CN1291992A - Isolated, polypeptides which bind to HLA-A29 molecules, nucleic acid, molecules encoding there, and uses thereof - Google Patents
Isolated, polypeptides which bind to HLA-A29 molecules, nucleic acid, molecules encoding there, and uses thereof Download PDFInfo
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- CN1291992A CN1291992A CN99803245A CN99803245A CN1291992A CN 1291992 A CN1291992 A CN 1291992A CN 99803245 A CN99803245 A CN 99803245A CN 99803245 A CN99803245 A CN 99803245A CN 1291992 A CN1291992 A CN 1291992A
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- C07K4/00—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
- C07K4/12—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof from animals; from humans
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- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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Abstract
公开了结合到HLA－A29分子上的肽。这些分子满足SEQIDNO:23、SEQIDNO:24或SEQIDNO:25定义的基元。还描述了编码本发明肽的小基因以及它们的应用。Peptides that bind to HLA-A29 molecules are disclosed. These molecules satisfy the motifs defined by SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25. Minigenes encoding the peptides of the invention and their use are also described.
Description
Related application
The application is that the sequence number of applying for September 21 nineteen ninety-five is 08/531,662 partial continuous application, the latter is again that the sequence number of applying for January 10 nineteen ninety-five is 08/370, the partial continuous application of 648 common co-pending application, the latter is again that the sequence number of applying on May 27th, 1994 is 08/250, the partial continuous application of 162 common unexamined patent application, the latter is again that the sequence number of application on July 22nd, 1993 is 08/096,039 partial continuous application.All these applications are incorporated herein by reference.
Invention field
The application relates to the nucleic acid molecule of codes for tumor rejection antigen precursor.Specifically, the present invention relates to gene, its tumor rejection antigen precursor is processed at least a Tumor rejection antigen by the HLA-Cw6 molecular presentation especially.It is irrelevant with the encoding sequence of other known cancer rejection antigen precursor that described gene seems.The invention still further relates to peptide and application thereof by the HLA-Cw6 molecular presentation.Peptide and application thereof by the HLA-A29 molecular presentation also are parts of the present invention.
Background technology
Immune system identification external source or dissimilar substances and with the process of its reaction be the process of a complexity.An importance of this system is that T lymphocyte or " T cell " are replied.This is replied and needs the T cell recognition to be called as the complex body of cell surface molecule of human leucocyte antigen (HLA) (" HLA ") or major histocompatibility complex (" MHC ") and peptide and interact with them.Described peptide derives from the macromole that the cell of also presenting the HLA/MHC molecule is processed into.In this referring to Male etc., immunology progress (Advanced Immunology) (J.P.Lipincott Company, 1987), particularly 6-10 chapter.The interaction of T cell and HLA/ peptide complex body is confined, need be specific to the T cell of the particular combinations of HLA molecule and peptide.If there is no even there is its pairing complex body (partner complex), there is not t cell response in specific T-cells yet.Similarly, if there is no specific compomers is not replied yet, but has the T cell.At immunity system the replying of allogenic material, autoimmunization pathology and unusual the replying of pair cell are all related to this mechanism.Extensive work concentrates on the mechanism that protein is processed into HLA bonded peptide.In this referring to, Barinaga, science 257:880 (1992); Fremont etc., science 257:919 (1992); Matsumura etc., science 257:927 (1992); Latron etc., science 257:964 (1992).Also referring to Engelhard, immunology year is summarized (Ann.Rev.Immunol.) 12:181-207 (1994).
The mechanism of T cell recognition cellular abnormality also and related to cancer.For example, (on November 26th, 1992 is open for the PCT application PCT/US92/04354 of application on May 22nd, 1992, be incorporated herein by reference) a kind of gene family disclosed, it is processed to peptide, this peptide is expressed at cell surface again, and this peptide can cause the tumour cell cracking by Specific CTL Cells solvability T lymphocyte (or be called hereinafter " CTL ").It is said, such genes encoding " tumor rejection antigen precursor " or " TRAP " molecule, the peptide that derives from them is called as " Tumor rejection antigen " or " TRA ".For the further information of this gene family is provided, referring to Traversari etc., immunogenetics (Immunogenetics) 35:145 (1992); Van der Bruggen etc., science 254:1643 (1991).Also being 807,043 U.S. Patent application referring to the sequence number of on December 12nd, 1991 application, is U.S. Patent number 5,342 now, 774.
At sequence number is 938,334 U.S. Patent application is United States Patent (USP) 5,405 at present, in 940 (its disclosure is incorporated herein by reference), wherein explain a kind of tumor rejection antigen precursor that is processed to by the nonapeptide of HLA-A1 molecular presentation of MAGE-1 genes encoding.This reference is pointed out, supposes the specificity of known concrete peptide to concrete HLA molecule, and people expect that a kind of concrete peptide is attached on a kind of HLA molecule, rather than on other molecule.This point is very important, because Different Individual has different HLA phenotypes.Consequently, although the counterpart that particular peptide is accredited as the specific HLA molecule has the auxiliary meaning of diagnosis and treatment, then they are only relevant with the individuality with this specific HLA phenotype.This field also requires further study, because cellular abnormality is not limited to a kind of specific HLA phenotype, targeted therapy needs some information about controversial abnormal cells phenotype.
Be to disclose such fact in 008,446 the U.S. Patent application (being incorporated herein by reference) at the sequence number of on January 22nd, 1993 application, promptly the MAGE-1 expression product is processed to the 2nd TRA.The 2nd TRA clones 10 molecular presentations by HLA-C.A kind of given TRAP of the disclosure text display can produce most TRA.
On December 22nd, 1992, the sequence number of application was 994,928 U.S. Patent application (being incorporated herein by reference) is pointed out: tyrosine oxidase, be a kind of molecule that some normal cell (for example melanophore) produces, in tumour cell, process, produce peptide by the HLA-A2 molecular presentation.
Be in 08/032,978 the U.S. Patent application (it is incorporated herein by reference in full), to point out that non-the 2nd TRA of tyrosine oxidase that derives from is by the HLA-A2 molecular presentation at the sequence number of on March 18th, 1993 application.This TRA derives from TRAP, but by non--MAGE genes encoding.The disclosure text shows that the specific HLA molecule can present the TRA of different sources.
Be in 08/079,110 the U.S. Patent application (being introduced into this paper as a reference), to have described a kind of incoherent tumor rejection antigen precursor, promptly so-called " BAGE " precursor at the sequence number of on June 17th, 1993 application.It doesn't matter for BAGE precursor and MAGE family.
The major part work that above-mentioned document, patent, patent application are described relates to MAGE gene family and incoherent BAGE gene.Yet, do not find other tumor rejection antigen precursor as yet by cell expressing.These tumor rejection antigen precursors are called as " GAGE " tumor rejection antigen precursor.They and MAGE gene family or BAGE gene do not have homology.Therefore, the gene of these TRAP that the present invention relates to encode, tumor rejection antigen precursor itself, and their application.
Therefore, another feature of the present invention is that length is 9 to 16 amino acid whose one group of peptides, and they comprise following sequence:
Xaa?Trp?Pro?Xaa?Xaa?Xaa?Xaa?Tyr
(sequence number: 23)
Wherein Xaa is an arbitrary amino acid, Xaa
(1,2)Mean the N-end that 1 or 2 amino acid may be the Trp residue.These peptide combinations, and/or be processed to be attached to peptide on the HLA-A29 molecule.
In the open text below the present invention is described in further detail.
The accompanying drawing summary
Fig. 1 has set forth the Study on Cleavage of using ctl clone 76/6.
Fig. 2 shows the release analysis of the tumour necrosis factor (" TNF ") that obtains with various transfection bodies and contrast.
Fig. 3 has compared the CTL inductive splitting action of clone CTL 76/6.Test the peptide of all lengths, comprised SEQ ID NO:4.
Fig. 4 provides the arrangement of the cDNA of six kinds of GAGE genes that the present invention discusses.Among the figure, same zone surrounds with frame.Translation initiation site and terminator have also been indicated.The primer of using in the polymerase chain reaction that embodiment describes marks with arrow.
Fig. 5 has set forth the arrangement of GAGE family member's putative amino acid sequence.Same area shows with frame, and has shown the antigen peptide of SEQ ID NO:4.
Fig. 6 has shown the result who obtains in the COS cell time with HLA-Cw6 cDNA transfection as each GAGE cDNA.Behind the twenty four hours, add CTL 76/6 sample, behind twenty four hours, detect TNF and discharge.
Fig. 7 has compared the COS-7 cytositimulation CTL 22/23 of HLA-A29 cDNA, MAGE, BAGE or the transfection of GAGE sequence shown in the usefulness.Provide control value by MZ2-MEL.43 and COS cell as stimulator.
Fig. 8 has shown and utilizes various peptides to comprise that SEQ ID NO:22 and the various peptides that derive from them carry out
51The result that the Cr releasing research draws.
Detailed description of the preferred embodiments
The application standard method is set up melanoma cell series from the melanoma cells of taking from patent MZ2, MZ2-MEL.This clone was applied on May 22nd, 1992 for example, described among disclosed PCT application on November 26th, 1992 PCT/US92/04354, and it is incorporated herein by reference in full.In case clone is shone its sample, so that it is not bred after setting up.Separate with these postradiation cells then it is had specific cytolysis T cell clone (" CTL ").
Peripheral blood lymphocytes (" PBMC ") sample is taken from patent MZ2, contacts with postradiation melanoma cells.Observe the melanoma cells splitting action of mixture, its peptide and HLA molecular complex that shows that existence is presented melanoma cells in the sample shows specific CTL.
Used cracking analysis is that the chromium of following Herin etc. (international journal of cancer (Int.J.Cancer) 39:390-396 (1987)) discharges and analyzes, and its disclosure is incorporated herein by reference.Yet, this analysis is still described among the present invention.The target melanoma cells is in growth in vitro, then with 10
7Cell/ml is suspended among the DMEM that adds 10mM HEPES and 30%FCS, then exist 200 μ Ci/mlNa (
51Cr) O
4Situation under cultivated 45 minutes in 37 ℃.With the DMEM that is added with 10mM HEPES labeled cell is washed three times.Then, labeled cell is resuspended in being added with among the DMEM of 10mM HEPES and 10%FCS, afterwards, 100 μ l is contained 10
3The aliquots containig of individual cell is assigned in the 96 hole microplates.Add the PBL sample that is scattered in the 100 μ l same medium, repeat this detection then.With flat board after under the 100g centrifugal 4 minutes, at 8%CO
2Cultivated 4 hours in 37 ℃ under the atmosphere.
The recentrifuge flat board is collected the aliquots containig of 100 μ l supernatant liquors and is also counted.Following calculating
51The release percentage of Cr:
Wherein ER is observed experiment
51Cr discharges, and SR is a single culture 10 in 200 μ l substratum
3The spontaneous release that individual labeled cell records, MR adds the maximum release that 100 μ 10.3%TritonX-100 obtain in target cell.
Those show that the active monokaryon blood sample of high CTL is exaggerated and clones through limiting dilution, utilizes with quadrat method then and screens once more.Separation of C TL clones MZ2-CTL 76/6 then.This is cloned in hereinafter and is called as " 76/6 ".
Use same method test purpose K562 cell and melanoma cell series.Fig. 1 shows identification of this ctl clone and cracking melanoma cell series, i.e. MZ2-MEL but be not K562.Test this clone transforms the B cell to other melanoma cell series and autologous EBV-effect in above-mentioned same mode then.What Fig. 1 showed that Epstein Barr virus (" EBV ") transforms does not have cracking from body B cell, and MZ2-MEL 3.0 is by ctl clone 76/6 cracking, clone MZ2-MEL.4F, and the varient of a kind of not antigen expressed F does not have cleaved.Therefore, this clone may have specificity to this antigen.
Top result displayed be can not determine any HLA molecular presentation TRA.Known cracked clone, promptly MZ2-MEL expresses HLA-A1, HLA-A29, HLA-B37, HLA-B44, HLA-Cw6 and HLA-C clone 10.Do not have record at this paper but use in the experiment of this embodiment scheme, tested the MZ2-MEL subbreed, it has lost the ability of expressing HLA molecule A29, B44 and C clone 10.This subbreed is cleaved, therefore shows that presenting molecule should be one of A1, B37 or Cw6.
Further study to determine when 76/6 contacts with target cell, whether also producing tumour necrosis factor (" TNF ").The method of using is the method that Traversari etc. (immunogenetics 35:145-152 (1992)) describes, and its disclosure is incorporated herein by reference.Briefly, the target cell sample mix of being studied in CTL clone sample and the substratum.After 24 hours, remove culture supernatants, on the WEHI of TNF-sensitivity cell, detect then.Clone MZ2-MEL.43 above and the subclone of a kind of MZ2-MEL clone of inquiring in the reference of quoting as proof, provides extremely intensive and replys, and therefore is used for following experiment.
The result that embodiment 2 obtains shows that MZ2-MEL.43 provides the target antigen of being studied.Based on this, it is used as total mRNA source in preparation cDAN library.
Separate total RNA from this clone.Utilize widow-dT binding reagents box, come separating mRNA according to technique known.In case after mRAN is protected, can utilize the oligo dT primer that contains the NotI site it to be transcribed into cDNA, be the synthetic of second chain then through reverse transcription.Then cDNA is connected on the BstXI conjugant,, carries out apart by Sephacryl S-500 HR post with NotI digestion, then with its non-directional be cloned on the BstXI and NotI site of pcDNAI/Amp.Then recombinant plasmid is imported in the DH5 α colibacillus by electroporation.100 recombinant bacterias that amount to 1500 aggregates are inoculated in the micropore.Every hole contains 100 cDNA that have an appointment, because nearly all bacterium contains the insertion body.
Each aggregate is expanded to saturated, the molten and potassium acetate precipitation extraction plasmid DNA by alkali, and without phenol extraction.
Behind embodiment 3 described preparation libraries, with the cDNA transfection in eukaryotic cell.Carry out transfection of the present invention in duplicate.With 15,000 cells/well the COS-7 cell sample is inoculated in the flat micropore of tissue culture that contains the D μ lbecco ' s improvement Eagles substratum (" DEME ") that adds 10% foetal calf serum.Cell 37 ℃ of overnight incubation, is removed substratum then, be replaced with the DMEM substratum in 50 microlitres/hole, described substratum contains 10%Nu serum, 400 μ g/mlDEAE-dextran and 100 μ M chloroquines, adds the 100ng plasmid again.As shown previously, Study on Cleavage is not determined any HLA molecular presentation antigen.The result is, the cDNA that uses various HLA molecules (A1, B37, Cw6) that can antigen-presenting individually is with the cotransfection cell.Specifically, use with the same scheme of using with the library transfection 28ng is encoded the gene clone of HLA-A1 in pCD-SR α, the cDNA of 50ng HLA-B37 is cloned among the pcDNAI/Amp or the cDNA of 75ngHLA-Cw6 is cloned among the pcDNAI-Amp.
In diplopore, carry out transfection, but only the HLA-Cw6 transfection body of 500 aggregates (pools) can be tested in single hole.Cultivate after four hours for 37 ℃, remove substratum, be replaced with the PBS that 50 μ l contain 10%DMSO.Remove this substratum after two minutes, be replaced with the DMEM that 200 μ l are added with 10%FCS.
After being right after this replacing substratum, the COS cell was cultivated 24-48 hour at 37 ℃.Discard substratum then, add 1000-3000 ctl clone 76/6 cell, it is scattered in the Iscove ' s substratum that 100 μ l contain 10% PHS who adds the 20-30U/ml recombinant il-2.Remove supernatant liquor after 24 hours, in a analysis of describing as (immunogenetics 35:145-152 (1992) are incorporated herein by reference its disclosure) such as Traversari, measure TNF content based on the WEHI cell.
With 1500 aggregates of HLA-A1 transfection, and stimulate TNF to be released into concentration with 1500 aggregates of HLA-B37 transfection to be respectively 15-20pg/ml or 2-6pg/ml.Most of HLA-Cw6 transfection bodies are produced 3-20pg/ml, except an aggregate production surpasses 60pg/ml TNF.Select this aggregate to further investigate.
Bacterium in the aggregate of selecting is cloned, tested 600 clones.From wherein extracting plasmid DNA, transfection detects the effect of this cytositimulation ctl clone 76/6 once more in new COS cell sample in the same way as described above.Find 94 positive colonies.A kind of cDNA of being called as clone 2D6 is wherein further tested.In comparison test, with cDNA clone 2D6 and HLA-Cw6 cDNA, HLA-Cw6 cDNA, cDNA 2D6 rotaring redyeing COS cell separately separately.Also having used control cells is MZ2-MEL F and MZ2-MEL F
+As above-mentioned, the TNF that is discharged in the CTL supernatant liquor is measured in the effect of WEHI cell by detecting it.Cell is after MTT cultivates, by the WEHI cell count of spectrodensitometry survival.Fig. 2 shows the COS cell with HLA-Cw6 and cDNA-2D6 transfection, clone MZ2-MEL F
+Stimulate ctl clone 76/6 to discharge TNF, show that HLA-Cw6 presents target TRA.
According to technology known in the art cDNA 2D6 is checked order.Sequence retrieval shows that plasmid inserts body and known or protein and do not have homology.SEQ ID NO:1 shows by the cDNA Nucleotide information of identified gene (hereinafter being referred to as " GAGE ").The open reading frame of inferring is positioned on the 51-467 bit base of this molecule.Therefore preceding two bases of this sequence are not the parts of cDNA self from the carrier that carries the cDNA sequence.
Embodiment 7
After 6 pairs of cDNA order-checkings of embodiment, whether the cell of determining healthy tissues that experimentizes expresses this gene.In order to determine this, tissue and the tumor cell line of pointing out below carried out the Northern engram analysis.Blot experiment is used the cDNA of SEQ ID NO:1 complete sequence.Use PCT then and confirm the result.
T cell-ctl clone 82/30-liver that the expression normal structure PHA of table 1. gene GAGE activates-muscle-lung-brain-kidney-Placento-heart-skin-testis+tumor cell line melanoma 7/16 lung cancer 1/6 sarcoma 0/1 first shape marrow carninomatosis 0/1 tumor sample melanoma 1/1
Embodiment 8
Use polymerase chain reaction (" PCR ") and above-mentioned GAGE gene information detailed analysis healthy tissues and tumour.
At first, use technology well known in the art and from specific sample, extract total RNA.It is used to prepare cDNA.The scheme that is used to prepare cDNA relates to mixes 4 μ l reversed transcriptive enzyme damping fluid 5x, the various dNTP of 1 μ l (10mM), 2 μ l dithiothreitol (DTT) (100mM), 2 μ l dT-15 primers (20 μ m), 0.5 μ l RNA enzyme inhibitors (RNasin) (40 units/μ l) and 1 μ l MoMLV reversed transcriptive enzyme (200 units/μ l).Then, add 6.5 μ l template ribonucleic acids (1 microgram/3.25 microliters of water, or the total template ribonucleic acid of 2 micrograms).The cumulative volume of mixture is 20 μ l.With mixed 42 ℃ of following insulations 60 minutes that are incorporated in of mixture, place cooled on ice then.Add the water that cumulative volume is 80 μ l then, to cumulative volume 100 μ l.-20 ℃ of these mixtures of storage are up to being used for PCR.
In order to carry out PCR, use the primer shown in SEQ ID NO:2 and 3 respectively:
5 '-AGA CGC TAC GTA GAG CCT-3 ' (sense strand) and
5 '-CCA TCA GGA CCA TCT TCA-3 ' (antisense strand).
Reagent comprises 30.5 μ l water, 5 μ lPCR damping fluid 10x, the various dNTP of 1 μ l (10uM), the various primers of 2.5 μ l (20 μ M) and 0.5 μ l polysaccharase Dynazyme (2 units/μ l).Cumulative volume is 45 μ l.The cDNA (it is corresponding to the total RNA of 100ng) that adds total amount 5 μ l.Blend mixture adds a mineral oil then on the upper strata.Mixture is transferred to the thermal cycler unit that is preheated to 94 ℃, carry out cyclic amplification then 30 times, each cyclic amplification comprises the following steps:
94 ℃ of the first step sex change, 4 minutes
94 ℃ of sex change, 1 minute
Anneal 55 ℃ 2 minutes
Extend 72 ℃, 3 minutes
72 ℃ of last extensions, 15 minutes
After circulation is finished, get 10 μ l sample aliquot and on 1.5% sepharose, carry out electrophoresis, use ethidium bromide staining then.
Utilize the cDNN of above-mentioned primer amplification to produce a kind of fragment with 238 base pairs.If there is contaminating dna, they are not amplified.
The result shows in following table 2.Unique healthy tissues that the result further confirms to express GAGE is a testis, and a lot of tumour, comprises that melanoma, lung, mammary gland, larynx, pharynx, sarcoma, seminoma of testis, bladder and colon express this gene.Therefore, can detect in these tumours any one to the GAGE expression of gene.
Healthy tissues
Heart-
Brain-
Liver-
Lung-
Kidney-
Spleen-
Lymphocyte-
Marrow-
Skin-
Mole-
Melanophore-
Inoblast-
Prostato-
Testis+
Ovary-
Mammary gland-
Suprarenal gland-
Muscle-
Placento-
Umbilical cord-
Tumour
The clone tumor sample
Lung cancer
Small cell lung cancer 6,/23 0/2
The neck tumour
Seminoma of testis 6/6 (100%)
Embodiment 9
The evaluation guiding further work of the nucleic acid molecule of mentioning among the embodiment of front, the target of further work is to determine by the HLA-Cw6 molecular presentation and Tumor rejection antigen that derive from the GAGE gene.
With the full cDNA of the GAGE among restriction endonuclease NotI and the SpHI digestion expression vector pcDNA/Amp, (Erase-a-base System is Promega) with exonuclease III digestion according to supplier's indication then.This is handled and produces a series of progressive disappearances since 3 ' end.
To lack product and reconnect among the pcDNAI/AMP, and utilize the known technology electroporation then in coli strain DH5 α ' IQ.With the penbritin (screening of 50 micrograms/ml) transformant.
From every kind of recombinant clone, extract plasmid DNA, then with the coding HLA-Cw6 the carrier transfection in the COS-7 cell.The scheme of using is followed such scheme.
Tested transformant in the TNF release test then.This can separate positive and negative clone.All negative clones show the disappearance of full GAGE sequence.Minimum positive colony contains preceding 170 Nucleotide of SEQ ID NO:1.The analysis of this sequence (the same) indication open reading frame originates in the 51st Nucleotide.Therefore, this fragment contains preceding 40 amino acid whose sequences of coding GAGE TRAP.
Carry out other experiment then, so that determine the zone of coding TRA peptide more accurately.Use polymerase chain reaction (" PCR ") this experiment of amplification carrying out.
Synthetic two primers.First primer be a kind of 22 polymers (22-mer) with plasmid vector pcDNAI/Amp in be positioned at the sequence complementary sequence of upstream, BamHI site.Second primer is a kind of sequence of 29 polymers, and its 3 ' end contains the 102-119 Nucleotide of SEQ ID NO:1, and 5 ' end contains an extension with 11 Nucleotide of XbaI restriction site.
After the amplification,, be cloned into the BamHI-XbaI site of plasmid pcDNA-3 then with BamHI and XbaI digestion PCR product.According to the method for embodiment 4, in the COS-7 cell, the same then as above-mentioned CTL of utilization 76/6 carries out TNF and discharges and analyze with the cDNA cotransfection of recombinant clone and coding HLA-Cw6.
Observe TNF and discharge, show that " minigene " is processed to TRA.Minigene, i.e. the 1-119 Nucleotide of SEQ ID NO:1, from 51 to 119 Nucleotide in its coding region, preceding 23 amino acid of the cDNA of coding SEQ ID NO:1.This information is as the basis of next group experiment.
Embodiment 11
Synthetic two kinds of peptides on preceding 23 amino acid bases of SEQ ID NO:1.They are:
Met Ser Trp Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg (SEQ IDNO:12) and
Thr?Tyr?Arg?Pro?Arg?Pro?Arg?Arg?Tyr?Val?Glu?Pro?Pro?Glu?Met?Ile(SEQ?IDNO：13)
Each peptide pulse in the COS-7 cell of using HLA-Cw6 cDNA transfection in advance, is made up to determine whether inducing TNF to discharge with CTL 76/6 then.24 hours COS-7 cell of HLA-Cw6 cDNA transfection has been used in peptide (20 μ g/ml) adding in advance.Cultivate after 90 minutes for 37 ℃, discard substratum, 3000CTL is added 100 microlitres contain in the substratum of 25 units/ml IL-2.After 18 hours, the toxicity by determining the WEHI-164-13 cell is produced detects the TNF content in the supernatant liquor.Find that second kind of peptide (SEQ ID NO:13) induce the TNF above 30pg/ml, and first kind of peptide (SEQ ID NO:12) only induced the TNF less than 10pg/ml.Second kind of peptide is used to next step experiment.
Synthetic various peptides are tested on the basis of SEQ ID NO:13, and some of them show below.In order to carry out described test, will
51The LB33-EBV cell of Cr mark, it is the HLA-Cw6 positive, cultivates with one of following peptide:
Tyr?Arg?Pro?Arg?Pro?Arg?Arg?Tyr(SEQ?ID?NO：4)
Thr?Tyr?Arg?Pro?Arg?Pro?Arg?Arg?Tyr(SEQ?ID?NO：5)
Thr?Arg?Pro?Arg?Pro?Arg?Arg?Tyr?Val(SEQ?ID?NO：6)
Thr?Tyr?Arg?Pro?Arg?Pro?Arg?Arg?Tyr?Val(SEQ?ID?NO：7)
Arg?Pro?Arg?Pro?Arg?Arg?Tyr?Val?Glu(SEQ?ID?NO：8)
Met?Ser?Trp?Arg?Gly?Arg?Ser?Thr?Tyr?Arg?Pro?Arg?Pro?Arg?Arg(SEQ?IDNO：12)
Peptide concentration changes as shown in Figure 3, and CTL:LB33-EBV (" effector: the ratio of target ") is 10: 1.Cultivate after 4 hours, measure for 37 ℃
51The release of Cr.Positive (" F
+", MZ2-MEL.3.1) and negative (" F "; MZ2-MEL.2.2.5) control cells cracking level as shown in Figure 3.
Be surprisingly found out that the octamer of SEQ ID NO:4 is best peptide, it may be a Tumor rejection antigen.The report that relates to the octamer by people MHC molecular presentation still belongs to the first time.As Engelhard report (the same), for the H-2K of mouse system 199
bAnd H-2K
kMolecule has some precedents.The nonamer of SEQ IDNO:5 and SEQ ID NO:6 is also induced the CTL cracking, although induce degree low than the octamer of SEQ ID NO:4.
Do not have in the results reported in the present invention, second kind of CTL (CTL 82/31) tested.The cell of MZ2-F is presented in known this CTL cracking.After the peptide pulse with SEQ ID NO:4, this CTL is cracking HLA-Cw6 positive cell also.
Embodiment 13
Whether unique in order to find out above-mentioned GAGE DNA, the cDNA library and the probe hybridization that derives from GAGE cDNA that will prepare with RNA from MZ2-MEL.43.This probe is the PCR fragment of 308 base pairs between the 20-328 position of SEQ IDNO:1.Obtain 20 positive cDNA.Wherein six quilts check order fully.They all with GAGE sequence height correlation, but different slightly with it.Among six clones two are identical, but other differ from one another.Therefore, identify five kinds of but new sequences of height correlation different with the GAGE sequence.They are called as GAGE-2,3,4,5 and 6 (Fig. 4), respectively shown in SEQ ID NO:14-18.Other is cloned in the order-checking of 5 ' end parts in 14, and their sequence is corresponding to one of six kinds of GAGE cDNA.
The key distinction between these cDNA and the GAGE-1 is to lack one section sequence of 143 bases of the 379-521 position that is positioned at SEQ ID NO:1GAGE sequence.Remaining sequence shows only has mispairing on 19 different positionss, except GAGE-3, its 5 ' end is fully different with preceding 112 bases of other GAGE.One section long tumor-necrosis factor glycoproteins and hairpin structure are contained in this district of GAGE-3 cDNA.
The GAGE-1 protein corresponding to tumor rejection antigen precursor of inferring is about 20 amino acid than other 5 kinds of protein, its last seven residues also different with the homology residue of GAGE-1 (Fig. 5).These proteinic all the other sequences only show 10 mispairing.One of them is positioned at the zone corresponding to the antigen peptide of SEQ IDNO:4.GAGE-3,4,5 and 6 peptide sequence are modified, so that present position 2 is W, rather than R.
Embodiment 14
For whether the change of estimating position 2 influences the antigenicity of peptide, with the cDNA of 6 kinds of GAGE respectively with the cDNA transfection of HLA-Cw6 in the COS cell, then as above-mentioned, test transfection body is by the identification of CTL 76/6.Only GAGE-1 and GAGE-2 transfectional cell are identified, and show in this experiment not have antigenicity by GAGE-3,4,5 and 6 modified peptides of encoding.7 sequences that contain coding for antigens peptide of above-mentioned 14 kinds of other clones' 5 ' terminal sequence analysis revealed in them therefore may be corresponding to GAGE-1 or GAGE-2.
Embodiment 15
Be used for above detecting GAGE PCR primer that tumor sample is expressed GAGE-1 or 2 and other four kinds not as broad as long between the GAGE cDNA of coding for antigens MZ2F.Prepared one group of new primer, its specific amplification GAGE-1 and 2, but the GAGE-3,4 that do not increase, 5 and 6.These primers are:
VDE44????5’-GAC?CAA?GAC?GCT?ACG?TAG-3’(SEQ?ID?NO：9)
VDE24????5’-CCA?TCA?GGA?CCA?TCT?TCA-3’(SEQ?ID?NO：10)
These primers such as above-mentionedly in the RT-PCR reaction of using polysaccharase, use according to following temperature condition:
94 ℃ 40 minutes
94 ℃ 1 minute, 56 ℃ 2 minutes, under 72 ℃ of conditions of 3 minutes the circulation 30 times
72 ℃ 15 minutes
This analytical results is as shown in table 3.
The expression of table 3GAGE gene in tumor sample and tumor cell line
| Histological type | The number of GAGE positive tumor | |
| All GAGE genes * | GAGE-1 and 2 ** | |
| Tumor sample melanoma primary injury shifts sarcoma lung cancer NSCLC Head and neck squamous cell carcinoma prostate cancer breast cancer carcinoma of urinary bladder surface infiltration seminoma of testis colorectal cancer leukaemia and lymthoma kidney tumor cell line melanoma sarcoma | ????5/39 ????47/132 ????6/20 ????14/65 ????13/55 ????2/20 ????18/162 ????1/20 ????5/26 ????6/6 ????0/43 ????0/25 ????0/46 ????45/74 ????1/4 | ????5/39(13％) ????36/131(27％) ????6/20(30％) ????12/64(19％) ????10/54(19％) ????2/20 ????14/162(9％) ????1/20 ????3/26 ????5/6 ????40/74(54％) ????1/4 |
| Lung cancer SCLC NSCLC celiothelioma Head and neck squamous cell carcinoma breast cancer carcinoma of urinary bladder colon cancer leukemia-lymphoma kidney | ????7/24 ????1/2 ????5/19 ????0/2 ????1/4 ????0/3 ????5/13 ????3/6 ????0/6 ????0/6 | ????7/24(29％) ????1/2 ????5/19(26％) ????0/4 ????5/13 ????1/6 |
*By RT-PCR, with primer VDE-18 and VDE-24, detect total RNA of all GAGE genes, the expression of test GAGE.If derive from the DNA of MZ2-MEL with those primer analyses, then do not observe the PCR product.
*By PR-PCR, detect total RNA with primer VDE-44 and VDE-24 (it can distinguish out GAGE-1 and 2 from four kinds of other GAGE genes), test the expression of GAGE-1 and 2.If derive from the DNA of MZ2-MEL with those primer analyses, then do not observe the PCR product.
In further work, designed the new primer of all GAGE genes that are used to increase, to guarantee that any one is not at normal tissue expression in them.These primers are
VDE43?5’-GCG?GCC?CGA?GCA?GTT?CA-3’(SEQ?ID?NO：11)
VED24?5’-CCA?TCA?GGA?CCA?TCT?TCA-3’(SEQ?ID?NO：10)
They only are used to use the PCR of VDE44 and VDE24 primer.The result is as shown in table 4.The result confirms that further all the other healthy tissuess are negative except testis.
The expression of table 4GAGE gene in normal adult and fetal tissue
| Adult's tissue | GAGE expresses * |
| Suprarenal gland benign nevus marrow |
| Brain mammary gland cerebellum colon heart kidney liver lung melanocyte muscle ovary prostate skin splenocyte stomach testis thymocyte bladder placenta uterina umbilical cord fetal tissue | ????- ????- ????- ????- ????- ????- ????- ????- ????- ????- ????- ????- ????- ????- ????- ????+ ????- ????- ????- ????- ????- |
| Inoblast brain liver spleen thymus gland testis | ????- ????- ????- ????- ????- ????+ |
*With primer VDE43 and VDE24, by RT-PCR total RNA that increases, detect all GAGE genes (Fig. 7), the expression of test GAGE."-" expression lacks the PCR product, and there is the PCR product in "+" expression.If this primer is used to analyze the DNA that derives from MZ2-MEL, then do not observe the PCR product.
*Fetal tissue derives from the fetus greater than 20 ages in week.
Embodiment 16
In the work that this paper does not report, determined that cytolysis T cell clone CTL 22/23 (Van den Eynde etc., international journal of cancer 44:634-640 (1989) is incorporated herein by reference) can not discern melanoma cells MZ2-MEL.3.1.(European Journal of Immunology, 24:2134-2140 (1994)) such as Van den Bruggen reports that this melanoma cell series lost expression MHC molecule HLA-A29, HLA-B24 and HLA-Cw
*1601 ability.Study to determine whether can make this clone to CTL 22/23 sensitivity with the transfection of one of these MHC molecules.HLA-A29 is first detected molecule.In order to carry out this test, use of the indication of commercially available extraction test kit, from HLA-A29 according to the manufacturer
+Extract poly A among the clone MZ2-MEL.43
+RNA.The application standard method changes into cDNA with mRNA then, carries out fractional separation by size, and non-directional inserts BstXI and the NotI site of plasmid pcDNA-I/Amp then.This plasmid is inserted coli strain DH5 α 5 ' IQ by electroporation, use penbritin (50 μ g/ml) screening then.Bacterium is bed board on the Nitrocellulose filter, repeats twice.The preparation filter in 6 * SSC/0.1%SDS/1 * Denhardt ' s solution, is used
32The probe of P mark, hybridization is spent the night under 40 ℃, and described probe is:
5’-ACTCCATGAGGTATTTC-3’(SEQ?ID?NO：19)
This probe is the sequence around most of HLA ATG code of sequence.
Filter rinsing twice in 6 * SSC at room temperature, each 5 minutes, in 43 ℃ of twice of rinsings in 6 * SSC.Used mark then
32The probe of P: 5 '-TTTCACCACATCCGTGT-3 ' (SEQ ID NO:20) screens positive sequence.Determine that by the sequence and the Protein Data Bank (being incorporated herein by reference) that have immune meaning in the reference Kabat database this sequence has specificity to HLA-A29.This database can be located to obtain by NCBI (USA), is perhaps obtained by Web Solte (Intemet) WWW.NCBI.NLM.NIH.GOV place.This filter at room temperature, in 6 * SSC, rinsing twice, each 5 minutes, then at 42 ℃, at 6 * SSC, rinsing twice, each 5 minutes.
Embodiment 17
After isolating positive HLA-A29 clone, be transfected among the COS-7 with DEAE-dextran chloroquine method (the same).Briefly, the plasmid pcDNA-I/Amp that contains HLA-A29 with 50ng, contain the cDNA of one of above-mentioned GAGE sequence with 100ng, or the cDNA of one of the prior art MAGE in plasmid pcDNA α-I/Amp or pcDSR-α or BAGE sequence handles 1.5 * 10 respectively
4The COS-7 cell.Cultivated transformant 24 hours at 37 ℃ then.
Use the test method that the foregoing description 4 endings place are described then, test the ability that these transformant stimulate TNF to produce through CTL.
The Fig. 7 that shows this result of study shows that any and HLA-A29 that uses among the GAGE-3,4,5 or 6 all produces high-caliber TNF as transformant.Comparatively speaking, GAGE-1 and GAGE-2 do not stimulate ctl clone 22/23, therefore draw such conclusion, i.e. the antigen that is processed to by the HLA-A29 molecular presentation of GAGE 3,4,5 and 6, and discerned by CTL 22/23.
Embodiment 18
GAGE-3,4,5 and 6 peptides that are processed to by the HLA-A29+ presented by cells, and GAGE-1 and GAGE-2 no true suggestion are checked GAGE3,4,5 and 6 total but putative amino acid sequences that GAGE-1 and GAGE-2 lack.Described Sequence Identification is:
Arg?Ser?Thr?Tyr?Tyr?Trp?Pro?Arg?Pro?Arg?Arg?Tyr?Val?Gln(SEQ?ID?NO：21)
Synthetic this peptide, freeze-drying is dissolved in 1 volume DMSO, the 9 volume 10mM acetic acid aqueous solutions then.Other synthetic peptide that this method is used for hereinafter discussing.
According to the method described above, exist
51In the Cr release experiment, detection of peptides (SEQ ID NO:21).
Find that this peptide does not promote cracking.Preparation consecutive miss body, the same application
51The Cr cracking performance is analyzed, and detects them and promotes the cracked ability.This research such as Fig. 8 describe.Find to promote that the small peptide of cracked is Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr (SEQ ID NO:22),
It is that GAGE-3 to 6 is common.Specifically, the 10-18 amino acids of GAGE-3 and GAGE-4,5 and 6 9-17 amino acids are corresponding to this peptide.
As shown in Figure 8, and by for example, the data inconsistent (" the HLA-A29 peptide combines primitive " that member and the Toubert of the peptide families of SEQ ID NO:21 and 22 representatives etc. provides, digest numbers 4183, the 9th international conference of immunology, 23-29 day July nineteen ninety-five, San Francisco, CA is incorporated herein by reference).According to Toubert etc., at least the three of all peptide that are attached on the HLA-A29 must be the Phe residues.And according to shown in the present, true really not so.
Separating of the nucleic acid molecule of the foregoing description code displaying tumor rejection antigen precursor and Tumor rejection antigen.Yet MAGE and the BAGE encoding sequence described in these molecules and any previously disclosed above-mentioned reference do not have homology.Therefore, an aspect of of the present present invention is a kind of isolated nucleic acid molecule, and it comprises any nucleotide sequence and fragment thereof that SEQ ID NO:1-6 describes, for example the Nucleotide 1-170 of SEQ ID NO:1, and 51-170, perhaps be processed to any other fragment of Tumor rejection antigen.SEQ ID NO:1-6 sequence is neither MAGE neither the BAGE encoding sequence, with any this genoid sequence of describing in this sequence and the reference of quoting as proof this point as can be seen of comparing.A part of the present invention also is those nucleic acid molecule, their also encode non-MAGE and non-BAGE tumor rejection antigen precursors, but they are hybridized under stringent condition with the nucleic acid molecule that contains the described nucleotide sequence of SEQ ID NO:1.The term that the present invention uses " stringent condition " refers to the conventional parameter of using in this area.More particularly, the stringent condition that the present invention uses refers in 1M NaCl, 1%SDS and 10% T 500, hybridizes 18 hours down in 65 ℃.Then at room temperature in 2 * SSC to filter rinsing 5 minutes, totally twice, then at 65 ℃, at 2 * SSC, among the 0.1%SDS, rinsing once, 30 minutes.Can also use other condition, reagent etc., it can produce same or higher strict degree.Those skilled in the art are very familiar to these conditions, therefore do not provide here.
From embodiment, can also see, the present invention includes the application of sequence in expression vector, and described sequence is used for conversion or transfection host cell and clone, and they can be prokaryotic cell prokaryocyte (for example colibacillus) or eukaryotic cell (for example, CHO or COS cell).Expression vector needs suitable sequence (being above-described those sequences) to be operably connected to promotor.Owing to have been found that human leucocyte antigen (HLA) HLA-Cw6 and HLA-A29 can present the Tumor rejection antigen that derives from these genes, expression vector also can comprise the nucleic acid molecule of one of coding HLA-Cw6 or HLA-A29.Contain at carrier under the situation of two kinds of encoding sequences, it can be used to transfection can not normal expression the two one of cell.If, for example, host cell expressed HLA-Cw6 and HLA-A29 the two one of or all the time, can use the tumor rejection antigen precursor encoding sequence separately.Certainly, for the concrete host cell that can use without limits.Because if desired, the carrier that contains two kinds of encoding sequences can be used to present the cell of HLA-A29 or HLA-Cw6, the gene of tumor rejection antigen precursor can be used to not express the host cell of HLA-A29 or HLA-Cw6.
The present invention also comprises so-called expression test kit, and it allows the technician to prepare required expression vector.This expression test kit comprises the independent sector of various encoding sequences previously discussed at least.Can add other composition as required, as long as the necessary sequence of mentioning is in the past included.
For the MAGE and the difference of BAGE material of nucleic acid molecule of the present invention and TRAP and description are in the past come, the present invention should be called as GAGE gene family and TRAP.Therefore, as long as use " GAGE " herein, promptly refer to tumor rejection antigen precursor by previously described sequence encoding.Similar terms such as " GAGE coding molecules " is used to describe nucleic acid molecule itself.
The present invention as herein described serves many purposes, and this paper has described the some of them purposes.At first, the present invention makes the technician can diagnostic characteristic be the disease of expressing TRAP or presenting Tumor rejection antigen, for example melanoma.These methods relate to the expression of determining the TRAP gene and/or deriving from their TRA (for example, the TRA that presents by HLA-Cw6 or HLA-A29).Under former instance, can utilize any standard nucleic acid measuring method, comprise the polymerase chain reaction or use the mark hybridization probe, carry out this and determine.Under latter instance, advantageous applications TRA and HLA complex body analyzes in conjunction with counterpart (for example antibody) especially.The TNF that a kind of replacement method that is used to determine is a above-mentioned type discharges detection method.In order to carry out this detection, preferably guarantee not exist testicular cell, because they express GAGE usually.Yet this is unimportant, because people can distinguish testicular cell and other cell type routinely.And it is impossible in practice that testicular cell appears in the non-testis sample.
The separation of TRAP gene also makes the TRAP molecule that separates TRAP molecule self, especially contains any amino acid sequence coded among the SEQ IDNO:2-6 become possibility.If these isolating molecules exist as TRA, or exist as the complex body (for example HLA-Cw6 or HLA-A29) of TRA and HLA, then they can combine with material such as for example adjuvant, are used for the treatment of the vaccine that is characterised in that the disease of expressing the TRAP molecule with preparation.
The example of adjuvant comprises complete Freund's adjuvant and incomplete Freund's adjuvant, dead Bordetella parapertussis thalline, " BCG " (or bacill calmette-guerin), Al (OH)
3, Muramyl dipeptide and derivative thereof, they can emulsification in metabolizable oil, for example squalene, single phosphatidyl lipid A (MPL), keyhole _ hemocyanin (KLH), Saponin/TSM extract for example QA-7, QA-19 and QA-21 (being also referred to as QS-21) (they are at the United States Patent (USP) 5 of Kensil etc., 057, (being incorporated herein by reference) described in 540), MTP-MF59, N-[1-(2,3-two oleoyl oxygen) propyl group]-N, N, N ,-trimethylammonium methylsulfuric acid ammonium (DOTAP), cationically ampholytic DOTMA, neutral phosphatide is DOPE and their combination for example.These that list are never comprehensive, and those skilled in the art can enlarge this tabulation.All other adjuvants are also included among the present invention.
In addition, can present the cell preparation vaccine of TRA/HLA complex body from cell surface, for example non-proliferative cancer cells, non-proliferative transformant etc.With under the situation of cell as vaccine, these cells can be the encoding sequence cells transfected that the necessary composition that CTL replys is provided with one or both, or not transfection and express the cell of these two kinds of molecules.In addition, can adopt TRAP molecule, its relevant TRA and the complex body of TRA and HLA to prepare antibody with standard method known in the art.
" disease " used herein refers to any pathological conditions of expressing tumor rejection antigen precursor.An example of this class disease is a cancer, particularly melanoma.Melanoma is known to be a kind of cancer of aberrant differentiation results in numerous.
As implied above, Tumor rejection antigen, for example the antigen shown in the SEQ ID NO:4 also is a part of the present invention.Polypeptide for example contains 8 to 16 amino acid whose molecules, and wherein this polypeptide contains the aminoacid sequence that SEQID NO:4 describes, and also is a part of the present invention.Pointed as embodiment, the cancer cells that those peptides longer than eight aggressiveness of SEQ ID NO:4 are presented HLA-Cw6 is processed into the Tumor rejection antigen of SEQID NO:4, and is therefore presented.This presents cracking behind the cell that the CTL contact that causes existing in the humoral sample presents this complex body.Equally, than the long peptide of SEQ ID NO:22, for example SEQ ID NO:21 is processed to suitable TRA, and is presented by cancer cell, for example the HLA-A29 positive cell.
Therefore, of the present invention another is characterised in that the peptide of 9-16 amino acid whose random length, comprises sequence:
Xaa Xaa Trp Pro Xaa Xaa Xaa Xaa Tyr (SEQ ID NO:23) or
Xaa Xaa Trp Xaa Arg Xaa Xaa Xaa Tyr (SEQ ID NO:24) or
Xaa?Xaa?Trp?Xaa?Xaa?Xaa?Xaa?Arg?Tyr(SEQ?ID?NO：25)
Wherein Xaa is any amino acid under every kind of situation.
Particularly preferably be such peptide, it follows SEQ ID NO:23,24 and 25 general formula, also satisfy one or more of following standard: the amino acid of N-terminal position is Tyr, second is Tyr, the 4th is Pro, and the 5th is Arg, and the 6th is Pro, the 7th is Arg, and the 8th is Arg.Certainly, the 4th fixing in SEQ ID NO:23, and the 5th also fixing already in SEQ ID NO:24, and the 8th fixing in SEQ ID NO:25.When all these standards all are satisfied, this peptide contains 9 amino acid, then is SEQ ID NO:22.Any or all of external concrete replacement can be incorporated in the peptide of requirement of the present invention, obeys SEQ ID NO:23,24 or 25 primitive and size are 9-16 amino acid.Especially preferred is that length is 9-14 amino acid, comprises SEQ ID NO:23,24 or 25, follows the peptide of above-mentioned preferred replacement.
A part of the present invention also is so-called " minigene ", it be among the coding SEQ ID NO:21,22,23,24 or 25 any, and comprise especially preferred embodiment SEQ ID NO:23,24 or 25 whole isolated nucleic acid molecule.Only there is a limited number of nucleic acid molecule to encode, SEQ ID NO:21 or 22 for example, they can infer out like a dream according to known codon degeneracy principle.The application of these minigenes code book invention peptide in standard body and in the in vitro method is also included within the scope of the invention.These peptides are in conjunction with the HLA-A29 molecule, and/or are processed to the peptide in conjunction with the HLA-A29 molecule.The fact that these peptides are processed to Tumor rejection antigen as shown in the Examples.
In other parameter area of determining pathological condition (for example cancer, particularly melanoma) diagnosis, can develop this character.For example, the investigator can study the antigen that is distributed in blood or the urine, observes physiological change, the CTL method of proliferating diagnosis melanoma of utilizing the present invention to describe then.
A part of the present invention also is the complex body of HLA-A29 molecule and one of peptide of listing above, preferably is soluble form.This solvable complex body can be by adding sample with it, and the reaction of mensuration and CTL then is used for determining the existence of sample (for example humoral sample) CTL.For example, Bousso etc. (immunology collection of thesis 59 (2): 85-91 (1997) is incorporated herein by reference) point out to use the solubility complex body elutriation CTL of MHC molecule and peptide.Complex body preferably is fixed, to impel the CTL enrichment.Sakita etc. (immunological method magazine (J.Immunol.Meth), 192:105-115 (1996) is incorporated herein by reference) also merit attention, and it illustrates this complex body can be used for body internal stimulus CTL.This is another feature of the present invention.In a particularly preferred embodiment, the solubility complex body of pointing out above is a polymer, most preferably the tetramer.Altman etc. (science, 274:94-96 (1996) is incorporated herein by reference) have described how to prepare this structure.
Peptide of the present invention itself can be used to carry out the HLA-phenotypic analysis.As everyone knows, carry out dermatoplasty, organ transplantation etc. if desired, people must carry out the HLA phenotypic analysis, so that the minimizing possibility of transplant rejection.Peptide of the present invention can be used for determining whether individuality belongs to the HLA-Cw6 or the HLA-A29 positive, so that select suitable donor.The type analysis operation is easy.With peptide contact target sample of the present invention, whether the indication sample source individuality that combines with cell in this sample belongs to the HLA-Cw6 or the HLA-A29 positive.People can mark peptide itself, and its binding substances perhaps combines them with the joint that is labeled, and it is first-class that they are fixed to solid phase, so that optimize this analysis.Other standard method is clearly for those skilled in the art, and this paper is not giving unnecessary details.
Prerequisite according to the treatment plan of present disclosure is to cause TRA to be delivery cell (for example HLA-A29 or HLA-Cw6 cell) cracked experimenter immunity system to reply.One of this scheme is the CTL that the paracytic experimenter's administration that has the dispute phenotype is specific to described complex body.This CTL external is formed in technician's the technical scope.Specifically, with cell sample (as hemocyte) with present this complex body and can impel the specific CTL proliferating cells to contact.Target cell can be the transfection body, for example the COS cell of the above-mentioned type.These transfection bodies present required complex body to its surface, when when required CTL combines, stimulate its propagation.The COS cell, those that use among the present invention for example, it is very wide to originate, as other host cell that is suitable for.
Be called as the transfer of adopting property (Greenberg, Journal of Immunology 136 (5): 1917 (1986) in order to describe in detail; Riddel etc., science 257:238 (7-10-92); Lynch etc., European Journal of Immunology 21:1403-1410 (1991); Kast etc., cell 59:603-614 (11-17-89)) methods of treatment makes the cell of presenting required complex body combine with CTL, causes this cell is had specific CTL propagation.The CTL that will breed delivers medicine to the experimenter of cellular abnormality then, and described cellular abnormality is characterised in that some presents the abnormal cells of specific complex body, and its mesocomplex contains relevant HLA molecule.CTL cracking abnormal cells is realized required therapeutic goal thus then.
Some abnormal cells at least of above-mentioned treatment supposition experimenter is presented relevant HLA/TRA complex body.This point is easy to determined, because those skilled in the art present a kind of cell of specific HLA molecule for evaluation and how to go to identify that the method for cell (being the GAGE sequence among the present invention) of the RNA that expresses this correlated series is very familiar.In case by above-mentioned screening method identify present the cell of this related complexes after, can be with they and patient's the sample mix that contains CTL.If the cell of presenting this complex body can suppose that so the Tumor rejection antigen of a kind of GAGE of deriving from is presented, and this experimenter is the suitable candidate of above-mentioned treatment plan by mixed CTL sample dissociation.
Adoptive transfer is not unique form of treatment provided by the invention.Can also use in the certain methods body and bring out CTL.The front had been described a method in detail, promptly used non-proliferative cell to express this complex body.The cell that uses in this method can be the cell of those these complex bodys of normal expression, the melanoma cells that for example shone or be used to present the cell of the necessary gene transfection of this complex body with one or both.Chen etc. (the scientific advance 88:110-114 of NAS (in January, 1991)) have exemplified this method, show to have used the transfectional cell of expressing HPV E7 peptide in the treatment plan.Can use various cell types.Equally, can use the carrier that carries one or both target genes.Especially preferred virus or bacteria carrier.In these systems, target gene passes through, for example vaccinia virus or bacterium BCG delivery, and this class material is " infection " host cell in fact.The presented by cells target complex body that produces, and discerned, this CTL propagation then by self CTL.By Tumor rejection antigen or its precursor being combined with adjuvant to promote it to mix to present the cell of HLA-Cw6, this presented by cells target HLA/ peptide complex body is realized similar effect then.The processed peptide counterpart of TRAP with production HLA molecule, and TRA does not need further processing to be presented.
Others of the present invention are conspicuous for ability and technician, and this paper does not need repetition.
Term and the phraseology used are used as descriptive language, rather than restriction, using these terms and phraseology is not to attempt to get rid of any equivalent or its part that is expressed and describes feature, will be appreciated that it is possible carrying out various changes within the scope of the present invention.
Sequence table (1) general information
(ⅰ) applicant: Van der Bruggen, Pierre; Van den Eynde, Benoit; DeBacker, Olivier; Boon-Falleur, Thierry
(ⅱ) invention exercise question: be attached to the isolated polypeptide on the HLA-A29 molecule, their the molecule-nucleic acid of encoding, and use
(ⅲ) sequence number: 25
(ⅳ) address:
(A) address: Fulbright ﹠amp; Jaworski L.L.P.
(B) street: Fifth Avenue 666
(C) city: New York
(D) state: New York
(E) country: the U.S.
(F) postcode: 10103-3198
(ⅴ) computer-reader form:
(A) medium type: floppy disk, 3.5 inches, 1.44kb internal memory
(B) computer: IBM PS/2
(C) operating system: PC
(D) software: Wordperfect
(ⅵ) current request for data:
(A) application number:
(B) applying date:
(C) classification number: 435
(ⅶ) priority application data:
(A) application number: 09/012,818
(B) applying date: on January 23rd, 1998
(C) classification number: 435
(ⅶ) priority application data:
(A) application number: 08/531,662
(B) applying date: September 21 nineteen ninety-five
(ⅶ) priority application data:
(A) application number: 08/370,648
(B) applying date: January 10 nineteen ninety-five
(ⅶ) priority application data:
(A) application number: 08/250,162
(B) applying date: on May 27th, 1994
(ⅶ) priority application data:
(A) application number: 08/096,039
(B) applying date: on July 22nd, 1993
(ⅷ) lawyer/proxy's information:
(A) name: Hanson, Norman D.
(B) number of registration: 30,946
(C) reference/case number: LUD 5531 PCT
(ⅸ) communication information:
(A) phone: (212) 318-3168
(B) fax: (212) 752-5958
(2) information of SEQ ID NO:1:
(ⅰ) sequence signature:
(A) length: 646 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
( ⅹⅰ ) ：SEQ ID NO：1CTGCCGTCCG GACTCTTTTT CCTCTACTGA GATTCATCTG TGTGAAATAT 50GAGTTGGCGA GGAAGATCGA CCTATCGGCC TAGACCAAGA CGCTACGTAG 100AGCCTCCTGA AATGATTGGG CCTATGCGGC CCGAGCAGTT CAGTGATGAA 150GTGGAACCAG CAACACCTGA AGAAGGGGAA CCAGCAACTC AACGTCAGGA 200TCCTGCAGCT GCTCAGGAGG GAGAGGATGA GGGAGCATCT GCAGGTCAAG 250GGCCGAAGCC TGAAGCTGAT AGCCAGGAAC AGGGTCACCC ACAGACTGGG 300TGTGAGTGTG AAGATGGTCC TGATGGGCAG GAGATGGACC CGCCAAATCC 350AGAGGAGGTG AAAACGCCTG AAGAAGAGAT GAGGTCTCAC TATGTTGCCC 400AGACTGGGAT TCTCTGGCTT TTAATGAACA ATTGCTTCTT AAATCTTTCC 450CCACGGAAAC CTTGAGTGAC TGAAATATCA AATGGCGAGA GACCGTTTAG 500TTCCTATCAT CTGTGGCATG TGAAGGGCAA TCACAGTGTT AAAAGAAGAC 550ATGCTGAAAT GTTGCAGGCT GCTCCTATGT TGGAAAATTC TTCATTGAAG 600TTCTCCCAAT AAAGCTTTAC AGCCTTCTGC AAAGAAAAAA AAAAAA 646 ( 2 ) SEQ ID NO：2：
(ⅰ) sequence signature:
(A) length: 18 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:2AGACGCTACG TAGAGCCT 18 (2) SEQ ID NO:3:
(ⅰ) sequence signature:
(A) length: 18 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:3CCATCAGGAC CATCTTCA 18 (2) SEQ ID NO:4:
(ⅰ) sequence signature:
(A) length: 8 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:4Tyr Arg Pro Arg Pro Arg Arg Tyr 5 (2) SEQ ID NO:5
(ⅰ) sequence signature:
(A) length: 9 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:5Thr Tyr Arg Pro Arg Pro Arg Arg Tyr 5 (2) SEQ ID NO:6
(ⅰ) sequence signature:
(A) length: 9 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:6Tyr Arg Pro Arg Pro Arg Arg Tyr Val 5 (2) SEQ ID NO:7
(ⅰ) sequence signature:
(A) length: 10 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:7Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val 5 10 (2) SEQ ID NO:8
(ⅰ) sequence signature:
(A) length: 9 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:8Arg Pro Arg Pro Arg Arg Tyr Val Glu 5 (2) SEQ ID NO:9
(ⅰ) sequence signature:
(A) length: 18 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:9GACCAAGACG CTACGTAG 18 (2) SEQ ID NO:10
(ⅰ) sequence signature:
(A) length: 18 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:10CCATCAGGAC CATCTTCA 18 (2) SEQ ID NO:11
(ⅰ) sequence signature:
(A) length: 17 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:11GCGGCCCGAG CAGTTCA 17 (2) SEQ ID NO:12
(ⅰ) sequence signature:
(A) length: 15 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:12Met Ser Trp Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg 5 10 15 (2) SEQ ID NO:13
(ⅰ) sequence signature:
(A) length: 16 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:13Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val Glu Pro Pro Glu Met Ile 5 10 15 (2) SEQ ID NO:14
(ⅰ) sequence signature:
(A) length: 538 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
( ⅹⅰ ) ：SEQ ID NO：14ACGCCAGGGA GCTGTGAGGC AGTGCTGTGT GGTTCCTGCC GTCCGGACTC 50TTTTTCCTCT ACTGAGATTC ATCTGTGTGA AATATGAGTT GGCGAGGAAG 100ATCGACCTAT CGGCCTAGAC CAAGACGCTA CGTAGAGCCT CCTGAAATGA 150TTGGGCCTAT GCGGCCCGAG CAGTTCAGTG ATGAAGTGGA ACCAGCAACA 200CCTGAAGAAG GGGAACCAGC AACTCAACGT CAGGATCCTG CAGCTGCTCA 250GGAGGGAGAG GATGAGGGAG CATCTGCAGG TCAAGGGCCG AAGCCTGAAG 300CTCATAGCCA GGAACAGGGT CACCCACAGA CTGGGTGTGA GTGTGAAGAT 350GGTCCTGATG GGCAGGAGAT GGACCCGCCA AATCCAGAGG AGGTGAAAAC 400GCCTGAAGAA GGTGAAAAGC AATCACAGTG TTAAAAGAAG ACACGTTGAA 450ATGATGCAGG CTGCTCCTAT GTTGGAAATT TGTTCATTAA AATTCTCCCA 500ATAAAGCTTT ACAGCCTTCT GCAAAGAAAA AAAAAAAA 538 ( 2 ) SEQ ID NO：15
(ⅰ) sequence signature:
(A) length: 560 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
( ⅹⅰ ) ：SEQ ID NO：15CTCATATTTC ACACAGATGA GTTGGCGAGG AAGATCGACC TATTATTGGT 50CTAGGCCAAT AATAGGTCGA TCTTCCTCGC CAACTCATAT TTCACACAGA 100TGAATCTCAG TAGAGGAAAA TCGACCTATT ATTGGCCTAG ACCAAGGCGC 150TATGTACAGC CTCCTGAAGT GATTGGGCCT ATGCGGCCCG AGCAGTTCAG 200TGATGAAGTG GAACCAGCAA CACCTGAAGA AGGGGAACCA GCAACTCAAC 250GTCAGGATCC TGCAGCTGCT CAGGAGGGAG AGGATGAGGG AGCATCTGCA 300GGTCAAGGGC CGAAGCCTGA AGCTGATAGC CAGGAACAGG GTCACCCACA 350GACTGGGTGT GAGTGTGAAG ATGGTCCTGA TGGGCAGGAG ATGGACCCGC 400CAAATCCAGA GGAGGTGAAA ACGCCTGAAG AAGGTGAAAA GCAATCACAG 450TGTTAAAAGA AGGCACGTTG AAATGATGCA GGCTGCTCCT ATGTTGGAAA 500TTTGTTCATT AAAATTCTCC CAATAAAGCT TTACAGCCTT CTGCAAAGAA 550AAAAAAAAAA 560 ( 2 ) SEQ ID NO：16
(ⅰ) sequence signature:
(A) length: 540 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
( ⅹⅰ ) ：SEQ ID NO：16CGCCAGGGAG CTGTGAGGCA GTGCTGTGTG GTTCCTGCCG TCCGGACTCT 50TTTTCCTCTA CTGAGATTCA TCTGTGTGAA ATATGAGTTG GCGAGGAAGA 100TCGACCTATT ATTGGCCTAG ACCAAGGCGC TATGTACAGC CTCCTGAAAT 150GATTGGGCCT ATGCGGCCCG AGCAGTTCAG TGATGAAGTG GAACCAGCAA 200CACCTGAAGA AGGGGAACCA GCAACTCAAC GTCAGGATCC TGCAGCTGCT 250CAGGAGGGAG AGGATGAGGG AGCATCTGCA GGTCAAGGGC CGAAGCCTGA 300AGCTGATAGC CAGGAACAGG GTCACCCACA GACTGGGTGT GAGTGTGAAG 350ATGGTCCTGA TGGGCAGGAG ATGGACCCGC CAAATCCAGA GGAGGTGAAA 400ACGCCTGAAG AAGGTGAAAA GCAATCACAG TGTTAAAAGA AGGCACGTTG 450AAATGATGCA GGCTGCTCCT ATGTTGGAAA TTTGTTCATT AAAATTCTCC 500CAATAAAGCT TTACAGCCTT CTGCAAAAAA AAAAAAAAAA 540 ( 2 ) SEQ ID NO：17
(ⅰ) sequence signature:
(A) length: 532 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
( ⅹⅰ ) ：SEQ ID NO：17AGCTGTGAGG CAGTGCTGTG TGGTTCCTGC CGTCCGGACT CTTTTTCCTC 50TACTGAGATT CATCTGTGTG AAATATGAGT TGGCGAGGAA GATCGACCTA 100TTATTGGCCT AGACCAAGGC GCTATGTACA GCCTCCTGAA GTGATTGGGC 150CTATGCGGCC CGAGCAGTTC AGTGATGAAG TGGAACCAGC AACACCTGAA 200GAAGGGGAAC CAGCAACTCA ACGTCAGGAT CCTGCAGCTG CTCAGGAGGG 250AGAGGATGAG GGAGCATCTG CAGGTCAAGG GCCGAAGCCT GAAGCTGATA 300GCCAGGAACA GGGTCACCCA CAGACTGGGT GTGAGTGTGA AGATGGTCCT 350GATGGGCAGG AGATGGACCC GCCAAATCCA GAGGAGGTGA AAACGCCTGA 400AGAAGGTGAA AAGCAATCAC AGTGTTAAAA GAAGGCACGT TGAAATGATG 450CAGGCTGCTC CTATGTTGGA AATTTGTTCA TTAAAATTCT CCCAATAAAG 500CTTTACAGCC TTCTGCAAAG AAAAAAAAAA AA 532 ( 2 ) SEQ ID NO：18
(ⅰ) sequence signature:
(A) length: 539 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
( ⅹⅰ ) ：SEQ ID NO：18GCCAGGGAGC TGTGAGGCAG TGCTGTGTGG TTCCTGCCGT CCGGACTCTT 50TTTCCTCTAC TGAGATTCAT CTGTGTGAAA TATGAGTTGG CGAGGAAGAT 100CGACCTATTA TTGGCCTAGA CCAAGGCGCT ATGTACAGCC TCCTGAAGTG 150ATTGGGCCTA TGCGGCCCGA GCAGTTCAGT GATGAAGTGG AACCAGCAAC 200ACCTGAAGAA GGGGAACCAG CAACTCAACG TCAGGATCCT GCAGCTGCTC 250AGGAGGGAGA GGATGAGGGA GCATCTGCAG GTCAAGGGCC GAAGCCTGAA 300GCTGATAGCC AGGAACAGGG TCACCCACAG ACTGGGTGTG AGTGTGAAGA 350TGGTCCTGAT GGGCAGGAGG TGGACCCGCC AAATCCAGAG GAGGTGAAAA 400CGCCTGAAGA AGGTGAAAAG CAATCACAGT GTTAAAAGAA GACACGTTGA 450AATGATGCAG GCTGCTCCTA TGTTGGAAAT TTGTTCATTA AAATTCTCCC 500AATAAAGCTT TACAGCCTTC TGCAAAAAAA AAAAAAAAA 539 ( 2 ) SEQ ID NO：19
(ⅰ) sequence signature:
(A) length: 17 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:19ACTCCATGAG GTATTTC 17 (2) SEQ ID NO:20
(ⅰ) sequence signature:
(A) length: 17 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:20TTTCACCACA TGCGTGT 17 (2) SEQ ID NO:21
(ⅰ) sequence signature:
(A) length: 14 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:21Arg Ser Thr Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr Val Gln 5 10 (2) SEQ ID NO:22
(ⅰ) sequence signature:
(A) length: 9 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:22Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr 5 (2) SEQ ID NO:23
(ⅰ) sequence signature:
(A) length: 9 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅸ) feature:
(D) out of Memory: every kind of Xaa can be any amino acid
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:23Xaa Xaa Trp Pro Xaa Xaa Xaa Xaa Tyr 5 (2) SEQ ID NO:24
(ⅰ) sequence signature:
(A) length: 9 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅸ) feature:
(D) out of Memory: every kind of Xaa can be any amino acid
(ⅹ ⅰ) sequence description: the information of SEQ ID NO:24Xaa Xaa Trp Xaa Arg Xaa Xaa Xaa Tyr 5 (2) SEQ ID NO:25
(ⅰ) sequence signature:
(A) length: 9 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ⅸ) feature:
(D) out of Memory: every kind of Xaa can be any amino acid
(ⅹ ⅰ) sequence description: SEQ ID NO:25Xaa Xaa Trp Xaa Xaa Xaa Xaa Arg Tyr 5
Claims (22)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US1281898A | 1998-01-23 | 1998-01-23 | |
| US09/012,818 | 1998-01-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1291992A true CN1291992A (en) | 2001-04-18 |
Family
ID=21756847
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99803245A Pending CN1291992A (en) | 1998-01-23 | 1999-01-12 | Isolated, polypeptides which bind to HLA-A29 molecules, nucleic acid, molecules encoding there, and uses thereof |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1047707A1 (en) |
| JP (1) | JP2002509859A (en) |
| KR (1) | KR20010024877A (en) |
| CN (1) | CN1291992A (en) |
| AU (1) | AU2318999A (en) |
| CA (1) | CA2317492A1 (en) |
| WO (1) | WO1999037665A1 (en) |
| ZA (1) | ZA99445B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001053833A1 (en) * | 2000-01-20 | 2001-07-26 | Ludwig Institute For Cancer Research | Mage antigenic peptides which bind hla-b35 and hla-b44 |
| EP1255833A2 (en) * | 2000-02-15 | 2002-11-13 | Curagen Corporation | Polypeptides and nucleic acids encoding same |
| EP1605045A3 (en) * | 2000-02-15 | 2006-03-01 | Curagen Corporation | Polypeptides and nucleic acids encoding same |
| US6506875B1 (en) * | 2000-09-26 | 2003-01-14 | Ludwig Institute For Cancer Research | Isolated peptides which bind to HLA-C molecules and uses thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5858689A (en) * | 1993-07-22 | 1999-01-12 | Ludwig Institute For Cancer Research | Isolated peptides derived from the gage tumor rejection antigen precursor and uses thereof |
| US5877155A (en) * | 1995-03-17 | 1999-03-02 | The Research Foundation Of State University Of New York | Mimotopes and anti-mimotopes of human platelet glycoprotein Ib/IX |
-
1999
- 1999-01-12 EP EP99903082A patent/EP1047707A1/en not_active Withdrawn
- 1999-01-12 CN CN99803245A patent/CN1291992A/en active Pending
- 1999-01-12 JP JP2000528586A patent/JP2002509859A/en active Pending
- 1999-01-12 CA CA002317492A patent/CA2317492A1/en not_active Abandoned
- 1999-01-12 AU AU23189/99A patent/AU2318999A/en not_active Abandoned
- 1999-01-12 KR KR1020007008054A patent/KR20010024877A/en not_active Application Discontinuation
- 1999-01-12 WO PCT/US1999/000775 patent/WO1999037665A1/en not_active Application Discontinuation
- 1999-01-21 ZA ZA9900445A patent/ZA99445B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| KR20010024877A (en) | 2001-03-26 |
| ZA99445B (en) | 1999-07-21 |
| WO1999037665A1 (en) | 1999-07-29 |
| EP1047707A1 (en) | 2000-11-02 |
| JP2002509859A (en) | 2002-04-02 |
| CA2317492A1 (en) | 1999-07-29 |
| AU2318999A (en) | 1999-08-09 |
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