CN1278865A - Tumor-specific antigens, methods for their production and their use for immunization and diagnosis - Google Patents

Tumor-specific antigens, methods for their production and their use for immunization and diagnosis Download PDF

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CN1278865A
CN1278865A CN98810896A CN98810896A CN1278865A CN 1278865 A CN1278865 A CN 1278865A CN 98810896 A CN98810896 A CN 98810896A CN 98810896 A CN98810896 A CN 98810896A CN 1278865 A CN1278865 A CN 1278865A
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安德烈亚·阿尼基尼
乔治·帕尔米亚尼
马里亚露依萨·森西
卡蒂亚·特拉韦萨里
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Roche Diagnostics GmbH
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Abstract

A tumor-specific polypeptidic antigen, which is coded at least partially by an intron of an exon-coded tumor antigen, and which is obtained by (a) reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic fraction of a tumor cell, whereby nucleic acid fragments which hybridize under stringent conditions with intron sequences of an exon-coded tumor antigens are used as a primer, (b) isolation of the product of said PCR, expression of said PCR product or of a fragment thereof in a host cell, and isolation of said tumor-specific antigen which is coded by said PCR product or a fragment thereof which hybridizes also with exon sequences of said antigen, is useful for diagnosis and therapeutic use in connection with tumor diseases.

Description

Tumour specific antigen, its preparation method and their application in immunity and diagnosis
The present invention relates to new tumour specific antigen, its preparation method, and they are in immunity, particularly at activating cytotoxic, tumour-specific T-lymphocyte, and to the application in the specific diagnosis of the tumour cell that presents said tumour specific antigen in the mhc class i complex body.
Immunity system plays an important role at the immunologic surveillance of cancer and tumor regression.The antineoplastic immune response can be cell-mediated by B and T, the tumour antigen of expressing on their tumor cells.From the cytotoxic T lymphocyte (CTL) that produces from the peripheral blood lymphocyte last several years of the tumour patient or neoplasm invasiveness lymphocyte (TIL) people can be assessed to the effect of T cell in people's tumor regression.
Neoplasm invasiveness lymphocyte and interleukin II (IL-2) but (Rosenberg etc., New Engl.J.Med.319 (1988) 1676-1680 of disappearing of cancer patients self Mediated Human tumour advanced in adoptive transfer; Rosenberg etc., J.Natl.Cancer Inst.86 (1994) 1159-1166), prompting T cell plays an important role in the tumor rejection process in vivo.Ability that the mediation in-vivo tumour disappears and the ability relevant (Barth etc., J.Exp.Med.173 (1991) 647-658) of TIL when mediation specificity cracking and release of cytokines when external and homogenic type tumour are cultivated altogether.
In order to understand the molecular basis of the cell-mediated antitumor response of T, the antigenic gene of multiple codes for tumor (Boon etc., immunology annual review 12 (1994) 337-365 have been identified by the T cell recognition; Houghton, The Journal of Experimental Medicine 180 (1994) 1-4; Tsomides and Eisen, PNAS 91 (1994) 3487-3489; Pardoll, nature 369 (1994) 357-358; Rosenberg, Cancer J.Sci.Am.1 (1995) 90-100).Based on their expression type, these antigens can be divided into several classes:
First kind tumour antigen comprises the total antigen of tumour of melanoma and other multiple types of organization, but be not present in healthy tissues, (for example, MAGE except spermary and placenta, BAGE and GAGE) (Van der Bruggen etc., science 254 (1991) 1643-1647; Boel etc., immunity 2 (1995) 167-175; Van der Bruggen etc., The Journal of Experimental Medicine 182 (1995) 689-698).Based on the antigenic clinical experiment (Marchand etc., international journal of cancer 63 (1995) 883-885 in progress that use the CTL identification that limits by different HLA I class allelotrope; Rosenberg, immunology today (1997) 175-182), that use is the patient who suffers from melanoma and other tumor disease.Consider these antigens and the allelic expression frequency of HLA I class, surpass Caucasian's melanoma patients of 60%, 40% head and neck and 28% bladder cancer patients are suitable for carrying out immunity with at least a antigen in this class.In the patient who is tried, do not detect side effect.Really, MAGE, BAGE and GAGE genetic expression usually occur in the spermary of picture spermatogonium and spermatocyte (II), they not antigen expressed present the mhc class i molecule (Haas etc. of required classics, thereby will do not hit Am.J.Reprod.Immunol.Microbio.18 (1988) 47-57), by the T cellular targets.
The second class tumour antigen contains the tissure specific antigen of expressing in the normal and tumour cell of melanocyte series.From the tyrosine oxidase (Brichard etc., The Journal of Experimental Medicine 178 (1993) 489-49513) on melanoma and the normal melanocyte of cultivating, MelanA Martl(Coulie etc., The Journal of Experimental Medicine 180 (1994) 35-42; Castelli etc., The Journal of Experimental Medicine 181 (1995) 363-368; Kawakami etc., PNAS 91 (1991) 3515-3519), gp100P Mel7(Bakker etc., The Journal of Experimental Medicine 179 (1994) 1005-1009; Kawakami etc., PNAS 91 (1994) 6458-6462), gp75 TRP1(Wang etc., The Journal of Experimental Medicine 181 (1995) 799-804) and TRP-2 (Wang etc., The Journal of Experimental Medicine 184 (1996) 2207-2216) CTL identification epitope can increase from many melanoma patients external.Thereby melanoma patients may be benefited from the immunotherapy of inducing at these antigenic T cellular response greatly.In fact, all express the antigen of differentiation in nearly all melanoma, and the major part in them is presented in immune effector by HLA-A2 allelotrope, this equipotential gene is present among each ethnic crowd with high frequency.But, must think better of in these treatments at the side effect that cross reactivity brought of healthy tissues (being dermal melanin cell and Pigmented retina cell).
The 3rd class tumour antigen only comprises by the antigen of isolating tumor cells expression.These antigens are not expressed in the tumor tissues of other normal or different sources, and epitope point mutation in the general expressed protein produces by occurring in usually.The tumour antigen that belongs to this monoid is in mouse system (Boon etc., immune blood annual review 12 (1994) 337-365) with at some tumour (Wolfel etc., science 269 (1995) 1281-1284; Coulie etc., PNAS 92 (1995) 7976-7980; Robbins etc., The Journal of Experimental Medicine 183 (1996) 1185-1192) obtain in describing.Make at the shortage of these antigenic natural tolerances to induce strong immune response, avoid occurring strong autoimmune reaction simultaneously.But before finding a series of sudden change of focus widely, these antigenic clinical applications will be limited at the patient (Wolfel etc., science 269 (1995) 1281-1284) that several or a few its tumour is carried given sudden change.
The 4th class tumour antigen results from the difference processing of transcript.Robbin etc. have described a kind of gp100 transcript at immune blood magazine 159 (1997) 303-308, and it is corresponding to the part of the 4th intron and be coded in non-existent 35 extra amino acid in the normal gp100 glycoprotein.But, this epitope in melanoma only with low expression level.Robbins etc. have described the clone of the antigenic gene of a kind of lymphocyte identification of encoding at immune blood magazine 154 (1995) 5944-5950, and this lymphocyte is the neoplasm invasiveness lymphocyte that is limited by melanoma specificity HLA-A24.This antigen is the segment of a full-length clone, can't help intron or its part the coding.
Fujii etc., Journal of Immunology 153 (1994) 5516-5524 described a kind of soluble form non--kill tumour HLA-G antigen, it is by the mRNA coding that contains intron 4.But this is also not know the proteinic special function of this solubility HLA-G.
Reactive type based on a series of ctl clones, proposed to exist the 5th class tumour antigen recently, this ctl clone can be discerned the allosome melanoma from body and HLA-coupling, but the target thing of nonrecognition melanocyte or other different tissue sources (Anichini etc., Journal of Immunology 156 (1996) 208-217).
An object of the present invention is to provide new tumour specific antigen, it is not expressed in normal cell and can specifically tumour cell be distinguished from normal cell.
Summary of the invention
The present invention includes a kind of tumour-specific polypeptides with antigenic effect, it is characterized in that, it partly is by from the intron sequences coding of the gene of a peptide species, this polypeptide is presented on the tumour cell (a kind of tumour antigen of exons coding) by MHC I class complex body and can obtains by reverse transcriptase PCR from a kind of mRNA, this mRNA is isolating from the soluble cell matter of tumour cell, wherein is used as primer with a kind of nucleic acid fragment of intron sequences hybridization of tumour antigen of exons coding under rigorous condition; And, if obtain the PCR product, in host cell, express, with separating of the tumour specific antigen of encoding by this PCR product by the separation of PCR product.
Described antigen can be used as a segment and presents cell (APC) by antigen and present, and responds with inducing specific CTL.
Another object of the present invention is a kind of this method with tumour-specific polypeptides of antigenic effect of differentiating, may further comprise the steps:
--carry out reverse transcriptase PCR from a kind of mRNA of tumour cell, wherein under rigorous condition, be used as primer with a kind of nucleic acid fragment of intron sequences hybridization of tumour antigen of exons coding;
--if obtain the PCR product: separate the PCR product, express in host cell, with the tumour specific antigen that separates by this PCR product coding, it is also hybridized with said antigenic exon sequence.
After separating will be hybridized product or its fragment inserting expressioning carrier, this carrier is shifted into appropriate host cell and express in said host cell.Subsequently, separate the recombinant polypeptide that produces.
In a preferred embodiment of the invention, the fragment of 8 of this hybridization product to 12 codons is used to express this antigen.
Thereby theme of the present invention is a kind of tumour-specific polypeptides antigen, and it partly is to be encoded by a kind of intron of tumour antigen of exons coding, and it obtains by following steps:
A) isolating mRNA carries out reverse transcriptase PCR from a kind of solubility kytoplasm composition of tumour cell, wherein is used as primer with a kind of nucleic acid fragment of intron sequences hybridization of tumour antigen of exons coding under rigorous condition;
B) separate said PCR product, express said PCR product or its fragment in host cell, with the tumour specific antigen that separates by this PCR product or its fragment coding, it is also hybridized with said antigenic exon sequence.
Another theme of the present invention is a tumour-specific polypeptides antigen of the present invention, and wherein, the fragment of 8 to 12 codons of said PCR product is used to express.
Another theme of the present invention is a tumour specific antigen of the present invention, and wherein, the tumour antigen of said exons coding is a kind of MAGE of CTL identification Antigens, and BAGE and GAGE are from tyrosine oxidase, MelanA Martl, gp100 Pmel7, gp75 TRP1Or the CTL of TRP-2 identification epitope.
The invention still further relates to a kind of tumour-specific polypeptides antigen by SEQ ID NO:1 coding.
Another theme of the present invention is the method for separation by a kind of mRNA of tumour specific antigen of intron coding of tumour antigen of exons coding, may further comprise the steps:
A) isolating mRNA in a kind of solubility kytoplasm composition of tumour cell is carried out reverse transcriptase PCR, wherein under rigorous condition, be used as primer with a kind of nucleic acid fragment of intron sequences hybridization of tumour antigen of exons coding; With
B) separate said PCR product, it is also hybridized with said antigenic exon sequence.
The invention still further relates to a kind of method of measuring the propagation of tumor-specific cytotoxicity T-cell, wherein, tumour specific antigen of the present invention is added in patient's the sample of body fluid, it contains antigen and presents cell and cytotoxic T cell, measure the propagation of cytotoxic T cell, preferably measure (measuring for example TNF, IFN γ, the cytokine of GM-CSF) by the release of cytokine.
Another theme of the present invention is that the nucleic acid of coding tumour specific antigen of the present invention is used in preparation is used for the treatment of the medicine of tumor disease.
The invention still further relates to tumour specific antigen of the present invention in vivo or vivo activation from the application in the cytotoxic T cell of T precursor cell.
Another theme of the present invention is the antigenic method of a kind of preparation tumour-specific polypeptides, and wherein said tumour specific antigen obtains by following steps:
A) isolating mRNA carries out reverse transcriptase PCR from a kind of solubility kytoplasm composition of tumour cell, wherein is used as primer with a kind of nucleic acid fragment of intron sequences hybridization of tumour antigen of exons coding under rigorous condition;
B) separate said PCR product, express said PCR product or its fragment in host cell, with the tumour specific antigen that separates by this PCR product or its fragment coding, it is also hybridized with said antigenic exon sequence.
Another theme of the present invention is the combination of two kinds of nucleic acid, they under rigorous condition with a kind of intron sequences hybridization of tumour antigen of exons coding, and they can be used as from the primer of the reverse transcriptase PCR of mRNA right.
Another theme of the present invention is the nucleic acid by SEQ ID NO:3 to SEQ ID NO:9 coding.
Tumour antigen of the present invention is not found in normal cell for example on the surface of melanocyte.But it is present in and surpasses 80%, for example on the melanoma cell, thereby is specific to tumour cell.
Thereby this peptide is specially adapted to patient's tumor cell specific immunity, and also can be used for diagnosing the differentiation of melanoma cell and normal melanocyte.
Can be from the lymphocytic CTL clone of metastasis melanin tumor peripheral blood of patients (CTL 128) with mode cracking autologous tumor and several allosome melanoma of HLA A*6801 qualification.Coding identifies that by the transfection of cDNA library this library is from make up the into allelic Cos-7 cell of expression HLA-A*6801 from body melanoma mRNA by the antigenic gene of CTL 128 identifications.Find unexpectedly, opposite with melanocyte, the splicing mistake takes place in the mRNA maturation.This produces a kind of peptide of being made up of the melanocyte differentiation antigen (TRP)-2 that partly splices form in translation, it contains exons 1-4 and keeps intron 2 and part intron 4 (TRP-2-INT2).Encode a kind of 238 amino acid whose protein of supposition of TRP-2-INT2, it uses the reading frame identical with TRP-2, it extends to termination site (Nucleotide 1113) the intron 2,18 Nucleotide places in downstream, peptide-coding region territory from 400 initiator codon.
Differentiation antigen TRP-2 is described in R.F.Wang, The Journal of Experimental Medicine 184 (1996) 2207-2216.TRP-2 be one of glycoprotein of high expression level in Pigmented melanocyte of people and melanoma (Wang etc., The Journal of Experimental Medicine 184 (1996), 2207-2216).It is positioned on the human chromosome 13, and shown it is a member of the relevant gene family of tyrosine oxidase, enjoy amino acid identity (Yokoyama etc., Biochim.Biophys.Acta.1217 (1994), the 317-321 of 40-45% with tyrosine oxidase and gp75/TRP-1; Robbins etc., Journal of Immunology 154 (1995), 5944-5950).One of TRP-2 coding has 519 amino acid whose protein, and has shown the activity with DOPA chromium tautomerase, its participation is melanic synthesizes (Bouchard etc., european journal of biological chemistry 219 (1994), 127-134).
Opposite with the TRP-2 mRNA that shears fully that is found in melanoma, normal skin melanocyte and the retina of description such as Wang, TRP-2-INT2 mRNA only detects in melanoma.These results show, may be by betiding in the tumour but the different shearing that does not betide in the normal cell is derived from known strain-relevant protein by the melanoma-associated antigen of CTL identification.This mechanism has produced one group of new antigen, and it can be considered to have real tumour-specific, and only exists in the tumour of homologue's type, and is not present in the healthy tissues of identical type.Thereby new antigenic of this group be characterised in that it is derived from known strain-relevant protein by tumour-specific and selectable shearing, and this shearing is not present in the normal cell.Normal melanocyte, skin samples and retina are proved to be negative in reversed transcriptive enzyme (RT) pcr analysis.These features define a kind of antigen that can be used for developing safer and effective immunotherapy scheme.
Thereby, the present invention relates to the tumour specific antigen of part intron coding, it is that reverse transcriptase PCR from the isolating mRNA of soluble cell matter component of tumour cell obtains.These antigens are discerned by T lymphocyte specific, and cracking presents antigenic tumour cell of the present invention in MHC I class complex body then.Find that unexpectedly the coding of enrichment antigenic mRNA of the present invention is a tumour-specific in the tenuigenin of this tumour cell.
" tumour specific antigen of intron coding " is meant a kind of tumour antigen, it is not only by a kind of exon sequence and is (preferably about or greater than 30% by part, more preferably from about or greater than 50%, most preferably from about or greater than 80%) the intron sequences coding, and it discerns the tumour antigen that presents by mhc class i specifically.The shearing mechanism that changes in the expression of the intron protein relevant with strain is relevant." part " is meant preferred 30-50% or 30-80% intron coding.The sequence with the intron coding of antigenic exons coding of the present invention directly links to each other in relevant genome sequence, because said antigen is to be caused by different shearings.
" antigen of exons coding " is meant the antigen of describing in preface part, for example, and MAGE, BAGE and GAGE (Van der Bruggen etc., science 254 (1991) 1643-1647; Boel etc., immunity 2 (1995) 167-175; Van der Bmggen etc., The Journal of Experimental Medicine 182 (1995) 689-698) or for example, from the CTL identification epitope (Brichard etc., The Journal of Experimental Medicine 178 (1993) 489-49513) of tyrosine oxidase, MelanA Martl(Coulie etc., The Journal of Experimental Medicine 180 (1994) 35-42; Castelli etc., The Journal of Experimental Medicine 181 (1995) 363-368; Kawakami etc., PNAS 91 (1991) 3515-3519), gp100 Pmel7(Bakker etc., The Journal of Experimental Medicine 179 (1994) 1005-1009; Kawakami etc., PNAS 91 (1994) 6458-6462), gp75 TRPl(Wang etc., The Journal of Experimental Medicine 181 (1995) 799-804) and TRP-2 (Wang etc., The Journal of Experimental Medicine 184 (1996) 2207-2216).
" peptide of the present invention or polypeptide " is meant a peptide species, and it is preferably by 8 to 12 amino acid, and most preferably at least 10 amino acid is formed, but also can contain proteinic all.This peptide also can be a proteinic part, for example a kind of fused protein.
" polypeptide " with antigenic effect be meant a kind of in vivo with external polypeptide of drawing specific immune response.
Term " is hybridized under rigorous condition " and is meant that two kinds of nucleic acid fragments can be described in Sambrook etc., " expression of cloned genes in intestinal bacteria ", In molecular cloning: laboratory manual (1989), cold spring port press, New York, mutual cross mutually under the standard hybridization conditions in the U.S., 9.47-9.62 and 11.45-11.61.
More particularly, " rigorous condition " used herein is meant in 6.0 * SSC, and the hybridization in the time of about 45 ℃ is used among 2.0 * SSC then 50 ℃ of washings.For rigorous degree is selected, in washing step, can select salt concn, for example, and from about 2.0 * SSC, 50 ℃ low preciseness, to about 0.2 * SSC, 50 ℃ high preciseness.In addition, the temperature in washing step can be from the low preciseness condition of room temperature, and about 22 ℃, to high preciseness condition, about 65 ℃.
For at protokaryon or eucaryon organism, for example prokaryotic host cell or eukaryotic host cell, middle expression, this nucleotide sequence is integrated in the suitable expression, the method for using those of ordinary skills to be familiar with.This expression vector preferably contains one can be regulated/inducible promoter.Then these recombinant vectorss are imported in the appropriate host cell and express, for example as the intestinal bacteria of prokaryotic host cell, or Saccharomyces cerevisiae, as the CHO or the COS cell of eukaryotic host cell, and the host cell that will transform or transduce is cultivated under the effable condition of permission heterology gene.The separation of peptide can be carried out in accordance with known methods from host cell or from the culture supernatant of host cell.These methods are described in, for example, and Ausubel I, FrederickM, Current Protocols in Mol Biol, (1992) John Wiley and Sons, New York.
" host cell " is meant protokaryon and eukaryotic cell, preferred COS or Chinese hamster ovary celI.Not too important as being proved activity of proteins by saccharification, preferably in prokaryotic cell prokaryocyte, prepare.
Coding can be discerned by the transfection of cDNA library by the gene of the tumour specific antigen of specific cytotoxic T lymphocyte (CTL) identification, this library makes up from autologous tumor mRNA, be fabricated into eukaryotic cell, preferred expression patient's suitable HLA allelotrope.
In order to measure HLA antigen, separate whole RNA from patient's tumour cell or tumor tissues, and obtain cDNA by reverse transcriptase PCR, the primer that uses is that specificity coding HLA is antigenic, and it is cloned into eukaryotic expression vector, for example pcDNA3 (InvitrogenCorporation, Oxon, UK).In order to measure the tumour specific antigen of intron coding of the present invention, separate poly-A+RNA from the soluble cell matter component separating mRNA of tumour cell, and set up the cDNA library by reverse transcriptase PCR, the primer that uses is the tumour antigen of specificity coding exons coding, for example, MAGE, BAGE and GAGE, from tyrosine oxidase, MelanA Martl, gp100, gp75 TRP1Epitope with the CTL of TRP-2 identification.To advance carrier for expression of eukaryon from the cDNA fragment cloning in cDNA library, for example, pcDNA3.1 (InvitrogenCorporation, Oxon, UK).Advance in the eukaryotic cell at this expression vector cotransfection, for example the Coa-7 cell (is expressed relevant mhc gene, it can present this antigenic peptide fragment that can activate specific tumour antigen T cell), people can measure those clones that tumor-specific cytotoxicity T lymphocyte caused stimulation then.Can stimulate by CTL the lymphocytic stimulatory effect of tumor-specific cytotoxicity T and to measure (mensuration TNF-α, IFN-γ, GM-CSF), and as Traversari etc., immunogenetics 35 (1992), 145-152 is described.
By this way, might obtain specificity and activate the cell clone that those cause corresponding tumour cell cracked cytotoxic T cell.
This cell clone is expressed a peptide species, its isolating can being applied to the patient with form purifying is carried out immunity/inoculation, perhaps use,, perhaps use with the form of the polypeptide fragment of brachymemma because this antigenic peptide of the present invention processes in vivo with the form of the polypeptide of total length.
In order to reach this purpose, the nucleic acid of coding polypeptide of the present invention is separated (Sambrook etc., molecular cloning (1989), press of cold spring harbor laboratory) and carried out brachymemma by restrictive diges-tion according to the method for having set up from this cell clone.Then, the fragment of these brachymemmas is transfected after the clone advances carrier for expression of eukaryon advances eukaryotic cell, for example, COS-7 cell (document is the same), stimulate the stimulation of measuring cytotoxic T cell in the test at CTL then, as Traversari etc., 35 (1992) 145-152 are described for immunogenetics.Coding nucleic acid can be limited on the necessary peptide antigen determining yl of stimulation of cytotoxic T cell by this way.
In a preferred embodiment, encode this nucleic acid sequences to proteins with can improve the nucleotide sequence coupling of the processing of this protein in host cell.
Simultaneously, more known sequences, for example ubiquitin can improve proteinic transportation, proteinic degraded and importing MHC I class complex body (Bachmair etc., science 234 (1986) 179-186; Gonda etc., journal of biological chemistry 264 (1989) 16700-16712; Bachmair etc., cell 56 (1989) 1019-1032).Protein by will containing antigenic peptide of the present invention and ubiquitin coupling mutually can make this proteinic degraded accelerate and more effective to the importing of MHC I class complex body.As a result, this peptide presenting on tumor cell surface is effective especially.In this respect, contain amino acid particularly important aspect the coupling of protein that contains antigen peptide and ubiquitin between antigen peptide and the ubiquitin.By selecting suitable amino acid, people can discern in the melanoma cell at specific cytotoxic t lymphocytes and be improved greatly in addition.
Use the segment of 8 to 12 codons of a PCR product in a preferred embodiment.
In another preferred embodiment, used a kind of tumour specific antigen TRP-2-INT2 (hereinafter being known as TRP-2-INT2 fragment or TRP-2-INT2 peptide) of part intron coding, it is discerned by T lymphocyte specific, their cracking present those melanotic tumor cells of polypeptide EVISCKLIKR in MHC I class complex body, and its aminoacid sequence is by the dna sequence encoding that is shown in SEQ IDNO:2.
Encoding sequence for the tumour specific antigen that obtains the TRP-2-INT2 peptide, from the kytoplasm of K-1735, separate coding HLA-A*6801 allelotrope and the antigenic cDNA of TRP-2-INT2, this clone is that the metastasis (metastases) that the surgical operation sample from a patient obtains is set up, this patient is that (Milan Italy) undergos surgery at Istituto Nazionale Tumori.Two cDNA are transfected advance in the COS-7 cell after, isolate a clone (TRP-2-INT2, HLA-A*6801), but the cellulotoxic effect of its irritation cell toxic T lymphocyte (CTL128).Dna sequence analysis has been found the antigenic cDNA fragment of the TRP-2 of an exons coding, and it contains exons 1-4, has kept intron 2 and part intron 4 (TRP-2-INT2).By TRP-2-INT2 DNA is obtained the segmental subfragment of TRP-2-INT2 with Restriction Enzyme according to the digestion of those of ordinary skill known method, Sambrook etc., " expression of cloned genes in the intestinal bacteria " molecular cloning: laboratory manual (1989), press of cold spring harbor laboratory, New York, the U.S., 5.3-5.10, perhaps obtain by pcr amplification with fragments specific primer (SEQ ID NO:6,7,8).After the COS-7 cell is advanced in transfection, stimulate measurements determination CTL-stimulatory effect by CTL, as Traversari etc., 35 (1992) 145-152 are described for immunogenetics.Measure the sequence of coding nucleic acid by dna sequence analysis.
Another theme of the present invention is the application that presents antigenic tumour cell of the present invention by MHC I class.
Another theme of the present invention is from patient's body fluid, and preferred blood or tissue sample detect the method for antigenic expression of the present invention.For this purpose, before treatment, use the tumour-specific cDNA of coding antigenic intron coding of the present invention in the therapeutic process and after the treatment, antigenic tumour cell of the present invention is expressed in diagnosis.
Before treatment, measure tumour antigen of the present invention and whether in patient's tumour cell, express, because, only expressed antigen of the present invention really in tumour, treatment just can be carried out.In the process that treatment is carried out, whether the of the present invention antigenic expression that people can monitor as the sign of the tumour cell that exists reduces, and after treatment, make people can monitor tumour cell to the mensuration of antigenic expression of the present invention and whether farthest reduced.
In a preferred embodiment, a kind of nucleic acid of codes for tumor specificity T RP-2-INT2 peptide is used to diagnose the melanoma cell that presents the TRP-2-INT2 peptide on its surface.
Express in melanoma on the sequence-specific ground of TRP-2-INT2 peptide.Thereby the TRP-2-INT2 sequence can be used as the sample of identifying tumour cell.Identify it to be by the hybridization sample of PCR or mark or by various tests based on nucleic acid probe well known in the prior art.
To the mensuration of the expression of TRP-2-INT2 is to carry out by reverse transcription PCR (RT-PCR) with by the hybridization with the TRP-2-INT2 probe of specific marker in the mRNA level.
In order to distinguish with TRP-2 mRNA, to the specific assay of TRP-2-INT2 mRNA be by:
1) is transcribed into cDNA;
2) use the intron Auele Specific Primer;
3) relatively the segmental length of cDNA of amplification (is determined to expand with cDNA exon to exon
The segmental length of cDNA exon-intron-exon that increases);
4) with the hybridization of intron specific probe.
Another theme of the present invention is a method of measuring the lymphocytic propagation of tumor-specific cytotoxicity T.This method is used to before treatment or detects in the therapeutic process can be by the cytotoxic T cell of antigen activates of the present invention.Before treatment, can select to have had can be by the patient of the cytotoxic T lymphocyte of antigen activates of the present invention.
But whether have the tumour-specific activated T cells among the patient in order to detect, separation T cell and antigen present cell from patient's blood, antigen of the present invention and antigen are presented cell contact (" pulsed ") in vitro.If can be present in the patients'blood by the T cell of antigen activates of the present invention, then specific cytotoxicity L lymphopoiesis, this can stimulate test to detect by for example CTL.But this activated T cells can be used to treatment after with antigenic stimulation of the present invention.Before treating, must carry out this diagnostic procedure.
In therapeutic process, another diagnostor is used as the lymphocytic contrast of cytotoxicity L of formation, make may quantitative assay tumour-specific t cell activation induce.
The propagation of cytotoxic T lymphocyte can stimulate by for example CTL to be measured.Irritation cell system is measured the ability that it induces TNF to produce by CTL, and as Traversari etc., 35 (1992) 145-152 are described for immunogenetics.TNF content be in MTT colour developing test (CTL stimulates test) by detecting (Espevic and Nissen-Meyer, immunological method magazine 95 (1986) 99-105) that its cellulotoxic effect on the WEHI-164.13 cell is measured.
In a preferred embodiment, the TRP-2-INT2 peptide is used to measure the propagation of TRP-2-INT2 specificity cell toxicity T lymphocyte.
Another theme of the present invention is that the nucleic acid of coding intron specific antigens of the present invention is used for the treatment of application in the medicine of tumor disease in preparation.
At this on the one hand, nucleic acid of the present invention can be used to gene therapy.This nucleic acid is imported in patient's body by means of virus or non-virus carrier, and wherein, encoding sequence should be expressed specifically, and peptide of the present invention should produce the specific cytotoxic t lymphocytes response by means of the combination that antigen presents cell.The immune response that is produced should be at all tumour cells at its cell surface expression peptide of the present invention.The form that the coded DNA sequence can naked DNA on the carrier is with liposome combination combination, or with suitable adjuvant (commonly known in the art), by using in subcutaneous, intramuscular or the tumour.
In a preferred embodiment, the TRP-2-INT2 peptide is used to gene therapy.
In another embodiment, peptide of the present invention can be used to tumour patient is carried out immunity and/or inoculation.This immunity presents the activation of antigen of the present invention to specific cytotoxic T cell based on present cell by antigen.Immunity can be in vitro or or body in carry out.
In this course, antigen presents cell (giant cell, dendritic cell, or B cell) separate and contact with antigen of the present invention (" pulsed ") with the T lymphocyte in vitro from patient's blood.These antigens of having equipped peptide of the present invention are presented the activation that cell causes specific cells toxicity T cell by this way, fail back patient's blood then.
Immunologic process can be in vivo used polypeptide of the present invention to the patient and is carried out by subcutaneous, wherein directly reaches the activation of specific cytotoxic T cell in patient's body.Peptide of the present invention and antigen present cell lip-deep corresponding HLA molecule combine the propagation that causes specific cytotoxic T lymphocyte.
Peptide of the present invention immunogen effect in use can strengthen by following manner:
1) with bacteriotoxin coupling (superantigen);
2) be used in combination with freund's adjuvant;
3) peptide of the present invention is mixed with liposome.
In a preferred embodiment, the TRP-2-INT2 peptide is used to immunity and inoculation melanoma patients.
Another theme of the present invention is the primer by the expression of RT-PCR detection specificity tumour antigen.At this on the one hand, this primer can be selected by following measure:
1) justice and antisense primer and two of tumour antigen different exon sequence hybridization are arranged.
2) have adopted primer and exon sequence hybridization, and antisense primer (oligomerization dT primer) with
Poly--the A of the mRNA sequence of tumour antigen is hybridization.
In a preferred embodiment, a specific primer is used to detect the segmental expression of TRP-2-INT, and its aminoacid sequence is by the dna sequence encoding that is shown in SEQ ID NO:2 (PRIT-3) and SEQ ID NO:4 (INT2-1260).
Following embodiment is provided, reference, sequence table and accompanying drawing further specify all respects of embodiment of the present invention, do not limit its scope.
Brief Description Of Drawings: Fig. 1: CTL 128 discerns from body melanoma (Me18732) in the mode that HLA-A*6801 limits.With target cell in that (A.:Me 18732+ does not have with fixed E:T ratio; B:Me 18732+HLA I class; C:Me18732+HLA-A2 ,-A69; D:ME 18732+HLA-A2 ,-A28; Me 18732+HLA-B ,-C) add before the effector at room temperature with shown in anti--HLA monoclonal anti body temperature bathe.Fig. 2: CTL 128 identifications are from body melanoma Me18732 and HLA-A*6801+ K-1735.In 25000 irritation cells, add 1500 CTL, and (melanoma: A:Me 18732+ does not have to the TNF content in the WEHI-164.13 cell detection supernatant liquor after 24 hours; B:Me 20842; C:Me 17697 D:Me 2559/1; E:ME 12657; F:Me 17088; G:Me 4023; H:LB-33; I: lung cancer Calu 3; K: mammary cancer SKBR3; L: ovarian cancer SKOV3).Fig. 3: by the stimulation of the Cos-7 cell of cDNA131 and HLA-A*6801 cDNA transfection to ctl clone 128.Cos-7 cell HLA-A*6801 and A255 or cDNA131 set transfection.The A255 set is 100 cDNA clones in one group of Me 18732 cDNA library, from wherein having extracted plasmid DNA.CDNA131 is the single clone from A255 set subclone.Cultivating the TNF that measured by CTL 128 generations in back 20 hours altogether with cells transfected, using the TNF sensitivity cell is WEHI-164.13.Cos-7 cell in contrast is with independent cDNA 131 or HLA-A*6801 transfection (A:Me18732; B:COS; C:COS+A*6801; D:COS+eDNA 131; E:COS+A*6801+cDNA 131).Fig. 4: cDNA 131 codings are by the antigen of CTL 128 identifications.(A) pcDNA3.1/cDNA 131 construct transfections are used in identification and (B) by the cracking of the CTL 128 of the Geneticin resistance colony of HLA-A*6801 K-1735 LB33 then.By the TNF of CTL 128 secretion mensuration after 1500 response cells are cultivated 24 hours altogether with 20000 irritation cells.The lytic activity of CTL 128 be 51On the target cell of Cr mark at different effector: measure after cultivating 4 hours altogether with CTL under target (E/T) ratio.Fig. 5: coding is by the evaluation of the sequence of the antigen peptide of CTL128 identification.The organizational form of the exon of CDNA131 is shown in the top of (A).Exon and intron are expressed as solid and hollow frame respectively, and terminal sea line is represented the pcDNA3.1 carrier, and the numbering of sequence is with respect to the 5 ' end of cDNA131.From the subfragment of cDNA 131 with the form of hollow frame be shown in cDNA 131 below, cloned into expression vector and with the transfected Cos-7 of the advancing cell of HLA-A*6801 cDNA.Screen the TRP-2 cDNA of the total length form that 18732 cDNA libraries obtain shearing with exon 8 specific oligonucleotide probes.TNF by CTL128 is released on the WEHI164.12 and assesses (B).Pointed out the peptide-coding sequence that in PCR fragment INT-2-166 and INT-2-107, exists.Fig. 6: by the HLA-A*6801 cell of CTL 128 cracking with synthetic antigen peptide pulse (pulsed). 51The HLA-A*6801 EBV-LCL (LB-EBV) of Cr-mark bathes with 20: 1 E/T ratio and CTL 128 temperature, exist simultaneously be shown in the left side shown in the synthetic peptide of concentration.Measure after 4 hours 51Cr-discharges.As negative contrast, used that be derived from can be in conjunction with the peptide (MAGE-3 of M3A1 of HLA-A1.Fig. 7: when the HLA allelotrope by the super type of A3-class is current, 128 couples of TRP-2-INT of CTL 221-231Identification. 51The EBV-LCL of Cr-mark and CTL 128 carry out the temperature bath with 20: 1 E/T ratio, have the peptide TRP-2-INT of different concns simultaneously 221-231Measure the release of chromium after 4 hours.C: do not have the negative contrast of peptide.Sequence description: SEQ ID NO:1: by the encoding sequence (nucleic acid) of the antigen peptide of CTL 128 identification.SEQ ID NO:2: by the encoding sequence (amino acid) of the antigen peptide of CTL 128 identification.SEQ ID NO:3: (PRIT-1) that be used to confirm the intron TRP2-INT of non-shearing has adopted primer; Be arranged in 5 ' UTR of TRP2-gene.SEQ ID NO:4: (INT2-1260) antisense primer that is used to confirm the intron TRP2-INT of non-shearing; Be arranged in the 5 ' UTR and the intron 2 of TRP2-gene.SEQ ID NO:5: be used to prepare the subfragment of cDNA 131 and be used for adopted primer being arranged from genomic dna cloning TRP-2-INT2 (KS-INT2); Be arranged in the exon 2 of TRP2-gene.SEQ ID NO:6: (INT2-as1) antisense primer that is used to prepare the subfragment INT2-107 of cDNA 131; Be arranged in the intron 2 of TRP2-gene.SEQ ID NO:7: (INT2-as2) antisense primer that is used to prepare the subfragment INT2-166 of cDNA 131; Be arranged in the intron 2 of TRP2-gene.SEQ ID NO:8: (Sp6) antisense primer that is used to prepare the subfragment INT2-434 of cDNA 131; Be arranged in the pCDNA1-plasmid.SEQ ID NO:9: be used for (PR2) antisense primer from genomic dna cloning TRP-2-INT2; Be arranged in exon 3.SEQ ID NO:10: the TRP-2 DNA that is used to increase (PR3) has adopted primer; Be arranged in exon 2.SEQ ID NO:11: (TPR-2L) antisense primer of the TRP-2 DNA that is used to increase; Be arranged in exon 8.SEQ ID NO:12: 5 ' end-1500 segmental nucleotide sequences that comprise the coding region of antigen peptide.Before the beginning of cDNA 131 and 45 the initial bases that belong to pcDNA3.1 be omitted.
128 pairs of identifications of embodiment 1CTL as the HLA-A*6801 of restrictive element
The K-1735 Me18732 that sets up from patient's metastasis (metastases), be fixed to LA-A2 and HLA-A28 by serological method, be fixed to HLA-A*0201 and HLA*68011 (being also referred to as HLA-A*6801) (Oh etc., genome 29 (1995) 24-34) by sequence specific oligonucleotide probes (SSOP) hypotype method then.As Anichini etc., Journal of Immunology 156 (1996) 208-217 are described to the antitumor CTL clone.
Ctl clone 128 is discerned from the body melanoma in the environment in HLA-A site, because its lysis activity is reduced by anti-HLA I class mAB W6/23, and not by anti-HLA-B ,-C mAb reduces (Fig. 1).HLA-A*0201 can be got rid of to come out by the antigenic molecule that presents of CTL 128 identification from being used for, because cracked is suppressed only using anti-HLA-A2,-A28 mAb observes during CR1 1.351, and is using anti-HLA-A2, does not observe during-A69 mAb BB7.2 (Fig. 1).Thereby the inhibition of CR1 1.351 is active to show that A28 (A*6801) is that the HLA of CTL128 presents molecule.Clone is cultivated
K-1735 Me18732 sets up from the metastasis (metastases) of patient's surgical operation sample, and this patient is from Istituto Nazionale Tumori (Milan, Italy).This patient's PBL is fixed to by serum; HLA-A2 ,-A28 ,-B44 ,-B51 ,-C2 ,-C5.Set up human transitivity (Me17697, Me2559/1, Me17088/1, Me4023) and primary (Me20842) K-1735 and being cultured among the 10%FCS/PRMI 1640.K-1735 LB-33, LV-40 and Cos-7 (ATCC:CRL 1651) clone is maintained among the 10%FCS/DMEM.Available from American type culture collection (ATCC, Rockville, cancerous cell line CALU3 MD), SDBR3 and SKOV3 are maintained among the 10%FCS/RPMI-1640.The C1RA*03301 transfectant, the LCL that the EBV-that isozygotys transforms, clone SCHU is fixed to HLA-A*0301, B*0702 ,-C7, AMA-1 is fixed to HLA-A*6802, B*5301 ,-C4 and WT-100-bis are fixed to HLA-A11 ,-B35 ,-C4.EBV-LCL JHAF (HLA-A*31011 ,-B51 ,-C8) and LB (HLA-A*68011, B*40011 ,-C2 ,-C3) derive from ATCC.EBV-LCL is maintained among the 10%FCS/RPMI-1640.Antigen-specific assessment to CTL 128
For on the tumour cell that is evaluated at other by the antigenic expression frequency of CTL 128 identification, one group of HLA-A*6801 melanocyte is tied up to a CTL to stimulate in the test and detects.5 in 8 K-1735s are induced TNF by CTL 128 and discharge (Fig. 2).From three different tissue-derived HLA-A*6801 cancerous cell lines, observe reactivity (Fig. 2).The negative melanoma of the HLA-A*6801 of other types of organization, melanocyte and tumor cell line can not stimulate the release cytokine by CTL128.Uncorrelated by the expression to the melanoma-associated antigen described in the reaction type of the K-1735 that detects and these cell types that CTL 128 shows, this assesses by RT-PCR.This point with the Cos-7 cell of plasmid pcDNA3/A*6801 cotransfection in the presence of the disappearance that discharges of TNF by CTL 128 be confirmed, this plasmid contains patient 18732 HLA-A*6801, and each gene of the total melanoma-associated antigen of known coded: Melan-A/MART-1, tyrosine oxidase, gp100, TRP-1 ,-2, MAGE-1,2 ,-3 ,-4,-12, BAGE-1 ,-2, GAGE-1 ,-2 ,-3,-4 ,-5 ,-6.These results are consistent with such hypothesis, i.e. the total neoantigen of CTL 128 one kind of multiple tumours of identification of HLA-A*6801 qualification.
Ctl clone 128 is that the blended lymphocyte tumour culture (MLTC) by ages in limiting dilution 4 week obtains, and to before cultivate (Anichini etc., Journal of Immunology 156 (1996) 208-217) under the similar condition of description.CTL 128 expresses CD3 +, CD4 +, CD8 +, TCR- +Phenotype, this is to assess by flow cytometry with specific monoclonal antibody.The active test of lysis:
The lytic activity of CTL 128 discharges in the test at a chromium and detects (Anichini etc., Journal of Immunology 156 (1996) 208-217) as previously mentioned.The result is expressed as follows: Wherein spontaneous release is to assess by target cell temperature in the presence of effector is bathed, and maximum release is that (existence UK) is measured down for BDH Biochemicals, Poole at the 1%NP-40 washing composition.At (the Anichini etc. that suppress from the melanomatous cracked of body to be to use the following monoclonal antibody of having reported to carry out, Journal of Immunology 156 (1996) 208-217): anti-HLA-A,-B,-C W6/32 (Parhan etc., Journal of Immunology 123 (1979) 342-349), anti-HLA-A2,-A69 BB7.2 (Parham and Brodsky, human immunity is learned 3 (1981) 277-299), anti-HLA-A2 ,-A28 CR11.351 (RUsso etc., immunogenetics 18 (1983) 23-35), with anti-HLA-B ,-C4E (Yang etc., immunogenetics 19 (1984) 217-231).The allelic subclone of HLA-A*6801
(Cinna/Biotecx, SouthLoop East is TX) from the total RNA of Me18732 cell preparation to use RNAzolz.B by guanidine-lsothiocyanates method.Strand cDNA is synthetic to be to use widow-dT primer and do not have the active Moloney murine leukemia virus of Rnase-H deutero-reversed transcriptive enzyme (MMLV-RT Rnase-H-Superscript on total RNA of 2 micrograms; GIBCO BRL, Gaithersburg MD) carries out according to the explanation of manufacturers.Use 1 U DynaZymeTM (Finnzymes OY, Espoo, Finland) and the primer of the allelic coding region of HLA-A that is suitable for specific amplification and directed cloning total length to by the cDNA of pcr amplification corresponding to the total RNA of 300 ng.After BamHI and HindIII digestion, the PCR product subclone of 1.1Kb is entered carrier for expression of eukaryon pcDNA3 (Invitrogen Corporation, Oxon, BamHI/EcorRV site UK).
Use the plasmid clone of diagnostic restriction enzyme identification code HLA-A*68011 or A*0201 (patient 18732 HLA-A28 and-A2 allelotrope).Then the HLA-A*68011 gene is checked order, to confirm and the correspondence of disclosed dna sequence dna.This plasmid is called as pcDNA3/HLA-A*6801.Embodiment 2 codings are by the cDNA clone of the melanoma-associated antigen of CTL 128 identifications
Use is from poly-(A) of Me18732 cell extraction +RNA is member cDNA library in suitable expression.Poly-(A) +RNA uses Fast Track mRNA extraction test kit (Invitrogen) isolating from the Me18732 cell.This library is to use SuperScript ChoiceSystem test kit, and (MD) also use contains poly-(A) of the widow-dT primer in a NotI site with 5 micrograms at 5 '-end for GIBCO BRL, Gaithersburg +RNA changes into the cDNA structure.Then the adaptive thing of cDNA and BstXI (adapter) (Invitrogen) is connected and digests with NotI.Behind size fractionation, cDNA is cloned the into BstXI/NotI site of mammalian expression vector pcDNA3.1 (Invitrogen) with single direction.The recombinant plasmid electroporation is advanced the DH5-intestinal bacteria and screens (100mg/ml) with penbritin.
The library is divided 1300 set of cloning into about 100cDNA.The set of each bacterium is expanded to saturated, extracts plasmid DNA and advance 1.2 * 10 with pcDNA3/A*6801 (100ng) transfection (100ng) 4The Cos-7 cell, use be DEAE-dextran-chloroquine method (Coulie etc., test medical journal 180 (1994) 35-42; Seed and Aruffe, PNAS 84 (1987) 3365-3369).Use identical technology, in other experiment, the Cos-7 cell is carried out cotransfection with the pcDNA3/A*6801 carrier of 100ng and the pcDNAI or the pcD SR plasmid that contain the 100ng of a kind of cDNA in the following melanochrome antigen: Melan A/MART-I, tyrosine oxidase, gp100, MAGE-1 ,-2,-3 ,-4 ,-12, BAGE-1 ,-2, GAGE-1,-2 ,-3 ,-4,-5 ,-6, TRP-1.By RT pcr amplification total length TRP-2 cDNA, employed primer is the Auele Specific Primer that is positioned at 5 '-untranslated zone (UTR) and exon 8 ends, it is cloned into pcDNA3 and check order, confirm consistence (Yokohama etc., Bioch.Bioph.Acta 1271 (1994) 317-321) with the cDNA sequence of having delivered.Stimulate test to detect the Cos-7 cell of transfection by CTL after 48 hours.CTL stimulates test
(Traversari etc., immunogenetics 35 (1992) 145-152) as previously mentioned detect transfectant or irritation cell system and are induced the ability that produces TNF by CTL 128.1500 CTL are added in the micropore that contains target cell, contain 100l IMDM (BioWhittaker, Walkersville, MD) and 10% the set human serum (PHS) and 25 U/ml r-hu-IL2 (EuroCetus, Amsterdam, The Netherlands).After 24 hours, collect supernatant liquor,, for example on the WEHI-164.13 (Espevic and Nissen-Meyer, immunological method magazine 95 (1986) 99-105), in a colour developing test, detect its cellulotoxic effect and measure its TNF content by at the WEHI cell.The WEHI-164.13 cell is held cultivation in 10%FCS/RPMI-1640.
Transfection is carried out in little cultivation to multiple, and screens the ability that it stimulates TNF to discharge by CTL 128 after two days.The generation (Fig. 3) that the DNA of (set A 255) in 1300 set has induced high-caliber TNF, this discovery is confirmed in second transfection experiment.The bacterium of positive set cloned and their plasmid and HLA-A*6801 construct carried out cotransfection as previously mentioned.Among 159 clones 25 have stimulated TNF release by CTL 128.With a result who obtains in them, promptly cDNA 131, are shown in Fig. 3.The transfection of K-1735
Be used in the cDNA 131 transfection K-1735 LB-33 that clone among the plasmid pcDNA3.1 (Invitrogen) by the calcium phosphate precipitation technology, it contains neomycin resistance gene.The ubcellular system that from the colony of G418-resistance transfection, separates a clone.Use identical method, with K-1735 LB-40, SK23-MEL and MZ2-MEL also remain on 10%FCS/DMEM) use HLA-A*6801 cDNA transfection, and select in the G418 kind.Confirm the allelic expression of HLA A*6801 of transfection in stable transfectant with monoclonal antibody specific by flow cytometry.
HLA-A*6801+ K-1735 LB-33 is not by CTL 128 identifications (Fig. 2), when changeing the performance (Fig. 4 A) that obtains to induce TNF release then with cDNA 131, and to the cracking sensitivity (Fig. 4 B) of CTL 128, show that antigenic identification can occur in the tumour cell, and do not rely on the high artificial expression level that in the Cos-7 cell, reaches.
It is long that the sequence of cDNA 131 is proved to be 3548 base pairs.Genbank finds by retrieval, Nucleotide (nt) 1-994 and two the discrete zone identical (Yokohamas etc. of nt 3081-3347 with the TRP-2 of cDNA coding, Bioch.Bioph.Acta 1217 (1994) 317-321), it is accredited as the melanoma-associated antigen (Wang etc., The Journal of Experimental Medicine 184 (1996) 2207-2216) of melanocyte strain recently.5 '-UTR of TRP-2 gene is contained in first zone, exons 1 and exon 2, and exon 3 and exon 4 are contained in second zone.The sequence in sequence between nt995-3080 and nt 3347 downstreams demonstrate with database in any sequence of writing down do not have significant homology.These two are present among the cDNA 131 but are not present in the intron sequences of zone for keeping among the TRP-2 cDNA.The fragment of the ten amino acid of one section side joint exon part fully with the sequence that exon-the intron junction is described at the TRP-2 gene be complementary (Sturm etc., genome 29 (1995) 24-34).And the length of the length of sequence 995-3080 (2086 Nucleotide) and the intron 2 of TRP-2 is compatible, and this infers (Sturm etc., Genomics29 (1995) 24-34) from disclosed Genome Atlas.Thereby the identity of Nucleotide 995-3080 is consistent with the intron of TRP-2 2.The sequence in Nucleotide 3347 downstreams of cDNA 131 provides one 5 ' donor shearing site sequence, it and the intron 4 identical (Sturm etc., Genomics29 (1995) 24-34) of TRP-2.Because it lacks 3 ' acceptor shearing site sequence, and its length is obviously shorter than the length of estimating from disclosed Genome Atlas, and this is the sequence of the intron 4 of TRP-2, and 200 places are blocked at Nucleotide.Thereby cDNA 131 is made up of the melanocyte differentiation antigen TRP-2 of part shear-form, and it contains exons 1-4, has kept the beginning part (Fig. 5) of intron 2 and intron 4.Dna sequencing and homology search
Carry out dna sequence analysis by the specificity initiation of using the synthetic oligonucleotide.Sequencing reaction is to use the dideoxy nucleotide of dye marker and ABIPRISMTM Dye Terminator Cycle Sequencing Ready Reaction test kit by the dideoxy-chain terminating method (Perkin Elmer, Foster City CA) carries out.The dna sequence dna of plasmid clone cDNA 131 is to use automatic dna sequencing instrument (ABI Prism 377DNA sequenator; Perkin Elmer) measures.The computer search of sequence homology is to use program FASTA EMBL-Heidelberg to carry out.Coding is by the evaluation of the sequence of the antigen peptide of CTL 128 identifications
For the sequence of location coding,, obtain being respectively the subfragment (5 ' among Fig. 5-end-1500,200 base pairs and 3 '-end) of 1500,200 and 2000 base pairs with HindII digested cdna 131 by the antigen peptide of CTL 128 identifications.The preparation of the subfragment of cDNA 131
By the subfragment (5 '-end-1500,5 '-end-800,200 base pairs and 3 '-end) that obtains cDNA 131 with HindII and BstUI digested plasmid.On sepharose, after the purifying, these fragment clonings are advanced the pcDNAI plasmid.From 5 '-end-1500, produce less fragment INT-2-434, INT-2-166 and INT-2-107 by pcr amplification.There is adopted primer KS-INT2 (SEQ ID NO:5) to be used to all segmental amplifications.This primer produces an ATG initiator codon (underscore), has a suitable Kozak consensus sequence, and its reading frame is identical with TRP-2's.Be respectively applied for the amplification INT-2-434 ,-166 and-107 segmental antisense primers are the SP6 (SEQ IDNO:8) that are arranged in the pcDNAI plasmid, be arranged in the INT2-as1 (SEQID NO:6) and the INT2-as2 (SEQ ID NO:7) of the intron 2 of TRP-2.Antisense primer INT2-asl and-as2 contains an XhoI restriction site that is used for directed cloning.For the ease of connecting, we have utilized single a 3 ' A-in segmental 5 ' the end existence of DynazymeTM amplification to hang, and this is to produce by the terminal enzyme (DNA) of archaeal dna polymerase is active.The pcDNAI plasmid is digested with EcoRV, then at each segmental 3 ' end by in the presence of 2 mM dTTP, bathing single thymus pyrimidine of adding with DynazymeTM temperature, as Marchuk etc., nucleic acids research 19 (1991) 1154 is described.T-carrier and PCR product are digested purifying and connection on sepharose with XhoI.After the connection, the plasmid electroporation is advanced the DH-5 intestinal bacteria, and select (50 mg/ml) with penbritin.Separating clone, the extracting plasmid DNA is also advanced the Cos-7 cell with the HLA-A*6801 gene transfection.
In this step, do not investigate the existence of 5 '-end fragment of the initiator codon of two translations of regulating them.With 5 '-the Cos-7 cell of end-1500 fragments (SEQ ID NO:12) transfection in the presence of TNF emission levels and be on close level (Fig. 5) of stimulating by cDNA 131 by CTL 128, show that antigen peptide encodes in this zone.The nucleotide sequence of this subfragment comprises exons 1, and the 410 initial base pairs of exon 2 and intron 2 are shown in SEQ IDNO:12.CTL 128 is not by the stimulation (Fig. 5) by the Cos-7 cell of TRP-2 cDNA that shears fully and the common transfection of HLA-A*6801.This shows that coding may be arranged in the intron part of the TRP-2 gene of cDNA 131 existence whole or in part by the antigenic sequence of CTL 128 identifications.This conclusion has obtained support, promptly uses 5 '-shortage (Fig. 5) of the identification of the Cos-7 cell of end-800 cDNA fragment transfections, and this fragment is blocked in the BstUI site from 5 '-end-1500 fragments and obtained, and contains exons 1 and exon 2 half.The location of the intron of the sequence of coding for antigens peptide is confirmed by the segmental ability of a pcr amplification, and this fragment comprises initial 434 base pairs (INT-2-434) of intron 2, and it can pass on antigenic expression (Fig. 5).In the reading frame identical with the exon of this regional front, observe the existence of the sequence of a coding decapeptide (EVISCKLIKR) (SEQ ID NO:2), it has anchor residues (2,9, with 10), corresponding to HLA-A*6801 peptide binding motif (Rammensee etc., immunogenetics 41 (1995) 178-228).
In order further to limit the zone of containing epitope, use has adopted primer KS-INT2 (SEQID NO:5), it produces ATG and an antisense primer INT2-asl (SEQ ID NO:6) or INT2-as2 (SEQ ID NO:7) amplification PCR fragment with suitable Kozak consensus sequence.First fragment (INT-2-166) comprises the peptide-coding sequence of whole suppositions, and this is excalation in second fragment (INT-2-107).The fragment INT-2-166 that only contains the peptide sequence of complete supposition can stimulate the TNF by CTL 128 to discharge (Fig. 5).Synthetic then 10-aggressiveness and 9-mer peptides, EVISCKLIKR (SEQ ID NO:2) and EVISCKLIK (difference of 9-mer peptides and 10-aggressiveness attitude (SEQ ID NO:2) is the disappearance of last amino acid (R)), and to isozygoty with HLA-A*6801 be that the LB temperature is bathed.But decapeptide EVISCKLIKR sensitization LB cell makes them can be by CTL 128 cracking, obtain maximum cracked half value in the concentration of about 100pM, and nonapeptide has very low effectiveness, (Fig. 6) that control peptide is negative.
For the epitope of getting rid of by CTL 128 identifications can produce by the sudden change that occurs in the tumour, by PCR from the genomic dna of CTL 128 with from a kind of different melanoma, MZ2 one mel, the fragment of one 2152 base pair of amplification, it is across complete intron 2 (the Nucleotide 995-3080 among the cDNA 131).Embodiment 3 is from genomic dna cloning TRP-2-INT2
(QIAGEN, Hilden is Germany) from MZ2 melanoma cell and CTL 128 purified genomic dnas to use QLAamp blood test kit.The KS-INT2 (SEQ ID NO:5) that use is arranged in exon 2 as the PR2 (SEQ ID NO:9) that adopted primer is arranged and be arranged in exon 3 as antisense primer by the intron 2 of PCR from the genomic dna amplification TRP-2 of 100 ng.As mentioned above the TRP-2-INT2 fragment cloning is advanced the pcDNAI carrier, order-checking is also advanced the Cos-7 cell with the transfection of HLA-A*6801 gene.
With the PCR product cloning, the Cos-7 cell is advanced in order-checking and transfection.All clones all have desired sequence, and transmit antigenic expression.
The RT-PCR that use is arranged in exons 1 and intron 2 has adopted primer PRIT-I (SEQ ID NO:3) and antisense primer INT2-1260 (SEQ ID NO:4) to carry out has only amplified the segment of one 977 base pair from the TRP2 transcript that keeps intron 2.The pcr analysis of the expression of embodiment 4TRP-2-INT2 and TRP2
From cultured cells system, fresh skin, retina and tumor sample use RNAzol B to prepare total RNA by guanidine-lsothiocyanates method.Use the DynazymeTM (Finnzymes) of 1 U and every kind of primer of 1 mM by PCR, cDNA corresponding and the total RNA of 300ng increases in the final volume of 50 ml.Reaction mixture is carried out 30 amplification cycles.For amplification TRP-2cDNA, use the TRP-2L (SEQ ID NO:11) be arranged in the PR3 (SEQ ID NO:10) of exon 2 and be positioned at exon 8 respectively conduct justice and antisense primer are arranged.PCR carries out 30 circulations (94 ℃ 1 minute, 58 ℃ 1 minute and 72 ℃ 1 minute).For the expression of the intron 2 that confirms non-shearing, using has adopted primer PRIT-1 (SEQ ID NO:13) and antisense primer INT2-1260 (SEQ ID NO:4), and they lay respectively among intron 2 and the 5 '-UTR.PCR carries out 30 circulations (94 ℃ 1 minute, 55 ℃ 1 minute and 72 ℃ 1 minute).When detecting TRP-2 by primer being positioned in the far-end exon, exist (Sturm etc., genome 29 (1995) 24-34) by the long introne 1 of 10 kb between exons 1 and the exon 2 when amplification TRP-2-INT2 have avoided the amplification of the genomic dna of pollution.By use Auele Specific Primer right-pcr amplification of exciting albumen cDNA checks the quality of RNA prepared product.
The TRP-2 courier's of Jian Qieing expression is used fully has adopted primer PR3 (SEQ ID NO:10) and antisense primer TRP-2L (SEQ ID NO:11) to detect, and they lay respectively in exon 2 and 8.
In the tumour that is detected, only melanoma confirms TRP-2 and TRP-2-INT2 antigen positive (table 1).When not existing, TRP-2 never observes the expression of TRP-2-INT2.This result is consistent with such conclusion, and promptly TRP-2 is a kind of melanocyte differentiation antigen (Wang etc., The Journal of Experimental Medicine 184 (1996) 2207-2216), and identical promoters driven common courier's is synthetic, thereby produces two kinds of antigens.Analyzed 69% fresh melanoma sample is expressed TRP-2,78% TRP-2 +Melanoma is also expressed TRP-2-INT2 (table 1).This also observes in K-1735, and wherein, the TRP-2 of normal form and reservation the sort of of intron 2 are expressed in 84% and 68% analyzed sample respectively.Healthy tissues to wherein known TRP2 existence is carried out the analysis that TRP-2-INT2 expresses.Three melanocyte systems in RT-PCR detects, four skin samples and (table 1) that retina is negative.Four skin samples are analyzed and compare with biopsy primary focus from identical patient, though TRP-2 is detected in all samples, TRP-2-INT2 exists only in the tumor sample.The expression of table 1 TRP-2-INT2 in tumour and healthy tissues
The number of sample of number/detection with sample of antigen presentation
TRP-2-INT2 ?TRP-2
K-1735 13／19(68％)* 16／19(84％)
Melanocyte system 0／3 3／3
Fresh melanoma sample 7／13(54％)* 9／13(69％)
Skin 0／4 4／4
Retina 0／1 1／1
Tumour (the plain knurl of non-black) 0／3 0／3
* only in TRP-2 male sample, detect the expression of TRP-2-INT2
Has the intensive dependency between the expression of discovery TRP-2-INT2 mRNA in K-1735 and their stimulations the ability by the TNF release of CTL 128.By presenting of the allelic antigen peptide of the super type of HLA-A3-class
HLA-A*6801, and A3, A11:A31 and A*3301 belong to the super type of the allelic A3-class of HLA-A, and they have similar peptide bonding properties (Sidney JM etc., immunology 154 (1995) 247-259).Whether can present in order to investigate peptide EVISCKLIR (SEQ ID NO:2), will express the target (Fig. 7) of this allelic EBV LCL with this peptide pulse (pulsing) afterwards as CTL 128 by the HLA-A86802 allelotrope phase CTL 128 of the super type of A3-class.
This analysis also comprises HLA-A*6802 allelotrope, a kind of hypotype that belongs to the lHLA-A28 of the super type of A2-class.In the super type allelotrope of A3-class, only A*3301 can present TRP-2-INT with the effectiveness identical with A*6801 222-223Peptide.When this peptide is current by A*6802, observe in higher concentration to have low-level identification.Embodiment 5 antigen peptide and CTL test
Use is used for Fmoc synthetic peptide on solid phase of the terminal protection of moment NH2-, and by mass spectrum identify (Primm, Milan, Italy).Analyze demonstration by HPLC, all peptides are pure greater than 90%.The concentration of cryodesiccated peptide with 10 mM is dissolved among the 10%DMSO, and-80 ℃ of preservations.Detect in a test at these peptides, wherein 51The target cell of Cr-mark at room temperature had on the 96 hole microwell plates of different concns temperature and was bathing 1 hour before adding CTL 128 with 20: 1 effector/target ratio.Measure cracking after 4 hours.In a TNF release test, also detect presenting of antigen peptide.In brief, with the at room temperature warm bath of the peptide of stimulator cell and fixed concentration 1 hour; Thorough washing adds CTL 128 and assessing TNF release after 18-20 hour on the WEHI-164.13 cell then.
Reference Anichini, A., R., Deng, J.Immunol. (Journal of Immunology) 156 (1996) 203-217Bachmair etc., Science (science) 234 (1986) 179-186Bachmair etc., Cell (cell) 56 (1989) 1019-1032Bakker, A., M., etc., J.Exp.Med. (The Journal of Experimental Medicine) 179 (1994) 1005-1009Barth, R.J., wait J.Exp.Med.173 (1991) 647-658 20Boel, P., Deng, Immunity (immunity) 2 (1995) 167-175Boon, T., etc., Annu Rev.Immuno1. (immunology annual review) 12 (1994) 337-365Boon, T., etc., Immunology Today (immunology today) 18 (1997) 267-268Bouchard, B., Deng, Eur.J.Biochem. (european journal of biological chemistry) 219 (1994) 127-134Brichard, V., Deng, J.Exp.Med.178 (1993) 489-495Castelli, C., etc., J.Exp.Med.181 (1995) 363-368Coulie, P.G., etc., J.Exp.Med.180 (1994) 35-42Coulie, P.G., Deng, Proc.Nat.Acad.Sci.USA (PNAS) 92 (1995) 7976-7980Espevik, T., and J.Nissen-Meyer, J.Immunol.Methods (immunological method magazine) 95 (1986) 99-105Fujii, T., etc., J.Immunol.153 (1994) 5516-5524Gonda etc., J.Biol.Chem. (journal of biological chemistry) 264 (1989) 16700-16712Haas, G., etc., Am.J.Reprod.Immunol.Microbiol.18 (1988) 47-51Houghton, A.N., J.Exp.Med.180 (1994) 1-4Kawakami, Y., Deng, Proc.Natl.Acad.Sci.USA 91 (1994) 3515-3519Kawakami.Y., etc., Proc.Natl.Acad.Sci.USA 91 (1994) 6458-6462Mandelboim, O., Deng, Nature (nature) 369 (1994) 67-71Marchand, M., Deng, Int.J.Cancer (international journal of cancer) 63 (1995) 883-885Monach, P., etc., Immunity 2 (1995) 45-49Niarchuk, D., etc., Nucl.Acids Res. (nucleic acids research) 19 (1991) 1154Oh, S., Deng, Tissue Antigenics (tissue antigen) 41 (1993) 135-142Pardoll, D.M., Nature:369 (1994) 357-358Parham, P., etc., J.Immunol.123 (1979) 342-349Parham, P., and F.Brodsky, Hum.Immurol. (human immunity) 3 (1981) 277-299Rammensee, H.G., Deng, Immunogeneties (immunogenetics) 41 (1995) 178-228Robbins, P., etc., J.Exp.Med.183 (1996) 1185-1192Robbins, P., etc., J.Immunol.154 (1995) 5944-5950Robbins, P., Deng, J.Immunol.159 (1997) 303-308Rosenberg, S.A., Cancer J.Sci.Am.1 (1995) 90-100Rosenberg, S., Immunol.Today 18 (1997) 175-182Rosenberg, S.A., Deng, J.Natl.Cancer Inst.USA 86 (1994) 1159-1166Russo, C., etc., Immunogenetics 18 (1983) 23-35Sambrook J., Fritsch E.F.1 989.Molecular cloning:A laboratorv manual.Cold SpringHarbor Laboratory Press (2 nd edition) New York USA (molecular cloning: laboratory manual, press of cold spring harbor laboratory (second edition) New York, the U.S.) Seed, B., and A.Aruffo, Proc.Natl.Acad.Sci.USA 84 (1987) 3365-3369Sidney, J., Deng, J.Immunol.154 (1995) 247-259Sturm, R., etc., Genomics (genome) 29 (1995) 24-34Takahashi, K., etc., Cancer Res. (cancer research) 55 (1995) 3478-3482Traversari, C., Deng, Immunogenetics 35 (1992) 145-152Tsomides, T.J., and Eisen, H.N., Proc.Natl.Acad Sci.USA 91 (1994) 3487-3489Van den Eynde, B., Deng, J.Exp.Med.182 (1995) 689-698Van der Bruggen, P., etc., Science 254 (1991) 1643-1647Wang, R., etc., J.Exp.Med.184 (1996) 2207-2216Wang, R., Deng, J.Exp.Med.181 (1995) 799-804Wolfel, T., Deng, Scienee 269 (1995) 1281-1284Yang, S., etc., Immunogenetics 19 (1984): 217-231Yokoyama, K., etc., Bioch.Bioph.Acta (biological chemistry biophysics annual report) 1217H (1994) 317-321
Sequence table (1) total information:
(ⅰ) applicant:
(A) title: BOEHRINGER MANNHEIM GMBH
(B) street: Sandhofer str.116
(C) city: Mannheim
(E) country: Germany
(F) postcode: D-68305
(G) phone: 08856/60-3446
(H) fax: 08856/60-3451
(ⅱ) denomination of invention: tumour specific antigen, its preparation method and their application in immunity and diagnosis
(ⅲ) sequence number: 12
(ⅳ) computer-reader form
(A) media type: floppy disk
(B) Computer I BM PC compatibility
(C) operating system PC-DOS/MS-DOS
(D) software: PatentIn Release #1.0, the information of Version #1.30B (EPO) (2) SEQ ID NO:1:
(ⅰ) sequence signature
(A) length: 30 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: cDNA
(ⅸ) characteristics:
(A) title/keyword: CBS
(B) site: 1..30
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:1:GAA GTA ATT TCA TGC AAG TTA ATT AAG AGA 30Glu Val Ile Ser Cys Lys Leu Ile Lys Arg1 5 10 (2) SEQ ID NO:2:
(ⅰ) sequence signature
(A) length: ten amino acid
(B) type: amino acid
(D) topological structure: linearity
(ⅱ) molecule type: protein
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:2:Glu Val Ile Ser Cys Lys Leu Ile Lys Arg1 5 10 (2) SEQID NO:3:
(ⅰ) sequence signature
(A) length: 17 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: other nucleic acid
(A) describe :/desc=" primer "
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:3:GGAGA AAAGT ACGACAG (2) SEQ ID NO:4:
(ⅰ) sequence signature
(A) length: 20 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: other nucleic acid
(A) describe :/desc=" primer "
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:4:ACCTCACCAA CTCACATCTT (2) SEQ ID NO:5:
(ⅰ) sequence signature
(A) length: 27 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: other nucleic acid
(A) describe :/desc=" primer "
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:5:GCCGCCATGT ATTCTGTTAG AGATACA (2) SEQ ID NO:6:
(ⅰ) sequence signature
(A) length: 27 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: other nucleic acid
(A) describe :/desc=" primer "
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:6:ATCCGCTCGA GCATGAAATT ACTTCCC (2) SEQ ID NO:7:
(ⅰ) sequence signature
(A) length: 25 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: other nucleic acid
(A) describe :/desc=" primer "
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:7:ATCCGCTCGA GGATAATTCT ACGAC (2) SEQ ID NO:8:
(ⅰ) sequence signature
(A) length: 18 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: other nucleic acid
(A) describe :/desc=" primer "
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:8:ATTTAGGTGA CACTATAG (2) SEQ ID NO:9:
(ⅰ) sequence signature
(A) length: 20 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: other nucleic acid
(A) describe :/desc=" primer "
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:9:GAGAAATCTA TGGCCCTGTA (2) SEQ ID NO:10:
(ⅰ) sequence signature
(A) length: 20 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: other nucleic acid
(A) describe :/desc=" primer "
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:10:TTCGGCAGAA CATCCATTCC (2) SEQ ID NO:11:
(ⅰ) sequence signature
(A) length: 27 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: other nucleic acid
(A) describe :/desc=" primer "
(ⅹ ⅰ) sequence description: the information of SEQID NO:11:ACCCTAGGCT TCTTCTGTGT ATCTCTT (2) SEQ ID NO:12:
(ⅰ) sequence signature
(A) length: 1409 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ⅱ) molecule type: DNA (genome)
(ⅸ) feature:
(A) title/keyword: exon
(B) site: 1..994
(ⅸ) feature:
(A) title/keyword: intron
(B) site: 995..1409
(ⅸ) feature:
(A) title/keyword: misc signal
(B) site: 399..402
(C) out of Memory :/function=" initiator codon "
(ⅸ) feature:
(A) title/keyword: misc signal
(B) site: 1111..1113
(C) out of Memory :/function=" first terminator codon "
(ⅸ) feature:
(A) title/keyword: mat peptide
(B) site: 1063..1093
( ⅹⅰ ) ：SEQ ID NO：12：AGCTAAGGAG GGAGGGAGAG GGTTTAGAAA TACCAGCATA ATAAGTAGTA TGACTGGGTG 60CTCTGTAAAT TAACTCAATT AGACAAAGCC TGACTTAACG GGGGAAGATG GTGAGAAGCG 120CTACCCTCAT TAAATTTGGT TGTTAGAGGC GCTTCTAAGG AAATTAAGTC TGTTAGTTGT 180TTGAATCACA TAAAATTGTG TGTGCACGTT CATGTACACA TGTGCACACA TGTAACCTCT 240GTGATTCTTG TGGGTATTTT TTTAAGAAGA AAGGAATAGA AAGCAAAGAA AAATAAAAAA 300TACTGAAAAG AAAAGACTGA AAGAGTAGAA GATAAGGAGA AAAGTACGAC AGAGACAAGG 360AAAGTAAGAG AGAGAGAGAG CTCTCCCAAT TATAAAGCCA TGAGCCCCCT TTGGTGGGGG 420TTTCTGCTCA GTTGCTTGGG CTGCAAAATC CTGCCAGGAG CCCAGGGTCA GTTCCCCCGA 480GTCTGCATGA CGGTGGACAG CCTAGTGAAC AAGGAGTGCT GCCCACGCCT GGGTGCAGAG 540TCGGCCAATG TCTGTGGCTC TCAGCAAGGC CGGGGGCAGT GCACAGAGGT GCGAGCCGAC 600ACAAGGCCCT GGAGTGGTCC CTACATCCTA CGAAACCAGG ATGACCGTGA GCTGTGGCCA 660AGAAAATTCT TCCACCGGAC CTGCAAGTGC ACAGGAAACT TTGCCGGCTA TAATTGTGGA 720GACTGCAAGT TTGGCTGGAC CGGTCCCAAC TGCGAGCGGA AGAAACCACC AGTGATTCGG 780CAGAACATCC ATTCCTTGAG TCCTCAGGAA AGAGAGCAGT TCTTGGGCGC CTTAGATCTC 840GCGAAGAAGA GAGTACACCC CGACTACGTG ATCACCACAC AACACTGGCT GGGCCTGCTT 900GGGCCCAATG GAACCCAGCC GCAGTTTGCC AACTGCAGTG TTTATGATTT TTTTGTGTGG 960CTCCATTATT ATTCTGTTAG AGATACATTA TTAGGTGGGT TTTTTCCTTG GCTGAAGGTA 1020TATTATTACA GGTTTGTGAT TGGGTTGAGG GTATGGCAGT GGGAAGTAAT TTCATGCAAG 1080TTAATTAAGA GAGCAACCAC AAGGCAGCCT TAGGTTTATG AAAGTCGTAG AATTATCAAA 1140TACCGCCTGG AGTTAGAAGG AAGCAGTTTC TTCCTGTGCA TTGGATGCAG ACACTTTAAA 1200TGTTCTCTCC TCTACCGTAT GTTCTTGGTT CAAAGTGTAA ACTTTTCTCT GTGAAGCTGT 1260TAATCATCAA AGATGTGAGT TGGTGAGGTG GAGGCGAATT CCTTTTGATT TCAGAAGAAA 1320ATATTTGCGA ATCTGGCCAT GGAAGCCCTC TCTGACCTTT TCTCCAAATT AGAGGAATTA 1380ACTGAACATG TGCTAAGGCA CATGAAGCT 1409

Claims (11)

1. tumour-specific polypeptides antigen, it partly is by a kind of intron coding of tumour antigen of exons coding, and it obtains by following steps:
A) isolating mRNA from a kind of solubility kytoplasm composition of tumour cell is carried out reverse transcriptase PCR, wherein under rigorous condition, be used as primer with a kind of nucleic acid fragment of intron sequences hybridization of tumour antigen of exons coding;
B) separate said PCR product, express said PCR product or its fragment in host cell, with the tumour specific antigen that separates by this PCR product or its fragment coding, it is also hybridized with said antigenic exon sequence.
2. according to the described tumour-specific polypeptides antigen of claim 1, wherein, the fragment of 8 to 12 codons of said PCR product is used to express.
3. according to claim 1 or 2 described tumour-specific polypeptides antigens, wherein, the tumour antigen of said exons coding is MAGE, and BAGE and GAGE are from tyrosine oxidase, MelanA Martl, gp100 Pmel7, gp75 TRP1Or the CTL of TRP-2 identification epitope.
4. tumour-specific polypeptides antigen by SEQ ID NO:1 coding.
5. the method for the mRNA of the separation tumour specific antigen of partly being encoded by a kind of intron of tumour antigen of exons coding may further comprise the steps:
A) isolating mRNA from a kind of solubility kytoplasm composition of tumour cell is carried out reverse transcriptase PCR, wherein under rigorous condition, be used as primer with a kind of nucleic acid fragment of intron sequences hybridization of tumour antigen of exons coding; With
B) separate said PCR product, it is also hybridized with said antigenic exon sequence.
6. method of measuring the propagation of tumor-specific cytotoxicity T-cell, wherein, any one tumour specific antigen is added in patient's the sample of body fluid in the claim 1 to 4, and it contains antigen and presents cell and cytotoxic T cell, measures the propagation of cytotoxic T cell.
7. the nucleic acid of coding any one tumour specific antigen in the claim 1 to 4 is used in preparation is used for the treatment of the medicine of tumor disease.
In the claim 1 to 4 any one tumour specific antigen in vivo or vivo activation from the application in the cytotoxic T cell of T precursor cell.
9. one kind prepares the antigenic method of tumour-specific polypeptides, and wherein said tumour specific antigen obtains by following steps:
A) isolating mRNA from a kind of solubility kytoplasm composition of tumour cell is carried out reverse transcriptase PCR, wherein under rigorous condition, be used as primer with a kind of nucleic acid fragment of intron sequences hybridization of tumour antigen of exons coding;
B) separate said PCR product, in host cell, express said PCR product or its fragment,, and under rigorous condition, hybridize with said antigenic exon sequence with the tumour specific antigen that separates by this PCR product or its fragment coding.
10. the combination of two kinds of nucleic acid, they under rigorous condition with a kind of intron sequences hybridization of tumour antigen of exons coding, and they can be used as from the primer of the reverse transcriptase PCR of mRNA right.
11. nucleic acid by SEQ ID NO:3 to SEQ ID NO:9 coding.
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