, potential neurological susceptibility peptide bond can be by relatively the amino acid sequence of MAGE-A12 immunogenic polypeptide and the known cracking site specificity of protease test group are determined. According to this test result, may can replace by the synthetic peptide bond with non-hydrolysable of external peptide by proteoclastic individual peptide bond.

The peptide bond of many non-hydrolysables and the synthetic method that contains the peptide of this peptide bond are well-known in this field. The non-hydrolysable peptide bond comprises-psi[CH₂NH]-the reducing amide peptide bond ,-psi[COCH₂]-ketone methylene peptide bond ,-psi[CH (CN) NH]-(cyanogen methylene) amino peptide bond ,-psi[CH₂CH (OH)]-the hydroxy ethylene peptide bond ,-psi[CH₂O]-peptide bond ,-psi[CH₂S]-the thio-methylene peptide bond.

The non-peptide analogues of peptide for example provides the non-peptide analogues of the biodegradation of stable structure or reduction also to take into account. Peptide analogy thing can prepare by form the one or more residues of replacement with non-peptide according to the MAGE-A12 HLA binding peptide of choosing. Preferably, non-peptide forms so that Toplink keeps its native conformation, or stable preferred such as the biologically active conformation. The example of a method for preparing non-peptide analogy thing from peptide is described among the Regul.Pept.57:359-370 (1995) people such as Nachman. Peptide analogues also can be chosen from synthetic compound (such as the combinatorial libraries of little organic molecule) or natural molecule library in conjunction with attribute and/or T cytositimulation attribute according to the HLA of such molecule. The method of testing of the analog of the MAGE-A12 immunogenic polypeptide that discriminating obtains from the library is as being well-known in conjunction with method of testing in this field. Peptide comprises all aforementioned things as used herein.

If variant comprises the variation of MAGE-A12 immunogenic polypeptide (SEQ ID NO:4,5 or 6), the functional variety that contains the MAGE-A12 immunogenic polypeptide that conservative amino acid replaces is normally preferred, namely, maintenance source amino acid character such as electric charge, hydrophobicity, the replacement of conformation etc. The example that the amino acid conservative is replaced is included in the replacement of implementing in the amino acid in following group: (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.

The additive method of the functional variety of discriminating MAGE-A12 immunogenic polypeptide is open in the PCT application (US/96/03182) of Strominger and Wucherpfennig. These methods depend on and manifest the amino acid sequence motif that potential epitope can compare with it. Every kind of primitive has been described limited amino acid sequence group, wherein may (a) be limited on the single residue at each (relatively) locational residue, (b) in restricted group of residue, can change, or (c) in all possible residue, can change. For example, the residue that primitive may specify on the primary importance can be the residue valine, leucine, isoleucine, methionine, or in the phenylalanine any one; The residue that may specify on the second place must be histidine; Specifying in the 3rd locational residue can be any amino acid residue; Specifying in the 4th locational residue can be the residue valine, leucine, isoleucine, methionine, phenylalanine, any one in tyrosine or the tryptophan; Specifying in the 5th locational residue must be lysine.

The sequence motif of MAGE-A12 HLA binding peptide functional variety can manifest by φt cell receptor (" the TCR ") contact point in conjunction with territory or binding pocket and/or MAGE-A12 immunogenic polypeptide described herein of analyzing ajor histocompatibility compound HLA Cw protein. By the detailed construction analysis of the residue relevant with forming HLA I class binding pocket is provided, the those of ordinary skill in this field can be to making a prediction in conjunction with the sequence motif of any HLA I proteinoid.

Use these sequence motifs as research, evaluation, or design standard, the those of ordinary skill in this field can be differentiated the peptide classification (functional variety of MAGE-A12 HLA binding peptide disclosed herein) with the reasonable possibility of being combined with concrete HLA molecule and reply with inducing T cell with the T cell interaction. As described herein, these peptides can synthesize and test active. Use these primitives, opposite from pure sequence homology (it does not comprise the similar but significantly different multiple peptide of sequence of antigenicity) or the sequence homology (it is included in the different multiple peptide in high conservative site) replaced with unrestriced " conservative ", represent that the those of ordinary skill in a kind of this field can evaluate with it method of peptide use possibility in disease treatment.

Strominger and Wucherpfennig PCT apply for (list of references that this paper quotes, the full content of document is with reference to this piece of writing of income), have described HLA II class and the TCT binding pocket of the residue of contact HLA II class peptide. Similarly, may be combined in the uniformity of the residue in HLA I class and/or the TCR binding pocket by maintenance, or only allow special replacement, the functional variety of the MAGE-A12 HLA binding peptide that maintenance is combined with HLA I class and φt cell receptor can prepare.

In protein sequence the location one or more antigenic peptides can obtain by according to set up in conjunction with the possibility principle (as, the people such as Parker, immunology periodical, 152:163,1994; The people such as Rammensee, immunogenetics 41:178-228,1995) hla peptide of making is auxiliary in conjunction with prediction. HLA can use by Internet in conjunction with prediction and carry out easily in the rule of transporting that the World Wide Web station of uniform resource locator (URL) http://bimas.dcrt.nih.gov obtains in (U.S.) NIH (Nation Institutes of Health) website.

The method of differentiating the MAGE-A12 immunogenic polypeptide is provided. Usually, method comprises chooses MAGE-A12 HLA binding peptide, in conjunction with the HLA I class binding molecule of MAGE-A12 HLA binding peptide, and the T cell that stimulates of the MAGE-A12 HLA binding peptide of being presented by HLA I class binding molecule. In preferred embodiments, the MAGE-A12 immunogenic polypeptide comprises SEQ ID NO:6 amino acid sequence. More preferably, peptide comprises SEQ ID NO:4, the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. The first amino acid residue of MAGE-A12 HLA binding peptide is suddenlyd change to prepare variant peptides. According to above-mentioned HLA and T cells contacting point principle, amino acid residue can be by mutagenesis. Any method for preparing variant peptides can be used, and such as synthesizing of variant peptides, uses mutant nucleic acid molecular recombination vegetation variant peptides, etc.

Determine the combination of variant peptides and HLA I class binding molecule and/or to the spread effect of T cell according to standard method. For example, example as shown below, variant peptides can contact with containing the HLA I quasi-molecule antigen presenting cell of being combined with MAGE-A12 HLA binding peptide, forms the compound of variant peptides and antigen presenting cell. Then can be with this compound and the T cells contacting of identifying the MAGE-A12 HLA binding peptide of being presented by HLA I class binding molecule. The T cell can be to express from suffering from characteristics the patient of the patient's condition of MAGE-A12 and obtain. The T cell can be determined by the indicator (such as the generation of TNF or IFN γ) of measuring the effect of T cytositimulation the identification of variant peptides.

The combination of variant peptides and HLA I class binding molecule and/or the variant peptides of being presented by HLA I class binding molecule are functional varieties to the spread effect explanation variant peptides of T cell. Method can comprise that also comparison MAGE-A12 HLA binding peptide determines that to the spread effect of T cell functional variety is to the step of the validity of T cytositimulation effect to spread effect and the functional variety of T cell. By MAGE-A12 HLA binding peptide and functional variety are compared, can prepare the peptide of the T cytositimulation attribute with increase.

If necessary, can the variant of the MAGE-A12 HLA binding peptide by the preparation of any preceding method be checked order, to determine amino acid sequence, release the nucleotide sequence of this variant of coding.

Therefore, coding MAGE-A12 immunogenic polypeptide or its variant, the nucleotide sequence that comprises allele variant also is a part of the present invention. In the examination to the nucleic acid of coding MAGE-A12 immunogenic polypeptide, can use³²The P probe is implemented nucleic acid hybridization under stringent condition, such as Southern trace or Northern trace. As used herein " stringent condition " refers to the parameter known in this field. The nucleic acid hybridization parameter can find in compiling the document of this method, such as molecular cloning: laboratory manual, J. the people such as Sambrook, second edition, publishing house of cold spring harbor laboratory, the cold spring port, New York 1989, or molecular biological current scheme, the people such as F.M.Ausubel edit, John Wiley ﹠ Sons Co., Ltd, New York. The stringent condition of example is included in 65 degrees centigrade of hybridization buffers (3.5 * SSC, 0.02%Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 25mM NaH₂PO ₄(pH7), 0.5%SDS, 2mM EDTA) middle hybridization. SSC is 0.15M sodium chloride/0.015M natrium citricum, pH7; SDS is lauryl sodium sulfate; EDTA is ethylenediamine tetra-acetic acid. After the hybridization, can wash DNA and transfer to film on it, as, in room temperature with 2 * SSC, then in room temperature to 68 degree centigrade with 0.1-0.5 * SSC/0.1 * SDS. After the DNA of flushing coding MAGE-A12 immunogenic polypeptide finally transfers to film on it, film is placed to measure radiated signal facing to x-ray film.

Can use in addition and can produce other conditions of similar stringency degree, reagent. The technical staff knows such condition, so these conditions here do not provide. Yet be appreciated that and think that the technical staff can operate these conditions in homologue and the allelic mode of nucleic acid that can clearly determine coding MAGE-A12 immunogenic polypeptide of the present invention. The technical staff also knows cell and the library that such molecule is expressed in examination, and separates routinely subsequently, then separates the method for relevant nucleic acid molecules and order-checking.

The present invention also comprises the application of nucleotide sequence of variable cipher of the same amino acid residue of coding MAGE-A12 immunogenic polypeptide. For example, as disclosed herein, peptide VRIGHLYIL (SEQ ID NO:4) is MAGE-A12 HLA binding peptide. Leucine residue can be by codon CUA, CUC, CUG, CUU, UUA and UUG coding. Each of six codons is equivalent for the coding leucine residue. Therefore, clearly, any leucine-coding nucleotide triplet can be used for directed protein synthesizer the those of ordinary skill in this field, in the body or the external leucine that mixes. Similarly, coding comprises that the nucleotide sequence triplet of other amino acid residues of the MGAE-A12 HLA binding peptide of SEQ ID NO:4 comprises: GUA, GUC, GUG and GUU (valine codon); GGU, GGA, GGG, GGC (codon glycine); UGC and UAU (tyrosine codon). Other amino acid residues can be encoded similarly by multiple nucleotide sequence. Therefore, the present invention includes since the degeneracy of genetic codon at the degeneracy nucleic acid that is different from natural MAGE-A12 immunogenic polypeptide code nucleic acid aspect the codon sequence.

The nucleic acid of preferred coding MAGE-A12 polypeptide is preferably to express MAGE-A12 immunogenic polypeptide, the as described herein nucleic acid of HLA binding peptide. MAGE-A12 nucleic acid of the present invention whole MAGE-A12 polypeptide of not encoding, but really comprise the nucleotide sequence of coding MAGE-A12 HLA binding peptide.

The present invention also provides and has comprised interpolation, replaces and lack the modified nucleic acid molecule of one or more Nucleotide.In preferred embodiments, the nucleic acid of these modifications and/or its encoded polypeptides keep the activity or the function of at least a unmodified nucleic acid molecule and/or polypeptide, as antigenicity, enzymic activity, receptors bind, by the mixture of MHC I class and the formation of II quasi-molecule binding peptide, or the like.In certain embodiments, the nucleic acid molecule encoding modified polypeptides of modification, preferably polypeptide contains conservative amino acid replacement as described elsewhere herein.Nucleic acid molecule with unmodified on the structural nucleic acid molecule of modifying is relevant, and in preferred embodiments, structurally the nucleic acid molecule with unmodified is relevant fully for the nucleic acid molecule of modifying, like this modification of nucleic acids and unmodified making nucleic acid molecular hybridization under the stringent condition known of the technician in this field.

For example, can prepare the modified nucleic acid molecule (preferably not being that those are the amino acid of HLA in conjunction with point of contact for example) that coding has the polypeptide of single amino acid variation.Each of these nucleic acid molecule is except the Nucleotide corresponding to the degeneracy of genetic codon described herein changes can to have one, two or three Nucleotide replacements.Similarly, the nucleic acid molecule that coding contains the modification of the polypeptide that two amino acid change can prepare, and such nucleic acid molecule has as 2-6 Nucleotide variation.Technician in this field can be contemplated to the nucleic acid of the multiple modification identical with these nucleic acid easily, comprises the replacement as Nucleotide in the codon of coded amino acid 2 and 3,2 and 4,2 and 5,2 and 6, or the like.In the aforementioned embodiment, every kind of combination of two seed amino acids all is included in modified nucleic acid molecule, and in the group of all Nucleotide replacements of coded amino acid replacement.So the those of ordinary skill in the field can be contemplated to easily, coding has other replacements (that is, 3 or a plurality of), interpolation or disappearance (as, by introducing terminator codon or splice site) other nucleic acid molecule of polypeptide also can prepare, and comprise in the present invention.Any aforesaid nucleic acid or polypeptide all can be by structural dependence and the active maintenance of normal experiment mensuration for nucleic acid disclosed herein and/or polypeptide.

Be understandable that equally, present invention resides in and use sequence in the expression vector, and transfection host cell and clone, as prokaryotic cell prokaryocyte (E.coli), or eukaryotic cell (as, dendritic cell, Chinese hamster ovary celI, the COS cell, recombinant baculovirus is expressed in yeast expression system and the insect cell).Expression vector needs correlated series, that is, described above those can be operated with promotor and combine.In addition, found human HLA-Cw ^*07 molecular presentation MAGE-A12 HLA I class binding peptide, expression vector also can comprise the nucleotide sequence of coding HLA-Cw*07 molecule.(, can use different HLA molecules for other I classes or II class binding peptide.) comprise in the situation of two kinds of encoding sequences that at carrier carrier can be used for transfection does not have any one cell of normal expression.When host cell has been expressed HLA-Cw ^*07 fen period of the day from 11 p.m. to 1 a.m, MAGE-A12HLA I class binding peptide encoding sequence can use separately.Certainly, if desired, do not expressing HLA-Cw to being used as to contain to use ^*The concrete host cell of the carrier of two encoding sequences in the host cell of 07 molecule is hard-core, and the nucleic acid of coding MAGE-A12 HLA I class binding peptide also can use at expression HLA-Cw ^*In the antigen presenting cell of 07 molecule.As used herein, " HLA-CW ^*07 molecule " comprise hypotype HLA-Cw ^*0701 (07011,07012), 0702,0703,0704,0705,0706,0707,0708,0709,0710,0711,0712,0713 and 0714.HLA-Cw ^*07 molecule also comprises can be people such as Bodmer, tissue antigen 49:297, the hypotype that can find in 1996.Definite at present HLA-Cw ^*07 hypotype inventory can find in the IMGT/HLA database among the Internet URL http://www.ebi.ac.uk/imgt/hla/.

Be understandable that equally, present invention resides in and use sequence in the expression vector, expression vector comprises plasmid recombinant, phagemid, virus etc., and use sequence transfection host cell and cell kind system, as prokaryotic cell prokaryocyte (E.coli), or eukaryotic cell (as, dendritic cell, Chinese hamster ovary celI, the COS cell, recombinant baculovirus is expressed in yeast expression system and the insect cell).。Expression vector needs correlated series, that is, described above those can be operated with promotor and combine.The expression vector that contains the MAGE-A12 sequence in vivo or external conveying can by known nucleic acid delivery system in this field (referring to, as people such as Allsopp, European immunology periodical, 26 (8): 1951-1959,1996).The recombinant vector that comprises the virus of choosing from following influenza virus group can use in such delivery system, as can be used as vaccine, the virus group comprises adenovirus, adeno-associated virus, poxvirus (comprising vaccine virus and attenuation poxvirus such as NYVAC), Semliki Forest virus, Venezuelan equine encephalitis (Venezuelan equineencephalitis) virus, retrovirus, Sindbis virus, with the Ty viruslike particle, plasmid (as " naked " DNA), bacterium.The analogue that other viruses, expression vector and being used for prepare vaccine is known to the those of ordinary skill in this field.The MAGE-A12 delivery system can be tested in the mode standard system, in mouse, to determine the validity of delivery system.System also can test in human clinical experiment.

In addition, non--MAGE-A12 tumor-associated peptides also can be taken to increase the immunne response by HLA I class and/or II class.Fully definite cancer can be expressed more than a kind of tumor-related gene.For the those of ordinary skill in this field, determine specifically to be tried body and whether express other tumor-related genes and belong in the normal experiment scope, and comprise from the expression product deutero-HLA I and/or the HLA II class binding peptide of this gene MAGE-A12 composition and vaccine.

Particularly preferably be the encode series epitope nucleic acid of (being called " multi-epitope ").A plurality of epitopes can be in a continuous manner or overlap mode arrange (referring to as, people such as Thomoson, Proc.Natl.Acad, Sci.USA 92:5845-5849,1995; People such as Gilbert, Nature Biotechnol.15:1280-1284,1997), being with or without natural flanking sequence, epi-position can be by incoherent joint sequence at interval if desired.Multi-epitope is processed into the individual epi-position of product that produces immunne response by immune system recognition.

Therefore, by the MHC molecular presentation and by the MAGE-A12 HLA binding peptide of CTL (or T th cell) identification, as SEQ ID NO:4,5 and 6, can make up to form " multi-epitope " with the peptide that obtains from other Tumor rejection antigens (as by preparation hybrid nucleic acid or polypeptide).Can take and induce or example tumor-associated peptides that enhancing immunity is replied is to derive from tumor-related gene and encoded protein matter, comprise 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), tyrosine oxidase, 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, the antigen peptide of tumour characteristics comprises in the following Table I listed.

Table I: example antigen

Gene	MHC	Peptide	The position	SEQ?ID?NO：
					MAGE-A1	HLA-A1	?EADPTGH ?SY	?161-169	?9
	HLA- Cw16	?SAYGEPR ?KL	?230-238	?10
					MAGE-A3	HLA-A1	?EVDPIGH ?LY	?168-176	?11
	HLA-A2	?FLWGPR ?ALV	?271-279	?12
						HLA-B44	?MEVDPIG ?HLY	?167-176	?13
BAGE	HLA- Cw16	?AARAVFL ?AL	?2-10	?14
					GAGE-1，2	HLA- Cw16	?YRPRPRR ?Y	?9-16	?15

?RAGE	?HLA-B7	?SPSSNRIR ?NT	11-20	?16
					?GnT-V	?HLA-A2	?VLPDVFI ?RC(V)	2-10/11	?17，18
?MUM-1	?HAL-B44	?EEKLIVV ?LF	Exon 2/intron	?19
							EEKLSVV LF (wild-type)		?20
?CDK4	?HLA-A2	?ACDPHSG ?HFV	?23-32	?21
							ARDPHSG HFV (wild-type)		?22
?β-catenin	?HLA-A24	?SYLDSGI ?HF	?29-37	?23
					Tyrosine oxidase		SYLDSGI HS (wild-type)		?24
	?HLA-A2	?MLLAVLY ?CL	?1-9	?25
						?HLA-A2	?YMNGTM ?SQV	?369-377	?26
	?HLA-A2	?YMDGTM ?SQV	?369-377	?42
						?HAL-A24	?AFLPWH ?RLF	?206-214	?27

	?HLA-B44	?SEIWRDI ?DF	?192-200	?28
						?HLA-B44	?YEIWRDI ?DE	?192-200	?29
	?HLA-DR4	?QNILLSN ?APLGPQF ?P	?56-70	?30
						?HLA-DR4	?DYSYLQ ?DSDPDSF ?QD	?448-462	?31
?Melan-A MART	?HLA-A2	(E)AAGIG ?ILTV	?26/27-35	?32，33
					?Gp100 Pmell17	?HLA-A2	?ILTVILGV ?L	?32-40	?34
	?HLA-A2	?KTWGQY ?WQV	?154-162	?35
						?HLA-A2	?ITDQVPF ?SV	?209-217	?36
	?HLA-A2	?YLEPGPV ?TA	?280-288	?37
						?HLA-A2	?LLDGTAT ?LR	?457-466	?38
	?HLA-A2	?VLYRYGS ?FSV	?476-485	?39
					?PRAME	?HLA-A24	?LYVDSLF ?EL	?301-309	?40
?MAGE-A6	?HLA- ?Cw16	?KISGGPRI ?SYPL	?292-303	?41

?NY-ESO-1	?HLA-A2	?SLLMWIT ?QCFL	?157-167	?43
						?HLA-A2	?SLLMWIT ?QC	?157-165	?44
	?HLA-A2	?QLSLLM ?WIT	?155	?45

Other examples of HLA I class HLA II class binding peptide to the those of ordinary skill in this field be known (as, referring to Coulie, stem cell 13:393-403,1995), and can use in the present invention with similar fashion described herein.According to molecular biological standard method, the those of ordinary skill in this field can prepare and comprise one or more MAGE-A12 peptides and one or more aforementioned tumor rejection peptide, or the polypeptide of the nucleic acid of this peptide species of encoding.

Therefore, multi-epitope is (as multi-joint, eclipsed) two or more the possible immunogenicities that can combine with multiple arrangement or the group of immunne response stimulator polypeptide.Multi-epitope (or nucleic acid of coding multi-epitope) can be taken with the standard immunoassay scheme, as taking to animal, is stimulating with the test multi-epitope, strengthens and/or promote the validity of immunne response aspect.

Can directly combine or by using the peptide of the formation multi-epitope that flanking sequence combines, and use multi-epitope as vaccine in this field be well-known (referring to, as people such as Thomson, Proc.Natl.Acad.Sci USA92 (13): 5845-5849; People such as Gilbert, Nature Biotechnol.15 (12): 1280-1284,1997; People such as Thomson, immunology periodical, 157 (2): 822-826,1996; People such as Tem, J.Exp.Med 171 (1): 299-306,1990).For example, Tam shows and to comprise that MHC I class and II class successfully produce antibody and protective immunity in conjunction with the multi-epitope of epi-position in mouse model.Tam has also demonstrated the multi-epitope processing that comprises epi-position " string " and has produced the individual epitope that the MHC molecular presentation is arranged and discerned by CTL.Therefore, the multi-epitope that contains the epitope of different quantities and combination can prepare, and the recognition reaction of test CTL, and the validity of test in increasing immunne response.

As everyone knows, tumour has been expressed one group of tumour antigen, and wherein only some subgroup can be expressed in any given patient's tumour.Can prepare multi-epitope corresponding to the various combination of the epitope of representing the Tumor rejection antigen subgroup of in concrete patient, expressing.The multi-epitope that also can prepare the known Tumor rejection antigen that will express by a tumor type of the wide spectrographic of reflection.Multi-epitope can be incorporated among the patient who needs this treatment such as polypeptide structure, as by use known nucleic acid delivery system in this field (referring to, as people such as Allsopp, European immunology periodical, 26 (8): 1951-1959,1996).Adenovirus, poxvirus, Ty-virus type particle, adeno-associated virus, plasmid, bacteriums etc. can be used in such delivery system.Can in mouse model, test the multi-epitope delivery system to determine the validity of delivery system.System also can test in human clinical experiment.

As has been found, human HLA-Cw ^*07 molecular presentation MAGE-A12 immunogenic polypeptide, expression vector also can comprise coding HLA-Cw ^*The nucleotide sequence of 07 molecule.Coding comprises and HLA-Cw ^*The nucleic acid of the solvable HLA/ peptide complex of strand of the 07 MAGE-A12 immunogenic peptide that merges can be as people's such as lone description preparation (J.Immunother.21:283-294,1998).

Comprise in the situation of two kinds of encoding sequences that at carrier carrier can be used for transfection does not have any one cell of normal expression.When host cell has been expressed HLA-Cw ^*07 fen period of the day from 11 p.m. to 1 a.m, MAGE-A12 HLA I class binding peptide encoding sequence can use separately.Certainly, if desired, do not expressing HLA-Cw to being used as to contain to use ^*The concrete host cell of the carrier of two encoding sequences in the host cell of 07 molecule is hard-core, and the nucleic acid of coding MAGE-A12 HLA I class binding peptide also can use at expression HLA-Cw ^*In the antigen presenting cell of 07 molecule.

As used herein, " carrier " can be any amount of nucleic acid, and wherein required sequence can be inserted in restricted connection, to transport between different genotypic environments or to express in host cell.Although the RNA carrier also can use but carrier generally includes DNA.Carrier includes but not limited to, plasmid, and phagemid, bacterium and viral genome as described herein, as adenovirus, poxvirus and BCG.Cloning vector is carrier that can duplicate in host cell or the carrier that duplicates after it is incorporated in the host cell gene group, its further characteristics are to have one or more endonuclease restriction sites, carrier can be can determine that mode is cut thereon, and required dna sequence dna can be connected to wherein, the ability that so new recombinant vector keeps it to duplicate in host cell.In the plasmid situation, when the number of copies of plasmid in host bacteria increased, duplicating of required sequence can take place repeatedly, or each host is taken place once before the host is by the mitotic division breeding.In the phage situation, duplicate and initiatively to take place or in the passive generation of molten source stage in cleavage stages.Expression vector is that wherein required dna sequence dna can insert so that it can operationally combine and can be expressed as the carrier of rna transcription body with the adjusting sequence in restricted connection.Carrier may further include one or more flags sequence that is suitable for being used for differentiating or also not had suppressed by vector transfection or cell transformed.Mark comprises, for example, coding increases or reduces the proteinic gene to microbiotic or other compound resistances or susceptibility, the gene of the enzyme that its activity of encoding can detect by known standard testing method in this field (as, β-version lactoside enzyme, luciferase or alkaline phosphatase), with and obviously influence transform or cells transfected, the host, the gene of the phenotype of colony or phage (as, green fluorescent protein matter).Preferred carrier is the energy self-replication and expresses to present and can operate the carrier of the structure gene product in the bonded dna fragmentation with them.

As used herein, when encoding sequence with regulate sequence with the expression of encoding sequence or transcribe when placing the control of regulating sequence and the mode covalent attachment under the influence, encoding sequence and to regulate sequence be " operationally " bonded.If desired encoding sequence is translated in the functional protein, if 5 ' the inducing of promotor of regulating in the sequence produces transcribing of encoding sequence, if and the connection between two dna sequence dnas does not have (1) to produce phase shift mutation to introduce, (2) disturb promoter region to instruct the ability of transcribing of encoding sequence, (3) the corresponding rna transcription body of interference is translated the ability in the protein, and two dna sequence dnas can be described as operationally and combine.Therefore, the product transcription may be translated in the required protein or polypeptide if promoter region can influence transcribing of dna sequence dna, and promoter region operationally combines in encoding sequence so.Point out that as top some preferred nucleic acid is only expressed the MAGE-A12 polypeptide fragment that comprises HLA binding peptide described herein.

The precise nature of the adjusting sequence that genetic expression is required can change between species or cell category, but should comprise usually, respectively with transcribe and translate relevant 5 '-non-transcribed sequence and 5 '-non-translated sequence, as the TATA frame, cap sequence, CAAT sequence or the like.Especially, 5 '-non-transcribed adjusting sequence will comprise the promoter region that contains the promoter sequence that can operate bonded genetic transcription control.Regulating sequence also can be by required enhancer sequence or the upstream activator sequence of comprising.Carrier of the present invention can at random comprise 5 ' leader sequence or signal sequence.Select and design the limit of power that the carrier that is fit to belongs to those of ordinary skill in this field.

Containing the expression vector that all of expression needs must the factor can buy, and is well-known to the technician in this field.Referring to, as people such as Sambrook, molecular cloning " laboratory manual, second edition, press of cold spring harbor laboratory, 1989.Allogeneic dna sequence DNA (RNA) by the MAGE-A12 immunogenic polypeptide of will encoding is incorporated into that pair cell carries out the genetic engineering design in the cell.Placing transcription factor to operate allogeneic dna sequence DNA (RNA) controls down so that allogeneic dna sequence DNA is expressed at host cell.

The vote that mRNA expresses in mammalian cell is (from Invitrogen as pcDNA3.1, Carlsbad, CA obtains), it contains selectable mark as having G418 the resistance gene and human cytomegalovirus (CMV) enhanser-promoter sequence of (helping choosing the cell kind system of stable transfection).Therefore, that be suitable for expressing in primates or Canis animals is pCEP4 carrier (Invitrogen), and it contains Epstein Barr virus (EBV) replication orgin, helps plasmid is remained the multiple copied extra-chromosomal genetic element.Another kind of expression vector is the pEF-BOS plasmid that contains the polypeptide protracting factor 1 α promotor, and it stimulates in-vitro transcription effectively.This plasmid is described by Mishizuma and Nagata (Nuc.Acids Res.18:5322,1990), and its application in the transfection test is by open as Demoulin (molecular cytobiology, 16:4710-4716,1996).Other preferred expression carriers are adenovirus of being described by Stratford-Perricaudet, and it lacks E1 and E3 protein (J.Clin.Invest.90:626-630,1992).Use gland virus expression protein to carry out immunization and described by people such as Warnier, anti-P1A immunization (Int.J.Cancer, 67:303-310,1996) is carried out in use subcutaneous injection in murine.

The present invention also comprises so-called expression test kit, expresses test kit and makes the technician can prepare required expression vector or multiple expression vector.Such expression test kit comprises isolating at least at least two kinds of materials previously discussed.Other compositions can add on demand.

As described herein, the present invention serves many purposes, and this paper describes a part wherein.At first, make the technician diagnosis characteristics be to express the pathology of MAGE-A12 immunogenic polypeptide.These methods are included in the expression of determining MAGE-A12 HLA binding peptide in the biological sample, or the expression of the mixture of definite MAGE-A12 HLA binding peptide and HLA I quasi-molecule.The expression of the expression of peptide or peptide and HLA I quasi-molecule mixture can be right by the combination group with peptide or mixture, determines as the test of antibody.The expression of MAGE-A12 also can be measured by the Standard PC R TRAP of using the MAGE-A12 primer in biological sample (as the knubble biological section).Embodiment this paper that tumour is expressed provides, and the further example condition and the primer of MAGE-A12 amplification can find in 422 documents at United States serial No.09/018.

Preferably, diagnostic method comprises from being tried the isolating biological sample of body and the special reagent of MAGE-A12 HLA binding peptide being contacted, has MAGE-A12 HLA binding peptide to detect in biological sample.As used herein, " contact " mean suitable condition (as concentration, temperature, time, ionic strength) under biological sample is fully placed near reagent, so that reagent and being present between the MAGE-A12 HLA binding peptide in the biological sample interacts.Usually, is that known to the skilled in this field helps the related thing with it of molecule in the biological sample (as protein and the related thing of device acceptor with reagent in the condition of biological sample contact, antibody and the related thing of its proteantigen, the related thing of nucleic acid and its complementary sequence) the special interactional condition between.The special interactional example condition that helps between molecule and its related thing is described in the U.S. Patent No. 5,108,921 of awarding to people such as Low.

Biological sample can be fixed in vivo or be external.For example, biological sample can be intravital tissue, the special reagent of MAGE-A12 immunogenic polypeptide be can be used to detect have such molecule in tissue.Replacedly, biological sample can be at external fixing (as blood sample, tumor biopsy, tissue extract).In particularly preferred embodiments, biological sample is to contain cell sample, more preferably contains the sample of tumour cell.

The present invention further comprises nucleic acid or comprises MAGE-A12 HLA binding peptide or the little arrangement of protein of the nucleic acid of this peptide of encoding.In this aspect of the invention, the standard technique of little permutation technology can be used to evaluate the expression of MAGE-A12 HLA binding peptide and/or differentiates biotic component in conjunction with this peptide.The composition of biological sample comprises antibody, the HLA molecule, and lymphocyte (particularly T lymphocyte), or the like.Little permutation technology, it is also referred to as other titles, comprise protein fragment technology (protein chiptechnology) and solid phase protein permutation technology, be that those of ordinary skill in this field is well-known, its according to but be not limited to, on immobilized substrate, obtain the peptide or the proteinic arrangement of discriminating, target molecule or biotic component are combined with peptide, evaluate such combination.Referring to, as G.MacBeath and S.L.Schreiber, " Printing Proteins asMicroarrays for High-Throughput Function Determination, " science, 289 (5485): 1760-1763,2000.Nucleic acid is arranged, and particularly the homotaxy rule in conjunction with MAGE-A12 HLA binding peptide also can be used to diagnosis.As be used for differentiating the body that tried of suffering from the patient's condition that characteristics are that MAGE-A12 HLA binding peptide is expressed.

Little arrangement substrate includes but not limited to, glass, silicon, aluminosilicate, borosilicate, metal oxide such as aluminum oxide and nickel oxide, multiple clay, nitrocotton, or nylon.Little arrangement substrate can apply with compound, to strengthen synthetic (peptide or the nucleic acid) of probe on substrate.Can be used for first Nucleotide or amino acid are covalently bound on the substrate at coupling reagent on the substrate or reagent set.Multiple coupling reagent or reagent set are well-known to technician Jie in this field.Peptide or nucleic acid probe can directly be synthesized on the substrate with the predetermined net that carries.Perhaps, peptide or nucleic acid can be put and fix on the substrate, and in such a case, substrate can be with compound coated with strengthening combining of probe and substrate.In these embodiments, the synthetic probe can be with accurately in advance, a predetermined amount and a year net pattern spread upon on the substrate, and the robot of the control that preferably uses a computer spreads upon probe on the substrate in contact-printing mode or in noncontact mode (as ink-jet or piezoelectric type-electron transport mode).Probe can with the substrate covalent attachment.

In some embodiments, one or more control peptides or nucleic acid molecule are attached on the substrate.Preferably, the contrast nucleic acid molecule can be determined as binding characteristic, reagent quality and validity, hybridization success ratio and factors such as analysis limit value and success ratio.

The present invention the technician can be treated suffer from characteristics be to express the MAGE-A12 immunogenic polypeptide pathology tried body.Treatment comprises takes the reagent that increases the mixture that is tried interior MAGE-A12 HLA binding peptide of body body and HLA I quasi-molecule, and takes the CD8+T lymphocyte special to this mixture.The reagent of use in aforementioned therapies comprises MAGE-A12 immunogenic polypeptide and its functional variant, the mixture of such peptide and HLA I class binding molecule (as HLA Cw*07), the antigen presenting cell that has the mixture of MAGE-A12 immunogenic polypeptide and HLA I class binding molecule, the solvable strand fusions of HLA and MAGE-A12 polypeptide, or the like.The present invention also makes the technician can optionally increase the T lymphocyte populations, the CD8+T lymphocyte special to MAGE-A12 HLA binding peptide.

Separating MAGE-A12 HLA binding peptide also makes the nucleic acid of separation or design coding MAGE-A12 HLA binding peptide become possibility.Nucleic acid can be used for external or in prokaryotic host cell or in eukaryotic host cell preparation MAGE-A12 HLA binding peptide or contain the protein of this peptide.The well-known several different methods of those of skill in the art can be used for obtaining isolated M AGE-A12 HLA binding peptide in this field.For example, expression vector can be incorporated in the cell to produce peptide.In another approach, the mRNA transcript can be incorporated into the peptide that produces coding in the cell by microinjection in cell or with other modes.Translation mRNA also can be used to produce peptide in not celliferous extract as in the skein cell cracking system.The peptide that comprises MAGE-A12HLA binding peptide of the present invention also can be external synthetic.In order to obtain isolated M AGE-A12 HLA binding peptide, the technician in this field also can use the method for separation known peptide easily.These methods include, but not limited to immunochromatographic method, HPLC, size exclusion chromatography method, ion exchange chromatography and immune-affinity chromatography.

These isolated M AGE-A12 HLA binding peptide, or peptide and HLA I quasi-molecule such as HLA-Cw ^*The mixture of 07 molecule, can with as the assistant agent combinations of substances, be applied to vaccine in the pathology that the treatment characteristics are to express the MAGE-A12 immunogenic polypeptide with preparation.In addition, vaccine can be from presenting the cell preparation of MAGE-A12HLA binding peptide/HLA mixture in its surface, as the dendritic cell of transfection, and the B cell of transfection, the transfectant of non-proliferative, or the like.All wherein cell be used as in the situation of vaccine, these cells can be the cells that one or both stimulate the required composition of CD8+ lymphocyte that has with the encoding sequence transfection, maybe can be to have expressed two kinds of molecules not need cells transfected.Vaccine also can comprise expression vector and naked DNA or RNA, coding MAGE-A12 HLA binding peptide, and its precursor, or its fused protein, vaccine can be in external preparation and by injection, partickle bombardment, snuffing is gone into and additive method is taken.The vaccine of " naked nucleic acid " type has demonstrated and can immune stimulatory originality reply, and comprises that generation is to the special CTL (science 259:1745-1748,1993) of peptide by the naked nucleic acid coding.

Use the standard technique in this field, MAGE-A12 HLA binding peptide, and the mixture of MAGE-A12 HLA binding peptide and HLA molecule also can be used to prepare antibody.The normative document works of listing the general principle of antibody producing comprises Catty, D., Antibodies, A Practical Approach (antibody, hands-on approach),1 volume, IRL press, Washington DC (1988); Klein, J., Immunology; The Science Of Cell-Non-Cell Discrimination (distinguish by immunology: cell-non--cell Science), John Wiley and Sons, New York (1982); Kennett, R. waits the people Monoclonal Antibodies, Hybridoma, A New Dimension In Biological Analyses (monoclonal antibody, hybridoma, the side in the biological analysis To), Plenum press, New York (1980); Campbell, A., M Onoclonal Antibody Technology, in Laboratory Techniques and Biochemistry And Molecular Biology is (in laboratory technique and biological activity and the molecular biology Monoclonal antibody technique), 13 volumes (Burdon, people EDS. such as R.), ElsevierAmsterdam (1984); And Eisen, H.N., Microbiology (microbiology),The third edition, Davis, people EDS such as B.D. (Harper ﹠amp; Rowe, Philadelphia (1980)).

Therefore, antibody of the present invention can prepare by several different methods, and comprise to animal and take protein, protein fragments, marking protein or its segmental cell and suitable HLA I quasi-molecule are to induce polyclonal antibody.MONOCLONAL ANTIBODIES SPECIFIC FOR can be according to well-known technology in this field.

Noticeable, as well-known in this field, only small portion antibody molecule (paratope) relevant with antibody with combining of its epitope (referring to, Clark, W.R. (1986) The Experimental Foundations of Modern Immunology (immunological testing basis now)Wiley ﹠amp; Sons company limited, New York; Roitt, I. (1991) Essential Immunology (basic immunology), the 7th edition, BlackwellScientific Publications.Oxford).For example, pFc ' and Fc zone are the effectors of complementary cascade, but combine irrelevant with antigen., or be prepared to and do not have pFc ' zone by the antibody of enzymatic lysis from the zone of pFc ' wherein, specified F (ab ') ₂Segmental antibody has kept two antigen binding sites of complete antibody.Similarly,, or be prepared to and do not have the Fc zone by the antibody of enzymatic lysis from Fc zone wherein, the segmental antibody of specified Fab has kept an antigen binding site of complete antibody.And the Fab fragment comprises that covalently bound light chain of antibody and a part of heavy chain of antibody are called Fd.The Fd fragment is the main determining factor (10 kinds of different light chains are relevant under the situation that does not change antibodies specific with nearly for single Fd fragment) of antibodies specific, and the Fd fragment has kept the epitope binding ability in separation.

In the antigen-binding portion thereof of antibody, so well-known in the field, complementarity determining region (CDR) is arranged, it directly interacts with antigenic epitope, and ramework region (FR), the tertiary structure of its maintenance paratope (referring to, as Clark, 1986; Roitt, 1991).In heavy chain Fd fragment and light chain IgG immunoglobulin (Ig), four ramework regions (FR1 is to FR4) are arranged, separated by three complementarity determining regions (CDR1 is to CDR3) respectively.CDR, particularly CDR3 zone, heavy chain CDR3 antagonist specificity plays great role more specifically.

What fully determine at present in this field is that in the specificity while of the epitope that keeps source antibody, the non-CDR zone of Mammals antibody can be replaced with common zone similarities special or different specific antibodies.This point is in development and use non-human CDR wherein to obtain the most clearly proving in " humanization " antibody that produces function antibody in human FR and/or the regional covalent attachment of Fc/pFc '.Referring to as, US Patent No 4,816,567,5,225,539,5,585,089,5,693,762 and 5,859,205.

Therefore, for example PCT international application serial WO92/04381 has taught preparation and has used to small part murine FR zone the humanization murine RSV antibody of having been replaced by people source FR zone.Such antibody comprises the fragment of the complete antibody with antigen binding capacity usually all being called " chimeric " antibody.

Therefore, clearly, the present invention also provides F (ab) the those of ordinary skill in this field ₂, Fab, Fv and Fd fragment; Wherein Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 zone are by the chimeric antibody of the homology mankind or the replacement of non-human sequence; Wherein FR and/or CDR1 and/or CDR2 and/or light chain CDR3 zone are by the chimeric F (ab) of the homology mankind or the replacement of non-human sequence ₂Fragment antibody; Wherein FR and/or CDR1 and/or CDR2 and/or light chain CDR3 zone are by the chimeric Fab fragment antibody of the homology mankind or the replacement of non-human sequence; Wherein FR and/or CDR1 and/or CDR2 zone are by the chimeric Fd fragment antibody of the homology mankind or the replacement of non-human sequence.The present invention also comprises so-called single-chain antibody and human monoclonal antibody, as the antibody that is produced by the murine with function human immunoglobulin gene seat.

Such antibody can be used to differentiate the tissue of marking protein or is used for purification of protein.For treat antibody also can be used for imaging the coupling of specific mark reagent or with the anti-tumor agent comprising salmosin coupling, include but not limited to, Methotrexate, the radioiodination compound, toxin such as Ricin, other cytostatic medicaments or cytolysis medicine, or the like.Antibody according to the present invention's preparation is equally preferably special to peptide described herein/HLA mixture.

When this paper uses " pathology " or " patient's condition ", be meant wherein any pathological condition of MAGE-A12 immunogenic polypeptide expression.Such pathology comprises cancer, comprises bladder cancer, malignant melanoma, esophagus cancer, lung cancer, head and neck cancer, breast cancer, colorectal carcinoma, myelomatosis, cerebral tumor, pernicious sarcoma, prostate cancer and kidney.Prerequisite according to some methods of treatment of disclosure is to induce the immunity system of being tried body that the MAGE-A12 immunogenic polypeptide is replying of delivery cell.A kind of such method is to take self the CD8+T cell special to the mixture of MAGE-A12 HLA binding peptide and HLA I quasi-molecule to the body that tried that suspection suffers from the abnormal cells phenotype.In the skill of this CD8+T cell of external development the technician.Usually, will be from being tried sample cell such as hemocyte that body obtains and presenting mixture and also can stimulate the lymphopoietic cells contacting of CD8+T.Target cell can be a transfectant, as the COS cell of transfection, or the antigen presenting cell that has HLA I quasi-molecule of transfection, as dendritic cell or B cell.These transfectants are at the required mixture of their surperficial transfection, and when when interesting CD8+T lymphocyte combines, stimulate its propagation.The COS cell is widely used other proper host cell.Give then and tried self CD8+T lymphocyte that body is taken clone's expansion.The CD8+T LS is tried the immunne response of body, thereby reaches required therapeutic purpose.

Choose existing description of another kind of method (people such as Altman, science 274:94-96,1996 of antigen-special ctl clone body; People such as Dunbar, Curr.Biol.8:413-416,1998), wherein use the fluorophore tetramer of MHC I quasi-molecule/peptide complex to detect special ctl clone body.Simply, solvable MHC I quasi-molecule is folded at the small sphaeroprotein of β 2-with in conjunction with the peptide antigen of I quasi-molecule external.After the purification, the MHC/ peptide complex is purified and use biotin labeling.Be mixed with the tetramer by avidin (as phycoerythrin) at 4: 1 with molar ratio with biotinylated peptide-MHC mixture and mark.Then the tetramer is contacted as peripheral blood or lymphatic node with the CTL source.The tetramer is in conjunction with the CTL of identification polypeptide antigen/MHC I class mixture.Can be by tetramer bonded cell by the fluorescence-activated cell sorter sorting with isolating active CTL.Isolating then CTL can use as described above in external expansion.

In order to describe methods of treatment in detail, be called method (Grenberg, the J.Immunol.136 (5): 1917,1986 of adoptive transfer; People such as Riddel, science 257:238,1992; People such as Lynch, European immunology periodical, 21:1403-1410,1991; People such as Kast, cell 59:603-614,1989), the cell of presenting required mixture contacts with CTL, causes its special CTL propagation.CTL with propagation takes for the body that tried of suffering from cellular abnormality then, and the characteristics of cellular abnormality are that some presents the cellular abnormality of concrete mixture.CTL cracking abnormal cells, thus reach required therapeutic purpose.

The supposition of aforementioned therapies method, at least some are tried the abnormal cells of body and are presented relevant HLA/TRA mixture.This point is easy to determine, because the method for the cell of presenting concrete HLA molecule very is familiar with differentiating in this field, and how to differentiate and express the cell of presenting relevant sequence DNA that correlated series is the tumor-related gene sequence in this case.In case identify the cell of presenting related complex by aforementioned examination technology, these cells can combine with the sample that obtains from the patient, and wherein sample contains CTL.If mixture is the mixed CTL sample dissociation of delivery cell, just can suppose that tumor-related gene deutero-TRA exists, being tried body is the suitable candidate people of above-mentioned methods of treatment.

Adoptive transfer is not the therapeutics that the present invention only can use.The CD8+T lymphocyte also can use several different methods to stimulate in vivo.A kind of method is to use nonproliferating cell to express mixture.The cell of use in this method can be the cell of normal expression mixture, and they can be dendritic cells or present the cell of the required gene transfection of mixture with one or both.People such as Chen (Proc.Natl.Acad.Sci.USA88:110-114,1991) example has illustrated this method, is presented to use cells transfected to express the HPV-E7 peptide in the therapeutics.Can use the various kinds of cell kind.Similarly, the carrier that has one or both interesting genes also can use.Virus or bacteria carrier are particularly preferred.For example, the nucleic acid of coding MAGE-A12 HLA binding peptide can operationally combine with the expression promoter and the enhancer sequence that instruct MAGE-A12 HLA binding peptide in some tissue or cell category.Nucleic acid can be incorporated in the expression vector.Expression vector can be the extrachromosomal nucleic acid of unmodified, and plasmid or structure or modification can be inserted the viral genome of exogenous nucleic acid (as the nucleic acid of coding MAGE-A12 HLA binding peptide).The nucleic acid of coding MAGE-A12 HLA binding peptide also can be inserted in the reverse transcription virus gene group, helps like this nucleic acid is incorporated in the genome of target tissue or cell category.In these systems, interesting is carried by microorganism, as VaccineVirus, retrovirus or bacterium BCG, and the material of " infection " host cell in fact.Present the cell of interest mixture,, then breed by self CD8+T cell recognition.

Be in the delivery cell and can obtain similar effect by MAGE-A12 HLA binding peptide being combined with assistant agent to impel it to be incorporated into HLA I class in vivo.If MAGE-A12 HLA binding peptide can be processed the MAGE-A12HLA binding peptide if desired greater than HLA I class bound fraction, right with the peptide group that produces the HLA molecule, when presenting, TRA do not need to do further processing.Usually, but tried the MAGE-A12 immunogenic polypeptide of body subcutaneous injection significant quantity.According to the standard immunoassay vaccination regimen in this field, predose can then advance dosage.

As the composition of some immunization composition, one or more cancer associated antigens or its pungency fragment can be taken with one or more assistant agents, with induce immune response or increase immunne response.Assistant agent is the material that is incorporated in the antigen that booster immunization replys or takes with the antigen that booster immunization is replied.Assistant agent can be by providing antigen storage tank (as in extracellular or scavenger cell), activated macrophage and stimulate special group of lymphocyte to come enhancing immunity originality to reply.Many kinds of assistant agents are well-known in this field.The specific examples of assistant agent comprises monophosphoryl lipid A (MPL, SmithKline Beecham), the isoplassont that the Salmonella minnesota Re595 lipopolysaccharides acid hydrolysis and the back of purifying obtain; Saponin/TSM comprises QS21 (SmithKlineBeecham), the pure QA-21 Saponin/TSM of purifying from Quillaja saponaria extract; The DQS21 (SmithKlineBeecham) that in PCT application WO96/33739, describes, QS-7, QS-17, QS-18, and QS-L1 people such as (, molecular cell 7:178-186,1997) So; Incomplete Freund assistant agent; Complete Freund assistant agent; Montanide; The immunostimulating oligonucleotide (referring to as, people such as Kreig are at natural 374:546-9, the CpG oligonucleotide of describing in 1995); The water-in-oil emulsion of multiple preparation from biodegradable oil (as shark alkene and/or vitamin-E).Preferably, peptide and DQS21/MPL are mixed and take, and the ratio of DQS21 and MPL was generally about 1: 10 to 10: 1, preferably about 1: 5 to 5: 1, was more preferably about 1: 1.Generally take for the mankind, DQS21 and MPL are present in the vaccine formulation, and scope arrives about 10ug at about 1ug.Other assistant agents also are known in this field, and can use in the present invention (referring to, as Goding, monoclonal antibody: principle and practice, second edition 1986).The preparation peptide and the mixture of assistant agent or the method for emulsion are well-known to the those of ordinary skill in the vaccine field.

Stimulate other reagent of the immunne response of being tried body also can take to being tried body.For example, because the lymphocyte of other cytokines is regulated attribute, they also can use in the vaccine scheme.Many other cytokines that are used for this purpose are well-known to the those of ordinary skill in this field; comprise the interleukin 12 (IL-12) that shows the protectiveness effect that can strengthen vaccine (referring to as; science 268:1432-1434,1995), GM-CSF and IL-18.Therefore, cytokine can combine with antigen and assistant agent and take to increase antigenic immunne response.

Many other immunne responses enhancing compounds that can use in the vaccine scheme are arranged.These compounds comprise with co stimulatory molecule multiple or that the nucleic acid form provides.Such co stimulatory molecule comprises B7-1 and B7-2 (being respectively CD80 and CD86), its be expressed in that dendritic cell (DC) goes up and with the CD28 interaction of molecules that is expressed on the T cell.This interaction provides the common stimulation (signal 2) of (signal 1) T cell that antigen/MHC/TCR is stimulated, and increases T cell proliferation and effector function.B7 also with the T cell on CTLA4 (CD152) interact, about the interaction energy between the B7-CTLA4 that studies show that of CTLA4 and B7 part strengthens antineoplastic immune and CTL propagation (Zheng, Deng the people, Proc.Natl.Acad.Sci.USA95:6284-6289,1998).

B7 is not expressed on the tumour cell usually, so they are not that effective antigens is delivery cell (APC) for the T cell.Inducing B7 to express can more effectively stimulate CTL propagation and effector function.The common combination that stimulates of B7/IL-6/IL-12 has demonstrated can induce IFN-γ and the distribution of Th1 cytokine in the T cell mass, causes further enhancing T cytoactive people such as (, immunity periodical, 154:5637-5648,1995) Gajewski.People such as Wang (J.Immnother.19:1-8,1996) use B7 transfection tumor cell in external CTL expansion to carry out having discussed in the adoptive transfer.Other delivery systems of B7 molecule will comprise nucleic acid (naked DNA) immunization (people such as Kim, Nature Biotechnol.15:7:641-646,1997) and recombinant virus such as adenovirus and poxvirus (people such as Kim, Gene Ther.4:726-735,1997).These systems also be suitable for B7 and other molecules of choosing as discussed herein the expression cassette of antigen or antigen fragment (comprising multi-epitope) or cytokine coexpression application and make up.These delivery systems can be used to external evoked suitable molecule and use in the vaccine inoculation situation in vivo.Use in anti--CD28 antibody body and also can consider with external direct stimulation T cell.Similarly, the induced costimulatory molecules ICOS that inducing T cell is replied exogenous antigen also can be modulated, for example by using anti--ICOS antibody people such as (, natural 397:263-266,199) Hutloff.

LFA-3 (LFA-3) is expressed on APC and some tumour cells, and with the T cell on the CD2 that expresses interact.This interaction inducing T cell IL-2 and IFN-γ produce, and therefore can be complementary singly replace, and B7/CD28 stimulates interaction (people such as Parra, immunity periodical, 158:637-642,1997 jointly; People such as Fenton, J.Immunother.21:95-108,1998).

LFA-1 (LFA-1) is expressed on the white corpuscle, and interacts with the ICAM-1 that expresses on APC and some tumour cells.This interaction inducing T cell IL-2 and IFN-γ produce, and therefore can be complementary singly replace, and B7/CD28 stimulates interaction people such as (, 1998) Fenton jointly.Therefore, LFA-1 is another example of the co stimulatory molecule that provides in many ways in the immunization scheme of the B7 that can discuss in the above.

Complete auxiliary (people such as Ridge, natural 393:474,1998 of the interactional Th cell between the CD40 molecule that need express by Th cell CD40L (CD40 part) molecule with by DC of CTL activation and effector function; People such as Bennett, natural 393:478,1998; People such as Schoenberger, natural 393:480,1998).The mechanism of this common stimulus signal may comprise that B7 is just regulating and the relevant IL-6/IL-12 that is produced by DC (APC).Therefore, CD-40-CD40L interaction complementary signal 1 (antigen/MGC-TCR) interact with signal 2 (B7-CD28).

Use anti-CD 40 antibodies directly to stimulate the expectation of CD cell can strengthen the replying of tumor associated antigen, tumor associated antigen runs into outside the inflammatory environment usually, or is presented by non--full-time APC (tumour cell).In these situations, Th is complementary not to be provided with the common stimulus signal of B7.This mechanism can be used in antigen pulsed D C base therapeutics, or uses in the Th epi-position also unqualified before the known cancer related antigen in the intravital situation.

When taking, medical composition of the present invention is taken with pharmaceutically acceptable preparation.Such preparation can contain the salt of pharmaceutically acceptable concentration routinely, buffer reagent, and sanitas, compatible carrier replenishes immunostimulant reagent, as assistant agent and cytokine, and any other treatment reagent." pharmaceutically acceptable " means not the nothing-toxicant of bioactive validity that can the interferon activity composition.The characteristics of carrier will depend on route of administration.

Therapeutant of the present invention can be taken by any classical pathway, comprises injection or passes through cumulative perfusion of for some time.Medication can be an oral medication, intravenously medication, intraperitoneal medication, intramuscular medication, Nasacort, chamber innerlich anwenden, subcutaneous medication, intradermal medication or through the skin medication.

The preparation of non-enterally administer comprises aseptic aqueous solution or non-aqueous solution, suspension and emulsion.Examples of non-aqueous comprises propylene glycol, polyoxyethylene glycol, and vegetables oil such as plam oil and injectable organic ester are as ethyl oleate.Water-soluble carrier comprises water, ethanol/water solution, emulsion or suspension.Comprise salt solution and buffering medium.The injection carrier comprises sodium chloride solution, Ringer dextrose, dextrose and sodium-chlor, newborn acidifying Ringer solution or fixed oil.Intravenously comprises liquid and nutritional supplement with drug carrier, electrolytic solution fill-in (as fill-in) based on the Ringer dextrose, or the like.Sanitas and other additives also can exist, as antimicrobial, and antioxidant, sequestrant and rare gas element or the like.

The present invention has also considered gene therapy.External enforcement gene therapy methods is in U.S. Patent No. 5,399, describes in 346 and in the exhibit of submitting in the submission process of this patent, and all documents all are the obtainable files of the public.Usually, gene therapy is included in external function copy with gene and is incorporated in the cell that is tried body of the defectiveness copy that contains this gene, and the cell that genetic engineering is designed turns back to and tried in the body then.It is to make gene under genetic engineering designs the control operated of the regulatory factor of expressing in the cell that the function of gene copies.Many transfections and transduction technology and suitable expression are well-known to the those of ordinary skill in this field, and some such technology are described in PCT application WO95/00654.According to the present invention, use the vivo gene treatment of carrier such as adenovirus also to consider.

Preparation of the present invention is taken with significant quantity.Significant quantity is meant separately or can stimulates the amount of the required medication preparation product of replying with further dosage.In the situation of treatment cancer, required replying is the development that suppresses cancer.This can only comprise and slows down advancing of disease temporarily, though more preferably, comprises stopping advancing of disease muchly.In the situation of induce immune response, required replying is special antibody or the T lymphocyte of MAGE-A12 immunogen that increases using.Replying that these are required can be monitored by ordinary method, or can monitor according to diagnostic method of the present invention described herein.

Use in the situation that medical composition immune stimulatory of the present invention replys at needs, this also can comprise stimulates humoral antibody to reply, humoral antibody is replied and is caused in the serum antigen titration degree to increase, the clonal expression of cytotoxic lymphocyte, or some other required immunogenic response.Can believe and think that scope will be that effectively immunogenic dosage depends on the mode of taking at 1 nanogram/kilogram to the immunogen dosage of 100 mg/kg.Preferred range is believed at 500 nanograms between 500 mg/kg.Absolute magnitude depends on multiple factor, comprises the selected material of taking, and takes single formulation or multi-form, and the individual patient parameter, comprises the age, physical appearance, build size, body weight, and the stage of disease.These factors are well-known to the those of ordinary skill in this field, and just can obtain by normal experiment.

Embodiment

Material and method

Cell kind system

(pT3, G3) the transitional cell bladder carcinoma cell line kind of deriving is LB831-BLC, LB831 (HLA-A to invade tumor of bladder from initial stage of 65 years old Caucasian patient ^*2403 ,-A3 ,-B ^*4403 ,-B ^*4901 ,-Cw ^*0401 ,-Cw ^*07).Cell kind in 7 performances of going down to posterity is that karyotype shows karyomit(e) quantity 56 to 144 variations, confirms that LB831 cell kind system is a tumour kind system.MI13443-MEL is a malignant melanoma cell kind system, and LE9211-RCC is a kidney cancer cell kind system.Two kind systems all are deutero-from the HLA-Cw7-negative patient.LB373-MEL from HLA-Cw7-negative patient deutero-malignant melanoma kind is.(Life Technologies, Gaithersburg cultivate in MD) at the Iscove substratum that contains 10% human serum or 10%FCS (Life Technologies) in the incubator of 8% carbonic acid gas with tumour cell.Use standard technique to use cyclosporin A (Cyclosporine the A) (Sandoz of 1ug/ milliliter, Basel, Switzerland) and the supernatant liquor of the B95-8 cell of 20% (volume ratio) EBV transfection to be derivatized to lymphocyte (lymphoblastoid) cell kind from the PBL of patient LB831 be LB-831-EBV.This cell kind is tied up to growth in containing the RPMI-1640 substratum of 10%FCS (Life Technologies) in the incubator of 5% carbonic acid gas.By with 1.0% (volume ratio) PHA (Difco) and 100U/ milliliter IL-2 (Eurocetus, Amsterdam, Netherlands) stimulate PBL to prepare the PBL-PHA cell, and the PBL-PHA cell is being contained in the incubator of 8% carbonic acid gas in the Iscove substratum of 10% human serum and cultivate.Replenish in the All Media and add L-arginine (116ug/ milliliter), altheine (36ug/ milliliter), L-glutaminate (216ug/ milliliter), Streptomycin sulphate (0.1 mg/ml) and penicillin (200U/ milliliter).

The antitumor CTL clone body

(Norway) density gradient centrifugation is separated the blood mononuclear cell of patient LB831 and is kept under-80 degrees centigrade for Nycomed, Oslo to use Lymphoprep.As previously described (people such as Gueguen, immunology periodical 160:6188-6194,1998), will mix as the B7-1 transfection LB831-BLC cell of the irradiation of stimulator with as the CD8+T lymphocyte of replying thing and implement self mixed lymphocytes-tumor cell culture.With with anti--covalently bound magnetic bead of CD8 antibody (MACS, Miltenyi BiotecGmbH) with the sorting of CD8+T lymphocyte.Non--CD8+ the cell that will shine in stimulating for the first time joins in the blended culture.At the 3rd day, add IL-2 (25U/ milliliter).After a week, stimulate 5 * 10 again with the tumour cell of the B7-1 transfection of shining and the IL-2 of 25U/ milliliter ⁵Lymphocyte.At the 28th day, the lymphocyte that from culture, obtains by limiting dilution in replenishing the Iscove substratum of IL-2 (50U/ milliliter).Implement ctl clone body long-term cultivation as previously described people such as (, Int.J.Cancer39:390-396,1987) Herin.

The cytotoxicity test

As previously described people such as (, J.Exp.Med.152:1184-1193,1980) Boon, discharge the lytic activity that detects CTL in the test at chromium.Simply, with the cell of 1000 chromium marks among the 100ul in 96-culture dish minitype plate with different effectors with target ratio and isopyknic CTL incubation.Measuring the chromium of incubation after four hours discharges.In order to carry out peptide test, with label L B831-EBV cell at 37 degrees centigrade with the peptide incubation of different concns 30 minutes.Add CTL then, measure chromium as mentioned above to discharge/

The CTL hormesis is measured

3000 CTL of total amount are joined contain in 10000 stimulator cells (in the Iscove substratum of the IL-2 that is supplemented with 10% human serum and 25U/ milliliter) microculture.After 24 hours, collect supernatant liquor, by MTT colony metrology and measurement (people such as Hansen, J.Immunol.Meth.119:203-210,1989) measure TNF content (Espevik and the Nissen-Meyer that the cytotoxic effect of 13 cells of WEHI-164 clone body is determined supernatant liquor in, J.Immunol.Methods 95:99-105,1986).By in test, adding the ascites of 1/20-1/30 dilution, with mAbsW6/32 (anti--HLA I class), Bi.23.3 (anti--HLA-B and-C), B9.4.1 (anti--CD8), 1B8.2 is (anti--CD4, donate by D.Olive, INSERM U119, Marseille, France), GAPA-3 (anti--HLA-A3), C7709A2.6 (resists-HLA-A24) implements restraining effect.

Transient transfection

Use LipofectAMINE ^TMReagent (Life Technologies) is implemented transient transfection.Simply, with 5 * 10 ⁴Individual 293-EBNA cell (expressing 293 cells of EBV nuclear antigen EBNA-1) in flat 96 culture dish plates with the DNA of 100ngMAGE-A12cDNA or be cloned into pcDNA3 (Invitrogen, Carlsbad, the CA) subgenomic fragment in, 50ng contains HLA-Cw ^*0701 plasmid pcDNA3, and 1.5ulLipofectAMINE ^TMTransfection.LB373-MEL cell (10,000) makes up and 1ul LipofectAMINE with 150ngHLA-Cw7 ^TMTransfection.After 24 hours, in CTL stimulation test, measure cells transfected.

As previously described people such as (, 1998) Gueguen, implement cDNA from LB831-BLC clone HLA-Cw7.Its sequence and allelotrope subtype C w ^*07011 is identical, and except on 1087 positions of encoding sequence, wherein G has replaced A.The variation of this Nucleotide causes in the kytoplasm inner compartment of molecule L-Ala to replace Threonine.At Cw ^*0704 and Cw ^*Identical difference has had description (Baurain and Coulie, tissue antigen 53:510-512,1999) between 0711 allelotrope.

The stable transfection of tumour cell kind system

(people such as Traversari, immunogenetics 35:145-152) as previously described comes transfected in human bladder cancer cell LB831-BLC by the calcium phosphate precipitation method.With 1 * 10 ⁶Individual cell contains plasmid pEF-BOS purine-PL3 transfection of cDNA B7-1 with 20ug.Simply, use from the cell kind be the cDNA that obtains of LB23-EBV as template, using in the PCT reaction has adopted primer 5 '-GGGTCCAAATTGTTGGCTTTCACT (SEQ ID NO:7) and antisense primer 5 '-GAAGAATGCCTCATGATCCCCA (the SEQ ID NO:8) B7-1 that increases.The PCR condition was described by people such as Gueguen in 1998.Then B7-1 is inserted body and be cloned among plasmid pEF-BOS purine-PL3, plasmid pEF-BOS purine-PL3 derives from pEF-BOS (Mishizuma and Nagata, nucleic acids research 18:5322,1990) by inserting puromycin resistance gene and polylinker.(Sigma, St.Louis choose in MO) tetracycline resistance LB831-BLC cell, pass through limited dilution cloning then at 0.8ug/ milliliter tetracycline.

The clone of the subgenomic fragment of gene M AGE-A12

The MAGE-A12cDNA that contains the complete open reading frame (ORF) of 945 base pairs is used as the template of pcr amplification (people such as DePlaen, immunogenetics 40:360-369,1994).At first eight fragments of 195,342,525,540,591,651,683 and 816 Nucleotide that contain MAGE-A12 ORP are used following primer amplification 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?IDNO：49)

5’-CCAGTCAGGTGACAAGGATG-3’(LHE10；SEQ?IDNO：50)

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

5’-CTCCTAAGGGGCACAGTCGC-3’(LHE8；SEQ?IDNO：52)

5’-TCAGATGCCTACAACACACT-3’(LHE5；SEQ?ID?NO：53)

5’-GGACCCTACAGGAACTCGTRA-3’(LHE6；SEQ?IDNO：54)。(TaKaRa Taq) is used for pcr amplification to the Taq archaeal dna polymerase.First denaturing step was implemented 5 minutes at 94 degrees centigrade, implement the amplification in 25 cycles then, comprise: 94 degrees centigrade of all primers 1 minute, 62 degrees centigrade of LHE3, LHE4 and LHE5 primer 2 minute or at 64 degrees centigrade of LHE2, LHE6, LHE8, LHE9 and LHE10 primer 2 minute, and 72 degrees centigrade of all primers 3 minutes.Circulating in 72 degrees centigrade of last extension steps of 10 minutes finishes.Use BidirectionalEukaryotic TOPO TA clone's test kit (Invitrogen) with the PCR product cloning in the pcDNA3 carrier.

The PCR test that MAGE-A12 expresses

Implement the expression that RT-PCR detects MAGE-A12 in the tumor tissues.As previously described people such as (, Int.J.Cancer 56:826-829,1994) Weynants, implement that total RNA purifies and cDNA synthesizes.Use has 1/40th amplification of the cDNA that adopted primer 5 '-CGTTGGAGGTCAGAAACAG-3 ' (SEQ ID NO:55) and antisense primer 5 '-GCCCTCCACTGATCTTTAGCAA-3 ' (SEQ ID NO:56) will produce from the total RNA of 2ug.For PCR, first denaturing step was implemented 4 minutes at 94 degrees centigrade, implemented the amplification in 32 cycles then, comprising: 94 degrees centigrade 1 minute, 62 degrees centigrade 2 minutes, and 72 degrees centigrade 3 minutes.Circulating in 72 degrees centigrade of last extension steps of 15 minutes finishes.

Embodiment 1: derive and by the HLA-of bladder cancer patients from gene M AGE-A12

Cw ^* The novel antigens peptide of 0701 Restricted CTL identification

LB831-BLC expresses at least three kinds of antigenic transitional cell bladder carcinoma cell line kind systems by self CTL identification.One in them was described (people such as Gueguen, immunology 160:618806194,1998).To mix as the B7-1 transfection LB831-BLC cell of the irradiation of in serum, cultivating of stimulator with as the CD8+T lymphocyte of replying thing and implement self mixed lymphocytes-tumor cell culture.With with anti--CD8 antibody (magnetic active cells sorter, MACS, Miltenyi Biotec, Gergisch-Gladbach, Germany) covalently bound magnetic bead is with the sorting of CD8+T lymphocyte.Non--CD8+ the cell that will shine in stimulating for the first time joins in the blended culture.At the 3rd day, add IL-2 (50U/ milliliter).After a week, stimulate 5 * 10 again with the tumour cell of the B7-1 transfection of shining and the IL-2 of 25U/ milliliter ⁵Lymphocyte.At the 28th day, the lymphocyte that from culture, obtains by limiting dilution in replenishing the Iscove substratum of IL-2 (50U/ milliliter).Obtain one group of special CTL-clone body of BL831-, wherein CTL 501D/19 identification self tumour kind is but the B-cell (that is, its identification is different from first three antigenic antigens, is called LB831-D) of nonrecognition self EBV-conversion.

In order to determine the restriction of MHC to these ctl clone bodies, the anti--HLAmAb of research is to the influence of clone body hormesis.When with the LB831-cytositimulation, ctl clone body 501D/19 produces TNF, exist anti--during HLA I class mAbW6/32 or when having the monoclonal antibody (mAb B1.23.2) of common determiner of directed opposing HLA-B and-C molecule, this output is blocked fully.Because the HLA class of LB831 has HLA-A ^*2403 ,-A ^*3 ,-B ^*4901 ,-Cw ^*0401 and-Cw ^*07, this presentation of results target gene is by HLA-B ^*44, B ^*49, CW ^*04 presents or by CW ^*04 presents.

In standard chromium release in 4 hours test, discern other target cells by CTL501D/19.Shown in Figure 1A, CTL 501D/19 is two kinds of allotype tumours of cracking kind system also, and malignant melanoma MI13443-MEL and kidney cancer cell kind are LE9211-RCC.Target is LB831-BLC (self transitional cell bladder carcinoma cell line kind system); LB831-EBV (self EBV-conversion B cell); LE9211-RCC (the positive kidney cancer cell kind of allotype HLA-Cw4 and HLA-Cw7-system); MI13443-MEL (the positive malignant melanoma cell kind of allotype HLA-Cw4 and HLA-Cw7-system); And K562 (NK cell target).Before being used as target, allotype and self tumour cell kind system to be anticipated 1 or 5 day with IFN γ respectively, the chromium of measuring after 4 hours discharges.

All aforementioned tumour cells are to B ^*44 and B ^*The 49th, negative, but to Cw ^*04 and Cw ^*The 07th, male.In order to confirm which kind of HLA presents tumour antigen, with the instantaneous Cw that uses of malignant melanoma ^*04 or Cw ^*07 transfection, and the recognition reaction of test CTL501D/19.Cw only ^*07 transfectant is discerned by CTL.Shown in Figure 1B, LB373-MEL (from HLA-Cw7 negative patient deutero-malignant melanoma cell kind system) is used HLA-Cw7 plasmid construction transient transfection.3,000 CTL are joined in the 10000 stimulator cells, measure the TNF that CTL produces after 24 hours.Because CTL501D/19 also discerns HLA-Cw ^*07-is positive, and the malignant melanoma cell kind is NaMe16, so can reach a conclusion, antigen LB831-D is by HLA-Cw ^*07 molecular presentation.The Cw of LB831-BLC ^*07 allelotrope hypotype is determined and found is HLA-Cw ^*07011.

In order whether to determine LB831-D antigen by CTL501D/19 identification by the genes encoding of having known, with the 293-EBNA cell with containing HLA-Cw ^*The expression vector of 0701cDNA and the MAGE that comprises discovery great expression in the tumor of bladder sample of patient LB831, BAGE, GAGE, RAGE, LAGE ^NY-ESOThe common transfection of series of genes of cDNA (and other).Measuring transfectant stimulates CTL501D/19 to produce the ability of TNF.After the transfection 24 hours, with the CTL501D/19 incubation cell of 3000 cells.After 24 hours, the cytotoxicity of WEHI-164-13 cell is measured TNF in the supernatant liquor by measuring TNF.The LB831-BLC cell is used as positive comparison.Only ought use HLA-Cw ^*07 and during the 293-EBNA cytositimulation of gene M AGE-A12 transfection CTL produce TNF (Fig. 2).Only do not observe hormesis with HLA-Cw7 or with the 293-EBNA cell of HLA-Cw7 and any other assortment of genes transfection.

By RT-PCR, can determine that all express MAGE-A12 by tumour cell kinds system of CTL501D/19 identification, confirm that antigen is by this genes encoding.Other HLA-Cw7 Restricted CTL clone body that are created among the MLTC have also been detected.Six kinds of other ctl clone bodies are found the 293-EBNA cell of identification with MAGE-A12 and HLA-Cw7 transfection.

MAGE-A12 elder generation sequence for the identifier number antigen peptide produces different lengths MAGE-A12 fragment by PCR.These subgenomic fragments are cloned among the pcDNA3 and and HLA-Cw ^*0701 makes up transfection together in the 293-EBNA cell.Implement CTL hormesis test (Fig. 3) with transfectant.Cells transfected is used CTL501D/19 incubation 24 hours, the toxicity of WEHI-164.13 cell is measured its output in supernatant liquor by measuring TNF.The segmental numbering of PCR is corresponding to the Nucleotide of coding region.

CTL501D/19 can be stimulated with 540 or more a plurality of base pair cells transfected, and CTL501D/19 can not be stimulated with shorter fragment cells transfected.This explanation, the end of the sequence of coding for antigens peptide is between the Nucleotide 525 and 540 of MAGE-A12 ORF.In aminoacid sequence, find two overlapping nonapeptides, EVVRIGHLY (the codon 168-176 of MAGE-A12 corresponding to Nucleotide 525-540; SEQ IDNO:3) and VRIGHLYIL (the codon 170-178 of MAGE-A12; SEQ IDNO:4), it is consistent in conjunction with primitive with the HLA-Cw7 peptide, that is, and and at the tyrosine or the leucine (people such as Rammensee, immunogenetics 41:178,1995) of C-terminal.It is the cracking sensitivity (Fig. 4) of LB-831-EBV to CTL501D/19 that decapeptide VVRIGHLYIL (SEQ ID NO:5), nonapeptide VRIGHLYIL (SEQ IDNO:4) and octapeptide RIGHLYIL (SEQ ID NO:6) make self lymphocytoblast kind.With self cell kind system with MAGE-A12 peptide VRIGHLYIL (SEQ ID NO:4), VVRIGHLYIL (SEQ ID NO:5), or RIGHLYIL (SEQ ID NO:6) load, and contact with CTL501D/19.With the cell of peptide (VRIGHLYIL (SEQ ID NO:4) or RIGHLYIL (SEQ ID NO:6)) the pulse chromium mark of prescribed concentration 30 minutes.Ratio with effector and target is 10 adding CTL501D/19.Measuring chromium after 4 hours discharges.Also measure lack the terminal leucic peptide of C-(as, EVVRIGHLY, SEQ ID NO:3), but not identification.Use the very nonapeptide VRIGHLYIL of lower concentration (SEQ ID NO:4) acquisition partly to measure maximum cracking, 100pM illustrates that CTL501D/19 can discern this peptide very effectively.

The expression of embodiment 2:MAGE-A12 in tumor sample

Determine that by RT-PCR MAGE-A12 expresses in the cell kind system of tumor sample and/or kinds of tumors.Use aforesaid MAGE-A12 special primer and condition to implement pcr amplification.The result of MAGE-A12 RT-PCR amplification provides in Table I.

The expression of the MAGE-A12 that detects by RT-PCR

Sample type	The specimen number	The MAGE-A12 positive
				?N?????????????????????????????????????????％
		On the malignant melanoma skin, nascent	83			?28	?34
Shift	243			?151	?62
			326			?179	?55

The esophagus squamous cell carcinoma	19	?5	?26
				Gland cancer	5	?2	?40
	24	?7	?29
				Squamous cell lung carcinoma	93	?26	?28
Gland cancer	43	?14	?33
					136	?40	?29
Head and neck squamous cell cancer	85	?23	?27
				The bladder cancer surface (＜T2)	70	?7	?10
Infiltrate (〉=T2)	53	?18	?34
					123	?25	?20
Breast cancer	50	?8	?16
				The rectum cancer	46	?5	?11
The myelomatosis I-II phase	11	?0	?0
				The III phase	27	?4	?15
	38	?4	?11
				Cerebral tumor	11	?1	?9
Sarcoma	13	?1	?8
				Prostate gland body of gland gland	22	?1	?5

Cancer
				Kidney	6	?0	?0
Uterus tumor	5	?0	?0
				Thyroid tumor	4	?0	?0
Mesothelioma of pleura	4	?0	?0
				Leukemia	112	?0	?0

Equivalent

Technician in this field will appreciate that, or only uses normal experiment just can determine many equivalents of the specific embodiments of invention described herein.Such equivalent comprises in the following claims.All reference disclosed herein are all taken in this piece of writing in full.

Sequence table

＜110〉The Ludwig Inst. of Cancer Research

＜120〉MAGE-A12 antigen peptide and application thereof

<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〉Windows of FastSEQ is 3.0 editions

<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?gag?gcc?caa?gga?gag?gcc?ctg?ggc?ttg?gtg?ggt??????3040Pro?Glu?Glu?Gly?Leu?Glu?Ala?Gln?Gly?Glu?Ala?Leu?Gly?Leu?Val?Gly

15??????????????????20??????????????????25gcg?cag?gct?cct?gct?act?gag?gag?cag?gag?act?gcc?tcc?tcc?tcc?tct??????3088Ala?Gln?Ala?Pro?Ala?Thr?Glu?Glu?Gln?Glu?Thr?Ala?Ser?Ser?Ser?Ser

30??????????????????35??????????????????40act?cta?gtg?gaa?gtc?acc?ctg?cgg?gag?gtg?cct?gct?gcc?gag?tca?cca??????3136Thr?Leu?Val?Glu?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?Ile?60??????????????????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?acg?agc?ttc?caa?gta??????3280Gln?Glu?Gly?Pro?Ser?Thr?Phe?Pro?Asp?Leu?Glu?Thr?Ser?Phe?Gln?Val

95?????????????????100?????????????????105gca?ctc?agt?agg?aag?atg?gct?gag?ttg?gtt?cat?ttt?ctg?ctc?ctc?aag??????3328Ala?Leu?Ser?Arg?Lys?Met?Ala?Glu?Leu?Val?His?Phe?Leu?Leu?Leu?Lys

110?????????????????115?????????????????120tat?cga?gcc?agg?gag?cca?ttc?aca?aag?gca?gaa?atg?ctg?ggg?agt?gtc??????3376Tyr?Arg?Ala?Arg?Glu?Pro?Phe?Thr?Lys?Ala?Glu?Met?Leu?Gly?Ser?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?tgc?ctg?ggc?ctc?tcc?tac?gct??????3520Ile?Gly?His?Leu?Tyr?Ile?Leu?Val?Thr?Cys?Leu?Gly?Leu?Ser?Tyr?Ala

175?????????????????180?????????????????185ggc?ctg?ctg?ggc?gac?aat?cag?atc?gtg?ccc?aag?aca?ggc?ctc?ctg?ata??????3568Gly?Leu?Leu?Gly?Asp?Asn?Gln?Ile?Val?Pro?Lys?Thr?Gly?Leu?Leu?Ile

190?????????????????195?????????????????200atc?gtc?ctg?gcc?ata?atc?gca?aaa?gag?ggc?gac?tgt?gcc?cct?gag?gag??????3616Ile?Val?Leu?Ala?Ile?Ile?Ala?Lys?Glu?Gly?Asp?Cys?Ala?Pro?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?gaa?aac?tac?ctg?gag?tac?cgg?cag?gtc?ccc?ggc?agt?gat?cct?gca??????3760Gln?Glu?Asn?Tyr?Leu?Glu?Tyr?Arg?Gln?Val?Pro?Gly?Ser?Asp?Pro?Ala

255?????????????????260?????????????????265tgc?tac?gag?ttc?ctg?tgg?ggt?cca?agg?gcc?ctc?gtt?gaa?acc?agc?tat??????3808Cys?Tyr?Glu?Phe?Leu?Trp?Gly?Pro?Arg?Ala?Leu?Val?Glu?Thr?Ser?Tyr

270?????????????????275?????????????????280gtg?aaa?gtc?ctg?cac?cat?ttg?cta?aag?atc?agt?gga?ggg?cct?cac?att??????3856Val?Lys?Val?Leu?His?His?Leu?Leu?Lys?Ile?Ser?Gly?Gly?Pro?His?Ile

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?Glu?Gln?Arg?Ser?Gln?His?Cys?Lys?Pro?Glu?Glu?Gly?Leu?1???????????????5??????????????????10??????????????????15Glu?Ala?Gln?Gly?Glu?Ala?Leu?Gly?Leu?Val?Gly?Ala?Gln?Ala?Pro?Ala

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?Gly?Ala?Ser?Thr?Leu?Pro?Thr?Thr?Ile?Asn?Tyr?Thr?Leu?Trp65??????????????????70??????????????????75??????????????????80Ser?Gln?Ser?Asp?Glu?Gly?Ser?Ser?Asn?Glu?Glu?Gln?Glu?Gly?Pro?Ser

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

100?????????????????105?????????????????110Met?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?Phe?Pro?Val?Ile?Phe?Ser?Lys?Ala?Ser?Glu?Tyr?Leu?Gln?Leu145?????????????????150?????????????????155?????????????????160Val?Phe?Gly?Ile?Glu?Val?Val?Glu?Val?Val?Arg?Ile?Gly?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?Ser?Asp?Gly?Arg?Glu?Asp?Ser?Val?Phe?Ala225?????????????????230?????????????????235?????????????????240His?Pro?Arg?Lys?Leu?Leu?Thr?Gln?Asp?Leu?Val?Gln?Glu?Asn?Tyr?Leu

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?Tyr??1??????????????5

<210>4

<211>9

<212>PRT

<213>Homo?sapiens

<400>4Val?Arg?Ile?Gly?His?Leu?Tyr?Ile?Leu?1???????????????5

<210>5

<211>10

<212>PRT

<213>Homo?sapiens

<400>5Val?Val?Arg?Ile?Gly?His?Leu?Tyr?Ile?Leu?1???????????????5??????????????????10

<210>6

<211>8

<212>PRT

<213>Homo?sapiens

<400>6Arg?Ile?Gly?His?Leu?Tyr?Ile?Leu?1???????????????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?Tyr??1???????????????5

<210>10

<211>9

<212>PRT

<213>Homo?sapiens

<400>10Ser?Ala?Tyr?Gly?Glu?Pro?Arg?Lys?Leu??1???????????????5

<210>11

<211>9

<212>PRT

<213>Homo?sapiens

<400>11Glu?Val?Asp?Pro?Ile?Gly?His?Leu?Tyr??1???????????????5

<210>12

<211>9

<212>PRT

<213>Homo?sapiens

<400>12Phe?Leu?Trp?Gly?Pro?Arg?Ala?Leu?Val??1???????????????5

<210>13

<211>10

<212>PRT

<213>Homo?sapiens

<400>13Met?Glu?Val?Asp?Pro?Ile?Gly?His?Leu?Tyr??1???????????????5??????????????????10

<210>14

<211>9

<212>PRT

<213>Homo?sapiens

<400>14Ala?Ala?Arg?Ala?Val?Phe?Leu?Ala?Leu??1???????????????5

<210>15

<211>8

<212>PRT

<213>Homo?sapiens

<400>15Tyr?Arg?Pro?Arg?Pro?Arg?Arg?Tyr??1???????????????5

<210>16

<211>10

<212>PRT

<213>Homo?sapiens

<400>16Ser?Pro?Ser?Ser?Asn?Arg?Ile?Arg?Asn?Thr??1???????????????5??????????????????10

<210>17

<211>9

<212>PRT

<213>Homo?sapiens

<400>17Val?Leu?Pro?Asp?Val?Phe?Ile?Arg?Cys??1?????????????????5

<210>18

<211>10

<212>PRT

<213>Homo?sapiens

<400>18Val?Leu?Pro?Asp?Val?Phe?Ile?Arg?Cys?Val??1???????????????5??????????????????10

<210>19

<211>9

<212>PRT

<213>Homo?sapiens

<400>19Glu?Glu?Lys?Leu?Ile?Val?Val?Leu?Phe??1???????????????5

<210>20

<211>9

<212>PRT

<213>Homo?sapiens

<400>20Glu?Glu?Lys?Leu?Ser?Val?Val?Leu?Phe??1???????????????5

<210>21

<211>10

<212>PRT

<213>Homo?sapiens

<400>21Ala?Cys?Asp?Pro?His?Ser?Gly?His?Phe?Val??1???????????????5??????????????????10

<210>22

<211>10

<212>PRT

<213>Homo?sapiens

<400>22Ala?Arg?Asp?Pro?His?Ser?Gly?His?Phe?Val??1???????????????5??????????????????10

<210>23

<211>9

<212>PRT

<213>Homo?sapiens

<400>23Ser?Tyr?Leu?Asp?Ser?Gly?Ile?His?Phe??1???????????????5

<210>24

<211>9

<212>PRT

<213>Homo?sapiens

<400>24Ser?Tyr?Leu?Asp?Ser?Gly?Ile?His?Ser??1???????????????5

<210>25

<211>9

<212>PRT

<213>Homo?sapiens

<400>25Met?Leu?Leu?Ala?Val?Leu?Tyr?Cys?Leu??1???????????????5

<210>26

<211>9

<212>PRT

<213>Homo?sapiens

<400>26Tyr?Met?Asn?Gly?Thr?Met?Ser?Gln?Val??1???????????????5

<210>27

<211>9

<212>PRT

<213>Homo?sapiens

<400>27Ala?Phe?Leu?Pro?Trp?His?Arg?Leu?Phe??1???????????????5

<210>28

<211>9

<212>PRT

<213>Homo?sapiens

<400>28Ser?Glu?Ile?Trp?Arg?Asp?Ile?Asp?Phe??1???????????????5

<210>29

<211>9

<212>PRT

<213>Homo?sapiens

<400>29Tyr?Glu?Ile?Trp?Arg?Asp?Ile?Asp?Phe??1???????????????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?Pro??1???????????????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?Asp??1???????????????5??????????????????10??????????????????15

<210>32

<211>10

<212>PRT

<213>Homo?sapiens

<400>32Glu?Ala?Ala?Gly?Ile?Gly?Ile?Leu?Thr?Val??1???????????????5??????????????????10

<210>33

<211>9

<212>PRT

<213>Homo?sapiens

<400>33Ala?Ala?Gly?Ile?Gly?Ile?Leu?Thr?Val??1???????????????5

<210>34

<211>9

<212>PRT

<213>Homo?sapiens

<400>34Ile?Leu?Thr?Val?Ile?Leu?Gly?Val?Leu??1???????????????5

<210>35

<211>9

<212>PRT

<213>Homo?sapiens

<400>35Lys?Thr?Trp?Gly?Gln?Tyr?Trp?Gln?Val??1???????????????5

<210>36

<211>9

<212>PRT

<213>Homo?sapiens

<400>36Ile?Thr?Asp?Gln?Val?Pro?Phe?Ser?Val??1???????????????5

<210>37

<211>9

<212>PRT

<213>Homo?sapiens

<400>37Tyr?Leu?Glu?Pro?Gly?Pro?Val?Thr?Ala??1???????????????5

<210>38

<211>10

<212>PRT

<213>Homo?sapiens

<400>38Leu?Leu?Asp?Gly?Thr?Ala?Thr?Leu?Arg?Leu??1???????????????5??????????????????10

<210>39

<211>10

<212>PRT

<213>Homo?sapiens

<400>39Val?Leu?Tyr?Arg?Tyr?Gly?Ser?Phe?Ser?Val??1???????????????5??????????????????10

<210>40

<211>9

<212>PRT

<213>Homo?sapiens

<400>40Leu?Tyr?Val?Asp?Ser?Leu?Phe?Phe?Leu??1???????????????5

<210>41

<211>12

<212>PRT

<213>Homo?sapiens

<400>41Lys?Ile?Ser?Gly?Gly?Pro?Arg?Ile?Ser?Tyr?Pro?Leu??1???????????????5??????????????????10

<210>42

<211>9

<212>PRT

<213>Homo?sapiens

<400>42Tyr?Met?Asp?Gly?Thr?Met?Ser?Gln?Val??1???????????????5

<210>43

<211>11

<212>PRT

<213>Homo?sapiens

<400>43Ser?Leu?Leu?Met?Trp?Ile?Thr?Gln?Cys?Phe?Leu??1???????????????5??????????????????10

<210>44

<211>9

<212>PRT

<213>Homo?sapiens

<400>44Ser?Leu?Leu?Met?Trp?Ile?Thr?Gln?Cys??1???????????????5

<210>45

<211>9

<212>PRT

<213>Homo?sapiens

<400>45Gln?Leu?Ser?Leu?Leu?Met?Trp?Ile?Thr??1???????????????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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