CN1280307C

CN1280307C - Cytotoxin T lymphocyte

Info

Publication number: CN1280307C
Application number: CNB02108565XA
Authority: CN
Inventors: 糠谷育卫; 冈泽一秀; 川一郎; 黑目彻; 竹迫一任; 加藤郁之进
Original assignee: Takara Bio Inc
Current assignee: Takara Bio Inc
Priority date: 2001-04-03
Filing date: 2002-03-29
Publication date: 2006-10-18
Anticipated expiration: 2022-03-29
Also published as: CN1379041A; KR20020079403A; KR100669065B1

Abstract

Disclosed are peptide having a specific sequence, a mutant thereof and a nucleic acid encoding the same. The peptide is an HLA-A24- restricted antigen peptide capable of inducing a CTL(cytotoxic lymphocyte) having a T cell receptor specifically recognizing a cell manifesting on the cell surface a complex between the peptide and HLA(human main histocompatibility antigen)-A24 molecule. Disclosed are also a CTL having the T cell receptor, a method for inducing a CTL using the HLA-A24-restricted antigen peptide, a CTL inducer and an antitumor agent containing the HLA-A24-restricted antigen peptide, an antitumor agent and a detecting agent for a CTL-sensitive cell containing the CTL, an antigen manifesting cell manifesting the complex, and a CTL inducing agent and an antitumor agent containing the antigen manifesting cell. Disclosed are also a method for detecting the CTL-sensitive cell and a detecting agent using the CTL, a method for detecting the HLA-A24 molecule, a target cell of the CTL, a preparation method therefore, and the like.

Description

Cytotoxic T lymphocyte

Technical field

The present invention relates to the disease that prostate specific membrane antigen (prostate specificmembrane antigen, below PSMA) slightly unconventionality expression causes, cancer, specifically for example is useful technology in the treatment of prostate cancer etc. and the diagnosis.In more detail, the present invention relates in cancer, be specially in the treatment of cancer of PMSA unconventionality expression such as prostate cancer and the diagnosis useful, cytotoxic T lymphocyte (cytotoxic T lymphocytes with TXi Baoshouti of the mixture that can discern antigen peptide and people's major histocompatibility antigen (HLA) molecule, following CTL slightly), this antigen peptide, useful CTL inductor such as when inducing this CTL, the induction method of this CTL, the anticarcinogen useful to above-mentioned cancer, the detection method of this CTL sensitive cells and detection reagent thereof, the detection method of HLA-A24 molecule and the target cell that can be killed and wounded by this CTL and preparation method thereof.

Background technology

Prostate cancer is the highest cancer kind of morbidity in the U.S. especially, but is also increasing rapidly in Asian countries such as Japan.Hormonotherapy and radiotherapy are best to the treatment of prostate cancer effect.But for a long time hormonotherapy can produce the non-responsiveness cancer of hormone, therefore is starved of a kind ofly to the more effective methods of treatment of this morbid state, and thirsts for carrying out immunotherapy.

In CTL, antigen peptide and major histocompatibility antigen gene composite (majorhistocompatibility genecomplex, following MHC slightly) the major histocompatibility antigen MHCI class of coding is (if object is a man-hour, be called human leucocyte antigen (HLA) I class (human leukocyte antigen class I), following HLA I class slightly) mixture in conjunction with forming of molecule can be by specific t-cell receptor (T cellreceptor, following TCR slightly) identification kills and wounds the cell of presenting this mixture on cell surface thus.This CTL only discerns and kills and wounds the target cell with identical MHC I quasi-molecule with this CTL itself, therefore, is called MHC I class Restricted CTL again.

Therefore, set up, must possess following two conditions: 1) have the CTL that carries specificity TCR in order to make this cell killing reaction; 2) in order to become the antigen peptide that to be presented by HLA I quasi-molecule and can be discerned by CTL, need to exist the antigen peptide that both can combine and can form the discernible mixture of TCR with MHC I class antigen molecule.

This antigen peptide, for example synthetic antigen etc. is degraded to little epitope peptide through the course of processing of endoplasmic reticulum in the cell of mammalian cell, and it further combines with the MHCI quasi-molecule then, presents on cell surface.That is, in the proteolytic enzyme mixture of being made up of a lot of subunits, protein is degraded to the peptide that 8～15 amino acid are formed, and wherein severally is transported to endoplasmic reticulum from tenuigenin by the TAP transporter.These peptides if can combine with I class/β2Wei Qiudanbai (microglobulin) heterodimer, then will keep stable as 3 molecular complexes, and be transported to cell surface by gorky's device on endoplasmic reticulum.The proteinic cancer cells of expressing tumor related antigen or tumour specific antigen can be presented the restricted antigen peptide of MHC I quasi-molecule that can be discerned by the T lymphocyte at tumor cell surface.

As the initial tumour specific antigen that can be discerned by the T lymphocyte, clones such as P.van derBruggen identified be called as melanoma (melanoma) antigen E (below, MAGE slightly) gene (P.van der Bruggen etc., Science, the 254th volume, 1643-1647 page or leaf (1991)).Uses such as P.van der Bruggen come from that several have the cell strain that cell obtained of CTL resistance in a series of melanoma cells strains, this cell strain of immunoscreening in the discernible same patient of ctl clone, this CTL of melanoma patient source, and isolate MAGE according to the cloning by expression method.

That is to say, by being made the 5kb dna segment by the melanoma cells strain that CTL discerns, then with this segment transfection to the cell strain that can not be discerned by CTL originally, thereby resulting transfection strain can be discerned by above-mentioned ctl clone.This dna segment coding is inferred the protein of molecular weight about 26,000.The gene of encoding such proteins is known as MAGE-1.In addition, by the mRNA analysis revealed, the cell strain that comes from non-metastatic early stage melanomatous 40% can be expressed above-mentioned MAGE-1.Deriving from the proteinic antigen of MAGE-1 is the restricted antigen of a kind of HLA-A1, MAGE-1 specific CTL clone can discern with HLA I quasi-molecule in the middle of the peptide (C.Traversari etc. that constitute of 9 amino-acid residues combining of a kind of HLA-A1 molecule, Journal of Experimental ofMedicine, the 17 6 volume, 1453-1457 page or leaf (1992)).

Known have a lot of antigens to can be used as tumour antigen, but as can tens of kinds of above MAGE family, gp100, tyrosine oxidase (tyrosinase), CEA, HER2/neu etc. such as above-mentioned MAGE-1 being arranged by the antigen of the positive CTL identification of CD8 is known.The peptide that can be discerned by CTL is the peptide that comes from tumor antigen protein matter, and as presenting with the mixture of HLA I quasi-molecule.

As everyone knows, HLA I quasi-molecule mainly contains HLA-A, HLA-B, HLA-C, form by 8-10 amino acid with the antigen peptide that these molecules combine and present, and because of the different feature differences that have on the certain structure of their HLA molecular speciess separately.For example, as the peptide that combines with frequency is the highest in the world HLA-A2.1 molecule, be studied maximum and be, the 2nd of N-terminal is Leu, and the C-terminal position is the peptide of Leu or Val, and the peptide of being made up of 9-10 amino acid.In addition, as conduct common in the Asia ethnic groups such as Japanese and HLA-A24 molecule bonded peptide, what study at most is, the 2nd of N-terminal is arbitrary amino acid among Tyr, Phe, Met or the Trp, and the C-terminal position is arbitrary amino acid among Leu, Ile, Trp or the Phe, and the peptide (A.Kondo etc., the Joural of Immunology that are made up of 9-10 amino acid, the 155th volume, 4307-4312 page or leaf (1995)).

Up to now, the tumour antigen that has identified as antigen peptide is, MAGE-1, MAGE-3 at HLA-A1, at MAGE-3, the MART1 of HLA-A2.1, tyrosine oxidase, gp100, HER2/neu, CEA etc., MAGE-3 at HLA-Cw1, at the MAGE-3 of HLA-B44 etc., at MAGE-1, the MAGE-2 of HLA-A24, MAGE-3, CEA, HER2/neu, tyrosine oxidase, beta-catenin white (catenin) etc.

In prostate cancer, the tumor marker that is called prostate specific membrane antigen (prostatespecific membrane antigen, below PSMA) slightly is found, and is desirable to provide with they antigen peptide as target.About antigen peptide from PSMA, certified have, form and form by the aminoacid sequence of sequence number 3 (PSM-P1:LLHETDSAV) by the aminoacid sequence of sequence number 4 (PSM-P2:ALFDIESKV), and be respectively 2 kinds of peptides of 9 amino-acid residues, it is the restricted antigen peptide of HLA-A2.1 (prostate gland (Prostate), the 28th volume, 65-69 page or leaf (1996)).

In addition, known PSMA both can also can express [Chang S.S. etc., cancer research (CancerResearch), the 59th volume, the 3192nd～3198 page (1999)] at the tumor cells expression of prostate cancer in the new vessel of tumor locus.

Most preparation process is in these tumor antigen peptides, at first with the I class Restricted CTL cell strainization of tumor cell, the tumour antigen of identifying this CTL then and being discerned, method by genetic engineering is sought the least unit in the tumor antigen protein matter that CTL can discern again, and then based on motif information in conjunction with HLA I quasi-molecule, from above-mentioned least unit, identify and have antigenic peptide (Y.Kawakami etc., Proc.Natl.Acad.Sci., USA the 91st volume, 3515-3519 page or leaf (1994)).In addition, be that the HLA I quasi-molecule binding peptide in the tumor antigen protein matter is sought on the basis at first with above-mentioned HLA I quasi-molecule binding peptide common motif structure, then, the antigen peptide of utilizing the antigen presenting cell screening can induce CTL, tumour cell is had anti-personnel CTL determine antigen peptide (E.Celis etc. at last by whether inducing again, Proc.Natl.Acad.Sci., USA the 91st volume, 2105-2109 page or leaf (1994); B.Gaugler, Journal of Experimental of Medicine, the 179th volume, 921-930 page or leaf (1994)).

Yet, in peptide molecule, have the HLA-A24 binding motif only to induce 1 required condition of CTL, and be difficult in fact infer by this motif whether peptide molecule can demonstrate the selective killing activity of pair cell.

On the other hand, HLA I quasi-molecule is divided into several hypotypes, and there is very big difference in the subtype category that carries between different ethnic groups.Modal in the world is to carry HLA-A2, accounts for whitely 45%, and progress is also best aspect the evaluation of the restricted antigen peptide of this HLA-A2.

In the Japanese, above-mentioned HLA-A2 accounts for 40%, and when analyzing its hypotype, the same HLA-A*0201 with white people is 20%, and other mostly is HLA-A*0206 greatly.These hypotype institute bonded peptides are also inequality, and the HLA-A2 of main research is HLA-A*0201, and on the other hand, HL A-A24 accounts for more than 60% in the Japanese, and other ethnic group of this HLA-A24 and Asia is compared, and ratio is very high.

With regard to present situation, the restricted antigen peptide that causes about above-mentioned HLA-A24, white (β-catenin) etc. of MAGE-1, MAGE-2, MAGE-3, CEA, HER2/neu, tyrosine oxidase, beta-catenin for example, but the analysis and research of comparing them with HLA-A 2.1 are also very backward, and the utmost point need be found the novel antigens peptide at various tumour antigen.Also have, also very backward to Asia ethnic group, especially research to Japanese CTL with tumor cell specific effect, be difficult to provide the CTL that is applicable to oncotherapy.

Summary of the invention

The objective of the invention is to, provide (the prostate specific membrane antigen of prostate specific membrane antigen among Asia ethnic group, the especially Japanese, following PSMA slightly) disease that causes of unconventionality expression, for example cancer, the treatment that is specially prostate cancer etc. and diagnosis useful technology for example provide CTL etc.In detail, for example, the object of the present invention is to provide the CTL that can satisfy one of following demand at least, these demands comprise the disease that treatment and diagnosis PSMA unconventionality expression cause, for example, cancer, be specially prostate cancer etc., alternative kills and wounds HLA-A24 and PSMA is positive cells, especially tumour cell, the cell medicine (anticarcinogen etc.) that can be used as cancer therapy checks in the sample that exsomatizes whether exist HLA-A24 and PSMA to be positive cells, especially tumour cell.Second, the object of the present invention is to provide restricted antigen peptide of the HLA-A24 that can satisfy one of following demand at least and coding nucleic acid thereof, these demands comprise that alternative induces above-mentioned CTL, the inductor that can be used as above-mentioned CTL becomes possibility based on the treatment of diseases such as cancer of inducing, make of above-mentioned CTL.The 3rd, the object of the present invention is to provide the antigen presenting cell that can satisfy one of following demand at least, these demands comprise the preparation that is used for above-mentioned CTL, become possibility based on the treatment of diseases such as cancer of inducing, make of above-mentioned CTL.The 4th, the object of the present invention is to provide can be efficiently and optionally obtain CTL induction method and the CTL inductor of CTL of the present invention.The 5th, the object of the present invention is to provide the CTL sensitive cells detection method and the detection reagent that can satisfy one of following demand at least, these demands comprise can be efficiently and optionally detect the CTL sensitive cells, and the classification of HLA, is specially the diagnosis of diseases such as prostate cancer at cancer.The 6th, the object of the present invention is to provide can people's major histocompatibility antigen not express cell and/or antigen protein not in the express cell expressing human major histocompatibility antigen and/or antigen protein, the target cell that can be killed and wounded by cytotoxic T lymphocyte and the preparation method that can efficiently obtain its this target cell.

The present invention relates to,

[1] a kind of human body major histocompatibility antigen (HLA)-restricted antigen peptide of A24 has following (a) or aminoacid sequence (b),

(a) aminoacid sequence shown in the sequence number 1 or 2, or

(b) in sequence number 1 or 2, have and be selected from the 1. following～3. aminoacid sequence of at least a sudden change,

1. lack 1 amino-acid residue at least,

2. having 1 radical amino acid replacement at least is other amino acid or amino acid analogue, and

3. additional at least 1 other amino-acid residue or amino acid analogue; And have the character that to induce CTL, but the mixture that peptide that this TXi Baoshouti specific recognition is formed the aminoacid sequence shown in above-mentioned sequence number 1 or 2 and HLA-A24 molecule form is presented the cell on cell surface with following TXi Baoshouti.

[2] a kind of cytotoxic T lymphocyte has following TXi Baoshouti, but this TXi Baoshouti specific recognition is presented cell on cell surface with the restricted antigen peptide of above-mentioned [1] described HLA-A24 and HLA-A24 molecule in conjunction with the mixture that forms.

[3] a kind of induction method of cytotoxic T lymphocyte is characterized in that, the restricted antigen peptide of above-mentioned by using [1] described HLA-A24 is come inducing cytotoxic T lymphocyte.

[4] above-mentioned [3] described induction method comprises following 3 steps,

(1) restricted antigen peptide of above-mentioned [1] described HLA-A24 and the a kind of cells contacting with antigen presentation ability that is selected from scavenger cell, B cell and the dendritic cell are obtained antigen presenting cell, with obtaining antigen presenting cell the cell with antigen presentation ability is carried out antigenic stimulation then, thus, obtain effector cell's step

(2) with above-mentioned steps (1) the effector cell that obtains with can be contacted by the target cell that above-mentioned [2] described cytotoxic T lymphocyte kills and wounds, detecting whether to have the step of cytotoxic activity, and

(3) in above-mentioned steps (2), screening presents the step of the effector cell of cytotoxic activity as cytotoxic T lymphocyte.

[5] above-mentioned [4] described induction method is characterized in that, under the condition of the existence of IL-7, and for 1 lymphocyte, the antigen presenting cell of 0.01-1 ratio of contact.

[6] above-mentioned [4] described induction method, under the condition of IL-7 and the existence of keyhole  hemocyanin, the restricted antigen peptide of above-mentioned [1] described HLA-A24 is added in the peripheral blood lymphocytes by the amount of adding 1ng-100 μ g in the every 1ml solution that contains peripheral blood lymphocytes.

[7] a kind of CTL inductor contains the restricted antigen peptide of above-mentioned [1] described HLA-A24 as effective constituent, and is used to obtain above-mentioned [2] described cytotoxic T lymphocytes (CTL).

[8] a kind of anticarcinogen contains the restricted antigen peptide of above-mentioned [1] described HLA-A24 as effective constituent.

[9] a kind of anticarcinogen contains above-mentioned [2] described cytotoxic T lymphocyte as effective constituent.

[10] a kind of antigen presenting cell can be presented the mixture of restricted antigen peptide of above-mentioned [1] described HLA-A24 and HLA-A24 molecule at cell surface.

[11] a kind of CTL inductor contains above-mentioned [10] described antigen presenting cell as effective constituent, and is used to obtain above-mentioned [2] described cytotoxic T lymphocytes (CTL).

[12] a kind of anticarcinogen contains above-mentioned [10] described antigen presenting cell as effective constituent.

[13] a kind of detection method of cytotoxic T lymphocyte sensitive cells, it is characterized in that following detection index, make above-mentioned [2] described cytotoxic T lymphocyte and cells contacting to be measured, thereby when causing the variation of the lysis, release of cytokines and the CTL propagation that are selected from CTL and cause, the cell (sensitive cells) of described cell to be measured for above-mentioned [2] described cytotoxic T lymphocyte is had susceptibility.

[14] a kind of detection agent of cytotoxic T lymphocyte sensitive cells contains above-mentioned [2] described cytotoxic T lymphocyte as effective constituent.

[15] detection method of people's major histocompatibility antigen (HLA)-A24 molecule in a kind of cell to be measured, it is characterized in that, under the condition that the restricted antigen peptide of above-mentioned [1] described HLA-A24 exists, make above-mentioned [2] described cytotoxic T lymphocyte and cells contacting to be measured.

[16] a kind of nucleic acid, the restricted antigen peptide of above-mentioned [1] the described HLA-A24 that encodes constitutes.

[17] preparation method of a kind of target cell that can be killed and wounded by cytotoxic T lymphocyte comprises the operation with the coding nucleic acid transfered cell of the coding nucleic acid of people's major histocompatibility antigen and/or antigen protein.

[18] above-mentioned [17] described preparation method, by adenovirus carrier with the nucleic acid transfered cell.

[19] above-mentioned [17] or [18] described preparation method, the coding nucleic acid of people's major histocompatibility antigen is the coding nucleic acid of HLA-A24.

[20] each described preparation method of above-mentioned [17]～[19], the coding nucleic acid of antigen protein is above-mentioned [16] described nucleic acid.

[21] a kind of target cell, each described preparation method obtains by above-mentioned [17]～[20], and can be killed and wounded by cytotoxic T lymphocyte.

The working of an invention mode

The present invention is based on the inventor's following research, with the peptide that has as the HLA-A24 binding motif in the psma protein matter of tumour antigen is the center, from numerous candidate's peptides, seek found that of antigen peptide, in numerous candidate's peptides, when the peptide of forming with aminoacid sequence shown in sequence number 1 or 2 carries out antigenic stimulation to lymphocyte, from normal people's peripheral blood lymphocytes of expressing HLA-A24, can kill and wound the cytotoxic T lymphocyte (CTL) of this tumour antigen express cell by induced selective, thereby finish the present invention.

In addition, in this manual,, use " target cell " and " antigen presenting cell " as the term of representing the mixture of restricted antigen peptide of HLA-A24 and HLA-A24 molecule is presented to the cell of cell surface.

Particularly, described " target cell " is meant the cell that can be killed and wounded by the cytotoxic T lymphocyte of this cell of specific recognition (following CTL slightly).On the other hand, described " antigen presenting cell " be meant, has antigen-presenting and MHC molecule simultaneously and activate the cell of the lymphocytic function of T, during no Special Circumstances, refers to that the MHC molecule is the cell of HLA-A24 molecule.

Above-mentioned " potential antigen presenting cell " be meant, forms mixture by MHC molecule and the antigen peptide that exists in this cell, can become the cell of antigen presenting cell, during no Special Circumstances, refers to the cell that this MHC molecule is the HLA-A24 molecule.

In this specification sheets, cancer is meant malignant tumour, comprising cancer knurl, sarcoma both.In this specification sheets, cancer is except tumour cell, also comprises new vessel in tumour region of interest, for example tumor locus etc.

One of feature of the restricted antigen peptide of HLA-A24 of the present invention is to have following (a) or aminoacid sequence (b):

(a) sequence number: the aminoacid sequence shown in 1 or 2, or

(b) at sequence number: in 1 or 2, have the aminoacid sequence that is selected from 1. following～at least a sudden change in 3.,

1. lack 1 amino-acid residue at least,

3. additional at least 1 other amino-acid residue or amino acid analogue; And have the character that to induce CTL, but the mixture that peptide that this TXi Baoshouti specific recognition is formed the aminoacid sequence shown in above-mentioned sequence number 1 or 2 and HLA-A24 molecule form is presented the cell on cell surface with following TXi Baoshouti.In addition, in this specification sheets, the peptide that the aminoacid sequence shown in the above-mentioned sequence number 1 or 2 is formed is also referred to as the PSMA antigen peptide sometimes.

The restricted antigen peptide of HLA-A24 according to the present invention, can induce useful HLA-A24 Restricted CTL in disease, for example treatment for cancer and the diagnosis that the PSMA unconventionality expression is caused, promptly, can bring into play and induce excellent effect, but the mixture that this TXi Baoshouti specific recognition forms above-mentioned antigen peptide and HLA-A24 molecule is presented the cell on cell surface with following TXi Baoshouti CTL.That is to say, the restricted antigen peptide of HLA-A24 of the present invention is restricted to HLA-A24 common among Asia ethnic group, the especially Japanese, therefore in treatment of diseases such as tumour, can bring into play good effect, for example, but specificity suppress Asia ethnic group, especially Japanese etc. tumour cell and/or tumour region of interest, be specially tumour cell and/or tumour region of interest that the PSMA unconventionality expression causes, but especially specificity suppresses prostate cancer cell and/or prostate cancer region of interest.

In addition, the present invention also comprises the CTL with following TXi Baoshouti, but the mixture that this TXi Baoshouti specific recognition forms the restricted antigen peptide of HLA-A24 of the present invention and HLA-A24 molecule is presented to the cell on the cell surface.

In this manual, so-called " antigen peptide " be meant, has combine with MHC I quasi-molecule and form the ability of mixture with this MHC I quasi-molecule, and formed mixture of while has can be by antigenic peptide that TCR discerned of specific CTL.

Also have, in this manual, so-called " the restricted antigen peptide of HLA " is meant that the MHCI quasi-molecule is the antigen peptide as the HLA molecule.

The restricted antigen peptide of HLA-A24 of the present invention can be the peptide that isozygotys that simple amino acid is formed, and also can be the T1249 that contains non-aminoacid component.Described amino acid can be natural type amino acid, also can be the amino acid through chemically modified.Above-mentioned peptide can be a monomer, also can be polymer.

As the restricted antigen peptide of HLA-A24 of the present invention, for example, at least 1 amino-acid residue in the aminoacid sequence shown in sequence number 1 or 2, being specially 1 or several amino acid produces and is selected from disappearance, displacement and additional at least 1 sudden change, and then it is different with the aminoacid sequence shown in this sequence number 1 or 2, but it can with the HLA-A24 molecule forming composite, the CTL that formed mixture of while can be had following TXi Baoshouti discerns, but this TXi Baoshouti specific recognition is presented cell on cell surface with the mixture of antigen peptide shown in sequence number 1 or 2 and the formation of HLA-A24 molecule.

As said mutation, be specially,

1. lack 1 amino-acid residue at least,

3. additional at least 1 other amino-acid residue or amino acid analogue etc.

This sudden change can be a kind of separately, also can be their combination.

As long as performance can be induced the CTL character with following TXi Baoshouti, but the mixture that peptide that this TXi Baoshouti specific recognition is formed the aminoacid sequence shown in sequence number 1 or 2 and HLA-A24 molecule form is presented the cell on cell surface, the length amino acid sequence of the restricted antigen peptide of HLA-A24 then of the present invention can be a 9-10 amino-acid residue, but is not particularly limited.For example, in the aminoacid sequence shown in the above-mentioned sequence number 1 or 2, when having the peptide of the aminoacid sequence composition that 2. suddenlys change, the length of aminoacid sequence can be 9-10 residue, but is not particularly limited.In the aminoacid sequence shown in the above-mentioned sequence number 1 or 2, when having the peptide of the aminoacid sequence composition that 3. suddenlys change, the length of aminoacid sequence can be 9-10 residue, but is not particularly limited.

The N-acylate of multiple amino acids, 0-acylate, carboxylate, amidate, alkylide etc. are arranged as above-mentioned amino acid analogue.

In addition; mixture with the formation of HLA-A24 molecule; as long as can be had the CTL of following TXi Baoshouti discerns; but the mixture that this TXi Baoshouti specific recognition forms the antigen peptide shown in sequence number 1 or 2 and HLA-A24 molecule is presented the cell on cell surface; then can also be on the N-terminal of this antigen peptide and the free amino in conjunction with formyl radical, ethanoyl, uncle-butoxy carbonyl (t-Boc) base etc., and can also be in conjunction with methyl, ethyl, tert-butyl, benzyl etc. on the C-terminal of antigen peptide and the free carboxyl.

In addition, the restricted antigen peptide of HLA-A24 of the present invention can be carried out various modifications, imports in the organism being easy to.

The restricted antigen peptide of HLA-A24 of the present invention, can screen by the CTL that use has a following TXi Baoshouti, but the mixture that this TXi Baoshouti specific recognition forms the antigen peptide shown in sequence number 1 or 2 and HLA-A24 molecule is presented the cell on cell surface.Above-mentioned screening for example, can be carried out according to method of following 1～3 etc.

Method 1

Mixing after the candidate substances that is not attached on the HLA-A24 molecule is removed in washing, makes the gained candidate substances in conjunction with HLA-A24 express cell and ctl response as the candidate substances and the HLA-A24 express cell of the restricted antigen peptide of HLA-A24 of the present invention.When cell-specific toxicity, release of cytokines and the proliferative response etc. of candidate substances are identified, can judge that above-mentioned candidate substances is the restricted antigen peptide of HLA-A24 of the present invention.

Method 2

Candidate substances is contacted with the potential antigen presenting cell, through the regular hour, for example take in the cell, and the mixture of antigen peptide and HLA molecule is presented the time that will need on cell surface through antigen, after making it reaction, make gained reactant and ctl response.When the candidate substances specific cell factor discharges or proliferative response when being identified, can judge that above-mentioned candidate substances is the restricted antigen peptide of HLA-A24 of the present invention.

Method 3

Peptide can be in the carrier of passing for the HLA-A24 molecule on the potential antigen presenting cell described later that can express, insert the coding nucleic acid of the aminoacid sequence of candidate substances, carry out transfection with the gained recombinant vectors, make the potential antigen presenting cell and the ctl response of transfection again.When the candidate substances specific cell factor discharges or proliferative response when being identified, can judge that above-mentioned candidate substances is the restricted antigen peptide of HLA-A24 of the present invention.

In the restricted antigen peptide of HLA-A24 of the present invention, as long as can being had the CTL of following TXi Baoshouti, the mixture that forms with HLA-A24 discerns, but the mixture that this TXi Baoshouti specific recognition forms the antigen peptide shown in sequence number 1 or 2 and HLA-A24 molecule is presented the cell on cell surface, for example in the aminoacid sequence shown in sequence number 1 or 2, in order to strengthen and the combining of HLA-A24 molecule, the amino acid of the 2nd of N-terminal can by with HLA-A24 molecule bonded peptide in distinctive amino acid Tyr, Phe, Met or Trp replace, and/or the amino acid of C-terminal can by with HLA-A24 molecule bonded peptide in distinctive amino acid: Leu, Ile, Trp or Phe replace.For example, the C-terminal amino acid (Phe) that can enumerate the aminoacid sequence of sequence number 2 has the peptide of the aminoacid sequence of the sequence number 5 that is replaced into Leu.

As in aminoacid sequence shown in sequence number 1 or 2 as having the peptide that 1 or several amino acid are formed by the metathetical aminoacid sequence, for example had the peptide that the amino acid of similar side chain or amino acid analogue have been replaced to the amino acid of replacing object separately, and have and HLA-A24 molecule bonded ability, the CTL identification that can be had following TXi Baoshouti again, this TXi Baoshouti can specific recognition be presented cell on cell surface with the antigen peptide shown in sequence number 1 or 2 and HLA-A24 molecule in conjunction with the mixture with the HLA-A24 molecule of the mixture that forms.

As the similar amino acid of side chain, for example, glycine (Gly) and L-Ala (Ala), Xie Ansuan (Val), Isoleucine (Ile), leucine (Leu) and methionine(Met) (Met); L-asparagine (Asn) and glutamine (Gln), aspartic acid (Asp) and L-glutamic acid (Glu), Serine (Ser) and Threonine (Thr); Methionin (Lys) and arginine (Arg), phenylalanine (Phe) and tyrosine (Tyr).

Specifically, as having the peptide that aminoacid sequence that 1 or several amino acid have been replaced is formed in the aminoacid sequence shown in sequence number 1 or 2, for example in sequence number 2, the amino acid (Asn) with N-terminal is replaced into the peptide that the aminoacid sequence shown in the sequence number 6 of Gln is formed; The 5th amino acids (Thr) with N-terminal is replaced into the peptide that the aminoacid sequence shown in the sequence number 7 of Ser is formed.

The peptide that has the aminoacid sequence that 1 or several amino acid have been replaced in the aminoacid sequence shown in the sequence number 1 or 2, as long as can be had the CTL of following TXi Baoshouti discerns, but the mixture that this TXi Baoshouti specific recognition forms antigen peptide shown in sequence number 1 or 2 and HLA-A24 molecule is presented the cell on cell surface, then also can with side chain similarly amino acid replace.For example, in the aminoacid sequence shown in the sequence number 2,1 or several amino acid are the peptides that are replaced into other amino acid or amino acid analogue, for example formed by Ala institute metathetical aminoacid sequence, and have and HLA-A24 molecule bonded ability, and with the mixture that the HLA-A24 molecule forms be the peptide that CTL discerned that can be had following TXi Baoshouti, but the mixture that this TXi Baoshouti specific recognition forms antigen peptide shown in sequence number 1 or 2 and HLA-A24 molecule is presented the cell on cell surface.

In the aminoacid sequence shown in sequence number 1 or 2, have the peptide that 1 or several amino acid are formed by the metathetical aminoacid sequence, for example be peptide with sequence number 8 or 9 arbitrary aminoacid sequences.Can might be replaced into and original different other amino acid or the amino acid analogues of amino acid by the 5th amino acids of Ala metathetical N-terminal and N-terminal in the above-mentioned peptide.

In addition, the restricted antigen peptide of HLA-A24 of the present invention can be the N-terminal or the additional amino acid whose peptide of C-terminal of sequence number 1 or 2, but the CTL that need be had following TXi Baoshouti discerns, but the mixture that this TXi Baoshouti specific recognition forms antigen peptide shown in sequence number 1 or 2 and HLA-A24 molecule is presented the cell on cell surface.In order to become the peptide of sequence number 1 or 2, additional suitable amino acid thus, more effectively is and passs HLA-A24 molecule these peptides in antigen presenting cell.For example, advantageous applications is from the peptide fragment of PSMA, and contains the aminoacid sequence shown in sequence number 1 or 2, and the peptide of Duoing than this aminoacid sequence amino-acid residue number, specifically, and the peptide of forming by 15-25 amino-acid residue.

The restricted antigen peptide of HLA-A24 of the present invention can be by the preparation of organic chemistry methods such as liquid phase or solid phase amino acid coupling method.In addition, the DNA recombinant technology of the coding nucleic acid of the aminoacid sequence that the restricted antigen peptide of above-mentioned HLA-A24 also can be by using the restricted antigen peptide of HLA-A24 prepares.Peptide by the DNA recombinant technology obtains in case of necessity, can adopt suitable organic chemistry or biochemical method etc. to modify.

The present invention also comprises the nucleic acid of the restricted antigen peptide of the above-mentioned HLA-A24 of coding.Promptly, in can being had the CTL identified range of following TXi Baoshouti, the polypeptide of nucleic acid encoding of the present invention can be the variant with various variations, this TXi Baoshouti characteristic is, but specific recognition is presented to the mixture of antigen peptide shown in sequence number 1 or 2 and HLA-A24 molecule in the cell of cell surface.In addition, this class nucleic acid can be the nucleic acid that has difference by merger with above-mentioned nucleic acid.

As the nucleic acid of coding HLA-A24 restricted antigen peptide, PSMA gene for example, this gene order is disclosed (R.S.Israeli etc., Cancer Research, the 53rd volume, 227-230 page or leaf (1993)).Can select and use the zone of the described peptide of coding from above-mentioned sequence.

One of feature of cytotoxic T lymphocyte of the present invention is, but has the TXi Baoshouti that specific recognition is presented the mixture of restricted antigen peptide of HLA-A24 of the present invention and the formation of HLA-A24 molecule in the cell on cell surface.The cell of can only specific killing presenting the mixture of restricted antigen peptide of HLA-A24 of the present invention and HLA-A24 molecule according to CTL of the present invention.That is to say, but according to CTL specific killing tumour cell, especially prostate cancer cell of the present invention.

Moreover, CTL of the present invention is restricted to HLA-A24 common among Asia ethnic group, the especially Japanese, therefore as oncotherapy, adopt this CTL specific killing Asia ethnic group, especially Japanese tumour cell, especially prostate cancer cell effectively.

CTL of the present invention can cause specific cell scission reaction or the release of cytokines reaction of target cell.In addition, the CTL in this specification sheets can discern the antigenicity of presenting the antigen peptide on the HLA-A24 molecule and have killing activity, and not limit by other antigenicity identification etc.

But CTL of the present invention since have specific recognition with the antigen peptide shown in sequence number 1 or 2, be the PSMA antigen peptide and the mixture of HLA-A24 molecule formation is presented the TXi Baoshouti of the cell on cell surface, so, but be male tumour cell specificity generation cytokine for HLA-A24 and PSMA.Therefore, can be used as cell drug, can also be used to sensitive cells that detects this CTL etc. for tumour.It also can be the cell that imports HLA-A24 or PSMA gene that HLA-A24 and PSMA are positive cells.

CTL of the present invention can be obtained by following method, the method is characterized in that, comes inducing cytotoxic T lymphocyte by using the restricted antigen peptide of HLA-A24.The induction method of this cytotoxic T lymphocyte is also contained among the present invention.

As the induction method of cytotoxic T lymphocyte of the present invention, specifically, for example contain the method for following steps.

(1) makes restricted antigen peptide of HLA-A24 of the present invention and a kind of the cells contacting that is selected from scavenger cell, B cell and the dendritic cell with antigen presentation ability, and obtain antigen presenting cell, with the gained antigen presenting cell cell with antigen presentation ability is carried out antigenic stimulation, and then acquisition effector cell's step

(2) make in the above-mentioned steps (1) resulting effector cell and can be contacted, whether have cell killing activity to detect by the target cell that cytotoxic T lymphocyte of the present invention kills and wounds, and

(3) in the above-mentioned steps (2), screening presents the effector cell of cell killing activity as cytotoxic T lymphocyte.

External (in vitro) for example uses the stripped sample that comes from the body that can express HLA-A24 when inducing this CTL, induces by stimulate the sample that should exsomatize with the restricted antigen peptide of HLA-A24 of the present invention.As above-mentioned " exsomatize sample ", for example blood, lymphatic node, the spleen extractd by operation etc., also have other various organs and tissues etc.In the present invention, preferably use lymphocyte or the lymphocyte infiltration that exists in these test portions.

For example, when using blood, can induce CTL to carrying out antigenic stimulation repeatedly by the peripheral blood lymphocytes of HLA-A24 male human blood preparation (peripheral blood mononuclear cells, below also PBMC slightly).

Above-mentioned antigenic stimulation is implemented by following process, for example in the cell with antigen presentation ability of lymphoblastic same blood preparation, use the restricted antigen peptide of HLA-A24 of the present invention is the cell (antigen presenting cell) of passing cell surface, uses the restricted antigen peptide of this HLA-A24 etc. as the mixture with the HLA-A24 molecule by preparation.

Derivative CTL can be by giving the stimulation of anti-cd 3 antibodies etc. further, or give various stimulations make it propagation in the presence of restricted antigen peptide of HLA-A24 of the present invention or antigen presenting cell.For example in the presence of IL-7, with respect to 1 lymphocyte that is made by PBMC, the mixed antigen presenting cell with 0.01-1 can carry out inducing of CTL.Induce mode as other, for example at IL-7, keyhole  hemocyanin (Keyhole Limpet Hemocyanin, KLH) exist down, in the PBMC cell culture fluid, be 1ng/ml-100g/ml, be preferably the restricted antigen peptide of amount interpolation HLA-A24 of the present invention of 10ng/ml-1 μ g/ml with every kind of antigen peptide, and then induce CTL.

Derivative CTL can have stable Cytotoxic lymphocyte form and keep by cloning.For example, derivative CTL can make it propagation by stimulations such as antigen, various cytokine, anti-cd 3 antibodies.

The detection of derivative specific CTL, can be by measuring lethal to the target cell of marks such as antigen peptide of inducing CTL and radioactive substance, can increase by the value-added antigen-specific of the CTL that the absorption of radiant is measured, or the cytokine amounts such as GM-CSF, INF-γ that discharged by CTL or target cell antigen-specific detect.For example, by measuring CTL to above-mentioned ⁵¹The anti-personnel method of the target cell of Cr mark is estimated.

Specifically, mixed containing in each hole of 96 porocyte culture plates etc. ⁵¹The target cell of Cr mark and be used to eliminate the active cell of non-specific killing that the NK cell of sneaking into the causes cell mixture and the CTL of (as, K562 cell etc.) through after the incubation of appropriate time, uses gamma counter to measure ⁵¹The burst size of Cr detects.Then, can calculate the specific cell cytotoxic activity according to following formula 1.

(in the formula, the hole of minimum releasing value for only containing target cell and being used for eliminating the active cell of non-specific killing ⁵¹The Cr amount, the expression target cell ⁵¹When Cr nature burst size, maximum releasing value are represented with destruction target cells such as tensio-active agents ⁵¹The Cr burst size)

In addition, also can directly determine the specific cell cytotoxic activity by the antigen peptide and the mixture of marks such as use fluorescein.At this moment, for example make CTL and with after CTL specific antibody link coupled the 1st fluorescent marker contacts, contact again with the 2nd fluorescent marker link coupled antigen peptide-MHC mixture, (fluorescence-activated cell sorting, FACS) existence of analysis double-tagging cell is carried out by the fluorescence-activated cell sorting method then.

On the other hand, in the body (in vivo) when inducing CTL, for example can in body, implement by the restricted antigen peptide of HLA-A24 of the present invention and the mixture of HLA-A24 molecule formation being presented offer medicine at the antigen presenting cell of cell surface.When the people is carried out administration, each adult's dosage common preferred 10 ⁴-10 ⁹Individual.Other method also has, and the restricted antigen peptide of HLA-A24 of the present invention is mixed with suitable adjuvant then organism is carried out administration, and then can induce this peptide is had specific CTL.

As above-mentioned adjuvant; for example 1. Fu Shi (Freund) Freund's complete adjuvant, 2. Freund's incomplete adjuvant, 3. inorganics such as aluminium hydroxide, alum gel, 4. tensio-active agent such as lysolecithin, bromination dimethyl stearyl ammonium, 5. polyanion, 6. peptide, 7. Ribi corporate system list phosphoryl lipid (monophosphoryl lipid, MPL) A etc. of muramyl peptide, its general element ( Off トシ Application) etc.s of T 500, poly-IC etc.When in body, inducing CTL, except the mixture of antigen peptide and adjuvant, also can mix the restricted helper cell antigen peptide of administration MHC II class.

In order to keep stable in body, the restricted antigen peptide of HLA-A24 of the present invention also can be by carrying out administration after suitable joint and higher fatty acid and the helper cell antigen peptide covalent attachment.When the people is carried out administration, each adult's dosage, with the densitometer of the restricted antigen peptide of HLA-A24, every kind of antigen peptide administration 0.1 μ g/kg-10mg/kg, preferred 1 μ g/kg-1mg/kg, more preferably 1 μ g/kg-100 μ g/kg.

When inducing CTL by the stimulation of the restricted antigen peptide of HLA-A24 of the present invention, the mixture that also can use restricted antigen peptide of this HLA-A24 and HLA-A24 molecule to form.At this moment, the not necessarily natural HLA-A24 molecule of HLA-A24 molecule also can be the fragment that maintains in essence with the associativity of the restricted antigen peptide of HLA-A24.These fragments for example can obtain by the protein degradation or the recombinant DNA technology of natural type HLA-A24 molecule.This mixture can with β ₂-microglobulin keeps stable but especially coexist with the antibody of recognition complex.

Because CTL of the present invention is by the restricted antigen peptide inductive of HLA-A24 of the present invention, contains this peptide as CTL inductor effective constituent, that be used to obtain cytotoxic T lymphocyte (CTL) so can be used as.This CTL inductor is also contained in this

In the scope of invention.

CTL inductor of the present invention, it for example can be the independent restricted antigen peptide of above-mentioned HLA-A24, perhaps with the form of mixtures of other molecule (helper cell antigen peptide and/or adjuvant), supply with to be suspended in physiological saline or the phosphate buffer normal saline form.The restricted antigen peptide of above-mentioned HLA-A24 also can be to use with the covalent conjunct agent of higher fatty acid and helper cell antigen peptide or with the composite form of HLA-A24 molecule.

The content of the restricted antigen peptide of HLA-A24 of the present invention in the CTL inductor of the present invention, so long as induce the required amount of CTL to get final product, for example antigen peptide content is 0.01-100 weight % by every kind of antigen peptide, preferred 0.1-95 weight %.Described content, for example the human body from the HLA-A24 antigen positive separates peripheral blood lymphocyte, under isolated condition, add the different reagent of the restricted antigen peptide content of HLA-A24 respectively, when cultivating, at this moment can discern in the reagent of CTL of HLA-A24 positive cell and determine as the content of the restricted antigen peptide of this HLA-A24 by inducing specific while stimulating.At this moment, have or not, for example can determine with the amount that conventional ELISA method etc. is measured the various cytokines that generate with target cell reaction back CTL (as, IFN-γ) for the CTL inductive.In addition, also can by measure CTL to ⁵¹The anti-personnel method of the target cell of Cr mark is determined.

CTL inductor of the present invention, the additive of substratum when can be used as in-vitro multiplication CTL of the present invention, and to be used for T lymphocyte proliferation activity, retardance skin allergy be the diagnosis etc. of the sensitization state of index.For example, when being used as the additive of substratum, usage quantity is in the peptide concentration in the substratum, and every kind of antigen peptide is 1ng/ml-100 μ g/ml, preferred 10ng/ml-1 μ g/ml.In addition, can also be used as following anticarcinogen.

The restricted antigen peptide of HLA-A24 according to the present invention, can induce CTL of the present invention, and this CTL is restricted to HLA-A24 common among Asia ethnic group, the especially Japanese, therefore, as oncotherapy, but can provide to the Asia ethnic group, especially to the especially effective anticarcinogen of specific killing tumour cell, especially prostate cancer cell of Japanese, for example provide above-mentioned CTL or based on the anticarcinogen of inducing CTL.Therefore, the present invention can provide (1) to contain the anticarcinogen of the restricted antigen peptide of HLA-A24 of the present invention as effective constituent, and (2) contain the anticarcinogen of CTL of the present invention as effective constituent.This anticarcinogen is also contained in the scope of the present invention.

Anticarcinogen of the present invention, owing to contain the restricted antigen peptide of (1) HLA-A24 of the present invention or (2) CTL of the present invention as effective constituent, but so the tumour cell aspect performance excellent characteristic of this anticarcinogen in cancer, especially prostate cancer that specific killing prostate specific membrane antigen (prostate specificmembrane antigen, below PSMA) slightly unconventionality expression causes.

The formulation of the medicine of anticarcinogen of the present invention can be 1) the simple restricted antigen peptide of HLA-A24 of the present invention or simple CTL of the present invention, 2) mixture of restricted antigen peptide of HLA-A24 of the present invention or CTL of the present invention and pharmaceutically acceptable carrier and/or thinner, perhaps 3) in case of necessity above-mentioned 1) or 2) in add auxiliary agent.As above-mentioned carrier, for example human serum albumin.As thinner, for example phosphoric acid buffer, distilled water, physiological saline, phosphate buffer normal saline, be suitable for substratum of preserving CTL of the present invention etc.As substratum, be generally substratum such as RPMI, AIM-V.As auxiliary agent, pharmaceutically useful above-mentioned adjuvant etc. for example.When anticarcinogen of the present invention is put into human body, can use the syringe administration or with methods such as sprayings from mucous membrane through form administrations such as skin absorptions.When using injection, can contain injection solvent (for example water, alcohol, glycerine etc.).

As long as the effective constituent amount satisfies following condition in the anticarcinogen of the present invention, institute just, by the restricted antigen peptide inductive of HLA-A24 of the present invention CTL amount or CTL of the present invention amount is that the required amount of tumour cell in killing prostate specific membrane antigen (prostate specificmembrane antigen, below PSMA) slightly the unconventionality expression cancer, the especially prostate cancer that cause gets final product.

Above-mentioned " amount that killing tumor cell is required " for example can be by measuring CTL to above-mentioned ⁵¹The anti-personnel method of Cr labels targets cell is estimated.

The pharmacological action of anticarcinogen of the present invention as previously mentioned, can also deliver medicine to anticarcinogen of the present invention mouse that suffers from cancer etc. according to the tumor cytotoxicity degree is estimated, and estimate as index with the variation of tumour size.

The formulation of anticarcinogen of the present invention can be an injection, but in order to improve and intravital affinity, can be Liposomal formulation also, is combined in diameter and is the granular preparation on the pharmaceutically acceptable pearl of several μ m, in conjunction with the formulations such as preparation of lipid etc.

Above-mentioned injection can be independent, perhaps with after conventional auxiliary agents such as glucose, amino acid mix is expelled in the passages through which vital energy circulates, also can be expelled to intramuscular, intracutaneous, subcutaneous or intraperitoneal separately in case of necessity.

Medication has intradermal injection, subcutaneous injection, intravenous injection, oral administration, nasal cavity administration etc.

In addition, anticarcinogen of the present invention also can be used as other anticarcinogens that can share in the immunotherapy.

The contained restricted antigen peptide of HLA-A24 of the present invention in the anticarcinogen of above-mentioned (1) in every kind of antigen peptide, is 0.01-100 weight %, preferred 0.1-95 weight %.Each adult's dosage in every kind of antigen peptide, is 0.1 μ g/kg-10mg/kg as peptide concentration, preferred 1 μ g/kg-1mg/kg, more preferably 1 μ g/kg-100 μ g/kg.The consistency of anticarcinogen of the present invention and human body is good.

The content of CTL of the present invention in the anticarcinogen of above-mentioned (2) in every kind of CTL, is 10 ⁴-10 ⁸Individual/ml, preferred 5 * 10 ⁵-5 * 10 ⁷Individual/ml.

When taking the anticarcinogen of above-mentioned (2) to the people, also can use injection and use the syringe administration, each adult's dosage is suitable selected according to patient's age, body weight, disease degree etc., and for example the CTL number is 10 in every kind of CTL ⁶-10 ¹⁰Individual.Above-mentioned scope is a benchmark, not limited by this.In anticarcinogen of the present invention, as the CTL of effective constituent come from human body to be administered, so anticarcinogen of the present invention is good to the consistency of human body.

The anticarcinogen of above-mentioned (2) can be offerd medicine to cancer patientss such as prostate cancers, and this patient has the tumour cell that expression can be used as the antigen peptide that CTL discerned of effective constituent.Therefore, contain the anticarcinogen of useful PSMA antigen peptide inductive CTL of the present invention as effective constituent, the immunotherapy that is the cancers such as prostate cancer that the male tumour cell causes for HLA-A24 and PSMA is useful.

And then the present invention can provide a kind of antigen presenting cell, and this antigen presenting cell can be with the restrictive antigen peptide of HLA-A24, be that the mixture of PSMA antigen peptide and HLA-A24 molecule is presented at cell surface.According to antigen presenting cell of the present invention, can be used for inducing CTL of the present invention.

Antigen presenting cell of the present invention can prepare by the cell that use has an antigen presentation ability.

As cell with antigen presentation ability, scavenger cell, B cell and the dendritic cell that the mixture of restricted antigen peptide of HLA-A24 of the present invention and HLA-A24 molecule can be presented on its cell surface for example arranged, and (dendritic cells DC) waits leukocyte cell etc.The antigen amount that above-mentioned DC cell reason individual cells is presented is many, and the expression amount of the needed cell surface molecule of antigen presentation (CD80, CD83, CD86 etc. are total to pungency signal (co-stimulatory signal) molecule etc.) is also high, so DC is preferred especially as the cell with antigen presentation ability.

Above-mentioned cell with antigen presentation ability can prepare from above-mentioned stripped sample.For example, above-mentioned DC can be from following three kinds of methods preparation.1) by the method for PSMA with separation purifying such as several cell surface marker things, 2) carry out the inductive method by monocyte with GM-CSF and IL-4,3) carry out the inductive method by the CD34 positive cell with cytokines such as GM-CSF, TNF-α, SCF.

As making the restricted antigen peptide of HLA-A24 of the present invention be presented to method on the cell surface with antigen presentation ability, after for example will having the cell and the restricted antigen peptide mixing of HLA-A24 of the present invention of antigen presentation ability, excess quantity is removed in washing as required, allows above-mentioned antigen peptide be carried on method on the HLA-A24 molecule etc.In addition, with with among the preparation method of above-mentioned cell with antigen presentation ability 1) the cell of method preparation the same, on the HLA-A24 molecule, presented the cell of the antigen peptide that has nothing to do with the present invention, can before the restricted antigen peptide of load HLA-A24, handle, and then remove the peptide that can not induce CTL of the present invention that is present on the HLA-A24 molecule with acid.

Antigen presenting cell of the present invention except the cell of the restricted antigen peptide of the single HLA-A24 of load, also can be the cell of the restricted antigen peptide of several HLA-A24 of each cell loading.

As additive method, for example for B cell and CD34 positive cell, the method for the cell by the above-mentioned antigen presentation ability that has of the expression vector transfection that can present the restricted antigen peptide of HLA-A24.

As this carrier, for example in commercially available plasmid such as pcDNA3, pMQMneo, pCEP4, imported the expression of gene plasmid vector of coding PSMA by the DNA recombinant technology.The gene of the aminoacid sequence that all right additional code of this expression vector is suitable is more effectively degraded by proteolytic enzyme in cell so that will be presented to the two ends of the antigen peptide on the cell surface in addition.Can utilize and import cell or its cell extract that the PSMA molecule is highly expressed in the transfection of HLA-A24 expression carrier as gained on the LNCaP cell of prostate cancer cell.Also can utilize cell or its cell extract of while transfection HLA-A24 expression vector and PSMA expression vector.And then, more preferably use retrovirus vector and adenovirus carrier.

Preferred antigen presenting cell is a nonproliferating cell.In order not make cell proliferation, available X ray isoradial irradiation or mitomycin medicines such as (mitomycin) are handled.

Whether the cell that is obtained by aforesaid method is antigen presenting cell, can estimate by the cytokine growing amount that detects CTL.

In addition, by the coding nucleic acid of people's major histocompatibility antigen and/or the coding nucleic acid of antigen protein are imported in the cell, can prepare can be killed and wounded by cytotoxic T lymphocyte, be that the mixture of the peptide of restrictive antigen protein is presented the target cell on cell surface with this people's major histocompatibility antigen with deriving to this antigen.The present invention also comprises preparation method of described target cell and the target cell that can be killed and wounded by cytotoxic T lymphocyte that is obtained by this preparation method.According to target cell of the present invention, can be in the past because of failing to determine that the cytotoxic T lymphocyte that target cell fails to estimate estimates.In addition,, can express arbitrary people's major histocompatibility antigen or antigen protein, so the target cell that might construction expression different with known target cell, people's major histocompatibility antigen and antigen protein make up according to the preparation method of target cell of the present invention.

As the nucleic acid of the above-mentioned people's major histocompatibility antigen of coding, the nucleic acid of coding HLA-A24 is for example arranged.This nucleotide sequence is open, its gene pool (GenBank) be numbered L47206.This nucleic acid can above-mentioned sequence information be the basis, waits and obtains by currently known methods, for example PCR method from suitable material.

By with the coding nucleic acid of the coding nucleic acid of people's major histocompatibility antigen and/or antigen protein by importing in the suitable cell, can obtain to express the cell of these two kinds of nucleic acid, and the cell that can be killed and wounded by cytotoxic T lymphocyte.

By with import the carrier that the object cell adapts, can in order to obtain high transfection efficiency, preferably carry out transfection in the nucleic acid transfered cell by adenovirus carrier.

When using adenovirus carrier, by methods such as common use homologous recombination the coding nucleic acid of people's major histocompatibility antigen and/or the coding nucleic acid of antigen protein are imported in the carrier, and then can prepare the adenovirus particles that contains the adenovirus carrier that imports this nucleic acid (D.M.Glover etc. for example, DNA Cloning4, Mammalian SystemsSecond Edition A Practical Approach (Japanese version, Takara Shuzo Co., Ltd's distribution)), for example can pass through COS-TPC method (S.Miyake etc., Proc.Natl.Acad.Sci.USA, the 93rd volume, the 1320th page (1966)) preparation.

By adenovirus carrier transfection nucleic acid, can adopt currently known methods, for example the adenovirus particles that contains above-mentioned adenovirus carrier of COS-TPC method preparation can be infected as the cell that imports object.

Above-mentioned carrier can have some necessary elements, so that the coding nucleic acid of the coding nucleic acid of people's major histocompatibility antigen and/or antigen protein is easy to express in cell.For example, at the regulating and controlling sequences such as promoter sequence of the additional regulatory transcription in the upstream of the nucleic acid of expressing object, translation initiation codon etc., additional translation stop codon in its downstream makes up the recombinant vectors that can duplicate and play a role thus in cell.

Specifically, when importing zooblast, SV40 gene promoter, adenovirus major late gene promotor, thymidine kinase gene promotor, metallothionein gene promotor, beta-actin gene promoter, CAG promotor, SR α promotor, EF1 α promotor, CMG promotor, PGK promotor etc. are arranged as promotor with adenovirus carrier.Consider that from the expression intensity angle the present invention preferably uses the CAG promotor.

When the coding nucleic acid by adenovirus carrier expressing human major histocompatibility antigen and/or the coding nucleic acid of antigen protein, as the cell that imports object, for example have an expressing human major histocompatibility antigen and antigen protein one of them or cell that the two is not all expressed.

The gained transfectant can be cultivated in suitable medium according to the needs of cell.

Whether the gained transfectant presents above-mentioned people's major histocompatibility antigen and/or antigen protein, can estimate by the cytokine amount that CTL generates.

Antigen presenting cell of the present invention is owing to present the mixture of restricted antigen peptide of HLA-A24 and HLA-A24 molecule on cell surface, and can induce CTL of the present invention, so this antigen presenting cell can be used as the CTL inductor.The present invention also comprises this CTL inductor.This inductor contains antigen presenting cell of the present invention as effective constituent.

CTL inductor of the present invention provides with the formulation that this antigen presenting cell is suspended in substratum, physiological saline or the phosphoric acid buffer physiological saline that is suitable for preserving cell.As substratum, substratum such as RPMI, AIM-V, X-VIVO10 are for example arranged.To keep stabilizing to purpose, also can add serum albumin etc. in above-mentioned substratum or the damping fluid.

The content of the antigen presenting cell in the CTL inductor of the present invention in every kind of antigen presenting cell, is 10 so long as induce the required amount of CTL to get final product ³-5 * 10 ⁶Individual/ml, preferred 10 ⁴-10 ⁶Individual/ml.Described content, for example the human body from the HLA-A24 antigen positive separates peripheral blood lymphocyte, under isolated condition, add the different reagent of antigen presenting cell content respectively, cultivate while stimulating, at this moment can determine by the content that inducing specific is discerned this antigen presenting cell in the reagent of CTL of HLA-A24 positive cell.

CTL inductor of the present invention, when can be used as in-vitro multiplication CTL of the present invention outside the additive of substratum, also can be used for the T lymphocyte proliferation activity is the diagnosis etc. of the sensitization state degree of index.When for example being used as the additive of substratum,, can the individual ratio of 0.01-1 use usually with respect to 1 lymphocyte that is used to induce CTL.

Antigen presenting cell of the present invention can be induced the restrictive CTL of HLA-A24 common among Asia ethnic group, the especially Japanese, so this antigen presenting cell itself just can be used as the anticarcinogen of cancer, especially prostate cancer.In the anticarcinogen of the present invention, also comprise and contain the anticarcinogen of (3) antigen presenting cell of the present invention as effective constituent.

The anticarcinogen of above-mentioned (3) can provide the formulation that antigen presenting cell of the present invention is suspended in the pharmaceutically acceptable diluent.Above-mentioned thinner for example is suitable for preserving substratum, phosphoric acid buffer, physiological saline of cell etc.As substratum, substratum such as RPMI, AIM-V, X-VIVO10 for example.In order to keep stability of drug, can also contain pharmaceutically acceptable carrier in the anticarcinogen of above-mentioned (3).As above-mentioned carrier, human serum albumin etc. for example.

The content (active constituent content) of the antigen presenting cell in above-mentioned (3), amount with this antigen presenting cell inductive CTL is, the required amount of tumour cell in cancer, the especially prostate cancer that killing prostate specific membrane antigen (prostate specific membrane antigen, below PSMA) slightly unconventionality expression causes gets final product.This effective constituent amount can equally with the anticarcinogen of above-mentioned (1) or (2) be determined.Specifically, above-mentioned effective constituent amount in every kind of antigen presenting cell, is 10 in anticarcinogen ⁵-10 ⁸Individual/ml, preferred 5 * 10 ⁵-5 * 10 ⁷Individual/ml.

The anticarcinogen of above-mentioned (3) is offerd medicine to man-hour, can use the syringe administration, each adult's dosage can be suitable selected according to patient's age, body weight, ill degree etc., for example, as the quantity of antigen presenting cell, in every kind of antigen presenting cell, preferred 10 ⁴-10 ⁹Individual.Above-mentioned scope only is a benchmark, not limited by this.In addition,, so good as the antigen presenting cell of effective constituent to the consistency of human body because come from the people who desires administration.

In addition, this anticarcinogen also can be used as other anticarcinogens that can share in the immunotherapy.

According to CTL of the present invention, can also detect cell to above-mentioned CTL sensitivity.The present invention also comprises the detection method of CTL sensitive cells.

The detection method of the sensitive cells of cytotoxic T lymphocyte of the present invention is characterised in that with following index and detects, promptly, make cytotoxic T lymphocyte of the present invention and cells contacting to be measured, when changing thus, the cell (sensitive cells) of this cell to be measured for cytotoxic T lymphocyte of the present invention is had susceptibility.

Above-mentioned " cell to be measured " is meant the people's cell or the strain cell of stripped samples such as deriving from peripheral blood lymphocyte, tissue." sensitive cells " in this specification sheets is meant and can be discerned by CTL, also can cause the abnormal cellss such as cancer cells of lysis or release of cytokines.

Contact and the variation that produces for example lysis that causes of CTL, release of cytokines, CTL propagation etc. with sensitivity cell in the cell to be measured by CTL.

The detection of lysis behind available radio isotope and the fluorescein-labelled cell to be measured, when contacting with CTL again, is measured the amount of the radioisotopic amount that discharges in the cell to be measured and fluorescein and to be finished.

The detection method of release of cytokines can be by finishing measuring from the burst size of GM-CSF, TNF in CTL or the cell to be measured, IFN-γ etc.

The detection of CTL propagation can be measured, wait with mensuration number of cells such as microscopic examinations and finish by the 3H-thymidine amount of pair cell picked-up.

By this variation, can detect the HLA-A24 and the PSMA that are present in the cell to be measured and be the male tumour cell.

In the above-mentioned detection method, the preferred use contained the detection reagent of CTL of the present invention as the CTL sensitive cells of effective constituent.This detection reagent is also contained among the present invention.

Detection reagent of the present invention, the formulation that can be suspended in the substratum, physiological saline or the phosphate buffer normal saline that are suitable for preserving above-mentioned CTL provides.

As substratum, substratum such as RPMI, AIM-V or X-VIVO10 for example.To stablize CTL is purpose, also can add serum albumin etc. in above-mentioned substratum or the damping fluid.

The content of CTL (active constituent content) in the above-mentioned detection reagent is so long as cause that above-mentioned CTL contacts the required amount of the variation that produced (for example, the lysis that causes of CTL, release of cytokines, CTL propagation etc.) and gets final product with sensitive cells in the cell to be measured.Specifically, the content of CTL, in every kind of CTL, preferred 10 ⁴-10 ⁸Individual/ml.

Detection reagent of the present invention can be used for also identifying whether the HLA of cell expressing to be measured is the classification of the HLA of HLA-A24 usually except being used for detecting the sensitive cells of CTL of the present invention in the cell to be measured.

Detection reagent of the present invention also can suitably contain and is useful on the reagent that detects above-mentioned variation, for example molecular marked compound etc.

The present invention also provides the method that detects people's major histocompatibility antigen (HLA)-A24 molecule in the cell to be measured.One of feature of the detection method of people's major histocompatibility antigen (HLA)-A24 molecule is, under the condition that the restricted antigen peptide of HLA-A24 exists, makes cytotoxic T lymphocyte of the present invention and cells contacting to be measured.

For example, the HLA-A24 molecule on the cell surface to be measured can CTL of the present invention and the variation that produced of cells contacting to be measured detect as index.The variation that is produced as CTL and cells contacting to be measured, for example propagation of the lysis, release of cytokines or the CTL that cause of CTL.This variation can be detected by aforesaid method.

According to the said determination result, not only can detect having or not of HLA-A24 developed by molecule, also can measure the expression amount of the HLA-A24 molecule on the cell to be measured.

In addition, whether express PSMA in to cell to be measured when not understanding fully, can detect the HLA-A24 molecule according to following method.In present method, in reaction solution, allow cell to be measured and the ultimate density be the CTL of the present invention coexistence of mixture 0.01-50 μ g/ml, that can discern the restricted antigen peptide of HLA-A24 of the present invention, especially this antigen peptide and HLA-A24 molecule, this CTL and cells contacting to be measured are got final product.

The present invention can detect the HLA-A24 molecule of tumour cell, and adopts the method that must have the complement activation step in the past and need to detect the HLA molecule with anti-hla antibody to be difficult to accomplish this point.

Embodiment

Below, in conjunction with the embodiments the present invention is more specifically illustrated, but the present invention is not subjected to the qualification of these embodiment.

The preparation of embodiment 1 PSMA antigen peptide specific CTL

In the present embodiment, CTL is by the revulsion preparation that utilizes antigen presentation with dendritic cell (DC).

(1) CTL induces the screening with peptide to reach synthetic

The aminoacid sequence of the psma protein matter of forming for 750 amino acid, (its N-terminal the 2nd amino acids residue is selected from Tyr, Phe, Trp and Met with peptide with HLA-A24 associativity motif structure, the C-terminal amino-acid residue is selected from Leu, Ile, Phe and Trp) be the center, 9 peptides shown in the screening table 1 are as candidate's peptide of PSMA antigen peptide.

[table 1]

Sequence number	Position among the PSMA	Title
Sequence number	Position among the PSMA	Title		10	298-306	PSMA24-1
11	624-632	PSMA24-2		10	298-306	PSMA24-1
11	624-632	PSMA24-2	1	227-235	PSMA24-3
12	606-614	PSMA24-4	1	227-235	PSMA24-3
12	606-614	PSMA24-4	2	178-186	PSMA24-5
13	74-83	PSMA24-6	2	178-186	PSMA24-5
13	74-83	PSMA24-6	14	565-574	PSMA24-7
15	699-708	PSMA24-8	14	565-574	PSMA24-7
15	699-708	PSMA24-8	16	624-633	PSMA24-9

In the table 1, the sequence number that is numbered the aminoacid sequence of each peptide of expression in the sequence table that " sequence number " is.Being numbered of " position among the PSMA " one begun several total number of atnino acid by psma protein matter N-terminal.The symbolic representation of " title " hurdle item is by the title of the peptide of inventor's name.With Peptide synthesizer (PSSM-8, Shimadzu Seisakusho Ltd.'s system), prepare these peptides by solid phase Fmoc method.

(2) effector cell's preparation

For the PSMA24-1～PSMA24-9 of preparation in above-mentioned (1) totally 9 kinds of peptides, induce the effector cell of each peptide of identification by the normal people's who carries HLA-A24 PBMC, and measure cell killing activity.Below, the narration preparation process.

1. the preparation of PBMC

Take a blood sample by composition from the normal people who carries HLA-A24.Blood sampling is added in the upper strata of Ficoll-Paque parting liquid (Pharmacia corporate system), 500 * g, centrifugal 20 minutes in room temperature.Reclaim the PBMC in middle layer, after the washing, be suspended in the preservation liquid of forming by 90% N of tire serum (FCS, Intergen corporate system) and 10% dimethyl sulfoxide (DMSO) (Sigma corporate system), and be stored in (preservation PBMC) in the liquid nitrogen with this state.

2. the preparation of target cell

The EBV that expresses HLA-A24 is transformed B cell TISI (WSN09042), measure the day before yesterday by (1) preparation, respectively contain any peptide among 10 μ g/ml PSMA24-1～PSMA24-9 5 kinds of substratum (below, being designated as TISI (+) at the TISI that cultivates in this substratum) or do not contain in the substratum (below, the TISI that cultivates in this substratum is designated as TISI (-)) of peptide and carry out incubated overnight.Measure the same day, with each 5 * 10 ⁶Individual TISI (+) and TISI (-) are respectively at the Na of 200 μ Ci ₂ ⁵¹CrO ₄37 ℃ were mixed 1 hour down in the solution, then with the RPMI1640 nutrient solution washing that contains 10%FCS, and conduct ⁵¹The target cell of Cr mark is used.

3. effector cell's preparation

The preservation PBMC of preparation in above-mentioned (1) is melted, after the washing, the cell suspending liquid of 15ml is joined in the T-75 flask (コ one ニ Application グ corporate system), in 37 ℃ place 1.5 hours after, add the 5H-RPMI that 25ml contains the IL-4 (ジエ Application ザイ system corporate system) of the GM-CSF (シエ one リ Application グ corporate system) of 1000U/ml and 1000U/ml to the flask adherent cell, in 37 ℃ at CO ₂Cultivate in the incubator, and induce DC.After 7 days, reclaim cell, be suspended in the peptide that contains 40 μ g/ml, the β of 1 μ g/ml ₂Among the PBS of microglobulin, 1% bovine serum albumin, and to make its final concentration be 3 * 10 ⁶Individual/ml, reaction is 4 hours under the room temperature.Use x-ray bombardment (5500Rad) then,, make its final concentration reach 1 * 10 with the 5H-RPMI dilution ⁶Individual/ml, as antigen presenting cell.

After will melting by the above-mentioned preservation PBMC that 1. obtains, in 4 ℃, be suspended in the phosphate buffer normal saline (PBS) that contains 1% people AB serum (below, 1H-PBS) slightly, and to make its cell concn be 2 * 10 ⁷Individual/ml, obtain PBMC suspension.Then, for 2 * 10 ⁷The PBMC of individual/ml added the pearl that combine anti-CD8 antibody (Dynabeads M450, ダイ Na Le corporate system) of 140 μ l through 1H-PBS washing, in 4 ℃, reaction 1 hour.

Used cell count 1 * 10 during then, with above-mentioned preparation PBMC suspension ⁸Among the individual 1H-PBS that is suspended in 0.9ml, add the 0.1ml cell in the suspension of gained and separate and smoke pearl (DETACHaBEAD, ダイ Na Le corporate system).Then, gained solution reclaims the cell of desorb in mixed at room temperature 1 hour, washs with 1H-PBS.

The gained washed cell is suspended among the 5H-RPMI, and to make its final concentration be 2 * 10 ⁶Individual/ml.Gained suspension respectively with PSMA24-1～PSMA24-9 in the DC suspension balanced mix of any peptide and co-cultivation, and be that 10ng/ml adds IL-7 by final concentration.Gained solution is injected in 48 well culture plates with every hole 0.5ml respectively, and at 37 ℃ CO ₂Cultivate in the incubator.Second day, adding contained the 5H-RPMI 50 μ l of the IL-10 of 100ng/ml in culture.

Remove the culture supernatant in each hole after one week,, obtain suspension the 5H-RPMI of cell suspension in 0.5ml.Then, will be by the β of any peptide and 1 μ g/ml in the PBMC gained adherent cell of x-ray bombardment (5500Rad) and the PSMA24-1～PSMA24-9 that contains 20 μ g/ml ₂The 1HSA-PBS (phosphate buffer normal saline that contains 1% human serum albumin) of trace sphaeroprotein placed 2 hours in room temperature, washed each hole, and containing at gained stimulates with adding above-mentioned suspension in each hole of antigen presenting cell again.

Second day, in each hole, add IL-10, and to make its final concentration be 10ng/ml.Then, in each hole, add every three days and contain the 5H-PRMI of rIL-2, and make its ultimate density reach 20IU/ml, and at CO ₂Cultivate a week in the incubator.After carrying out 3 same stimulations more again, measure in each hole the effector cell to the cell killing activity of target cell.

PSMA24-3 and PSMA24-5 effector cell to the showed cell killing activity breed with anti-cd 3 antibodies respectively.Proliferating cells is suspended among the 5H-RPMI, measures cell killing activity target cell with the method for following (3).

(3) mensuration of the cell killing activity of CTL

With 5H-RPMI dilution above-mentioned (2) gained effector cell, and to make its final concentration be 3 * 10 ³～1 * 10 ⁶Individual/ml.Gained dilution 100 μ l/ holes join respectively in each hole of 96 well culture plates.Then, interpolation contains 1 * 10 in each hole ⁴Individual ⁵¹The target cell of Cr mark and 3 * 10 ⁵100 μ l cell suspending liquids of individual K562 cell obtain cell mixture.Above-mentioned K562 cell is used to eliminate the non-specific killing activity that the NK cell of sneaking into causes.

With above-mentioned cell mixture after 400 * g is centrifugal 1 minute, at 37 ℃ CO ₂Placed 5 hours in the incubator.Then, get the culture supernatant in 100 each hole of μ l, discharge with gamma counter mensuration ⁵¹The Cr amount.

The specific cell killing activity can be by calculating with following formula 1.

In the above-mentioned formula 1, minimum releasing value is for hole that target cell and K562 cell only are housed ⁵¹The Cr amount is in the expression target cell ⁵¹The natural burst size of Cr.Maximum releasing value is, add tensio-active agent TrionX-100 in the target cell after, when cell is destroyed ⁵¹The Cr burst size.

Fig. 1 and Fig. 2 show by PSMA24-3 or PSMA24-5 inductive effector cell specific cell killing activity curve TISI (-) and TISI (+), when the various E/T.

Result when Fig. 1 represents to use by PSMA24-3 inductive effector cell.Result when Fig. 2 represents to use by PSMA24-5 inductive effector cell.In addition, white box () expression with TISI (-) as target cell, solid black circle () expression with TISI (+) result during as target cell.In addition, among Fig. 1 and 2, ordinate zou is that specific cell killing activity (%), X-coordinate are the E/T ratio.

The result shows, by PSMA24-3 and PSMA24-5 inductive effector cell the TISI (+) that has added various peptides is demonstrated the specific cytotoxicity of antigen peptide.

By The above results as can be known, by PSMA24-3 (sequence number 1) and PSMA24-5 (sequence number 2) inductive effector cell for having the specific Cytotoxic CTL of antigen peptide.

The preparation of embodiment 2 PSMA antigen peptide functional derivatives

(1) the PSMA24-5 variant is synthetic

According to synthetic PSMA24-5 in embodiment 1 (1), 7 kinds of derivatives shown in the table 2 have been designed.

Table 2

Sequence number	Title
Sequence number	Title	8	PSMA24-5-1A
17	PSMA24-5-4A	8	PSMA24-5-1A
17	PSMA24-5-4A	9	PSMA24-5-5A
18	PSMA24-5-6A	9	PSMA24-5-5A
18	PSMA24-5-6A	19	PSMA24-5-7A
20	PSMA24-5-8A	19	PSMA24-5-7A
20	PSMA24-5-8A	5	PSMA24-5-9L

Prepare the totally 7 kinds of peptides of the PSMA24-5-1A～PSMA24-5-9L shown in the table 2 with Peptide synthesizer.

(2) evaluation of PSMA24-5 functional derivatives

Confirm for the peptide of gained CTL in the foregoing description 1 of the mixture that can discern PSMA24-5 and HLA-A24 molecule shown in whether can Identification Lists 2 and the mixture of HLA-A24 molecule.

At first, for discerning, right by embodiment 1 (2) CTL with the PSMA24-5 of quadrat method preparation ⁵¹Cr mark TISI (-) cell and with the peptide that is selected from PSMA24-5-1A～PSMA24-5-9L and by embodiment 1 (2) 7 kinds with the quadrat method preparation ⁵¹Whether Cr mark TISI (+) cell can showed cell toxicity be confirmed.

The result shows, can discern the CTL of PSMA24-5, to the culture medium culturing gained that adds a kind of peptide being selected from PSMA24-5-1A, PSMA24-5-5A and PSMA24-5-9L ⁵¹Cr mark TISI (+) demonstrates cytotoxicity.In addition, this CTL is right ⁵¹Cr mark TISI (-) cell does not demonstrate cytotoxicity.Show that thus PSMA24-5-1A (sequence number 8), PSMA24-5-5A (sequence number 9) and PSMA24-5-9L (sequence number 5) are the functional derivatives of PSMA24-5.

Embodiment 3 utilizes adenovirus evaluate CT L

(1) prepares gene by adenovirus and import target cell

As the target cell of endogenous antigen-presenting, structure can be expressed the tumour cell of HLA-A24 and PSMA.That is, to expressing PSMA, not expressing the prostate cancer cell strain LNCaP cell of HLA-A24, with changeing HLA-A24 gene adenovirus carrier with the gene transfered cell.To expressing HLA-A24, not expressing cancer cells MKN45,888mel, SW480, the T.T of PSMA, with changeing PSMA gene adenovirus carrier with PSMA gene transfered cell.

The transgenosis adenovirus carrier prepares according to the COS-TPC method.

At first, with 5 * 10 ⁵The cell suspending liquid 4ml of individual/ml is seeded in the 6 porocyte culture plates, incubated overnight.After the cultivation,, cultivated again 2 days with changeing HLA-A24 gene adenovirus, changeing PSMA gene adenovirus or the above-mentioned cell of not genetically modified adenovirus infection (MOI=20-30).Reclaim the cell of adenovirus infection, the RPMI1640 nutrient solution washing with containing 10%FCS prepares target cell.

(2) utilize the functional evaluation of the CTL of transgenosis target cell

Whether the target cell that reacts as the antigen-specific of evaluate CT L for the transgenic cell with Adenovirus Transfection can play a role, and confirms with the cytokine generative capacity of CTL.

(2) by embodiment 1 are diluted to 1 * 10 with quadrat method inductive effector cell with 5H-RPMI ⁵Individual/ml, the gained dilution is added in respectively in each hole of culture plate at the bottom of the 96 hole U types with 100 μ l/ holes.What then, prepare in the adding above-mentioned (1) in each hole contains 1 * 10 ⁴The 100 μ l cell suspending liquids of individual target cell.

With the CO of culture plate at 37 ℃ ₂Place in the incubator and spend the night.Then, get the culture supernatant 100 μ l in each hole, personnel selection IFN-γ measures with ELISA test kit (ジエ Application ザイ system テ Network ノ corporate system) and measures IFN-γ.IFN-γ growing amount is obtained according to the typical curve of the IFN-γ standard substance of measuring simultaneously.

To importing the prostate cancer cell strain LNCaP of HLA-A24 gene, PSMA24-3 or PSMA24-5 inductive CTL have generated IFN-γ, and to not importing the LNCaP of HLA-A24 gene, do not generate IFN-γ.

In addition, to importing HLA-A24 expression cell line 888mel, the SW480 of PSMA gene, PSMA24-3 inductive CTL has generated IFN-γ.To importing HLA-A24 expression cell line MKN45, the T.T of PSMA gene, PSMA24-5 inductive CTL has generated IFN-γ.To HLA-A24 non-expressing cell strain MKN28 and 526mel, all CTL all do not generate IFN-γ.

Above result shows, PSMA24-3 specific CTL and PSMA24-5 specific CTL to importing the target cell of HLA-A24 gene or importing PSMA expression of gene HLA-A24, expression PSMA, all can generate IFN-γ; Thus, utilize the transgenic cell of adenovirus to can be used for the functional evaluation of CTL; And then these transgenic cells are because of presenting antigen peptide of the present invention on the HLA-A24 molecule, so can be used as antigen presenting cell.

The sequence table explanation

Sequence number 1 is the sequence (PSMA24-3) of the restricted antigen peptide of synthetic HLA-A24.

Sequence number 2 is the sequence (PSMA24-5) of the restricted antigen peptide of synthetic HLA-A24.

Sequence number 3 is the sequence (PSM-P1) of the restricted antigen peptide of HLA-A2.1.

Sequence number 4 is the sequence (PSM-P2) of the restricted antigen peptide of HLA-A2.1.

Sequence number 5 is the sequence (PSMA24-5-9L) of derivative peptide of the peptide of sequence number 2.

Sequence number 6 is the sequence (PSMA24-5-1Q) of derivative peptide of the peptide of sequence number 2.

Sequence number 7 is the sequence (PSMA24-5-5S) of derivative peptide of the peptide of sequence number 2.

Sequence number 8 is the sequence (PSMA24-5-1A) of derivative peptide of the peptide of sequence number 2.

Sequence number 9 is the sequence (PSMA24-5-5A) of derivative peptide of the peptide of sequence number 2.

Sequence number 10 is the sequence (PSMA24-1) with synthetic peptide of HLA-A24 binding motif.

Sequence number 11 is the sequence (PSMA24-2) with synthetic peptide of HLA-A24 binding motif.

Sequence number 12 is the sequence (PSMA24-4) with synthetic peptide of HLA-A24 binding motif.

Sequence number 13 is the sequence (PSMA24-6) with synthetic peptide of HLA-A24 binding motif.

Sequence number 14 is the sequence (PSMA24-7) with synthetic peptide of HLA-A24 binding motif.

Sequence number 15 is the sequence (PSMA24-8) with synthetic peptide of HLA-A24 binding motif.

Sequence number 16 is the sequence (PSMA24-9) with synthetic peptide of HLA-A24 binding motif.

Sequence number 17 is the sequence (PSMA24-5-4A) of derivative peptide of the peptide of sequence number 2.

Sequence number 18 is the sequence (PSMA24-5-6A) of derivative peptide of the peptide of sequence number 2.

Sequence number 19 is the sequence (PSMA24-5-7A) of derivative peptide of the peptide of sequence number 2.

Sequence number 20 is the sequence (PSMA24-5-8A) of derivative peptide of the peptide of sequence number 2.

The effect of invention

The invention provides among Asia ethnic group, the especially Japanese, cancer, particularly, the treatment of the cancer of the unconventionality expression PSMA such as prostate cancer and diagnose useful technology. Specifically, CTL of the present invention can bring into play following excellent results, namely, can treat and diagnose cancer, the cancer that is specially the PSMA unconventionality expressions such as prostate cancer, alternative is killed and wounded all positive tumour cells of HLA-A24 and PSMA, and can be used as the cell medicine (anticarcinogen) for the treatment of cancer, and all detections of having or not of positive tumour cell of the HLA-A24 in the sample that can exsomatize and PSMA. The restricted antigenic peptides alternative of HLA-A24 of the present invention is induced above-mentioned CTL. Thus, the restricted antigenic peptides of HLA-A24 of the present invention can be used as derivant and the anticarcinogen of CTL. Antigen presenting cell of the present invention can be used among the preparation method of this CTL and useful as anti-cancer agents. According to CTL abductive approach of the present invention, can reach efficiently and optionally obtain the effect of CTL of the present invention. According to CTL inducer of the present invention, can be efficiently and optionally induce CTL of the present invention, and can show Asia ethnic group, especially Japanese cancer, be specially the useful advantageous property for the treatment of of the cancer of the unconventionality expression PMSA such as prostate cancer. According to CTL sensitive cells detection method of the present invention and detection reagent, can reach efficiently and optionally detect the good effect of CTL sensitive cells. And then, according to CTL sensitive cells detection method of the present invention and detect reagent, can reach HLA is classified, and be applied to cancer, be specially the excellent results of the diagnosis etc. of prostate cancer etc. According to nucleic acid of the present invention, can reach the excellent results of the restricted antigenic peptides of efficient supply HLA-A24 of the present invention. According to the preparation method of the target cell that can be killed and wounded by cytotoxic T lymphocyte of the present invention, can reach the excellent results of construction expression target cell different from known target cell, people's major histocompatibility antigen and antigen protein combination. And then, according to the target cell that can be killed and wounded by cytotoxic T lymphocyte of the present invention, can reach the excellent results because failing to determine that cytotoxic T lymphocyte that target cell fails to estimate is estimated in the past.

The simple declaration of accompanying drawing

Result when Fig. 1 represents to use by PSMA24-3 inductive effector cell.White box () expression with TISI (-) as target cell, solid black circle () expression with TISI (+) result during as target cell.Ordinate zou is that specific cell killing activity (%), X-coordinate are the E/T ratio.

Result when Fig. 2 represents to use by PSMA24-5 inductive effector cell.White box () expression with TISI (-) as target cell, solid black circle () expression with TISI (+) result during as target cell.Ordinate zou is that specific cell killing activity (%), X-coordinate are the E/T ratio.

Sequence table

＜110〉Takara Shuzo Co., Ltd (Takara Shuzo Co.Ltd.)

＜120〉cytotoxic T lymphocyte

<130>02-013CN

<150>JP2001-104693

<151>2001-04-03

<160>20

<170>PatentIn Ver.2.1

<210>1

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

＜223〉description of artificial sequence: the sequence of the restricted antigen peptide of synthetic HLA-A24

<400>1

Leu Tyr Ser Asp Pro Ala Asp Tyr Phe

1 5

<210>2

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>2

Asn Tyr Ala Arg Thr Glu Asp Phe Phe

1 5

<210>3

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

＜223〉description of artificial sequence: the sequence of the restricted antigen peptide of HLA-A2.1

<400>3

Leu Leu His Glu Thr Asp Ser Ala Val

1 5

<210>4

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>4

Ala Leu Phe Asp Ile Glu Ser Lys Val

1 5

<210>5

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

＜223〉description of artificial sequence: the sequence of derivative peptide that is used for the peptide of composition sequence numbers 2

<400>5

Asn Tyr Ala Arg Thr Glu Asp Phe Leu

1 5

<210>6

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>6

Gln Tyr Ala Arg Thr Glu Asp Phe Phe

1 5

<210>7

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>7

Asn Tyr Ala Arg Ser Glu Asp Phe Phe

1 5

<210>8

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>8

Ala Tyr Ala Arg Thr Glu Asp Phe Phe

1 5

<210>9

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>9

Asn Tyr Ala Arg Ala Glu Asp Phe Phe

1 5

<210>10

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

＜223〉description of artificial sequence: be used for synthetic sequence with peptide of HLA-A24 binding motif

<400>10

Gly Tyr Tyr Asp Ala Gln Lys Leu Leu

1 5

<210>11

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>11

Thr Tyr Ser Val Ser Phe Asp Ser Leu

1 5

<210>12

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>12

Lys Tyr Ala Asp Lys Ile Tyr Ser Ile

1 5

<210>13

<211>10

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>13

Leu Tyr Asn Phe Thr Gln Ile Pro His Leu

1 5 10

<210>14

<211>10

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>14

Phe Tyr Asp Pro Met Phe Lys Tyr His Leu

1 5 10

<210>15

<211>10

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>15

Lys Tyr Ala Gly Glu Ser Phe Pro Gly Ile

1 5 10

<210>16

<211>10

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>16

Thr Tyr Ser Val Ser Phe Asp Ser Leu Phe

1 5 10

<210>17

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>17

Asn Tyr Ala Ala Thr Glu Asp Phe Phe

5

<210>18

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>18

Asn Tyr Ala Arg Thr Ala Asp Phe Phe

5

<210>19

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>19

Asn Tyr Ala Arg Thr Glu Ala Phe Phe

5

<210>20

<211>9

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<400>20

Asn Tyr Ala Arg Thr Glu Asp Ala Phe

5

Claims

1. a human body major histocompatibility antigen (HLA)-restricted antigen peptide of A24, it has the aminoacid sequence shown in sequence number 1,2,5,8 or 9.

2. cytotoxic T lymphocyte, it has following TXi Baoshouti, but this TXi Baoshouti specific recognition is presented cell on cell surface with the restricted antigen peptide of the described HLA-A24 of claim 1 and HLA-A24 molecule in conjunction with the mixture that forms.

3. the external evoked method of a cytotoxic T lymphocyte is characterized in that, comes external evoked cytotoxic T lymphocyte by using the restricted antigen peptide of the described HLA-A24 of claim 1.

4. the described external evoked method of claim 3 comprises following 3 steps,

(1) restricted antigen peptide of the described HLA-A24 of claim 1 and the a kind of cells contacting with antigen presentation ability that is selected from scavenger cell, B cell and the dendritic cell are obtained antigen presenting cell, with obtaining antigen presenting cell the cell with antigen presentation ability is carried out antigenic stimulation then, thus, obtain effector cell's step

(2) with above-mentioned steps (1) the effector cell that obtains with can be contacted by the target cell that the described cytotoxic T lymphocyte of claim 2 kills and wounds, detecting whether to have the step of cytotoxic activity, and

5. the described external evoked method of claim 4 is characterized in that, under the condition that IL-7 exists, and for 1 lymphocyte, the antigen presenting cell of 0.01-1 ratio of contact.

6. the described external evoked method of claim 4, under the condition of IL-7 and the existence of keyhole  hemocyanin, with the restricted antigen peptide of the described HLA-A24 of claim 1, add in this peripheral blood lymphocytes by the amount of adding 1ng-100 μ g in the every 1ml solution that contains peripheral blood lymphocytes.

7. CTL inductor, it contains the restricted antigen peptide of the described HLA-A24 of claim 1 as effective constituent, and is used to obtain the described cytotoxic T lymphocyte of claim 2 (CTL).

8. anticarcinogen, it contains the described cytotoxic T lymphocyte of claim 2 as effective constituent.

9. antigen presenting cell is presented the mixture of restricted antigen skin of the described HLA-A24 of claim 1 and HLA-A24 molecule at cell surface and is formed.

10. CTL inductor, it contains the described antigen presenting cell of claim 9 as effective constituent, and is used to obtain the described cytotoxic T lymphocyte of claim 2 (CTL).

11. the detection method of a cytotoxic T lymphocyte sensitive cells, it is characterized in that, it detects index, make described cytotoxic T lymphocyte of claim 2 and cells contacting to be measured, thereby when causing the variation of the lysis, release of cytokines and the CTL propagation that are selected from CTL and cause, the cell (sensitive cells) of described cell to be measured for the described cytotoxic T lymphocyte of claim 2 is had susceptibility.

12. the detection method of people's major histocompatibility antigen (the HLA)-A24 molecule in the cell to be measured, it is characterized in that, under the condition that the restricted antigen peptide of the described HLA-A24 of claim 1 exists, make described cytotoxic T lymphocyte of claim 2 and cells contacting to be measured.

13. a nucleic acid, the restricted antigen peptide of its coding described HLA-A24 of claim 1.

14. the external preparation method of the target cell of being killed and wounded by cytotoxic T lymphocyte comprises that nucleic acid with the restricted antigen peptide of the coding described HLA-A24 of claim 1 is in the external step that imports in the cell.

15. a target cell of being killed and wounded by cytotoxic T lymphocyte, described preparation method obtains by claim 14.