CN1179180A - RAGE tumer rejection antigens - Google Patents

Abstract

The present invention describes the RAGE tumor rejection antigen precursor family, including nucleic acids encoding such tumor rejection antigen precursors, tumor rejection antigen peptides or precursors thereof and antibodies relating thereto. Methods and products also are provided for diagnosing and treating conditions characterized by expression of a RAGE tumor rejection antigen precursor.

Description

The RAGE Tumor rejection antigen

The technical field of the invention

The present invention relates to Tumor rejection antigen and its precursor are carried out nucleic acid molecules encoding.Particularly be machined into the tumor rejection antigen precursor at least a Tumor rejection antigen that is presented by the HLA molecule.This nucleic acid molecule, by the protein of this nucleic acid molecule encoding, the peptide of therefrom deriving out, relevant antibody and cytotoxic lymphocyte, be particularly useful in the diagnosis and the treatment aspect.

Technical background of the present invention

Immune system is very complicated to the identification and the reaction process of allogenic material and foreign matter.In this system, the T cell is a very important part.The T cell can discern other cell and with its reaction, this is that cell surface mixture or the main compatible mixture (" MHC ") of histology formed by molecule that is known as human leucocyte antigen (HLA) (" HLA ") on other cell and peptide are realized.Described peptide is to derive out from the macromole of being processed by cell and present the HLA/MHC molecule.Referring to people's such as Male Senior immunology[Male et al., Advanced Immunology(J.P.LipincottCompany, 1987)], 6-10 chapter particularly.Reaction between T cell and the HLA/ peptide complex is restrictive, and specific T cell needs the specific HLA molecule and the mixture of peptide.If lack specific T cell, even there is its counterpart to exist, t cell responses can not take place yet.Equally, if lack specific mixture, even there is the T cell to exist, t cell responses can not take place yet.This mechanism relates to the reaction of immunity system to allogenic material, autoimmunity pathology, and the unusual reaction of pair cell.

The mechanism of T cell recognition allogenic material is also involved in cancer.Described have some directly opposing from CTL (CTL) clone of autologous melanoma.In some cases, by the antigen of these clone identifications by characterization.In on November 26th, 1992 disclosed PCT patent application PCT/US92/04354, having disclosed tumour is specific one group of gene, i.e. " MAGE " group.These expression of gene products are processed into peptide, and then, these peptides are expressed on cell surface again.The molten born of the same parents of this tumour cell that can cause specific CTL to cause.These genes are considered to coding " tumor rejection antigen precursor " or " TRAP " molecule, and therefrom deutero-protein is called as " Tumor rejection antigen " or " TRA ".Referring to people's such as Traversari article, " immunogenetics " [Traversari et al., Immunogenetics 35:145 (1992)]; People's such as van derBruggen article, " science " [van der Bruggen et al., Science 254:1643 (1991)] can obtain further the information about this group gene.Can also be referring to United States Patent (USP) the 5th, 342, No. 774.

At United States Patent (USP) the 5th, 405, in No. 940, disclosed MAGE nonapeptide (nonapeptide), they are presented by the HLA-A1 molecule.If know specificity to a certain peptide of a certain HLA molecule, so, people just can know a certain peptide can with a certain HLA molecular reaction, and different other molecular reaction.This point is extremely important, because different individualities has different HLA phenotypes.The result is, though a certain peptide is had diagnostics and therapeutic application as the evaluation of the object of a certain specific HLA molecule,, they are just relevant with those individualities with this kind HLA phenotype.Therefore, in this field, exist the needs of further investigation because cell be not restricted to certain specific HLA phenotype unusually, and the goal treatment method also needs the understanding to paracytic phenotype.

Be found that also the MAGE expression product is processed into second kind of TRA again.This second kind of TRA clones 10 molecules by HLA-C and presents.Therefore, a certain given TRAP can obtain multiple TRA.

In disclosed PCT WO94/14459 on the 7th patent application July in 1994, tyrosine oxidase has been described to tumor rejection antigen precursor.This piece document illustration the molecule that produced of some normal cell (for example melanophore) Tumor rejection antigen that is machined in the tumour cell and obtains being presented by the HLA-A2 molecule.

In disclosed PCT WO94/21126 on the 29th patent application September in 1997, pointed out that a kind of is not to be presented by HLA-A2 by the 2nd tyrosinase derived TRA.This TRA derives from TRAP, but by non-MAGE genes encoding.This piece document shows that a certain HLA molecule can present the TRA that derives out from the difference source.

In disclosed PCT WO95/00159 on the 5th patent application in January nineteen ninety-five, the uncorrelated tumor rejection antigen precursor of a kind of being known as " BAGE " precursor has been described.Also described by TRAP deutero-TRA.They and MHC molecule HLA-C-clone 10 form mixture.

In disclosed PCT WO95/03422 on the 2nd patent application in February nineteen ninety-five, the irrelevant tumor rejection antigen precursor that another is known as " GAGE " precursor has been described.This GAGE precursor and BAGE or MAGE group all have nothing to do.

Various documents, the work of being introduced in patent and the patent application mostly is at the MAGE genome, BAGE gene and GAGE gene.These genes are expressed in some tumours, and still, they but are quiet in other healthy tissues except testis.They are not expressed in kidney.

Found another genome now, i.e. " RAGE " genome, Tumor rejection antigen and its precursor that their codings are other.This RAGE genome does not demonstrate and MAGE genome, BAGE genome and the genomic homology of GAGE.The RAGE genome obtains expressing in kidney cancer cell, but does not express in normal nephrocyte.This RAGE genome is also expressed in some other tumour cell.

Below give further description to the present invention.

General introduction of the present invention

The present invention relates to isolated nucleic acid molecule, contain the expression vector of these molecules and by the host cell of these molecule transfections.The present invention also provides isolating protein and peptide, and the antibody of these protein and peptide.In addition, also provide the test kit that contains above-mentioned molecule.Above-described material may be used to be characterized as the various diagnosis and the treatment of the disease that RAGE TRA and RAGE TRAP express.

According to an aspect of the present invention, provide separated polypeptide.Which comprises at least the aminoacid sequence that sequence numbering is 40 (SEQ.ID.NO.40), and it is RAGETRA.In preferred embodiments, separated polypeptide comprises SEQ.ID.NO.43 aminoacid sequence at least.In other embodiments, separated peptide can be made up of the aminoacid sequence of SEQ.ID.NO.40 or SEQ.ID.NO.43 basically or fully.

According to a further aspect in the invention, provide separated nucleic acid molecule.This molecule encoding comprises the polypeptide in a group that is made up of SEQ.ID.NO.40 and SEQ.ID.NO.43.This separated nucleic acid can comprise SEQ.ID.NO.44, and preferably includes SEQ.ID.NO.45.In other embodiment, separated nucleic acid is made up of SEQ.ID.NO.44 or SEQ.ID.NO.45 basically or fully.

According to a further aspect in the invention, separated nucleic acid molecule is provided, this nucleic acid molecule is hybridized under stringent condition with the molecule that contains nucleic acid sequence SEQ .ID.NO.1, SEQ.ID.NO.4, SEQ.ID.NO.6, SEQ.ID.NO.10, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15, SEQ.ID.NO.17, SEQ.ID.NO.23 and/or SEQ.ID.NO.35 of coding RAGE TRA or TRAP, and condition is do not encode TRA or the TRAP of MAGE, GAGE or BAGE of separated nucleic acid molecule.In preferred embodiments, separated nucleic acid molecule is mRAN molecule or cDNA molecule.In a kind of embodiment therein, separated nucleic acid molecule is that each Nucleotide of 269-832 of 185-247, SEQ.ID.NO.14 of 217-276, SEQ.ID.NO.13 of 273-449, SEQ.ID.NO.12 of 444-665, SEQ.ID.NO.12 of 313-399, the SEQ.ID.NO.1 of 204-326, SEQ.ID.NO.1 to SEQ.ID.NO.1 is complementary.In another embodiment, separated nucleic acid molecule is made up of nucleic acid sequence SEQ .ID.NO.1, SEQ.ID.NO.4, SEQ.ID.NO.6, SEQ.ID.NO.10, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15, SEQ.ID.NO.17, SEQ.ID.NO.23, SEQ.ID.NO.35, SEQ.ID.NO.44 and/or SEQ.ID.NO.45 basically.

According to a further aspect in the invention, expression vector and the host cell that comprises this expression vector are provided.This expression vector comprises any or multiple of above-mentioned separated nucleic acid molecule.In one embodiment, expression vector comprises separated nucleic acid molecule SEQ.ID.NO.44 or SEQ.ID.NO.45.Other expression vector according to the present invention comprises above-mentioned separated nucleic acid molecule and encodes and TRA of the present invention can be presented to the nucleic acid molecule of the HLA molecule of T cell.Its example is HLA-B7.Described host cell can endogenous expression this HLA molecule, for example HLA-B7.

The separated nucleic acid molecule of the characteristic fragment of SEQ.ID.NO.1, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14 or their complement is provided according to a further aspect in the invention.These characteristic fragments are used to identify or the above-described nucleic acid of selective amplification.When these characteristic fragments were used to identify the expression of above-mentioned nucleic acid molecule, then the length of this characteristic fragment was preferably between 200-1310 the Nucleotide, between the 200-1234 Nucleotide, between the 200-2050 Nucleotide or between the 200-1167 Nucleotide.When these characteristic fragments were used to increase said nucleic acid molecule, then the length of this characteristic fragment was between the 12-32 Nucleotide.It should be understood that amplification program is not unique program that can be used for identifying nucleic acid molecule.

According to a further aspect in the invention, provide whether having TRA or TRAP to express the test kit that detects.This test kit comprises the above-mentioned nucleic acid molecule of two or more, is contained in respectively in the mutual isolated container, and is in the packing.In a kind of test kit therein, a pair of amplimer is provided, the length that each primer wherein consists essentially of SEQ.ID.NO.1 be the length in abutting connection with fragment or its complement, SEQ.ID.NO.12 of 12-32 Nucleotide be the length in abutting connection with fragment or its complement, SEQ.ID.NO.13 of 12-32 Nucleotide be the length in abutting connection with fragment or its complement, SEQ.ID.NO.14 of 12-32 Nucleotide be 12-32 Nucleotide in abutting connection with fragment or its complement, and wherein not overlapping in abutting connection with fragment.Preferred amplimer is the PCR primer, one of them primer be the Watson chain in abutting connection with fragment, another primer is the segmental complement of the adjacency of Crick chain.In some embodiments, for selective amplification and/or identify in the RAGE group one, for example the part of RAGE1 or RAGE1 etc. has been carried out corresponding structure and arrangement to primer.For example, a pair of primer can be in the RAGE1 gene in abutting connection with fragment and its equipotential modification, but can not be in RAGE2,3 and 4 genes in abutting connection with fragment.Example is more specifically, first primer is to contain any one nucleic acid among the SEQ.ID.NO.50-57, and second primer can be the length of SEQ.ID.NO.1 basically be 12-32 Nucleotide in abutting connection with fragment or its complement, specifically to decide according to selected first primer.

Another kind of test kit according to the present invention is to express test kit, and this test kit comprises coding RAGE TRAP placed apart or comprises the separated nucleic acid molecule of RAGE TRA molecule and stimulate the HLA of the mixture of t cell responses to present molecule with TRA formation.A kind of nucleic acid that comprises the peptide of coding SEQ.ID.NO.40 or SEQ.ID.NO.43 in this test kit, and the nucleic acid molecule of coding HLA-B7.Another kind of test kit according to the present invention is the expression test kit that is placed with separated nucleic acid molecule respectively, this separated nucleic acid molecule under stringent condition with the molecular hybridization of forming by the nucleotide sequence of SEQ.ID.NO.1, SEQ.ID.NO.4, SEQ.ID.NO.6, SEQ.ID.NO.10, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15, SEQ.ID.NO.17, SEQ.ID.NO.23 and/or the SEQ.ID.NO.35 of coding RAGE TRAP, and the nucleic acid molecule of the HLA-B7 that encodes.

The separated multiple TRAP of above-mentioned molecule and its useful fragment coding is provided according to a further aspect in the invention.Also provide to these molecules and to the antibody of the mixture of HLA and multiple RAGE TRA.

According to a further aspect in the invention, provide be expressed as the diagnostic method of the disorder of feature with RAGE TRAP.A kind of method wherein relates to RAGE TRAP, and it is processed into RAGE deutero-TRA, and can with the HLA molecule forming composite.This method comprises the biological sample that will obtain and can contact with mixture bonded reagent, combines to determine disorder by what determine mixture and reagent then.In one embodiment, this method is determined combining of reagent and RAGE TRA and HLA-B7 mixture.In this embodiment, RAGE TRA can be selected from the peptide of SEQ.ID.NO.40 and SEQ.ID.NO.43.Another kind method relates to the biological sample that will obtain and is that specific reagent contacts to RAGE nucleic acid molecule or its expression product.Determine the interaction of reagent and nucleic acid or its expression product then, this interaction is disorderly index.This reagent can be under stringent condition with one group of nucleotide sequence forming by the nucleotide sequence of SEQ.ID.NO.1, SEQ.ID.NO.4, SEQ.ID.NO.6, SEQ.ID.NO.10, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15, SEQ.ID.NO.17, SEQ.ID.NO.23 and SEQ.ID.NO.35 of coding TRAP in selected making nucleic acid molecular hybridization, condition is the do not encode TRAP of MAGE, GAGE and BAGE of separated nucleic acid molecule.Another kind method relate to biological sample be that specific reagent contacts to the RAGE tumor rejection antigenic peptide, determine that then disorder is determined in the interaction between peptide and the reagent.In one embodiment, peptide is selected among one group that comprises SEQ.ID.NO.40 and SEQ.ID.NO.43.

According to a further aspect in the invention, provide separated biology preparation.Said preparation comprises that the mixture to HLA molecule and RAGE TRA is specific molten born of the same parents' property T cell.In the embodiment therein, molten born of the same parents' property T cell is that the mixture to HLA-B7 molecule and TRA is specific.In this embodiment, antigen can be the peptide that is selected from a group that is made up of the peptide of the peptide of SEQ.ID.NO.40 and SEQ.ID.NO.43.

According to another aspect of the present invention, provide that to include HLA molecule and RAGETRA mixture be the method that the T cell population quantity of specific molten born of the same parents' property T cell is carried out selective enrichment.This method comprises the isolating a group T cell that contains molten born of the same parents' property T cell is contacted with reagent, obtains the mixture that RAGE TRA and HLA present molecule, and its quantity is to be enough to make the T cell population that contains described molten born of the same parents' property T cell to obtain selective enrichment.In a preferred embodiment, the HLA molecule is HLA-B7, and RAGETRA then is selected among one group that is formed with amino acid whose peptide that contains SEQ.ID.NO.40 and the amino acid whose peptide that contains SEQ.ID.NO.43.

According to another aspect of the present invention, provide the disorder that be expressed as feature of treatment with RAGE TRA or TRAP.A kind of method wherein is the reagent that imposes effective dose to the patient of this treatment of needs, and this reagent can optionally strengthen the reaction of T cell and this mixture on one's body the patient who exists HLA and RAGETRA mixture, and is enough to slow down described disorder.This reagent comprises various RAGE TRAP and the reconstitution cell of expressing HLA and RAGE TRA mixture.In one embodiment, this reagent comprises to HLA-B7 with the peptide of SEQ.ID.NO.40 or the cell of expressing with the mixture that the peptide that the peptide of SEQ.ID.NO.43 constitutes is formed.Another kind method comprise to patient's sufficient amount from the molten born of the same parents' property T of body cell to slow down disorder, wherein be that mixture to HLA molecule and RAGE TRA is specific from the molten born of the same parents' property T of body cell.

Interrelate with above-mentioned any separated coding TRAP or the nucleic acid of TRA, the present invention also comprise to only since the degeneracy of genetic code and with isolating nucleic acid different nucleic acid and complement of above-mentioned any nucleic acid on the codon sequence.

The present invention also comprises the functional variant and the equivalents of all above-mentioned molecules.

The invention still further relates to discovery and be separated in various TRAP and the TRA that expresses in the tumour cell, particularly in kidney tumor cell, expressed, and the TRAP and the TRA that in normal nephrocyte, are not expressed.Before the present invention, though identified other TRAP and TRA of many other cell types,, TRAP as herein described and/or TRA but are unknown and not certified for kidney.Surprisingly, RAGE-1 gene of the present invention only obtains in the example in 57 routine kidneys expressing, and best antigen peptide to specific cells toxicity T cell clone to be found to be ten poly-peptides, rather than nine poly-peptides.Therefore, according to a further aspect in the invention, the invention provides TRAP, TRA, the nucleic acid of coding TRAP and TRA, they are not TRAP, TRA and the nucleic acid of MAGE, BAGE and GAGE, they are expressed in tumour cell, particularly in kidney cancer cell, expressed, but they are not expressed in normal nephrocyte, and can obtain by following method:

From the patient, separate kidney tumor cell;

Isolated lymphocytes from the patient;

External this kidney tumor cell is contacted with this lymphocyte;

From with the lymphocyte of kidney tumor cell reaction isolated cell toxicity T cell clone;

Prepare expression library from the mRNA of kidney tumor cell;

Be cloned in this expression library with cytotoxic T lymphocyte and filter out and cell

The library member of toxicity T cell clone reaction;

Separate described and the library member cytotoxic T cell cloning reaction; And

To described library member's nephrocyte nucleic acid sequencing, described nephrocyte nucleic acid is compiled

Sign indicating number TRAP (or its part) comprises coding TRA.

The present invention also provide have with the sequence of these nucleic acid homologous and coding TRAP relevant and TRA with kidney, under stringent condition with degeneracy thing, TRAP and the TRA itself of sequence, complement, characteristic fragment and aforementioned each material of TRAP these nucleic acid hybridizations and that coding and kidney are correlated with and TRA and the functional modification thing and the equivalent of aforementioned any material, they can be considered to RAGE nucleic acid, TRAP and TRA.

The present invention also provides the reagent of selectivity raising HLA/RAGE TRA mixture in object as drug use.This preparation includes but not limited to, RAGE TRA and/or RAGE TRAP, expresses the reconstitution cell of RAGE TRA and/or RAGE TRAP and expresses the reorganization of suitable HLA molecule or be not the cell of reorganization and the functional modification thing and the equivalent of aforementioned each material.Concrete example comprises the RAGE TRA of SEQ.ID.NO.43; Any fragment that contains the TRA of SEQ.ID.NO.43 of the RAGE TRAP of SEQ.ID.NO.5; The RAGE TRAP of SEQ.ID.NO.5; Express the TRA of SEQ.ID.NO.43 and the reconstitution cell of HLA-B7; And/or any other RAGE TRA; RAGE TRAP or its functional fragment and/or express the cell of these molecules.

The present invention also provides the reagent of selectivity raising HLA/RAGE TRA mixture in object to be used for the medicine of production for treating cancer.These reagent include but not limited to RAGETRA and/or RAGE TRAP; Express the reconstitution cell of RAGE TRA and/or RAGE TRAP and express the reorganization of suitable HLA molecule or the cell of not recombinating; And the functional modification thing and the equivalent of aforesaid each material, concrete example comprises any fragment of the TRA that comprises SEQ.ID.NO.43 of RAGE TRAP of RAGE TRA, the SEQ.ID.NO.5 of SEQ.ID.NO.43; The RAGE TRAP of SEQ.ID.NO.5; Express the TRA of SEQ.ID.NO.43 and the reconstitution cell of HLA-B7; And/or any other RAGE TRA, RAGE TRAP or their functional fragment, and/or the express cell of these molecules.

It is that specific cytotoxic T cell is as drug use that the present invention also provides for HLA and RAGE TRA mixture.A kind of example of indefiniteness is to be specific autogenous cell toxicity T cell to the tumour cell of expressing HLA-B7 and RAGE TRA mixture.

It is the medicine that specific cytotoxic T cell is used for the production for treating cancer that the present invention also provides HLA and RAGE TRA mixture.A kind of example of indefiniteness is to be specific autogenous cell toxicity T cell to the tumour cell of expressing HLA-B7 and RAGE TRA mixture.

The present invention also provides the pharmaceutical preparation that contains the medicine described in any or multiple above-mentioned and this specification sheets.These pharmaceutical preparations can contain pharmaceutically acceptable dilution carrier or vehicle.

These and other purpose of the present invention will be given more careful description in conjunction with following detailed description.

Brief description of drawings

Fig. 1 shows is ctl clone 263/17 and combined the tumour necrosis factor level that is then produced by the COS cell of the cDNA transfection of HLA-B7 cDNA and coding RAGE TRAP.

Fig. 2 is the structure iron of RAGE-1,2,3 and 4 cDNA.The part indication that is full of stain is three differences of reading each ORF in the sign indicating number.Dash area representative in RAGE-2,3 and 4 cDNA and the irrelevant sequence of RAGE-1 comprise two insertion portions.Represent by dotted portion by 5 ' the terminal sequence that PCR obtains.Overlapping 5 ' the terminal sequence of 3 ' end of this PCR sequence and RAGE-2,3 and 4 cDNA is identical.Shown coded antigen peptide by RAGE-1.

Fig. 3 shows be ctl clone 263/17 with combined by the COS cell of the minigene transfection of the ORF2 of the cDNA of HLA-B7 cDNA and coding RAGE TRAP or coding RAGE TRAP after the tumour necrosis factor level that produced.

What Fig. 4 showed is that ctl clone 263/17 combines the tumour necrosis factor level that the back is produced with the COS cell of peptide fragment of being encoded by RAGE gene ORF 2 and HLA-B7 transfection.

What Fig. 5 showed is 267/17 pair of molten cytoactive through the HLA-B7 LA23-EBVB cell after the concentration that improves the peptide that comprises RAGE TRA of ctl clone.

The concise and to the point description of sequenceSEQ.ID.NO.1 is the nucleotide sequence of RAGE-1 cDNA.SEQ.ID.NO.2 reads sign indicating number 1 (ORF1) beginning of the cDNA of SEQ.ID.NO.1.SEQ.ID.NO.3 is the aminoacid sequence that SEQ.ID.NO.2 translates.SEQ.ID.NO.4 reads sign indicating number 2 (ORF2) beginning of the cDNA of SEQ.ID.NO.1.SEQ.ID.NO.5 is the aminoacid sequence that SEQ.ID.NO.4 translates.SEQ.ID.NO.6 reads sign indicating number 3 (ORF3) beginning of the cDNA of SEQ.ID.NO.1.SEQ.ID.NO.7 is the aminoacid sequence that SEQ.ID.NO.6 translates.SEQ.ID.NO.8 reads sign indicating number 4 (ORF4) beginning of the cDNA of SEQ.ID.NO.1.SEQ.ID.NO.9 is the aminoacid sequence that SEQ.ID.NO.8 translates.SEQ.ID.NO.10 reads sign indicating number 5 (ORF5) beginning of the cDNA of SEQ.ID.NO.1.SEQ.ID.NO.11 is the aminoacid sequence that SEQ.ID.NO.10 translates.SEQ.ID.NO.12 is the nucleotide sequence of the cDNA of RAGE-2.SEQ.ID.NO.13 is the nucleotide sequence of the cDNA of RAGE-3.SEQ.ID.NO.14 is the nucleotide sequence of the cDNA of RAGE-4.SEQ.ID.NO.15 reads sign indicating number 2 ' (ORF2 ') beginning of the cDNA of SEQ.ID.NO.12.SEQ.ID.NO.16 is the aminoacid sequence that SEQ.ID.NO.15 translates.SEQ.ID.NO.17 reads sign indicating number 3 ' (ORF3 ') beginning of the cDNA of SEQ.ID.NO.12.SEQ.ID.NO.18 is the aminoacid sequence that SEQ.ID.NO.17 translates.SEQ.ID.NO.19 reads sign indicating number 4 (ORF4) beginning of the cDNA of SEQ.ID.NO.12.SEQ.ID.NO.20 is the aminoacid sequence that SEQ.ID.NO.19 translates.SEQ.ID.NO.21 reads sign indicating number 5 (ORF5) beginning of the cDNA of SEQ.ID.NO.12.SEQ.ID.NO.22 is the aminoacid sequence that SEQ.ID.NO.21 translates.SEQ.ID.NO.23 reads sign indicating number 6 (ORF6) beginning of the cDNA of SEQ.ID.NO.13.SEQ.ID.NO.24 is the aminoacid sequence that SEQ.ID.NO.23 translates.SEQ.ID.NO.25 reads sign indicating number 2 ' (ORF2 ') beginning of the cDNA of SEQ.ID.NO.13.SEQ.ID.NO.26 is the aminoacid sequence that SEQ.ID.NO.25 translates.SEQ.ID.NO.27 reads sign indicating number 3 ' (ORF3 ') beginning of the cDNA of SEQ.ID.NO.13.SEQ.ID.NO.28 is the aminoacid sequence that SEQ.ID.NO.27 translates.SEQ.ID.NO.29 reads sign indicating number 4 (ORF4) beginning of the cDNA of SEQ.ID.NO.13.SEQ.ID.NO.30 is the aminoacid sequence that SEQ.ID.NO.29 translates.SEQ.ID.NO.31 reads sign indicating number 5 (ORF5) beginning of the cDNA of SEQ.ID.NO.13.SEQ.ID.NO.32 is the aminoacid sequence that SEQ.ID.NO.31 translates.SEQ.ID.NO.33 reads sign indicating number 2 ' (ORF2 ') beginning of the cDNA of SEQ.ID.NO.14.SEQ.ID.NO.34 is the aminoacid sequence that SEQ.ID.NO.33 translates.SEQ.ID.NO.35 reads sign indicating number 3 beginning of the cDNA of SEQ.ID.NO.14 " (ORF3 ").SEQ.ID.NO.36 is the aminoacid sequence that SEQ.ID.NO.35 translates.SEQ.ID.NO.37 reads sign indicating number 4 ' (ORF4 ') beginning of the cDNA of SEQ.ID.NO.14.SEQ.ID.NO.38 is the aminoacid sequence that SEQ.ID.NO.37 translates.SEQ.ID.NO.39 is the ten dimerization peptides that contain the RAGE Tumor rejection antigen relevant with Fig. 4.SEQ.ID.NO.40 is nine poly-peptide (1-9 amino acid) fragments of the peptide described in SEQ.ID.NO.39.SEQ.ID.NO.41 is nine poly-peptide (2-10 amino acid) fragments of the peptide described in SEQ.ID.NO.39.SEQ.ID.NO.42 is nine poly-peptide (3-11 amino acid) fragments of the peptide described in SEQ.ID.NO.39.SEQ.ID.NO.43 is ten poly-peptide (1-10 amino acid) fragments of the peptide described in SEQ.ID.NO.39.SEQ.ID.NO.44 is the nucleotide sequence of DNA of the peptide of coding SEQ.ID.NO.40.SEQ.ID.NO.45 is the nucleotide sequence of DNA of the peptide of coding SEQ.ID.NO.43.SEQ.ID.NO.46 is used for the meaningful primer that RAGE TRAP expresses test.SEQ.ID.NO.47 is the antisense primer that is used for the PCR test of RAGE TRAP expression.Various

All identical in the RAGE gene of testing.SEQ.ID.NO.48 be used for RAGE-1 TRAP genetic expression PCR test to RAGE-1

Be specific antisense primer.SEQ.ID.NO.49 represents RAGE gene pairs ORF2 insertion point and is appointed as the flanking region of the insertion of N.SEQ.ID.NO.50-57 is useful on the PCR primer of identifying RAGE1.Detailed description of the present invention

By the antigen from the renal cell carcinoma that body CTL is discerned of HLA-B7 restriction enzyme digestion be by before ignorant genes encoding.This gene all is reticent in all healthy tissuess (comprising testis tissue), except in retina, and is expressed in several routine tumor samples.Most preferred embodiment of the present invention 1: the LE9211 patient's of embodiment anti-renal cell carcinoma ctl clone

Tumour is that LE9211-RCC is decided to be the renal cell carcinoma clone of deriving out in LE9211 number the tumor sample of female patient.Sample does not breed it through irradiation.Then, use the cell of these illuminated mistakes to go separation that it is specific molten born of the same parents' property T cell clone (" CTL ").

Peripheral blood lymphocytes (" the PBMC ") sample that obtains from LE9211 patient contacts with the cancer cells of illuminated mistake.After 14 days, observe the molten born of the same parents' situation of cancer cells of this mixture, being illustrated in of molten born of the same parents has peptide that cancer cells is brought in the sample and the mixture of HLA molecule is specific CTL.

Employed molten born of the same parents' detection is the chromium release detection according to people such as Herin [Herin et al., Int.J.Cancer39:390-396 (1987)].Briefly being described as of this detection, the vitro culture targeted cancerous cells is suspended in 10 then ⁷In the Eagles substratum (DMEM) of the Dulbecco modified of cells/ml and mend with 30%FCS, 37 ℃ of Na with 200 μ Ci/ml ( ⁵¹Cr) O ₄Cultivated 45 minutes.Wash the cell 3 times that mark crosses with DMEM.Be resuspended to then among the DMEM that is furnished with 10mM Hepes and 10% calf serum (FCS), after this, will contain 10 ³The sample aliquot of 100 microlitres of individual cell is distributed in the 96 hole droplet dishes.Be added in the lymphocyte in the same substratum of 10 microlitres, detect and carry out double.With 100g centrifugal 4 minutes, at 37 ℃ 8%CO to the droplet dish ₂Cultivated 4 hours in the gas.

Centrifugal to the droplet dish once more, collect the supernatant liquor sample aliquot of 100 microlitres and also count.To what discharged ⁵¹The percentile of Cr is calculated as follows:

Wherein ER is the test that observes ⁵¹Cr discharges; SR cultivates 10 in 200 microlitre substratum ³The mensuration that discharges in the time of the cell of mark; MR is by adding 100 microlitre 0.3%Triton X-100 resulting maximum release in the target cell.

Those show the active monokaryon blood sample of height CTL and increases by the restriction dilution and clone, and make to use the same method and screen once more.Separate then and obtain first ctl clone.After this, this clone is called 263/17.Obtained to be called second ctl clone of 361A/17 with same experimental technique, and used in the indeterminate growth failure of CTL 263/17, concrete use is seen below described.

Ctl clone 263/17 and 361A/17 can the molten born of the same parents' autologous tumor cells of specificity, but NK-target K562 cell is not taken place by molten born of the same parents.NK-target K562 cell can obtain from the ATCC in Maryland, USA Luo Keweier city.

After stimulating with autologous tumor cell, ctl clone 263/17 produces TNF.To provide antigenic HLA molecule in order identifying to ctl clone 263/17, to have carried out suppressing experiment, wherein, the production to TNF under the condition that the monoclonal antibody that anti-HLA molecule or anti-CD4/CD8 accessory molecule are arranged exists experimentizes.Discovery has four kinds of monoclonal antibodies to suppress CTL263/17 to produce TNF, and they are: (1) monoclonal antibody W6/32, the I type molecule of its directly anti-all HLA class (Parham et al., 1979, J.Immunol., 123:342); (2) antibody B1.23.2, and its identification HLA-B and C molecule (Rebai et al., 1983, TissueAntigens, 22:107); (3) the antibody ME-1 of specific recognition HLA-B7 (Ellis et al., 1982, Hum.Immunol., 5:49); And the antibody B9.4.1 of (4) anti-CD8.Do not find antibody (the L243:Lampson et al. of direct anti-HLA class II DR molecule, 1980, J.Immunol. is 125:293) with anti-HLA-A3 (GAPA 3:Berger et al., 1982Hybridoma, the restraining effect of the antibody of antibody 1:87) or anti-CD4 (13B.8.82).Conclusion is that CTL263/17 is the CD8 type, and discerns the antigen that is presented by HLA-B7.

In order to determine the tumour-specific of this ctl clone, the normal nephrocyte that obtains from the patient of other HLA-B7 (PTEC-HLA-B7 cell) is used for experiment.These cells are obtained from the proximal tubular epithelial cell, and this site is the originating point of renal cell carcinoma.The PTEC-HLA-B7 cell shows that not by the molten born of the same parents of CTL antigen is specific expressed on tumour.

The renal cell carcinoma that is obtained from another one HLA-B7 patient be MZ-1851 also by the molten born of the same parents of CTL, show that this antigen is by independently tumour is common. Embodiment 2: the cDNA that the antibody that CTL263/17 discerned is directly expressed clones

Separation

The A.cDNA library

Separated RNA from LE-9211-RCC, poly--A-RNA by widow-dT in conjunction with purification.The preparation of cDNA is with the widow-dT reverse transcription that contains Not I site, synthetic then second chain (Superscript Choice System, BRL, Life Technologies).This cDNA is connected with BstX I connector again, with Not I digestion, according to big or small portioning (SephacrylS-500 HR post, BRL, Life Technologies), and is cloned into BstXI and the Not I site of pcDNA-I-Amp (Invitrogen) with single direction.The plasmid electroporation that to recombinate then is in DH5 α E.coli.1500 samples to 100 recombinant bacterias increase, and obtain the plasmid DNA of each sample by the molten born of the same parents of alkali, potassium acetate precipitation and phenol extraction.

The transfection of B.COS cell

The cDNA of plasmid DNA from different samples and the HLA-B7 of 60ng (cDNA that comes from another one HLA-B7 patient clones and insert plasmid vector pcDSRalpha through PCR) cotransfection is to the COS cell.Double is carried out in this transfection.In brief, sample COS-7 cell is inoculated into flat tissue culture droplet dish with the quantity of 15,000 cells of every well, uses the DMEM that is equipped with 10% calf serum.37 ℃ of incubated overnight cells, remove substratum, with the amount adding DMEM substratum of 50 microlitres/well, this substratum contains 10%Nu-serum (Collaborative Research, Bedford, MA), the plasmid of DEAE-dextran, the green Kui of 100 μ M and the 100ng of 400 μ g/ml.After 4 hours, remove substratum 37 ℃ of cultivations, add the PBS that contains 10% methyl-sulphoxide (DMSO) of 50 μ l.After two minutes, remove this substratum and replacement to be furnished with the DMEM200 μ l of 10%FCS.

Changed after the substratum, the COS cell was cultivated 24-48 hour at 37 ℃.Then with CTL263/17 screening transfection thing.After for the first time removing substratum, add 3000CTL 263/17 cell that is placed in the 100 μ l substratum that contain 25U/ml IL-2 in every well.Measure the TNF quantity that exists in the supernatant liquor with cytotoxicity experiment then to WEHI 164.13 cells.The result of most of samples is that the concentration of TNF is lower than 10pg/ml.It is high density (24-37pg/ml) that result in the double The Small Well of two samples (1157 and 1319) is arranged.Behind the bacterial clone of 1319 samples, obtain 1200 clones.Extract their plasmid DNA and with the HLA-B7 transfection in the COS cell.With CTL263/17 screening transfection thing.A cDNA clone (9H3) is arranged to CTL263/17 high yield TNF.Fig. 1 shows be when this cDNA (60ng) after the COS cell is arrived in HLA-B7 cDNA (60ng) transfection, use the CTL263/17 results of screening.

This cDNA also is stabilized a kind of clone of ground transfection in the HLA-B7 sarcoma system, promptly in the LB23-SAR cell.Ctl clone 361A/17 with identification and ctl clone 263/17 same antigen carries out molten born of the same parents' test then.To these identification and COS-HLA-B7-cDNA 9H3 cell identical of cells transfected stably. The sequence of embodiment 3:cDNA 9H3

The length of cDNA clone 9H3 is 1130 base pairs.This cDNA is incomplete, because it is littler than observed mRNA (1.6kb) in Northern blot.5 ' the terminal of this cDNA is cloned by RACE-PCR, and has determined its whole sequence.This whole sequence is shown in SEQ.ID.NO.1.Relatively show with the sequence of being reported in the database, in 3 ' terminal and the short sequence of still not knowing 235 base pairs that are called " expressed sequence mark " of its function high homology (I) is arranged, 5 ' terminal then with the restricted property of the antisense strand homology (on 95 bases, having 75%) of two endogenous retrovirals of people that are known as RTVL-H2 and RGH2 (2,3).

To tumor of kidney AntiGEn, this gene is called RAGE.

This sequence contains 5 openings reads sign indicating number, i.e. ORF1:99 base pair, the protein of 32 residues of coding; ORF2:123 base pair, the protein of 40 residues of coding; ORF3:87 base pair, the protein of 28 residues of coding; ORF4:288 base pair, 95 the residue protein of encoding; ORF5:222 base pair, the protein (being respectively SEQ.ID.NO.2,4,6,8 and 10) of 73 residues of coding.The SEQ.ID.NO.4 TRAP that encodes, deriving from it with the antigen peptide of CTL263/17 (as the HLA-B7/ peptide complex) reaction obtains. Embodiment 4: the evaluation of other RAGE gene

Present embodiment has been described the evaluation of three kinds of other RAGE genes, and has determined to have only the RAGE gene of identifying among the top embodiment, promptly is known as the genes encoding of RAGE-1 and the RAGE TRA of CTL263/17 reaction now.

Prepare probe from RAGE-1 cDNA, be used at the other RAGE gene of the cDNA library of LE9211-RCC screening.Separated three kinds and be labeled as RAGE-2 (SEQ.ID.NO.12) respectively, RAGE-3 (SEQ.ID.NO.13) and RAGE-4 (SEQ.ID.NO.14) with RAGE homologous cDNA.With standard method to RAGE-2,3 and 4 crto gene sequences.Nucleotide sequence and the RAGE-1 cDNA of these RAGE cDNA are compared, show that existing the new opening of blocking in the RAGE cDNA that new evaluation is come out reads sign indicating number (ORF).RAGE-2, RAGE-3 and RAGE-4 contain the insertion of 37 base pairs of 249 positions of RAGE1 [with the corresponding sequence of ORF2 (SEQ.ID.NO.4) of RAGE-1 within].For the cDNA of RAGE-2, comparison shows that with cosmid this inserts the beginning corresponding to exon.The reason that the cDNA of RAGE-1 does not contain it may be to use the acceptor site of other downstream side.In addition, RAGE-2,3 different with RAGE-1 with 4 lacks the Nucleotide of the 192nd position of RAGE-1.These have changed and the ORF2 of RAGE-1 and 3 homologous RAGE-2,3 and 4 ORF with being changed significantly.In addition, RAGE-3 also has the insertion of one 47 base pair in addition at 5 ' end.Except these differences, RAGE-1,2 and 3 sequence are equal fully.

The length of RAGE-4 is approximately than long 800 base pairs of other RAGE cDNA.Its 5 ' terminal sequence is identical with RAGE-2's, and still, from 434 to poly--A afterbody, the sequence of RAGE-4 is different fully with other RAGE cDNA.RAGE-4 cDNA does not demonstrate to mosaic.The zero position of 3 ' irrelevant sequence is corresponding with the boundary of the exon-intron of RAGE genome sequence, and this 3 ' irrelevant sequence is present in 3 ' end of RAGE gene.Therefore, this RAGE-4 cDNA likes the result of the difference shearing of RAGE-2 gene well.Fig. 2 has provided the ordering of four kinds of cDNA.In the cDNA of four kinds of RAGE, have 17 kinds of ORF.Have in these 17 kinds 10 kinds inequality.These ORF are as follows:

Gene ORF Nucleotide SEQ.ID.NO.

RAGE-1 ORF1 173-271 2

ORF2 204-326 4

ORF3 313-399 6

ORF4 323-610 8

ORF5 444-665 10

RAGE-2 ORF2’ 217-276 15

ORF3’ 273-449 17

ORF4 373-660 19

ORF5 494-715 21

RAGE-3 ORF6 185-247 23

ORF2’ 274-333 25

ORF3’ 330-506 27

ORF4 430-717 29

ORF5 551-772 31

RAGE-4 ORF2’ 213-272 33

ORF3” 269-832 35

ORF4’ 369-557 37

The cDNA of RAGE-2, RAGE-3 and RAGE-4 is cloned in the expression vector with standard technique, and according to described with the HLA-B7 transfection in the COS-7 cell, thereby determine the antigen whether these cDNA encode and discerned by CTL 263/17.Carry out parallel control experiment simultaneously, use be RAGE cDNA (being referred to as RAGE-1 now) and LE9211-RCC cell.

Behind the LE9211-RCC cell or the cultivation of COS-7 cell process by RAGE-1 and the HLA-B7 cotransfection that has CTL 263/17, CTL263/17 causes the strong release of TNF.The cotransfection that carries out with RAGE-2, RAGE-3 or RAGE-4 and HLA-B7 is not caused the release of TNF.Therefore, have only RAGE-1 to shift expression to the antigen of CTL263/17 identification. Embodiment 5: to the evaluation of the ORF that contains the RAGE Tumor rejection antigen

The insertion of 37 base pairs in RAGE-2,3 and 4 has just stopped ORF2 prematurely in these three genes.Therefore, it is believed that the antigen peptide of being discerned by CTL263/17 is coded by the 3 ' end of ORF2.In order to check this theory, will be corresponding to the dna sequence dna of the ORF2 of RAGE-1, be cloned in the expression vector corresponding to the dna sequence dna of the ORF2 ' of RAGE-2 and RAGE-3, and according to above-mentioned with the HAL-B7 transfection in the COS-7 cell.As positive control, being the COS-7 cell of RAGE-1 cDNA and HLA-B7 cotransfection or using the LE2911-RCC cell of use.These transfection things or LE2911-RCC cell are used to evoke CTL 263/17 and discharge TNF.In ORF transfection thing, have only the ORF2 that comes from RAGE-1 successfully to stimulate of the release (Fig. 3) of CTL 263/17 cell to TNF.This experimental verification the RAGE antigen peptide of CTL 263/17 cell recognition be by 3 ' the end coding of the ORF2 of RAGE-1. Embodiment 6: identify the RAGE tumor rejection antigenic peptide

Synthesize 3 ' the synthetic peptide of holding, and test it discharges TNF to the CTL263/17 cell stimulation corresponding to the ORF2 of RAGE-1.According to the above-mentioned HLA-B7 transfection COS-7 cell of using, and will add in the culture corresponding to the synthetic peptide that 3 ' of ORF2 holds.Add the CTL263/17 cell, after 18 hours, measure the production (Fig. 4) of TNF.Peptide SPSSNRIRNTST (SEQ.ID.NO.39) has stimulated the release of 263/17 couple of TNF of CTL effectively.Because the peptide that the I type molecule of HLA class is presented generally all is 9 amino acid whose length, so having tested from the front, we are used to stimulate CTL 263/17 cell that 10 poly-peptide (SEQ.ID.NO.39) institute deutero-9 of the release of TNF are gathered peptides (SEQ.ID.NO.40,41 and 42).These result of experiment see Fig. 4.One of them peptide (SPSSNRIRN SEQ.ID.NO.40) is discerned by CTL 263/17, but its identification degree well below identifications to 10 poly-peptides, this just shows that 9 poly-peptides are not best peptides.(SPSSNRIRNT SEQ.ID.NO.43) can be discerned by CTL263/17 10 poly-peptides very effectively. The activity of embodiment 7:RAGE Tumor rejection antigen nine poly-peptides and ten poly-peptides

Present embodiment shows the induce relative reactivity that with nine poly-peptides and ten gather peptides of RAGE TRA peptide to the molten born of the same parents of HLA-B7 express cell.

They induce the molten born of the same parents of CTL 263/17 cell to the HLA-B7-LB23-EBVB cell to nine poly-peptides of RAGE peptide (being respectively SEQ.ID.NO.40 and 43) and ten poly-peptide tests in dose response detects.Freeze dried peptide being dissolved among the DMSO of 20mg/ml, is 2mg/ml and in-80 ℃ of preservations with 10mM acetate dilution.Target cell, i.e. the lymphoblast of HLA-B7-EBV-transfection (LB23-EBV cell) is used ⁵¹Cr, fully cleans in 37 ℃ of marks 1 hour then according to aforementioned, removes unconjugated marker.In 96 well droplet dishes of the peptide that different concns is arranged, cultivated the LB23-EBV cell 30 minutes in 37 ℃.Be that 10: 1 ratio adds the CTL 263/17 be in the equal-volume substratum with working substance to target then.Measure after 4 hours ⁵¹The release of Cr.Fig. 5 has provided the result of metering reaction detection.Concentration is maximum molten born of the same parents' half value that the SPSSNRIRNT peptide (SEQ.ID.NO.43) of 30ng/ml has guided the LB23-EBV cell. Embodiment 8:RAGE-1 expression of gene

With the expression of PCR test RAGE, the primer of use is as follows:

SEQ.ID.NO.46

-GTG TCT CCT TCG TCT CTA CTA (adopted primer is arranged)

SEQ.ID.NO.47

-GGT GTG CCG ATG ACA TCG (antisense primer identical) to all RAGE genes

SEQ.ID.NO.48

-GAG GTA TTC CTG ATC CTG (is specific antisense primer to RAGE-1)

At first, from specific sample, obtain full RNA with routine techniques.Prepare cDNA with this RNA.The procedure operation for preparing this cDNA comprises the 5x ThermoScript II damping fluid that merges 4 μ l, the various dNTP (10mM) of 1 μ l, the dithiothreitol (DTT) of 2 μ l (100mM), the dT-15 primer of 2 μ l (20 μ M), 0.5 the M-MLV ThermoScript II (200 units/μ l) of the RNasin of μ l (40 units/μ l) and 1 μ l; The template ribonucleic acid (1 μ l/3.25 μ l water, or the full template ribonucleic acid of 2ug) that adds 6.5 μ l then; The cumulative volume of mixture is 20 μ l; Mix the back and cultivated 60 minutes, be positioned over cooled on ice then at 42 ℃; Adding then, totally 80 μ l water make volume reach 100 μ l.With this mixture-20 ℃ of preservations until being used in PCR.

The reagent that uses among the PCR comprises:

The 10x DyanZyme damping fluid of ■ 5 μ l

Each 20pmole of every kind of primer of ■

Each 5nanomole of every kind of dNTP of ■

■ 1 unit polysaccharase " Dynazyme " (2 units/μ l)

The cDNA of ■ 5 μ l (corresponding to the full RNA of 100ng)

■ water, adding to final volume is 50 μ l

Combined mixture is with a mineral oil layering.Mixture is transferred in the thermo cycler that is preheated to 94 ℃, and amplification is included in 94 ℃ and carries out a circulation of 15 minutes, and it is as follows to carry out 33 circulations then:

■ 94 ℃ 1 minute

■ is 56 ℃ or 60 ℃ 2 minutes (referring to following)

■ is 72 ℃ of 15 minutes prolongation steps of carrying out 72 ℃ in 3 minutes at last.All RAGE expression of gene have the pcr amplification of adopted primer (SEQ.ID.NO.46) and antisense primer (SEQ.ID.NO.47) to detect with pan-RAGE, have wherein adopted 60 ℃ cancellation in 2 minutes.The RAGE-1 expression of gene has the pcr amplification of adopted primer (SEQ.ID.NO.46) and RAGE-1 specific antisense primer (SEQ.ID.NO.48) to detect with pan-RAGE separately, has wherein adopted 56 ℃ cancellation in 2 minutes.Containing on the sepharose of ethidium bromide (1.5%) product of can naked eyes seeing PCR, the i.e. product of 194 base pairs (general) and 239 base pairs (is specificity to the RAGE-1 gene) to all RAGE genes that tried.

This gene is a tumour-specific.In the healthy tissues of all experiments, this gene all is reticent, except in retina.Particularly, this gene all is reticent in suprarenal gland, bladder, marrow, brain, breast, cerebellum, colon, heart, kidney, liver, lung, melanophore, muscle, nerve, ovary, placenta, prostate gland, skin, splenocyte, stomach, testis, thymocyte, uterus and callus.Yet this gene is found in and expresses (referring to table 1) in tumor cell line and the neoplasmic tissue sample.And this gene is also expressed in the tumour that do not list here, though frequency is very low.

The expression of table 1.RAGE-1 gene in tumor sample

The tumour number of expressing

Histological type

Whole RAGE RAGE-1

Tumor sample

Kidney 2,/57 1/57

Sarcoma 5,/25 3/25

-0,/29 0/29 of bladder cancer surface

-3,/37 3/37 of infiltration

Melanoma primary-2,/60 2/60

Transitivity-8/,177 6/177

Head and neck cancer 2,/50 1/50

Mastocarcinoma 3/,128 1/128

Prostate cancer 0,/22 0/22

Colorectal carcinoma 0,/48 0/48

Leukemia 0,/19 0/19

Lung cancer (NSCLC ¹) 0,/59 0/59

(SCLC) 0/5 0/5

Carcinoma mesothelial 1/3 0/3

The cancer of the brain 0,/11 10/11

Esophagus cancer 0/7 0/7

Ovarian cancer 0/3 0/3

Tumor cell line

Kidney 8,/19 7/19

Bladder cancer 3/3 3/3

Carcinoma mesothelial 11,/19 8/19

Head and neck cancer 3/7 1/7

Sarcoma 2/6 1/6

Melanoma 11,/78 7/78

Colorectal carcinoma 1,/17 1/17

Lung cancer (NSCLC ¹) 0/2 0/2

(SCLC) 0/26 0/26

Leukemia/lymphatic cancer 0,/11 0/11

The cancer of the brain 0/1 0/1

Cancer of the stomach 0/2 0/2

¹NSCLA: nonsmall-cell lung cancer

The foregoing description has provided the separation to the nucleic acid of coding TRAP.Yet the encoding sequence that has disclosed in this TRAP coding molecule and the above-mentioned document of quoting does not all have homology.Therefore, one aspect of the present invention is to separate to comprise the whole nucleic acid of SEQ.ID.NO.1, SEQ.ID.NO.4, SEQ.ID.NO.6 or SEQ.ID.NO.10 or the nucleic acid molecule of differentiated part nucleic acid wherein.That can also expect can be discerned by other CTL clone beyond the CTL 263/17 by the antigen that SEQ.ID.NO.12,13 and 14 coded RAGE ORF derive out.Therefore, another aspect of the present invention relates to one or more in the RAGE group gene, comprise the differentiated part that it is separated, for example encode from the various TRAP of its deutero-and TRA, various RAGE TRAP and RAGE TRA, and relative diagnosis and methods of treatment.Aforesaid sequence is not MAGE, BAGE or GAGE sequence, can seeing when MAGE, BAGE described in itself and various document or GAGE sequence compare.

In addition, the present invention be on the other hand the non-MAGE of encoding those, non-BAGE and non-GAGE tumor rejection antigen precursor but can be under stringent condition and the nucleotide sequence that contains the making nucleic acid molecular hybridization of above-mentioned nucleotide sequence.Term " stringent condition " refers to technical parameter well-known to those skilled in the art in this article.Specifically, it refers to hybridization buffer (3.5xSSC, 0.02%Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 25mM NaH at 65 ℃ in this article ₂PO ₄(pH7), 0.5%SDS, 2mM EDTA) in the hybridization carried out.SSC is 0.15M sodium-chlor/0.15M Trisodium Citrate, pH7; SDS is a sodium lauryl sulphate; EDTA is an ethylenediamine tetraacetic acid (EDTA).After the hybridization, the film that has shifted DNA is cleaned with 2xSSC in room temperature, clean with 0.1xSSC/0.1xSDS at 65 ℃ then.

Can obtain the condition and the reagent that also have other of same stringent condition.Those skilled in the art is familiar with them very much, therefore there is no need to give unnecessary details at this.Appreciated by those skilled in the artly screen the method that cell particularly screens cancer cells in addition, express the method for described molecule, conventional separation method, and subsequently to the separation method of nucleic acid.

When screening RAGE group membership, can carry out Southern blot, use aforesaid condition, and adopt ³²The P probe.DNA being transferred at last after film on it cleans, carry out X-illumination with this film and check radiated signal mutually.

Therefore, the present invention also provides unique fragment or its complement of separated SEQ.ID.NO.1.Unique fragment refers to the fragment of RAGE gene for ' signature '.For example, its length guarantees that enough its accurate sequence can not be found in the molecule beyond the RAGE group, described as 23 of claims the.Preferred unique fragment is those fragments of only seeing in ORF2 or its complement.These unique fragments can be identified the member that those comprise the RAGE group of expressing ORF2 as probe in Southern blot detects, perhaps can be used for augmentation detection, for example are used for the detection that those have used PCR.As well known by the skilled person in the art, in some applications, for example in Southern blot, preferably use bigger probe, 200 probes that base pair is above for example, less fragment then is preferred for as among the PCR.As can be by those skilled in the art recognized, the big young pathbreaker of unique fragment be depended on its conservative property in genetic code.Therefore, SEQ.ID.NO.1, SEQ.ID.NO.12, some zones of SEQ.ID.NO.13 and SEQ.ID.NO.14 will need long unique fragment, other the relatively shorter fragment of then requirement, be generally 12-32 base pair (for example, 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31 and/or 32 base pairs are long).In fact, SEQ.ID.NO.1 any to have the fragment of 18 or more a plurality of Nucleotide all be unique.Those skilled in the art be familiar with to select the method for these sequences very much, all can be according to the character of unique fragment and optionally with them with non-this group membership separately.Usually neededly exactly this fragments sequence is compared with known database, though also can carry out external affirmation hybridization and sequential analysis.

For the structure of any one couple of PCR primers of selective amplification RAGE-1 with arrange to use the Auele Specific Primer of RAGE-1.Such primer is the continuous prolongation of RAGE-1, the two ends hybridization of the insertion point of it and ORF2, this ORF2 be inserted into other the RAGE gene Nucleotide and changed.Such Auele Specific Primer will be merely able to hybridize fully with the continuous prolongation of the Nucleotide of RAGE-1, still, will be only can and those RAGE genes of not sharing ORF2 part hybridization takes place.For effect that causes for PCR and the evaluation of RAGE1, the structure of RAGE 1 Auele Specific Primer and arrange make its 3 ' end effectively to hybridize with other RAGE gene beyond the RAGE 1.In order to reach this point, primer can be described to have two end points: one 5 ' end, with a side of insertion point in abutting connection with and complementary, and be directly connected to one 3 ' end, and this 3 ' to hold be opposite side adjacency and complementary with the insertion point.It is longer than 3 ' end that 5 ' of primer is held, and make 3 ' end lack (being 1-4 Nucleotide), and then Za Jiao kinetics is hybridized in holding 5 ' with regard to strong tendency.At this moment, only all with behind the nucleic acid hybridization just begin to take place 3 ' initial PCR prolongation, promptly only exist when inserting and take place in ORF2 at 5 ' end and 3 ' end.

As mentioned above, the RAGE-1 Auele Specific Primer can cause any one the synthesizing in the dna double coiled strand, is described as Watson chain and Crick chain at this.The sequence that flank among the RAGE 1 covers the insertion point is 5 '-CAAACANGGATCA-3 ' (SEQ.ID.NO.49; Watson chain, N are the Nucleotide that inserts).For the RAGE-1 Auele Specific Primer that designs of Watson chain of amplification RAGE-1 generally contains 12, preferred 15 or 3 ' the relevant Nucleotide of holding of Nucleotide of the Watson chain of more a plurality of and insertion point.The rest part of primer can be that 1-4 Nucleotide is long, and is complementary to 5 ' sequence of insertion point.Such primer will with complete complementation of the complementary portion in RAGE-1 and adjacency.Prolongation will be hybridized and begin to 3 ' end of this primer with the complementary portion of Watson chain.In the RAGE gene beyond the RAGE-1, insert of the hybridization of 3 ' end of incomplementarity Nucleotide meeting basically eliminate RAGE-1 Auele Specific Primer in the ORF2 insertion point to the Watson chain 5 ' end of insertion point.What this 3 ' end at primer took place during with the RAGE gene recombination that is not RAGE-1 mismatches, and will get rid of effective amplification of these genes.The sequence of the primer of illustrative is formed as follows substantially, wherein N be 0, on suitable Watson or Crick chain 1 or more a plurality of in abutting connection with Nucleotide: 5 '-NTATTCCTGATCCT-3 ' is (SEQ.ID.NO.50); 5 '-NTATTCCTGATCCTG-3 ' (SEQ.ID.NO.51); 5 '-NTATTCCTGATCCTGT-3 ' (SEQ.ID.NO.52); 5 '-NTATTCCTGATCCTGTT-3 ' (SEQ.ID.NO.53); 5 '-NCAAGTTCAAACAG-3 ' (SEQ.ID.NO.54); 5 '-NCAAGTTCAAACAGG-3 ' (SEQ.ID.NO.55); 5 '-NCAAGTTCAAACAGGA-3 ' (SEQ.ID.NO.56); And 5 '-NCAAGTTCAAACAGGAT-3 ' is (SEQ.ID.NO.57).

The expression of RAGE-1 can also be detected by PCR, and the primer of use is in the initial prolongation of a relative side of insertion point.To the analysis of amplified production can be by amplified production relatively RAGE-1 amplified production and non-RAGE-1 amplified production being made a distinction of length.Because the RAGE-1 gene does not contain the insertion that exists in other RAGE gene, therefore, will be from RAGE-1 deutero-amplified production than the amplified production weak point (short approximately 37 base pairs) of other RAGE gene.This difference can make a distinction with the ordinary method of this area.In addition, for those skilled in the art, the method for the basic homologous nucleotide sequence of other difference, for example LCR (ligase chain reaction) or the like is clearly.The Auele Specific Primer that can prepare RAGE 2,3 and 4 with similar methods.

The present invention also comprises and containing the application of nucleotide sequence of codon that coding is same as the amino-acid residue of RAGE coded by said gene.For example, disclosed as the foregoing description 7, ten poly-peptide SPSSNRIRNT (SEQ.ID.NO.43) are the RAGE Tumor rejection antigens.Serine residue (the 1st, 3 and 4 amino acid of SEQ.ID.NO.40) can be encoded respectively by for example codon TCA, AGT and TCA.Except TCA and AGT, serine residue can also be by codon TCC, TCG, TCT and AGC coding.In these six codons any one equates when the encoding serine residue.Therefore, for those skilled in the art, the nucleotide triplet of any encoding serine can be used to instruct in vivo or external protein synthesis adds serine residue.Equally, other nucleotide sequence triplet that contains the amino-acid residue of RAGE Tumor rejection antigen of encoding comprises: CCA, CCC, CCG and CCT (proline(Pro) codon); CCA, CGC, CGG, CGT, AGA and AGG (arginine codon); ACA, ACC, ACG and ACT (Threonine codon); AAC and AAT (l-asparagine codon); With ATA, ATC and ATT (Isoleucine codon).Other amino-acid residue also can be by similar situation by multi-joint nucleotide sequence coded.Therefore, the present invention includes the degeneracy of nucleic acid different nucleic acid on the codon sequence that those degeneracies owing to genetic code separate itself and biology to obtain.

Above embodiment has provided the separation of this class peptide of RAGE TRA.These representational peptides are to be processed by the translational product process of the nucleic acid of SEQ.ID.NO.1 to obtain.Therefore, to those skilled in the art, the translational product that is used for being processed into the final expression-form of RAGE TRA can be the sequence that contains RAGE TRA of any length.As what the foregoing description disclosed, the peptide of all size and protein, for example, little of 9,10 or 12 amino acid whose peptides or protein, big to by the ORF1 amino acid sequence coded etc., can suitably be processed, present by HLA-B7, and discern by CTL263/17.The peptide of SEQ.ID.NO.23 can add 1,2,3,4,5,6,7,8,9,10 even more a plurality of amino acid at its two ends respectively or simultaneously.The antigenic portions of this peptide can present by 1 type molecule of cause HLA class under the physiological condition down cut.

The derive protein of RAGE TRA and the aminoacid sequence of peptide can be natural or non-natural, that is to say, they can contain natural RAGE TRAP molecule or contain sequence through modifying, as long as its aminoacid sequence remains with when being presented in cell surface the Tumor rejection antigen sequence that can be discerned by CTL.For example, RAGE Tumor rejection antigen herein can be the protein of RAGE Tumor rejection antigen and irrelevant aminoacid sequence syzygy, RAGE-1 gene ORF 2 translate polypeptide, SEQ.ID.NO.39, the peptide of the synthetic peptide of shown aminoacid sequence, mark in 40 and 43, from the isolating peptide of the patient who suffers from kidney, from the isolating peptide of cell culture of expressing RAGE-1, for example the non-peptide molecule bonded peptide some drug delivery systems and other contain the molecule of the aminoacid sequence of SEQ.ID.NO.40 with non-peptide molecule.

It can also be seen that from above embodiment the present invention also comprises the application of sequence in expression vector, and the host cell of transfection and clone, comprise that prokaryotic cell prokaryocyte (for example, intestinal bacteria) and eukaryotic cell (for example, Chinese hamster ovary celI, COS cell, yeast expression system and at the recombinant baculovirus of expressed in insect cells).These expression vectors require relevant sequence, and promptly above-mentioned sequence is connected on the promotor by exercisable.As above find, people's HLA-B7 presents the TRA that derives out from these genes, and expression vector can also comprise the nucleotide sequence of the HLA-B7 that encodes.When carrier contains these two kinds of encoding sequences, just can come transfection not express the cell of one of these two kinds of sequences usually with it.TRAP and TRA encoding sequence can be used alone, for example, and when host cell is expressed HLA-B7.Certainly, the host cell of concrete use does not have special restriction.Because in the time of needs, the carrier that contains two kinds of encoding sequences can be used to HLA-B7 and present in the cell, so the nucleotide sequence of coding TRAP or TRA just can be used to not express in the host cell of HLA-B7.

The present invention also comprises so-called expression test kit, and it can make those skilled in the art prepare needed one or more expression vectors.This expression test kit comprises two kinds in the above-described material placed apart at least.The composition that can also contain simultaneously, other needs.

For nucleic acid molecule of the present invention is distinguished with former MAGE class, BAGE gene and GAGE gene mutually with TRAP, then to RAGE genoid and the TRAP of just being decided to be of the present invention.Therefore, part appears in herein " RAGE ", just MAGE, BAGE and GAGE gene, gene product, TRAP and TRA.

The present invention serves many purposes, and wherein some have given description at this.At first, the invention enables those skilled in the art to diagnose to the disorder that is expressed as feature with TRAP.These methods relate to the TRAP gene and/or from its deutero-TRA, and for example, the expression of the TRA that is presented by HLA-B7 is determined.Under the former situation, this definite can finishing by the detection of nucleic acids of any standard comprises polymerase chain reaction, or has the detection of the hybridization probes of mark.Under latter event, then preferably use to comprise TRA and HLA complex body and for example to use detection of antibodies as the detection of binding partner.The another kind of method of determining is that TNF discharges detection, as previously discussed.

To the separation of TRAP gene make to TRAP molecule itself with or by the possibility that is separated into of its deutero-TRA, particularly contain the TRAP or the TRA molecule of SEQ.ID.NO.1 or 4 amino acid sequence coded.This method described in detail can also separate other by SEQ.ID.NO.1,12,13 and 14 coded discerned by other ctl clone also/or the other TRAP and the TRA that are presented by other HLA molecule.(those skilled in the art has known multiple HLA molecule, including but not limited to that those are by HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G genes encoding) the known method of a variety of those skilled in the art can be used for separating the TRAP molecule, and/or the TRA that is derived and by it.From the proteinic cell of natural generation, can separate protein.In addition, expression vector can be incorporated into and cause proteinic production in the cell.In another approach, mRNA translates thing and can be incorporated in cell or with other method by microinjection and cause production encoded protein matter in the cell.In not celliferous extract for example in the erythrocytic molten born of the same parents of knitmesh system translation mRNA also can produce protein.The peptide that contains TRA of the present invention also can be external synthetic.Those skilled in the art can obtain isolating TRAP and/or by its deutero-TRA according to the separation known method of protein.Concrete method includes but not limited to immune chromatograph, HPLC, size exclusion chromatogram, ion-exchange chromatography and immune affinity chromatographic.Processed and with TRA or with TRA and HLA when for example the form of the mixture of HLA-B7 is presented when these molecules, the material that they can also be combined with other is as being used for the treatment of attached dose that vaccine with the disorder that is expressed as feature of TRAP molecule uses for preparation.In addition, the preparation of vaccine also can be used the cell that has the TRA/HLA mixture to present from the teeth outwards, the cancer cell of non-breeding for example, transfection thing of non-breeding or the like.Use in the vaccine of cell at all these, cell can be the encoding sequence institute cells transfected through needed one or both compositions of stimulation oversaturation ctl response, or does not need transfection just can express the cell of these two kinds of molecules.Vaccine also comprises the naked DNA or the RNA of coding RAGE TRA or its precursor, and they can be external generations, and the method for use can be injection, particle bombardment, nasal spray or other method.The vaccine of " naked nucleic acid " type has been proved to be can immune response stimulating, comprises that stimulation is the generation (Science 259:1745-1748,1993) of specific CTL to the coded peptide of naked nucleic acid.

The complex body of TRAP molecule, relative TRA and TRA and HLA can prepare antibody by standard method known in the art.The document of the main governing principle of introducing the antibody producing standard technique has, Catty.D., Antibodies.A Practical Approach.Vol.1.IRL Press.Washington DC (1988); Klein.J., Immunology:The Science of Cell-Non-Cell Discrimination.John Wileyand Sons.New York (1982); Kennett, R.et al., Monoclonal Antibodies, Hybridoma.A New Dimension In Biological Analyses,Plenum Press, New York (1980); Campbell.A., Monoclonal Antibody Technology,In Laboratory Techniques and Biochemistry and Molecular Biology,Vol.13 (Burdon, R.et al.EDS.), Elsevier Amsterdam (1984); And Eisen.H.N., Microbiology,Third edition, Davis, B.D.et al.EDS.[Harper﹠amp; Rowe, Philadelphia (1980)].

The preparation of antibody of the present invention is to use any suitable method to bring out the generation of polyclonal antibody for protein, proteinic fragment, the cell etc. of expressing these protein or protein fragments animal.The generation of monoclonal antibody is according to method well known in the art.As described in detail herein, such antibody can be used for identifying the tissue or the protein purification of marking protein.Antibody can also be combined with labelled reagent or the antineoplastic agent that is used for imaging, include but not limited to methotrexate, radioiodinated compound, toxin for example ricin, other cell suppress or cytolytic medicine or the like.Antibody prepared in accordance with the present invention is specific to TRA/HLA mixture as herein described preferably.

Term used herein " disorder " refers to the pathological state that any tumor rejection antigen precursor is expressed, for example cancer, particularly kidney.

Prerequisite according to methods of treatment more of the present invention is the reaction of patient's immune system, causes TRA to present the dissolving of cell, for example HLA-B7 cell.A kind of in this method is that the patient to the improper cell with described phenotype uses mixture is specific from body CTL.Those skilled in the art can be at this CTL of external preparation.Generally speaking, fetch cell sample on one's body from the patient earlier, for example hemocyte contacts with the cell that presents described mixture and can stimulate CTL to breed then.Target cell can be the transfection thing, for example the COS cell of the above type.These transfection things are presenting required mixture in its surface, and when combining with required CTL, stimulate its breeding.The COS cell, COS cell for example described herein can be from a lot of local acquisitions, and other suitable host cells also is like this.Production to ctl clone has provided concrete description in the above.To give the patient from body CTL through what the clone expanded then.Other RAGE-1 is specific CTL or is that specific CTL can separate and uses with similar methods to RAGE TRA by RAGE-2,3 or 4 codings.

For realizing concrete methods of treatment, the method that for example is called acquired transfer is [referring to Greenberg.J.Immunol.136 (5): 1917 (1986); Riddel et al., Science257:238 (7-10-92); Lynch et al., Eur.J.Immunol.21:1403-1410 (1991); Kast et al., Cell 59:603-614 (11-17-89)], the cell that will present required mixture combines with CTL, causes the breeding to this specific CTL.CTL with breeding gives the patient of suffering from cellular abnormality subsequently, and this is unusual is characterized as the cellular abnormality that presents concrete mixture.Then, CTL just dissolves abnormal cells, thereby reaches required therapeutic goal.

The theory of above-mentioned treatment is that at least some patients' abnormal cells presents relevant HLA/TRA mixture.This point is very easy to determine that those skilled in the art are familiar with identifying the method for the cell that presents a certain HLA molecule, and the method for identifying the cell of expressing the DNA with correlated series, is the RAGE sequence at this.In case after having identified the cell that presents relevant mixture by above-mentioned screening method, they are contacted with patient's the sample that contains CTL.If present the sample dissolution of the mixed CTL of cell of mixture, so just can suppose the RAGE derivation, presented TRA, then the patient is exactly the patient who is suitable for using above-mentioned methods of treatment.

Acquired transfer is not unique methods of treatment that the present invention allows.Many methods can make CTL obtain in vivo stimulating.The non-propagated cell of the expression mixture that one of method had described in detail above just being to use.The cell of Shi Yonging can be the cell of expressing mixture usually in the method, for example passes through the radiating tumour cell, perhaps uses for presenting the cell that mixture is necessary one or both gene transfections.People such as Chen are published in the article of Proc.Natl.Acad.Sci.USA 88:110-114 (January, 1991), have provided the example of this method, introduced the use cells transfected and express the HPVE7 peptide in therapeutic process.Operable cell has multiple.Similarly, can use the carrier that contains one or both required genes.Preferred virus and bacteroidal carrier.For example, be operably connected to the nucleic acid of coding RAGE TRA on the promotor or be connected on the enhancement sequences of in some tissue or cell, directly expressing RAGE TRA.Nucleic acid be directed in the expression vector.Expression vector can be not modified exogenous chromosome nucleic acid, plasmid, or in order to insert exogenous nucleic acid for example the encode nucleic acid of RAGE TRA and genome through modifying and making up.The nucleic acid of coding RAGE TRA can also be inserted in the retroviral gene group, thereby promotes the integration of this nucleic acid in the genome of destination organization or cell.In these systems, needed gene is carried by microorganism, for example, VacciniaVirus, retroviral or bacterium BCG, and the material that in fact " infects " host cell.Resulting cell presents required mixture, is discerned from body CTL, makes the CTL breeding then.

Combine with promoting the assistant agent of introducing HLA-B7 in vivo by TRAT or its irritating fragment, also can obtain similar effects.TRAP obtain the complementary peptide of HLA molecule, and presenting of TRA does not need further processing through after processing.Generally speaking, the patient can accept the RAGE TRAP of intradermal injection effective dose, and/or by its deutero-TRA.After the predose, can strengthen dosage, carry out according to the routine immunization handbook of this area.

As the part of immune handbook, can cause that immunoreactive material can be used with the nucleic acid of cancer vaccine or the composition of peptide.Thisly can cause that immunoreactive compound can be classified as assistant agent and cytokine.Assistant agent can come the enhancing immunity reaction by antigenic storehouse (cell external source or in scavenger cell) is provided, activated macrophage and stimulate specific lymphocyte.Many known assistant agents are arranged in this area, concrete example has MPL (SmithKline Beecham), through purification and the acidic hydrolysis of Salmonella minnesota Re 595 fat saccharans is obtained different be homogenic, QS21 (SmithKlineBeecham), the QA-21 Saponin/TSM of purifying from Quillja saponaria extract, and by the multiple biological level oil water-in-oil emulsion of shark alkene and/or tocopherol preparation for example.Cytokine is useful owing to have the lymphocytic character of stimulation in immune handbook.Those skilled in the art knows the cytokine of multiple this class, comprises plain-12 (IL-12) of human body Jie, and it has shown the enhancing protective nature (Science 268:1432-1434,1995) to vaccine.

When in use, therapeutic composition of the present invention is with the form administration of the acceptable preparation of pharmacology.This class preparation can contain the salt of the acceptable concentration of pharmacology usually, buffer reagent, and preservatives, compatible supporting agent, potential dose of complementary immunity, for example assistant agent and cytokine can also contain or not contain other medicament.

Preparation of the present invention uses with effective dose.Effective dose refers to the of a kind of medicament own or stimulates the dosage that obtains required reaction with other medicament.In the treatment cancer, required reaction is the development that suppresses cancer.Can be the advancing of disease of temporarily slowing down, even more ideal be permanently to stop advancing of disease.Method with routine just can detect these results, or reaches by diagnostic method of the present invention.

When needs the time, can comprise the raising that stimulates the humoral antibody reaction to obtain antibody titer in the serum, clone's property expansion cytotoxic lymphocyte, or other required immunological response with pharmaceutical composition immune response stimulating of the present invention.It is believed that immunogenic dosage is effective at 1 nanogram/kilogram according to the method for using to 100 milligrams/kilogram scope.The preferred dosage scope is the scopes of 500 nanogram/kilograms to 500 microgram/kilograms.Actual dosage will depend on numerous factors, comprise the material of selecting for use, single dose or multidose, patient's parameter, age for example, physical appearance, height, body weight, and the degree of disease.These factors are all known those skilled in the art, and they can make them clear according to the experiment of routine.

Other aspect of the present invention is all understood those skilled in the art, need not give unnecessary details.

Term used herein and expression all are descriptive, rather than determinate, use these terms and express the meaning of the equivalent that does not repel any above-mentioned feature and part, it should be understood that within the scope of the invention to also have numerous modification.

Document

1. " (chromosomal localization the library of extracting and the sequence mark of expression from retinal pigment epithelium " (Gleser, L., and Swaroop, A.1992.Expressed sequencetags and chromosomal localization of cDNA clones from asubtracted retinal pigment epithelium library. Genomics 13.873-876.)

2. " have and the relevant endogenous retroviral of people similar genome C type pol sequence and the gag sequence of human T-cell's lymph virus " (Mager, D., and Freeman, J.D.1987.Human endogenous retrovirus-like genome with Type C polsequences and gag sequences related to human T-cell lymphotropicviruses.J.Virol.61., 4060-4066.)

3. " existence of env gene among the RTVL-H group membership in human endogenous retroviral analogous components " (Hirose, Y., Takamatsu.M., Harada, F.1993, Presence of envgenes in members of the RTVL-H family of human endogenousretrovirus-like elements.Virology 192,52-61.)

Sequence table (1) background information

(i) applicant:

(A) title: LUDWIG ICR

(B) street address: No. 1345, Americas street

(C) city: New York

(D) state name: New York

(E) country: the U.S.

(F) postcode: 10105

(i) applicant:

(A) title: LEIDEN university

(B) street address: No. 46, Stationsweg street

(C) city: Leiden

(D) country: Holland

(E) postcode: 2312AV

(ii) denomination of invention: RAGE Tumor rejection antigen (RAGE TUMOR

REJECTION?ANTIGENS)

(iii) sequence number: 57

(iv) mailing address:

(A) addressee: WOLF, GREENFIELD ﹠amp; SACKS, P.C.

(B) street address: No. 600, street, the Atlantic Ocean

(C) city: Boston

(D) state name: Massachusetts

(E) country: the U.S.

(F) postcode: 02210

(v) computer reading method

(A) media types: floppy disk

(B) computer: IBM PC compatibility

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, #1.25 version

(vi) apply for present situation:

(A) application number:

(B) submission date:

(C) classification:

(vii) in the first to file situation:

(A) application number: US 08/401,015

(B) submission date: March 21 nineteen ninety-five

(vii) in the first to file situation:

(A) application number: US 08/530,569

(B) submission date: September 20 nineteen ninety-five

(viii) lawyer/proxy's information:

(A) name: GATES, EDWARDR.

(B) herd number: 31,616

(C) search/file number: L0461/7002WO

(ix) telecom number:

(A) phone: 617-720-3500

(B) fax: 617-720-2441 (2) the 1st sequence data: (i) sequence signature:

(A) length: 1311 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) originate:

( A ) ： ( xi ) ：1：GGTGAGCAGC CAAAGCAGGC ATCCCCGCAG TTGACTTGCC ACCAAGGGAA TGTGGGTGAA 60TGACCAAGGC AGGCATCCTC GCGGTGATCA GACACCAATG GAGTGTGGGT GAATAATCAG 120GCAGGCATCC CCGCAGTGAT TAAACACCAA GAGAAGACTA TTCCTGAGTC TGTGACTGGT 180GCTGGAGTTT TGAGTCCACA GATAAAATGT GTCTCCTTCG TCTCTACTAG AGAGGAAAAA 240GAACTGGAAT TGGAAGAACA GGGAGACTGA AGGGTAGCAA GAGAGGCTGG AGAAGAGAGT 300GAAAAGACCG CTTACCTGAT TTGAAATTGT CTGCAGCCCC TCTTTCCTGG AGTAAATGAA 360CTGGACCAAA TCTCAAAAAA TCCACGATGT CATCGGCACA CCCGCTCAGA AGATCCTCAC 420CAAGTTCAAA CAGGATCAGG AATACCTCTA CTAACAACCA ATTTGTCCCC ACAATGCCTC 480TCCCTCCTGC ACGCAATGGT GGCCTATGAT CCCGATGAGA GAATCGCCGC CCACCAGGCC 540CTGCAGCACC CCTACTTCCA AGAACAGAGA AACAGTCCCT AAAGCAAGAG GAGGACCGTC 600CCAAGAGACG AGGACCGGCC TATGTCATGG AACTGCCCAA ACTAAAGCTT TCGGGAGTGG 660TCAGACTGTC GTCTTACTCC AGCCCCACGC TGCAGTCCGT GCTTGGATCT GGAACAAATG 720GAAGAGTGCC GGTGCTGAGA CCCTTGAAGT GCATCCCTGC GAGCAAGAAG ACAGATCCGC 780AGAAGGACCT TAAGCCTGCC CCGCAGCAGT GTCGCCTGCC CACCATAGTG CGGAAAGGCG 840GAAGATAACT GAGCAGCACC GTCGTCTCGA CTTCGGAGGC AACACCAAGC CCGACCGGGC 900CAGGCCTGGG TGATCTGCTG CTGAGACGCC ACGGAGGGCT GGGGATGCGC CTGCGTCCGT 960TTCGCGCTGG CCGGGGCTCT GGGTGCTGCC CTGCGCCCTG CCGCACCCGC GGCCCGCGCA 1020GCTGCCTAGG ATGTTCTGGG CTAATATACT TGTAAAACCA CCGCATTCTA GGGTTTTCTT 1080TCATTTTCGT TAAGAATTTG GGGCAGGAAA TACTTTGTAA CTTTGTATAT GAATCAAAAC 1140AAACGAGCAG GCATTTCTGT GATGTGTTGG GCGTGGTTGG AAGGTGGGTT CTGCGTGTCC 1200CTTCCCAGCG CTGCTGGTCA GTCGTGGAGC GCCATCATGT CTTACCAGTG ACGCTGCTGA 1260CACCCCTGAC TTTTATTAAA GAATAAGCTG TCGTTAAAAA AAAAAAAAAA A 1311 ( 2 ) 2： ( i ) ：

(A) length: 99 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..99, (xi) sequence description: the 2nd sequence: ATG AAC TGG ACC AAA TCT CAA AAA ATC CAC GAT GTC ATC GGC ACA CCC 48Met Asn Trp Thr Lys Ser Gln Lys Ile His Asp Val Ile Gly Thr Pro 15 10 15GCT CAG AAG ATC CTC ACC AAG TTC AAA CAG GAT CAG GAA TAC CTC TAC 96Ala Gln Lys Ile Leu Thr Lys Phe Lys Gln Asp Gln Glu Tyr Leu Tyr

20 25 30TAA, 99 (2) the 3rd sequence datas: (i) sequence signature:

(A) length: 32 amino acid

(B) type: amino acid

(D) conformation: linear (ii) molecule pattern: protein (xi) sequence description: No. 3 sequence: Met Asn Trp Thr Lys Ser Gln Lys Ile His Asp Val Ile Gly Thr Pro 15 10 15Ala Gln Lys Ile Leu Thr Lys Phe Lys Gln Asp Gln Glu Tyr Leu Tyr

20 25 30 (2) the 4th sequence datas: (i) sequence signature:

(A) length: 123 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..123, (xi) sequence description: the 4th sequence: ATG TCA TCG GCA CAC CCG CTC AGA AGA TCC TCA CCA AGT TCA AAC AGG 48Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Arg 15 10 15ATC AGG AAT ACC TCT ACT AAC AAC CAA TTT GTC CCC ACA ATG CCT CTC 96Ile Arg Asn Thr Ser Thr Asn Asn Gln Phe Val Pro Thr Met Pro Leu

20 25 30CCT CCT GCA CGC AAT GGT GGC CTA TGA 123Pro Pro Ala Arg Asn Gly Gly Leu

35 40 (2) the 5th sequence datas: (i) sequence signature:

(A) length: 40 amino acid

(B) type: amino acid

(D) conformation: linearity is the molecule pattern (ii): protein (xi) sequence description: No. 5 sequence: Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Arg 15 10 15Ile Arg Asn Thr Ser Thr Asn Asn Gln Phe Val Pro Thr Met Pro Leu

20 25 30Pro Pro Ala Arg Asn Gly Gly Leu

35 40 (2) the 6th sequence datas: (i) sequence signature:

(A) length: 87 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..87, (xi) sequence description: the 6th sequence: ATG GTG GCC TAT GAT CCC GAT GAG AGA ATC GCC GCC CAC CAG GCC CTG 48Met Val Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu 15 10 15CAG CAC CCC TAC TTC CAA GAA CAG AGA AAC AGT CCC TAA 87Gln His Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro

20 25 (2) the 7th sequence datas: (i) sequence signature:

(A) length: 28 amino acid

(B) type: amino acid

(D) conformation: linearity is the molecule pattern (ii): protein (xi) sequence description: No. 7 sequence: Met Val Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu 15 10 15Gln His Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro

20 25 (2) the 8th sequence datas: (i) sequence signature:

(A) length: 288 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iV) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..288, (xi) sequence description: the 8th sequence: ATG ATC CCG ATG AGA GAA TCG CCG CCC ACC AGG CCC TGC AGC ACC CCT 48Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro 15 10 15ACT TCC AAG AAC AGA GAA ACA GTC CCT AAA GCA AGA GGA GGA CCG TCC 96Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser

20 25 30CCA GAG ACG AGG ACC GGC CTA TGT CAT GGA ACT GCC CAA ACT AAA GCT 144Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala

35 40 45TTC GGG AGT GGT CAG ACT GTC GTC TTA CTC CAG CCC CAC GCT GCA GTC 192Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val

50 55 60CGT GCT TGG ATC TGG AAC AAA TGG AAG AGT GCC GGT GCT GAG ACC CTT 240Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu 65 70 75 80GAA GTG CAT CCC TGC GAG CAA GAA GAC AGA TCC GCA GAA GGA CCT TAA 288Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro

85 90 95 (2) the 9th sequence datas: (i) sequence signature:

(A) length: 95 amino acid

(B) type: amino acid

(D) conformation: linearity is the molecule pattern (ii): protein (xi) sequence description: No. 9 sequence: Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro

1 5 10 15Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala ATg Gly Gly Pro Ser

20 25 30Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala

35 40 45Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val

50 55 60Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu?65 70 75 80Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro

85 90 95 (2) the 10th sequence datas: (i) sequence signature:

(A) length: 222 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..222, (xi) sequence description: the 10th sequence: ATG GAA CTG CCC AAA CTA AAG CTT TCG GGA GTG GTC AGA CTG TCG TCT 48Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser 15 10 15TAC TCC AGC CCC ACG CTG CAG TCC GTG CTT GGA TCT GGA ACA AAT GGA 96Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly

20 25 30AGA GTG CCG GTG CTG AGA CCC TTG AAG TGC ATC CCT GCG AGC AAG AAG 144Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys

35 40 45ACA GAT CCG CAG AAG GAC CTT AAG CCT GCC CCG CAG CAG TGT CGC CTG 192Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu

50 55 60CCC ACC ATA GTG CGG AAA GGC GGA AGA TAA 222Pro Thr Ile Val Arg Lys Gly Gly Arg, 65 70 (2) the 11st sequence datas: (i) sequence signature:

(A) length: 73 amino acid

(B) type: amino acid

(D) conformation: linear (ii) molecule pattern: protein (xi) sequence description: o.11 sequence: Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser 15 10 15Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly

20 25 30Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys

35 40 45Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu

50 55 60Pro Thr Ile Val Arg Lys Gly Gly Arg, 65 70 (2) the 12nd sequence datas: (i) sequence signature:

(A) length: 1168 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

( A ) ： ( xi ) ：12：CTGTGACTGG TGCTGGAGTT TTGAGTCCAC AGATAAAATG TGTCTCCTTC GTCTCTACTA 60GAGAGGAAAA AGAACTGGAA TTGGAAGAAC AGGGAGACTG AAGGGTAGCA AGAGAGGCTG 120GAGAAGAGAG TGAAAAGACC GCTTACCTGA TTTGAAATTG TCTGCAGCCC CTCTTTCCTG 180GAGTAAATGA ACTGGACCAA ATCTCAAAAA TCCACGATGT CATCGGCACA CCCGCTCAGA 240AGATCCTCAC CAAGTTCAAA CAGTCGAGAG CTATGAATTT TGATTTTCCT TTTAAAAAGG 300GATCAGGAAT ACCTCTACTA ACAACCAATT TGTCCCCACA ATGCCTCTCC CTCCTGCACG 360CAATGGTGGC CTATGATCCC GATGAGAGAA TCGCCGCCCA CCAGGCCCTG CAGCACCCCT 420ACTTCCAAGA ACAGAGAAAC AGTCCCTAAA GCAAGAGGAG GACCGTCCCA AGAGACGAGG 480ACCGGCCTAT GTCATGGAAC TGCCCAAACT AAAGCTTTCG GGAGTGGTCA GACTGTCGTC 540TTACTCCAGC CCCACGCTGC AGTCCGTGCT TGGATCTGGA ACAAATGGAA GAGTGCCGGT 600GCTGAGACCC TTGAAGTGCA TCCCTGCGAG CAAGAAGACA GATCCGCAGA AGGACCTTAA 660GCCTGCCCCG CAGCAGTGTC GCCTGCCCAC CATAGTGCGG AAAGGCGGAA GATAACTGAG 720CAGCACCGTC GTCTCGACTT CGGAGGCAAC ACCAAGCCCG ACCGGGCCAG GCCTGGGTGA 780TCTGCTGCTG AGACGCCACG GAGGGCTGGG GATGCGCCTG CGTCCGTTTC GCGCTGGCCG 840GGGCTCTGGG TGCTGCCCTG CGCCCTGCCG CACCCGCGGC CCGCGCAGCT GCCTAGGATG 900TTCTGGGCTA ATATACTTGT AAAACCACCG CATTCTAGGG TTTTCTTTCA TTTTCGTTAA 960GAATTTGGGG CAGGAAATAC TTTGTAACTT TGTATATGAA TCAAAACAAA CGAGCAGGCA 1020TTTCTGTGAT GTGTTGGGCG TGGTTGGAAG GTGGGTTCTG CGTGTCCCTT CCCAGCGCTG 1080CTGGTCAGTC GTGGAGCGCC ATCATGTCTT ACCAGTGACG CTGCTGACAC CCCTGACTTT 1140TATTAAAGAA TAAGCTGTCG TTAAAAAA 1168 ( 2 ) 13： ( i ) ：

(A) length: 1235 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

( A ) ： ( xi ) ：13：ATTCCTGAGT CTGTGACTGG TGCTGGAGTT TTGAGTCCAC AGATAAAATG TGTCTCCTTC 60GTCTCTACTA GAGAGGAAAA AGAACTGGAA TTGGAAGAAC AGGGAGACTG AAGGGTAGCA 120AGAGAGGCTG GAGAAGAGAG TGAAAAGACC GCTTACCTGA TTTGAAATTG ATGGTGGCGT 180GGGAATGAAG AATGTGATAT ACATCTTTGG AGTCTGTTCT GCAGCCCCTC TTTCCTGGAG 240TAAATGAACT GGACCAAATC TCAAAAATCC ACGATGTCAT CGGCACACCC GCTCAGAAGA 300TCCTCACCAA GTTCAAACAG TCGAGAGCTA TGAATTTTGA TTTTCCTTTT AAAAAGGGAT 360CAGGAATACC TCTACTAACA ACCAATTTGT CCCCACAATG CCTCTCCCTC CTGCACGCAA 420TGGTGGCCTA TGATCCCGAT GAGAGAATCG CCGCCCACCA GGCCCTGCAG CACCCCTACT 480TCCAAGAACA GAGAAACAGT CCCTAAAGCA AGAGGAGGAC CGTCCCAAGA GACGAGGACC 540GGCCTATGTC ATGGAACTGC CCAAACTAAA GCTTTCGGGA GTGGTCAGAC TGTCGTCTTA 600CTCCAGCCCC ACGCTGCAGT CCGTGCTTGG ATCTGGAACA AATGGAAGAG TGCCGGTGCT 660GAGACCCTTG AAGTGCATCC CTGCGAGCAA GAAGACAGAT CCGCAGAAGG ACCTTAAGCC 720TGCCCCGCAG CAGTGTCGCC TGCCCACCAT AGTGCGGAAA GGCGGAAGAT AACTGAGCAG 780CACCGTCGTC TCGACTTCGG AGGCAACACC AAGCCCGACC GGGCCAGGCC TGGGTGATCT 840GCTGCTGAGA CGCCACGGAG GGCTGGGGAT GCGCCTGCGT CCGTTTCGCG CTGGCCGGGG 900CTCTGGGTGC TGCCCTGCGC CCTGCCGCAC CCGCGGCCCG CGCAGCTGCC TAGGATGTTC 960TGGGCTAATA TACTTGTAAA ACCACCGCAT TCTAGGGTTT TCTTTCATTT TCGTTAAGAA 1020TTTGGGGCAG GAAATACTTT GTAACTTTGT ATATGAATCA AAACAAACGA GCAGGCATTT 1080CTGTGATGTG TTGGGCGTGG TTGGAAGGTG GGTTCTGCGT GTCCCTTCCC AGCGCTGCTG 1140GTCAGTCGTG GAGCGCCATC ATGTCTTACC AGTGACGCTG CTGACACCCC TGACTTTTAT 1200TAAAGAATAA GCTGTCGTTA CAGTATTGCA AAAAA 1235 ( 2 ) 14： ( i ) ：

(A) length: 2051 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

( A ) ： ( xi ) ：14：GACTGGTGCT GGAGTTTTGA GTCCACAGAT AAAATGTGTC TCCTTCGTCT CTACTAGAGA 60GGAAAAAGAA CTGGAATTGG AAGAACAGGG AGACTGAAGG GTAGCAAGAG AGGCTGGAGA 120AGAGAGTGAA AAGACCGCTT ACCTGATTTG AAATTGTCTG CAGCCCCTCT TTCCTGGAGT 180AAATGAACTG GACCAAATCT CAAAAATCCA CGATGTCATC GGCACACCCG CTCAGAAGAT 240CCTCACCAAG TTCAAACAGT CGAGAGCTAT GAATTTTGAT TTTCCTTTTA AAAAGGGATC 300AGGAATACCT CTACTAACAA CCAATTTGTC CCCACAATGC CTCTCCCTCC TGCACGCAAT 360GGTGGCCTAT GATCCCGATG AGAGAATCGC CGCCCACCAG GCCCTGCAGC ACCCCTACTT 420CCAAGAACAG AGAACCCAGA ACGGAAGCGA GGATGAAGGC CTCAGCCGTC CTCCTCCCCA 480TTCAAACACG TTCATCCCTC AACCCTCTGC TGAGCACCTG CATGCTGCCC GGCCGCAGTG 540TCACCCTTCT TGTGTGAGCC TACCCTCATC CACCCACCTC ACCCTCCTGA CCTTAAAGAA 600GACACCGGGC AGAAGCACAG GGGAGCCCAG TCACACCCCA CACTGGCGGG GGCAGGCCTT 660GCAGGGAGAA GCAGTAAGCA GCCATCTCCA TCAGCCATTT CCATCTGGCA CTCAGACGTG 720CACGTCTTCG TGTGACAGGC GGCAGCAGTG CGACCGTGAC CTCCCATCTG CTCTGCTGTC 780CCCACACCTG CGGTGCAGCC AGCCTGCCAC AAGGCAGCTA GAGTCCAGCT AGACCCACCC 840CTGGCACGGC CGACCTCTTC CTGGCTTCTT CTGGGCCTAA TCCCCGTGCA TTCTCCAACG 900CCAGAAGTGT AAGAAAGTGC AAGGCAACAA GTGAGAAGAG CAAACCCAAA TCGTACCAGG 960GAAGCTAGTC TTTCCAGGGC ACCTGAGTGA GGGCATGACC AGCCTTGACG CTGCCTCGCT 1020ACCATCTGCC CAGGGCCTGC TGAATGCTTG AGTCCATGGT GACAGTGGTG GGAACAGTTA 1080CGAGGCAGTT AGATTTTGGA AGTCATGTTG GCCCACTTGG CTACAGAGCA GTCTTAGGAA 1140CAGCACCATA AAAATAAAGA CTTATTCCTG ACACACATGC ATCTAGAGTA AACTGGGGCG 1200TATCTGACAG CGTTAGTACA GTGATGGCCA AATGCAAACT GCATTCCAGA ACCAGCGAAG 1260GGTGACAGAC TGGGCTGAGG CAGAGCTAGG ACTAACCATC TCGAGTGATG CCATCTCGGG 1320GCCAACAAAA GTTTTGGACA CGGCTGGATC ATCTGACCAA ACTGCTCAAA TCTTTACACA 1380ATTATTGTCC TGGTATTAAA CTTTCACCTG CCACTTCCAA CAAACAGGAG ACAGAATAAG 1440GAGATGACCA GGAAGATGGC TGGATTAAGA ATTCTAGACT TGGCCGGGTG CGGTGGCTCA 1500CACCTGTAAT CCTAGCATCT TGGGAGGCTG AGGCAGGAGG ATCGCTTGAG CCCAGAGTTT 1560GAGACCAGCC TAGGCAACAT AAGGAGACCC CATCTCTACA AAATATCAAA AAATTACCCA 1620GGTATGGTGG CACACACGTG TGATTCCAGC TACTCGGGAG GCTGAGATGG GAGGATCACT 1680TGAACCCAGG AGGTTGGGGC TACAATGAGC TATGATCGCA CCACTTCATT CCAGCCTGGA 1740TGACAGAAGA CTCACTCCAT AGTTCATGGC CCCGTGATCC AGAGTCCCTG CTGGCGCCTT 1800CGAGTGGGGC AGGCTGAGAA CTCAAGCTGT AACTAATGTC TCCTCCGAAG AAAACTAAAC 1860CGAGGGCTGA GCTGATGTGA AGTTTTCCGT GGCTGCATTC ATACAAATGG TGAAAATGTA 1920GCATACCTCC CCTCAAAAGC CTGAAAGTAA AGACATGCCC CCAATTTAAT GTGATGAATT 1980AGAGAAATAG GTTTCAGACA CTTCATGGTT TAAAGTCTCA CAAAATAAAG CTTTCGAAGG 2040AAAAAAAAAA A 2051 ( 2 ) 15： ( i ) ：

(A) length: 60 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..60, (xi) sequence description: the 15th sequence: ATG TCA TCG GCA CAC CCG CTC AGA AGA TCC TCA CCA AGT TCA AAC AGT 48Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser 15 10 15CGA GAG CTA TGA 60Arg Glu Leu, (2) the 16th sequence datas:, (i) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) conformation: linearity is the molecule pattern (ii): protein (xi) sequence description: No. 16 sequence: Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser 15 10 15Arg Glu Leu (2) the 17th sequence data: (i) sequence signature:

(A) length: 177 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..177, (xi) sequence description: the 17th sequence: ATG AAT TTT GAT TTT CCT TTT AAA AAG GGA TCA GGA ATA CCT CTA CTA 48Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu 15 10 15ACA ACC AAT TTG TCC CCA CAA TGC CTC TCC CTC CTG CAC GCA ATG GTG 96Thr Thr Asn Leu Ser Pro Gln Cys Leu Ser Leu Leu His Ala Met Val

20 25 30GCC TAT GAT CCC GAT GAG AGA ATC GCC GCC CAC CAG GCC CTG CAG CAC 144Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His

35 40 45CCC TAC TTC CAA GAA CAG AGA AAC AGT CCC TAA 177Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro

50 55 (2) the 18th sequence datas: (i) sequence signature:

(A) length: 58 amino acid

(B) type: amino acid

(D) conformation: linear (ii) molecule pattern: protein (xi) sequence description: No. 18 sequence: Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu 15 10 15Thr Thr Asn Leu Ser Pro Gln Cys Leu Ser Leu Leu His Ala Met Val

20 25 30Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His

35 40 45Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro

50 55 (2) the 19th sequence datas: (i) sequence signature:

(A) length: 288 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..288, (xi) sequence description: the 19th sequence: ATG ATC CCG ATG AGA GAA TCG CCG CCC ACC AGG CCC TGC AGC ACC CCT 48Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro 15 10 15ACT TCC AAG AAC AGA GAA ACA GTC CCT AAA GCA AGA GGA GGA CCG TCC 96Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser

20 25 30CAA?GAG?ACG?AGG?ACC?GGC?CTA?TGT?CAT?GGA?ACT?GCC?CAA?ACT?AAA?GCT 144Gln?Glu?Thr?Arg?Thr?Gly?Leu?Cys?His?Gly?Thr?Ala?Gln?Thr?Lys?Ala

35 40 45TTC?GGG?AGT?GGT?CAG?ACT?GTC?GTC?TTA?CTC?CAG?CCC?CAC?GCT?GCA?GTC 192Phe?Gly?Ser?Gly?Gln?Thr?Val?Val?Leu?Leu?Gln?Pro?His?Ala?Ala?Val

50 55 60CGT GCT TGG ATC TGG AAC AAA TGG AAG AGT GCC GGT GCT GAG ACC CTT 240Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu 65 70 75 80GAA GTG CAT CCC TGC GAG CAA GAA GAC AGA TCC GCA GAA GGA CCT TAA 288Glu Val His Pro Cys Glu Gin Glu Asp Arg Ser Ala Glu Gly Pro

85 90 95 (2) the 20th sequence datas: (i) sequence signature:

(A) length: 95 amino acid

(B) type: amino acid

(D) conformation: linearity is the molecule pattern (ii): protein (xi) sequence description: No. 20 sequence: Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro 15 10 15Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser

20 25 30Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala

35 40 45Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val

50 55 60Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu?65 70 75 80Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro

85 90 95 (2) the 21st sequence datas: (i) sequence signature:

(A) length: 222 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..222, (xi) sequence description: the 21st sequence: ATG GAA CTG CCC AAA CTA AAG CTT TCG GGA GTG GTC AGA CTG TCG TCT 48Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser 15 10 15TAC TCC AGC CCC ACG CTG CAG TCC GTG CTT GGA TCT GGA ACA AAT GGA 96Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly

20 25 30AGA GTG CCG GTG CTG AGA CCC TTG AAG TGC ATC CCT GCG AGC AAG AAG 144Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys

35 40 45ACA GAT CCG CAG AAG GAC CTT AAG CCT GCC CCG CAG CAG TGT CGC CTG 192Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu

50 55 60CCC ACC ATA GTG CGG AAA GGC GGA AGA TAA 222Pro Thr Ile Val Arg Lys Gly Gly Arg, 65 70 (2) the 22nd sequence datas: (i) sequence signature:

(A) length: 73 amino acid

(B) type: amino acid

(D) conformation: linear (ii) molecule pattern: protein (xi) sequence description: No. 22 sequence: Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser 15 10 15Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly

20 25 30Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys

35 40 45Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu

50 55 60Pro Thr Ile Val Arg Lys Gly Gly Arg, 65 70 (2) the 23rd sequence datas: (i) sequence signature:

(A) length: 63 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..63 (xi) sequence description: the 23rd sequence: ATG AAG AAT GTG ATA TAC ATC TTT GGA GTC TGT TCT GCA GCC CCT CTT 48Met Lys Asn Val Ile Tyr Ile Phe Gly Val Cys Ser Ala Ala Pro Leu 15 10 15TCC TGG AGT AAA TGA 63Ser Trp Ser Lys

20 (2) the 24th sequence datas: (i) sequence signature:

(A) length: 20 amino acid

(B) type: amino acid

(D) conformation: linearity is the molecule pattern (ii): protein (xi) sequence description: No. 24 sequence: Met Lys Asn Val Ile Tyr Ile Phe Gly Val Cys Ser Ala Ala Pro Leu 15 10 15Ser Trp Ser Lys

20 (2) the 25th sequence datas: (i) sequence signature:

(A) length: 60 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..60, (xi) sequence description: the 25th sequence: ATG TCA TCG GCA CAC CCG CTC AGA AGA TCC TCA CCA AGT TCA AAC AGT 48Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser 15 10 15CGA GAG CTA TGA 60Arg Glu Leu, (2) the 26th sequence datas:, (i) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) conformation: linearity, (ii) molecule pattern: protein, (xi) sequence description: No. 26 sequence: Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser 15 10 15Arg Glu Leu, (2) the 27th sequence datas:, (i) sequence signature:

(A) length: 177 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..177, (xi) sequence description: the 27th sequence: ATG AAT TTT GAT TTT CCT TTT AAA AAG GGA TCA GGA ATA CCT CTA CTA 48Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu 15 10 15ACA ACC AAT TTG TCC CCA CAA TGC CTC TCC CTC CTG CAC GCA ATG GTG 96Thr Thr Asn Leu Ser Pro Gln Cys Leu Ser Leu Leu His Ala Met Val

20 25 30GCC TAT GAT CCC GAT GAG AGA ATC GCC GCC CAC CAG GCC CTG CAG CAC 144Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His

35 40 45CCC TAC TTC CAA GAA CAG AGA AAC AGT CCC TAA 177Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro

50 55 (2) the 28th sequence datas: (i) sequence signature:

(A) length: 58 amino acid

(B) type: amino acid

(D) conformation: linear (ii) molecule pattern: protein (iii) sequence description: No. 28 sequence: Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu 15 10 15Thr Thr Asn Leu Ser Pro Gln Cys Leu Ser Leu Leu His Ala Met Val

20 25 30Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His

35 40 45Pro Tyr Phe Gln Glu Gln Arg Asn Ser Pro

50 55 (2) the 29th sequence datas: (i) sequence signature:

(A) length: 288 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..288, (xi) sequence description: the 29th sequence: ATG ATC CCG ATG AGA GAA TCG CCG CCC ACC AGG CCC TGC AGC ACC CCT 48Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro 15 10 15ACT TCC AAG AAC AGA GAA ACA GTC CCT AAA GCA AGA GGA GGA CCG TCC 96Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser

20 25 30CAA GAG ACG AGG ACC GGC CTA TGT CAT GGA ACT GCC CAA ACT AAA GCT 144Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala

35 40 45TTC GGG AGT GGT CAG ACT GTC GTC TTA CTC CAG CCC CAC GCT GCA GTC 192Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val

50 55 60CGT GCT TGG ATC TGG AAC AAA TGG AAG AGT GCC GGT GCT GAG ACC CTT 240Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu 65 70 75 80GAA GTG CAT CCC TGC GAG CAA GAA GAC AGA TCC GCA GAA GGA CCT TAA 288Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro

85 90 95 (2) the 30th sequence datas: (i) sequence signature:

(A) length: 95 amino acid

(B) type: amino acid

(D) conformation: linear (ii) molecule pattern: protein (iii) sequence description: No. 30 sequence: Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro 15 10 15Thr Ser Lys Asn Arg Glu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser

20 25 30Gln Glu Thr Arg Thr Gly Leu Cys His Gly Thr Ala Gln Thr Lys Ala

35 40 45Phe Gly Ser Gly Gln Thr Val Val Leu Leu Gln Pro His Ala Ala Val

50 55 60Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu?65 70 75 80Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro

85 90 95 (2) the 31st sequence datas: (i) sequence signature:

(A) length: 222 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..222, (xi) sequence description: the 31st sequence: ATG GAA CTG CCC AAA CTA AAG CTT TCG GGA GTG GTC AGA CTG TCG TCT 48Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser 15 10 15TAC TCC AGC CCC ACG CTG CAG TCC GTG CTT GGA TCT GGA ACA AAT GGA 96Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly

20 25 30AGA GTG CCG GTG CTG AGA CCC TTG AAG TGC ATC CCT GCG AGC AAG AAG 144Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys

35 40 45ACA GAT CCG CAG AAG GAC CTT AAG CCT GCC CCG CAG CAG TGT CGC CTG 192Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu

50 55 60CCC ACC ATA GTG CGG AAA GGC GGA AGA TAA 222Pro Thr Ile Val Arg Lys Gly Gly Arg, 65 70 (2) the 32nd sequence datas: (i) sequence signature:

(A) length: 73 amino acid

(B) type: amino acid

(D) conformation: linear (ii) molecule pattern: protein (xi) sequence description: No. 32 sequence: Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser 15 10 15Tyr Ser Ser Pro Thr Leu Gln Ser Val Leu Gly Ser Gly Thr Asn Gly

20 25 30Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys

35 40 45Thr Asp Pro Gln Lys Asp Leu Lys Pro Ala Pro Gln Gln Cys Arg Leu

50 55 60Pro Thr Ile Val Arg Lys Gly Gly Arg, 65 70 (2) the 33rd sequence datas: (i) sequence signature:

(A) length: 60 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..60 (xi) sequence description: the 33rd sequence: ATG TCA TCG GCA CAC CCG CTC AGA AGA TCC TCA CCA AGT TCA AAC AGT 48Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser 15 10 15CGA GAG CTA TGA 60Arg Glu Leu (2) the 34th sequence data: (i) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) conformation: linearity, (ii) molecule pattern: protein, (xi) sequence description: No. 34 sequence: Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser 15 10 15Arg Glu Leu, (2) the 35th sequence datas:, (i) sequence signature:

(A) length: 564 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..564, (xi) sequence description: the 35th sequence: ATG AAT TTT GAT TTT CCT TTT AAA AAG GGA TCA GGA ATA CCT CTA CTA 48Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu 15 10 15ACA ACC AAT TTG TCC CCA CAA TGC CTC TCC CTC CTG CAC GCA ATG GTG 96Thr Thr Asn Leu Ser Pro Gln Cys Leu Ser Leu Leu His Ala Met Val

20 25 30GCC TAT GAT CCC GAT GAG AGA ATC GCC GCC CAC CAG GCC CTG CAG CAC 144Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His

35 40 45CCC TAC TTC CAA GAA CAG AGA ACC CAG AAC GGA AGC GAG GAT GAA GGC 192Pro Tyr Phe Gln Glu Gln Arg Thr Gln Asn Gly Ser Glu Asp Glu Gly

50 55 60CTC AGC CGT CCT CCT CCC CAT TCA AAC ACG TTC ATC CCT CAA CCC TCT 240Leu Ser Arg Pro Pro Pro His Ser Asn Thr Phe Ile Pro Gln Pro Ser 65 70 75 80GCT GAG CAC CTG CAT GCT GCC CGG CCG CAG TGT CAC CCT TCT TGT GTG 288Ala Glu His Leu His Ala Ala Ala Pro Gln Cys His Pro Ser Cys Val

85 90 95AGC CTA CCC TCA TCC ACC CAC CTC ACC CTC CTG ACC TTA AAG AAG ACA 336Ser Leu Pro Ser Ser Thr His Leu Thr Leu Leu Thr Leu Lys Lys Thr

100 105 110CCG GGC AGA AGC ACA GGG GAG CCC AGT CAC ACC CCA CAC TGG CGG GGG 384Pro Gly Arg Ser Thr Gly Glu Pro Ser His Thr Pro His Trp Arg Gly

115 120 125CAG GCC TTG CAG GGA GAA GCA GTA AGC AGC CAT CTC CAT CAG CCA TTT 432Gln Ala Leu Gln Gly Glu Ala Val Ser Ser His Leu His Gln Pro Phe

130 135 140CCA TCT GGC ACT CAG ACG TGC ACG TCT TCG TGT GAC AGG CGG CAG CAG 480Pro Ser Gly Thr Gln Thr Cys Thr Ser Ser Cys Asp Arg Arg Gln Gln 145 150 155 160TGC GAC CGT GAC CTC CCA TCT GCT CTG CTG TCC CCA CAC CTG CGG TGC 528Cys Asp Arg Asp Leu Pro Ser Ala Leu Leu Ser Pro His Leu Arg Cys

165 170 175AGC CAG CCT GCC ACA AGG CAG CTA GAG TCC AGC TAG 564Ser Gln Pro Ala Thr Arg Gln Leu Glu Ser Ser

180 185 (2) the 36th sequence datas: (i) sequence signature:

(A) length: 187 amino acid

(B) type: amino acid

(D) conformation: linearity is the molecule pattern (ii): protein (xi) sequence description: No. 36 sequence Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu 15 10 15Thr Thr Asn Leu Ser Pro Gln Cys Leu Ser Leu Leu His Ala Met Val

20 25 30Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gln Ala Leu Gln His

35 40 45Pro Tyr Phe Gln Glu Gln Arg Thr Gln Asn Gly Ser Glu Asp Glu Gly

50 55 60Leu Ser Arg Pro Pro Pro His Ser Asn Thr Phe Ile Pro Gln Pro Ser?65 70 75 80Ala Glu His Leu His Ala Ala Alg Pro Gln Cys His Pro Ser Cys Val

85 90 95Ser Leu Pro Ser Ser Thr His Leu Thr Leu Leu Thr Leu Lys Lys Thr

100 105 110Pro Gly Arg Ser Thr Gly Glu Pro Ser His Thr Pro His Trp Arg Gly

115 120 125Gln Ala Leu Gln Gly Glu Ala Val Ser Ser His Leu His Gln Pro Phe

130 135 140Pro Ser Gly Thr Gln Thr Cys Thr Ser Ser Cys Asp Arg Arg Gln Gln145 150 155 160Cys Asp Arg Asp Leu Pro Ser Ala Leu Leu Ser Pro His Leu Arg Cys

165 170 175Ser Gln Pro Ala Thr Arg Gln Leu Glu Ser Ser

180 185 (2) the 37th sequence datas: (i) sequence signature:

(A) length: 189 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (ix) characteristics:

(A) title/legend: CDS

(B) location: 1..189, (xi) sequence description: the 37th sequence: ATG ATC CCG ATG AGA GAA TCG CCG CCC ACC AGG CCC TGC AGC ACC CCT 48Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro 15 10 15ACT TCC AAG AAC AGA GAA CCC AGA ACG GAA GCG AGG ATG AAG GCC TCA 96Thr Ser Lys Asn Arg Glu Pro Arg Thr Glu Ala Arg Met Lys Ala Ser

20 25 30GCC GCT CTC CTC CCC ATT CAA ACA CGT TCA TCC CTC AAC CCT CTG CTG 144Ala Val Leu Leu Pro Ile Gln Thr Arg Ser Ser Leu Asn Pro Leu Leu

35 40 45AGC ACC TGC ATG CTG CCC GGC CGC AGT GTC ACC CTT CTT GTG TGA 189Ser Thr Cys Met Leu Pro Gly Arg Ser Val Thr Leu Leu Val

50 55 60 (2) the 38th sequence datas: (i) sequence signature:

(A) length: 62 amino acid

(B) type: amino acid

(D) conformation: linear (ii) molecule pattern: protein (iii) is supposed: do not have (xi) sequence description: the 38th sequence Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro 15 10 15Thr Ser Lys Asn Arg Glu Pro Arg Thr Glu Ala Arg Met Lys Ala Ser

20 25 30Ala Val Leu Leu Pro Ile Gln Thr Arg Ser Ser Leu Asn Pro Leu Leu

35 40 45Ser Thr Cys Met Leu Pro Gly Arg Ser Val Thr Leu Leu Val

50 55 60 (2) the 39th sequence datas: (i) sequence signature:

(A) length: 12 amino acid

(B) type: amino acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): peptide is (iii) supposed: do not have (iv) clip types: interior fragment (v) originate:

(A) organism: people (xi) sequence description: the 39th sequence: Ser Pro Ser Ser Asn Arg Ile Arg Asn Thr Ser Thr1 5 10 (2) the 40th sequence datas: (i) sequence signature:

(A) length: nine amino acid

(B) type: amino acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): peptide is (iii) supposed: do not have (iv) clip types: interior fragment (v) originate:

(A) organism: people (xi) sequence description: the 40th sequence: Ser Pro Ser Ser Asn Arg Ile Arg Asn 15 (2) the 41st sequence datas: (i) sequence signature:

(A) length: nine amino acid

(B) type: amino acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): peptide is (iii) supposed: do not have (iv) clip types: interior fragment (v) originate:

(A) organism: people (xi) sequence description: the 41st sequence: Pro Ser Ser Asn Arg Ile Arg Asn Thr 15 (2) the 42nd sequence datas: (i) sequence signature:

(A) length: nine amino acid

(B) type: amino acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): peptide is (iii) supposed: do not have (iv) clip types: interior fragment (v) originate:

(A) organism: people (xi) sequence description: the 42nd sequence: Ser Ser Asn Arg Ile Arg Asn Thr Ser 15 (2) the 43rd sequence datas: (i) sequence signature:

(A) length: ten amino acid

(B) type: amino acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): peptide is (iii) supposed: do not have (iv) clip types: interior fragment (v) originate:

(A) organism: people (xi) sequence description: the 43rd sequence: Ser Pro Ser Ser Asn Arg Ile Arg Asn Thr 15 10 (2) the 44th sequence datas: (i) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 44th sequence: TCACCAAGTT CAAACAGGAT CAGGAAT 27 (2) the 45th sequence datas: (i) sequence signature:

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 45th sequence: TCACCAAGTT CAAACAGGAT CAGGAATACC 30 (2) the 46th sequence datas: (i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 46th sequence: GTGTCTCCTT CGTCTCTACT A 21 (2) the 47th sequence datas: (i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: exist (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 47th sequence: GGTGTGCCGA TGACATCG 18 (2) the 48th sequence datas: (i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: exist (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 48th sequence: GAGGTATTCC TGATCCTG 18 (2) the 49th sequence datas: (i) sequence signature:

(A) length: 13 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 49th sequence: CAAACANGGA TCA 13 (2) the 50th sequence datas: (i) sequence signature:

(A) length: 14 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 50th sequence: NTATTCCTGA TCCT 14 (2) the 51st sequence datas: (i) sequence signature:

(A) length: 15 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 51st sequence: NTATTCCTGA TCCTG 15 (2) the 52nd sequence datas: (i) sequence signature:

(A) length: 16 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 52nd sequence: NTATTCCTGA TCCTGT 16 (2) the 53rd sequence datas: (i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: do not have (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 53rd sequence: NTATTCCTGA TCCTGTT 17 (2) the 54th sequence datas: (i) sequence signature:

(A) length: 14 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: exist (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 54th sequence: NCAAGTTCAA ACAG 14 (2) the 55th sequence datas: (i) sequence signature:

(A) length: 15 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: exist (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 55th sequence: NCAAGTTCAA ACAGG 15 (2) the 56th sequence datas: (i) sequence signature:

(A) length: 16 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: exist (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 56th sequence: NCAAGTTCAA ACAGGA 16 (2) the 57th sequence datas: (i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain number: strand

(D) conformation: linearity is the molecule pattern (ii): cDNA (iii) supposes: do not have (iv) antisense strand: exist (v) clip types: interior fragment (vi) source:

(A) organism: people (xi) sequence description: the 57th sequence: NCAAGTTCAA ACAGGAT 17

Claims (40)

1. separated polypeptide, it comprises the aminoacid sequence of SEQ.ID.NO.40.

2. separated polypeptide according to claim 1, wherein said separated polypeptide comprises the aminoacid sequence of SEQ.ID.NO.43.

3. separated polypeptide according to claim 1, wherein said separated polypeptide comprise in fact by SEQ.ID.NO.40 and SEQ.ID.NO.43 formed in one group selected aminoacid sequence.

4. separated polypeptide according to claim 1, wherein said separated polypeptide comprises selected aminoacid sequence in a group that is made up of SEQ.ID.NO.40 and SEQ.ID.NO.43.

5. a separated coding is selected from the nucleotide sequence of the polypeptide in the group of being made up of the polypeptide of the polypeptide of the polypeptide of the polypeptide of claim 1, claim 2, claim 3 and claim 4.

6. nucleic acid according to claim 5, wherein said nucleic acid comprises SEQ.ID.NO.44.

7. expression vector, it comprises the separated nucleic acid of the claim 5 of the promotor that is operably connected.

8. expression vector according to claim 7, wherein said nucleic acid comprises SEQ.ID.NO.44.

9. according to claim 7 or 8 described expression vectors, also further comprise the nucleic acid of the HLA-B7 that encodes.

Expression vector institute transfection in the group formed of the expression vector of the expression vector of the expression vector of a selected free claim 7, claim 8 and claim 9 or transformed host cells.

11. the expression vector institute transfection in the group that the expression vector of a selected free claim 7, the expression vector of claim 8 are formed or transformed host cells, wherein said host cell expression HLA-B7.

12. one kind is used to detect the test kit that tumor rejection antigen precursor is expressed, this test kit comprises:

First primer, be selected among the SEQ.ID.NO.50-57 any one nucleic acid and

Second primer is to make up and arrange according to the part of the RAGE1 gene expression characteristics among the ORF2 that optionally increases with first primer.

13. one kind with being the method that the deliquescent T cell of specific cell optionally increases the T cell quantity to the RAGE Tumor rejection antigen, this method comprises:

Separated a group T cell is contacted with the reagent that HLA presents the mixture of molecule with presenting the RAGE Tumor rejection antigen, and employed quantity is to be enough to described separated T cell mass is optionally increased with described cytolysis T cell.

14. method according to claim 13, it is that HLA-B7 and described RAGE Tumor rejection antigen are selected from down group that wherein said HLA presents molecule: the amino acid whose polypeptide and the amino acid whose polypeptide that contains SEQ.ID.NO.43 that contain SEQ.ID.NO.40.

15. the method for the disorder of the expression that a diagnostic characteristic is the RAGE tumor rejection antigenic peptide, this method comprises:

With patient's biological sample with to the RAGE tumor rejection antigenic peptide be specific reagent contact and

Determine the interaction of this reagent and RAGE tumor rejection antigenic peptide, thereby determine this disorder.

16. method according to claim 15, wherein said peptide is selected from down group:

The amino acid whose peptide and the amino acid whose peptide that contains SEQ.ID.NO.43 that contain SEQ.ID.NO.40.

17. a diagnostic characteristic is and the method for the disorder of the RAGE tumor rejection antigenic peptide of HLA-B7 molecule forming composite that this method comprises:

Patient's biological sample is contacted with reagent in conjunction with this mixture; With determine this mixture and this reagent combine conduct definite to this disorder.

18. method according to claim 17, wherein said peptide is selected from down group:

The amino acid whose peptide and the amino acid whose peptide that contains SEQ.ID.NO.43 that contain SEQ.ID.NO.40.

19. a treatment is characterized as the method for the disorder of RAGE Tumor rejection antigen expression, this method comprises:

Impose the reagent of the mixture quantity that can optionally increase HLA-B7 and RAGE Tumor rejection antigen of the dosage of the described disorder of enough changes to the patient.

20. method according to claim 19, wherein said Tumor rejection antigen is selected from down group:

The amino acid whose peptide and the amino acid whose peptide that contains SEQ.ID.NO.43 that contain SEQ.ID.NO.40.

21. a treatment suffers from patient's method of the disorder that is characterized as the expression of RAGE Tumor rejection antigen, this method comprises:

Impose the cytolysis T cell of the dosage that enough slows down described disorder to the patient, this cytolysis T cell has the specificity to the mixture of HLA-B7 molecule and RAGE Tumor rejection antigen.

22. method according to claim 21, wherein said RAGE Tumor rejection antigen is selected from down group:

The amino acid whose peptide and the amino acid whose peptide that contains SEQ.ID.NO.43 that contain SEQ.ID.NO.40.

23. a separated nucleic acid molecule, it can be selected from hydridization under the stringent condition (a)

SEQ.ID.NO.1、 SEQ.ID.NO.4、 SEQ.ID.NO.6、

SEQ.ID.NO.10、 SEQ.ID.NO.12、 SEQ.ID.NO.13、

SEQ.ID.NO.14、 SEQ.ID.NO.15、 SEQ.ID.NO.17、

The molecule of the codes for tumor rejection antigen precursor of SEQ.ID.NO.23 and/or SEQ.ID.NO.35, condition are that this separated nucleic acid molecule is not encoded to MAGE, GAGE, BAGE tumor rejection antigen precursor; And (b) with (a) in nucleic acid molecule since the degeneracy of genetic code and on the codon sequence different nucleic acid molecule phase hydridization.

24. separated nucleic acid molecule according to claim 23, wherein said separated nucleic acid are cDNA molecule or mRNA molecule.

25. separated nucleic acid molecule according to claim 23, wherein said separated nucleic acid molecule encoding by SEQ.ID.NO.1, SEQ.ID.NO.12, SEQ.ID.NO.13 and/or SEQ.ID.NO.14 form one group in a selected coded tumor rejection antigen precursor.

26. separated nucleic acid molecule according to claim 23, wherein said separated nucleic acid molecule comprise that the Nucleotide to the 269-832 of the 185-247 of 217-276, the SEQ.ID.NO.13 of 273-449, the SEQ.ID.NO.12 of 444-665, the SEQ.ID.NO.12 of 313-399, the SEQ.ID.NO.1 of 204-326, the SEQ.ID.NO.1 that is selected from SEQ.ID.NO.1 and/or SEQ.ID.NO.14 is the complementary nucleic acid molecule.

27. a separated nucleic acid molecule, it comprise be selected from SEQ.ID.NO.1,

SEQ.ID.NO.4、 SEQ.ID.NO.6、 SEQ.ID.NO.10、

SEQ.ID.NO.12、 SEQ.ID.NO.13、 SEQ.ID.NO.14、

SEQ.ID.NO.15、 SEQ.ID.NO.17、 SEQ.ID.NO.23、

Selected nucleotide sequence in one group that SEQ.ID.NO.35 and/or SEQ.ID.NO.45 formed.

28. an expression vector, it contain the promotor that is operably connected according to claim 23,24,25,26 or 27 described separated nucleic acid molecule.

29. one kind with the transfection of the described expression vector of claim 28 institute or institute's transformed host cells.

30. a separated nucleic acid, it contains the sequence fragment of selected uniqueness in a group that is formed with the complement of the complement of the complement of the complement of SEQ.ID.NO.1, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.1, SEQ.ID.NO.12, SEQ.ID.NO.13 and/or SEQ.ID.NO.14.

31. separated nucleic acid according to claim 30, wherein said segmental length is between 200-2050 Nucleotide.

32. separated nucleic acid according to claim 30, wherein said fragment are length is 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31 and/or 32 Nucleotide.

33. one kind is detected the test kit that tumor rejection antigen precursor exists, it comprises a pair of separated nucleic acid molecule that makes up and arrange for the separated nucleic acid molecule of the claim 23 that optionally increases.

34. test kit according to claim 33, wherein said a pair of separated nucleic acid molecule is the PCR primer.

35. one kind by claim 23,24,25,26 or 27 the coded separated tumor rejection antigen precursor of nucleic acid molecule.

36. the method for the disorder of the expression that a diagnostic characteristic is the RAGE tumor rejection antigen precursor, wherein said antigen precursor be processed into RAGE deutero-Tumor rejection antigen and with HLA molecular composition mixture, this method comprises:

Patient's biological sample is contacted with reagent in conjunction with described mixture; With determine described mixture and described reagent combine to determine described disorder.

37. a diagnostic characteristic is that this method comprises by the method for the disorder of the expression of the coded RAGE tumor rejection antigen precursor of a kind of nucleic acid:

With patient's biological sample be that specific reagent contacts to described nucleic acid or its expression product, wherein, described nucleic acid can be selected from SEQ.ID.NO.1 in hydridization under the stringent condition, SEQ.ID.NO.4, SEQ.ID.NO.6, SEQ.ID.NO.10, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15, SEQ.ID.NO.17, SEQ.ID.NO.23, and/or the molecule of the codes for tumor rejection antigen precursor of SEQ.ID.NO.35, condition is that this separated nucleic acid molecule is not to MAGE, GAGE, the BAGE tumor rejection antigen precursor is encoded; And

Determine that the interaction of described reagent and described nucleic acid or described expression product determines described disorder.

38. according to the described method of claim 37, wherein said nucleic acid molecule is selected from component of being made up of the unique fragment of the unique fragment of the unique fragment of the unique fragment of SEQ.ID.NO.1, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.1, SEQ.ID.NO.12, SEQ.ID.NO.13 and SEQ.ID.NO.14.

39. a treatment suffers from patient's the method for the disorder of the expression that is characterized as the RAGE tumor rejection antigen precursor, this method comprises:

Be enough to slow down the reagent of the dosage of described disorder for described patient, this reagent selectivity ground increases by the tumor rejection antigen precursor institute deutero-Tumor rejection antigen of claim 23,24,25,26 or 27 described molecule encodings and the mixture of HLA.

40. a treatment suffers from patient's the method for the disorder of the expression that is characterized as the RAGE tumor rejection antigen precursor, this method comprises:

The autogenous cell solvability T cell of being enough to slow down the dosage of described disorder for described patient, this cytolysis T cell is to being specific by the tumor rejection antigen precursor institute deutero-Tumor rejection antigen of claim 23,24,25,26 or 27 described molecule encodings and the mixture of HLA molecule.

Images