CN105163754B

CN105163754B - Prostate specific tumour antigen and application thereof

Info

Publication number: CN105163754B
Application number: CN201380056563.6A
Authority: CN
Inventors: 王荣福
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-09-20
Filing date: 2013-09-18
Publication date: 2018-01-05
Anticipated expiration: 2033-09-18
Also published as: WO2014047085A2; CN105163754A; WO2014047085A3

Abstract

21 kinds of PSGR derived peptides are predicted by Immunoinformatics method based on HLA A2 binding motifs, have detected the ability of the peptide-specific T-cell response in its inducing peripheral blood monocyte (PBMCs), PMBC is obtained from HLA A2⁺Healthy donors or HLA A2⁺Patients with prostate cancer.It also tested the HLA A2 positives and express the identification of PSGR LNCaP cells.3 kinds of peptides, PSGR3, PSGR4 and PSGR14 often induce peptide-specific T-cell response in the PBMCs from healthy donors and patients with prostate cancer, and by CD8 in a manner of HLA A2 dependences⁺T cell identifies.These peptide specific T cells are with HLA I classes restrictive one identification HLA A2⁺And PSGR⁺Tumour cell, and kill LNCaP prostate gland cancer cells.The PSGR derived peptides of these identifications can be used as the Immune target of diagnosis marker and anti-cancer vaccine.

Description

Prostate specific tumour antigen and application thereof

Background technology

Prostate cancer has become most common cancer in American male, and is second largest cancer cause of the death of American male [1].It is operation and/or radiotherapy to the processing of most of patients with prostate cancer standards.However, up to 30% corrective surgery or radiotherapy Palindromia still occurs afterwards.Although male sex hormone blocking treatment is effective treatment method of anti-recurrence disease, these most of trouble Person finally develops into the refractory type prostate cancer of male sex hormone, and it is insensitive to traditional treatment.Therefore, there is an urgent need to it is more effective and compared with The therapy of small toxicity.Through showing that immunization therapy is promising prostate cancer therapy method, particularly to metastatic castration The patient [2-4] of refractory prostate cancer, it is promising cancer treatment method that control immune system, which eliminates malignant cell, but directly Fragmentary clinical success [4-6] is also only obtained to nearest its.Nearest Food and Drug Admistraton (FDA) approval is based on immunization therapy Vaccine/medicine sipuleucel-T (Provenge) and ipilimumab (Yervoy) are represent in cancer immunotherapy field Journey upright stone tablet [7,8].In addition, the gp100 peptides III phases clinical trial of the melanoma also very encouraging clinical effectiveness of output [9].However, the clinical benefit of these medicaments of report does not reach complete response and permanent healing far.With regard to sipuleucel- For T, it is only 4.1 months that the existence of patient, which is benefited, without the retroversion of objective tumour or PSA (PSA) water Flat substantial variation.Tumour specific antigen, which is further disclosed, using the nearest research of animal model is inducing confrontation development Importance [10] in the immune response of tumour, this has promoted more effort to differentiate that it is anti-that such cancer immunotherapy is used It is former.Further, since some main rejection antigens may be selected and killed because of T cell and lose or change [11], best plan Slightly it is come immunization therapy for kinds of tumors antigen present on individual tumors.

So far, it specify that a variety of prostate specific tumour antigens, including PSA [12,13], prostein [14, 15], prostate stem cell antigen (PSCA) [16], PSMA (PSMA) [17-19], prostatic acid phosphatase Enzyme (PAP) [20], and transient receptor potential p8 (trp-p8) [21].Further, it is described that derived from these tumour antigens HLA I classes restricted epitopes [22].One shortcoming of the immunization therapy based on single tumour antigen is that immunologic escape possible occur. Therefore it is used for metastatic, it is necessary to differentiate other PSA to develop the vaccine of more effective and antigentic specificity Patients with prostate cancer.

The content of the invention

Prostate specific G-protein coupling receptor (PSGR) is the member that G-protein couples olfactory receptor family, and is compared Normal prostate cell altimeter in prostate gland cancer cell reaches [23-25], and this shows that can be directed to PSGR develops anti-prostate The new Immunotherapy Strategy of cancer.

We determined that PSGR is identified by T cell, and describe T cell identification PSGR used and derive t cell epitope.Choosing 21 kinds of peptides that prediction is combined with HLA-A2 molecules are selected and synthesized, and measured interferon is examined based on ELISA or ELISPOT Its ability for stimulating T cell in the PBMCs from health objects and patients with prostate cancer is have evaluated outside γ (IFN-γ) releaser. It was found that 3 kinds of peptides, i.e. PSGR3, PSGR4 and PSGR14 (seeing below and table 1) induction are outer from health objects and prostate patient IFN-γ release in all T cells.Importantly, these peptide-specific T-cells can identify HLA- in a manner of HLA I class dependences A2⁺, express PSGR LNCaP cells.

Therefore, in one embodiment, the invention provides composition, including PSGR3, PSGR4 or PSGR14 ammonia The polypeptide of base acid sequence composition, or the combination of two or more in these three peptides, and pharmaceutical acceptable carrier.

In one aspect, present disclose provides the method for treating or preventing prostate cancer, including its patient applies to needs With the composition of the invention of effective dose.

In embodiments, present disclose provides the method that prostate cancer is treated in people patient, including apply to patient Effectively stable or reduction serum PSA (PSA), the step of the disclosure composition of amount horizontal particularly PSGR Suddenly.In some aspects, disclosed method further comprises applying granulocyte macrophage colony stimulating factor (GM-CSF). Some aspects, composition and GM-CSF are co-administered, and the composition described in further embodiment and GM-CSF It is administered simultaneously, and the composition described in further embodiment and GM-CSF are sequentially applied.

In some embodiments, PSGR is by load the composition of the dendritic cells of PSGR peptides to apply respectively, such as In multiple injection.

The administration of the composition or vaccine of the disclosure can be intracutaneous in every respect.Therefore, the disclosure also provides epidemic disease Seedling, including：(i) polypeptide of PSGR3, PSGR4 or PSGR14 amino acid sequence composition, or two or more in these three peptides The combination of kind, and (ii) pharmaceutical acceptable carrier.In some aspects, vaccine further comprises that granular leukocyte macrophage colony stimulates The factor (GM-CSF).Further, vaccine further comprise being effectively increased the toll samples of the amount of T cell immune response by Body 9 (TLR9) activator.In in a specific aspect, TLR9 activators are CpG- oligodeoxynucleotides (CpG-ODN).

In further embodiment, vaccine further comprises the CTLA4 suppressions for being effectively increased the amount of T cell immune response Preparation, and CTLA4 inhibitor is monoclonal antibody in specific aspect.

In other embodiment, vaccine further comprises that the PD-1 for being effectively increased the amount of T cell immune response suppresses Agent.PD-1 inhibitor is monoclonal antibody in specific aspect.

The disclosure also provides the method to individual vaccine inoculation, including the sheet of the effectively amount of inoculation individual is applied to individual Invention vaccine.In some aspects, vaccine of the invention is co-administered with GM-CSF, and further in multiple injection Middle co-administration.Further, PSGR peptides are administered simultaneously with GM-CSF, and in further itself and GM- of aspect CSF is sequentially applied.

Brief description of the drawings

Fig. 1 shows that PSGR derived peptides induce peptide-specific T-cell.The PSGR peptides that test amplification is detected by ELISA are special Specific T cell has the identification (A) of the T2 cells of titration concentration peptide (0-20 μ g/ml) to preload.PSGR3T cells (the B of amplification And E), PSGR4T cells (C and F) and PSGR14T cells (D and G) by respectively with single T2 cells in complete medium (CM) (1x10⁴Cells/well), or have corresponding peptide (5 μ g/mL) or common as the T2 cells of the control peptide of negative control with preload It is incubated.Cell is incubated 18-24 hours, passes through IFN-γ secretion (B, C and D) in ELISA detection assay supernatants.Pass through ELISPOT detections count IFN-γ spot formation cell (SFC) (E, F and G).Data are pressed average ± SD and drawn.As a result represent to Few three independent experiments.*P<0.05,**P<0.01,***P<(single T2 cells have loaded control peptide for 0.001 pair of control T2 cells).

Fig. 2 is shown to PSGR derived peptides specific T cell identification HLA-A2 positives PSGR expression LNCaP prostate cancers Cell.The expression (A) of PSGRmRNA in different cell lines is determined by RT-PCR.It is special that test PSGR derived peptides are detected by LDH Cytotoxicity (B) of the specific T cell to PC3 and LNCaP.Data from B are pressed average ± SD and drawn.As a result at least three are represented Independent experiment.*P<0.05, to control.

Fig. 3 shows that the t cell response that PSGR derives inducing peptide is CD8⁺T cell dependence is simultaneously limited by HLA-I.PSGR Derivative peptide-specific T-cell is by with loading the T2 cells with and without given peptide in the presence of GolgiStop on 48 orifice plates 37 DEG C are cultivated 4 hours.Cell then analyzes (A) by with anti-CD8 and the dyeing of anti-IFN-γ on FACScalibur instruments.PSGR Derivative peptide-specific T-cell by with single LNCaP cells in culture medium, or in anti-HLA-I monoclonal antibodies (W6/32), HLA-II It is incubated jointly with LNCaP cells in the presence of monoclonal antibody or control monoclonal antibody (anti-CD19 monoclonal antibodies).After being incubated 4 hours, detected and surveyed by LDH The fixed cytotoxicity (B) to LNCaP.Data from B are pressed average ± SD and drawn.As a result at least three independent experiments are represented.*P< 0.05, to control.

Specific embodiment

Clear and definite CD8⁺T cell plays a crucial role in tumor development and progress.Derived from tumor associated antigen (TAAs) peptide epitopes can be identified as antigen [32,33] in the presence of MHC-I molecules by T cell.T cell identification TAAs and The discriminating of its peptide is most important to developing effective Theratope.

The target of this research is to differentiate CD8 in health objects and patients with prostate cancer PBMCs⁺The HLA-A2 of T cell identification With reference to PSGR derive epitope.Use three kinds of different computer forecast algorithms including BIMAS, SYFPEITHI, and Rankpep Scan the PSGR protein sequences of the HLA-A2 binding peptides based on HLA-A2 binding motifs.Only calculated by 3 different computer forecasts The peptide of at least two success prediction is just included into method.21 kinds of nine amino acids or ten amino acid are selected according to the standard in our current research Peptide.All these peptides, which have been tested it, stimulates the ability from the PBMCs of health objects or patients with prostate cancer release IFN-γs. In 21 kinds of peptides, 3 peptides often obtained from the PBMCs of health objects or cancer patient induce peptide-specific T-cell response, and this A little peptide-specific T-cells also identify HLA-A2⁺, the LNCaP cells of PSGR expression, it is prostate gland cancer cell nature to show these peptides Processing.

PSGR has the prostata tissue specific gene of homology to couple olfactory receptor gene family with G-protein, and Its specific expressed in human prostate tissue [23-25].PSGR expression in human prostate intraepithelial neoplasia and tumor of prostate It is significantly higher than in normal structure [25].It is interesting that although PSGR has been considered to the novel targets of prostate cancer immunization therapy, PSGR derives t cell epitope and is not authenticated also.As far as we know, this is to differentiate and characterize CD8⁺The PSGR of T cell identification spreads out First report of raw epitope.It is developing cancer epidemic disease that the discriminating of the PSGR derivative epitopes of T cell identification, which further demonstrates PSGR, Seedling has prospect target spot.

Most of TAAs are from antigen [34], accordingly, it is intended to may occur when protecting individual to develop from autoimmunity certainly Body is resistant to.This is considered as the major obstacle of the TAA specific T-cells of the internal tumor eradication of induction energy.However, grinding at us In studying carefully, although PSGR is expressed in normal prostate tissue, due in the PBMCs from health objects or patients with prostate cancer The t cell response for deriving epitope to PSGR can be often detected, PSGR immune tolerance can be destroyed.

Use tumor lysis thing, TAA albumen, TAA peptides and encode TAA RNA or DNA carried out it is a large amount of based on exempting from The immunization therapy clinical trial of epidemic disease inoculation.However, these most of experiments are without the preferable result of acquirement.One reason is to come Expression from these TAAs in the tumour of different patients is heterogeneous, and can also be changed between the transfer obtained from a patient Become [35,36], therefore immunologic escape may occur when immunotherapy method is based only upon a kind of TAA.In order to avoid immunologic escape, Target the most important for developing successful cancer vaccine based on the Immunotherapy Strategy of vaccine of multiple different tumour antigens.Cause This, although authenticated in recent years including PSA [12,13], PSCA [16], PSMA [17-19], PAP [20], Prostein [14,15], many prostate specific tumour antigens including trp-p8 [21], it is still desirable to differentiate and other be used to be based on T The prostate specific tumour antigen of cellular immunotherapy.

Being studied based on the III phases, FDA has been recently approved the cancer vaccine for treating advanced prostate cancer patients, Sipuleucel-T[8].Sipuleucel-T is prepared from the autologous PBMCs containing antigen presenting cell, and autologous PBMCs is the same as by even The recombinant protein for having connect the PAP compositions of macrophage colony stimulatory factor (GM-CSF) is incubated.Speculate Sipuleucel-T parts It is by increasing PAP specific Cs D8⁺T cell response works, this further demonstrates that the tumour antigen of cancer vaccine induction Specific C D8⁺The importance of T cell.Up to now, Sipuleucel-T be FDA ratify for cancer patient treatment first Kind cellular immunotherapeutic agents.FDA approvals Sipuleucel-T not only demonstrates cancer immunotherapy as therapeutic cancer vaccine The effect of, also provide powerful promotion [37] for Cancer Immunol field.Therefore, differentiate and develop including PSGR and peptide derivative More new TAAs of CTLs identifications including thing, effective cancer epidemic disease to promoting following anti-prostate cancer and other types cancer Seedling development is definitely most important.

In addition, CD8⁺The epitope of T cell identification is used as diagnostic tool, for monitoring individual during immunization therapy Peptide specific CD8⁺T cell, thus differentiate treatment in the optimal immunization therapy period, including anti-tumor immunity decline when Whether body needs subsequent immunization therapy.

In a word, we authenticated 3 new PSGR derivative CTL epitopes.Because PSGR expression is strong in human prostata cancer Strong up-regulation, PSGR derived peptides may be used as diagnostic tool, or individually or with derived from other PSAs The immunotherapeutic targets of the united anti-cancer vaccine of other epitopes.

Illustrate the present invention by the following examples, embodiment is not configured to limit in any way.

Embodiment

Materials and methods

Healthy donors and patients with prostate cancer

10 HLA-A2⁺Prostate patient and 10 HLA-A2⁺Health objects participate in this after Written informed consent is obtained Research.Before beginning one's study all schemes by Baylor College Medicine (Baylor College of Medicine) evaluation committee of institute (IRB) ratify.20ml peripheral bloods are obtained from everyone, and use Lymphoprep (Nycomed Pharma AS；Oslo, Norway) by density gradient centrifugation come separating periphery blood monocytic cell (PBMCs).The PBMCs newly separated is frozen -140 1ml containing 90%FCS and 10% dimethyl sulfoxide (DMSO) at DEG C is freezed in culture medium then to use.To mark HLA- with FITC A2 monoclonal antibodies BB7.2 flow cytometry carrys out (BD Pharmingen, San Diego, CA, USA) checking and is obtained from cancer patient and is good for HLA-A2 developed by molecule on the PBMCs of health object.

Cell line

T2 cells (HLA-A2⁺TAP deficient cells system), PC3 cells (HLA-A2 feminine genders prostate cancer cell line), and LNCaP cells (HLA-A2 positive prostate cancer cells system) are purchased from American type culture collection (American Type Culture Collection)(ATCC；Manassas,VA,USA).All cell lines are maintained at added with 10%FBS, and 1% L glutamine, and the RPMI-1640 culture mediums (Mediatech of 1% penicillin and streptomysin；Manassas,VA,USA) In.

Peptide

BIMAS (http are used based on HLA-A2 binding motifs://www-bimas.cit.nih.gov/molbio/hla_ Bind/), SYFPEITHI (http://www.syfpeithi.de/), and Rankpep (http:// Bio.dfci.harvard.edu/Tools/rankpep.html 21 kinds of PSGR derived peptides (table 1)) are predicted.Only by these The epitope that at least two is predicted in algorithm is selected to further test.Peptide uses peptide synthesizer (AApptec, Inc.； Louisville, KY, USA) by Solid phase synthesis, purified by RPLC and verified by mass spectrum.Synthesis Peptide be dissolved in DMSO and be stored at -80 DEG C until further use with 10mg/mL concentration.

The stimulated in vitro of peptide-specific T-cell in PBMCs

PBMCs (1x10 from health objects or patients with prostate cancer⁵Cells/well) by the mark with every kind of μ g/mL of peptide 20 Quasi- peptide concentration is incubated on 96 hole U bottom plates on (BD, Franklin Lakes, NJ, USA) in 200 μ LT cell culture mediums (TCM) [26-28], T cell culture medium is by RPMI 1640 (Mediatech, Manassas, VA, USA), 10% people's AB serum (Valley Biomedical, Winchester, USA), 50 μM of 2 mercapto ethanols, 100U/mL proleulzins (IL-2), and 0.1mM MEM Nonessential amino acid solution (Invitrogen, Grand Island, NY, USA) forms.Remove within every 5 days half TCM and with containing peptide The fresh TCM of (20 μ g/mL) is replaced.After culture 14 days, harvesting simultaneously detects its response T2 cells (1x10⁴Cells/well) The ability of IFN-γ is produced, T2 cells are loaded with PSGR peptides (5 μ g/mL) or (unrelated as the control peptide of negative control in advance HLA-A2 binding peptides：NLLTHVESL).After 18 hours are incubated, supernatant is collected, is discharged by ELISA detection assays IFN-γ.

The HLA-A2 binding peptides derived from prostate specific G-protein coupling receptor (PSGR) that table 1. is predicted

The rapid amplifying method (REP) of PSGR peptide-specific T-cells

[29] are subject to minor modifications as previously described, and PSGR peptide-specific T-cells are expanded by rapid amplifying method (REP). In short, the 0th day in T25 bottles with added with 10% people's AB serum, 50 μM of 2 mercapto ethanols, and 30ng/mL OKT antibody The 20mL RPMI-1640 of (Ortho Biotech, Bridgewater, NJ), with 20x 10⁶Irradiation allogenic PBMCs with And 5x10⁶The Epstein epstein-Barr virus (EBV) of irradiation cultivates 0.1-0.5x10 together⁶PSGR peptide-specific T-cells.Bottle is 37 5% CO at DEG C₂In be uprightly incubated.IL-2 (300IU/mL) was added on the 1st and the 5th, is removed on half cell culture Clear and with the IL-2 containing 300IU/mL fresh culture supplements.REP start after 14 days, harvesting is frozen to afterwards Experiment.

ELISA is detected

By with the anti-human IFN-γs of 1 μ g/mL (Pierce Biotechnology；Rockford, IL, USA) 96 holes of coating Elisa plate (Thermo Fisher Scientific；Rochester, NY, USA) after measuring cytokine release yesterday.Plate quilt 6 times are cleaned to remove uncombined coated antibody with the PBS (cleaning fluid) containing 0.05%Tween-20, and with 1%BSA/PBS in room The lower closing of temperature 2 hours.Afterwards, 50 μ L of supernatant are added into each hole and are incubated 1 hour at room temperature, then add 50 μ L Biotinylated anti-human IFN-γ (the Pierce Biotechnology of 0.5 μ g/mL；Rockford, IL, USA), plate is at room temperature It is incubated again 1 hour.After incubation, plate is cleaned and with PBS/1%BSA 1:More-HRP streptomysins (Thermo of 5000 dilutions Fisher Scientific；Rochester, NY, USA) it is incubated 30 minutes.Plate is cleaned and 100 μ L are added into each hole Tmb substrate solution (Sigma-Aldrich Co.；St.Louis,MO,USA).Use 2N H₂SO₄Color development stopping is reacted and used Elisa plate readout instrument reads plate in 450nm.

IFN-γ ELISPOT is detected

After amplification in vitro as previously described [27], IFN-γ ELISPOT detections are carried out to drench with quantitation of peptides specificity cell toxicity T Bar cell (CTLs).In short, 96 hole ELISPOT plates (Millipore；Bedford, MA, USA) by anti-human with 7.5 μ g/mL IFN-γ(Pierce Biotechnology；Rockford, IL, USA) it is coated with overnight at 4 DEG C.With sterile PBS board-washings 6 times with Remove uncombined coated antibody.1x10⁵Cell per well be inoculated with T cell, and with single T2 cells, be loaded with PSGR peptides (5 μ G/mL the T2 cell incubations of the unrelated peptide) or as negative control.With 5 μ g/mL OKT3 antibody (Ortho Biotech； Bridgewater, NJ, USA) stimulate cell be previously used positive control.5% CO at 37 DEG C₂Middle samples of incubation 18-20 is small Shi Hou, plate are cleaned with cleaning fluid.Add biotinylated anti-human IFN-γ (the Pierce Biotechnology of 0.75 μ g/mL； Rockford, IL, USA), plate is incubated 2 hours at room temperature.After incubation, plate cleaned with cleaning fluid and with PBS/1%BSA 1:More-HRP streptomysins (Thermo Fisher Scientific of 1000 dilutions；Rochester, NY, USA) it is further incubated for 1 hour.Plate is cleaned and chloro- 1- naphthols substrate (the Sigma-Aldrich Co. of 200 μ L 4- are added into each hole； St.Louis,MO,USA).Finally, plate is being flowed underwater cleaning and is being dried at room temperature for.Use ELISPOT readout instruments (C.T.L.Technologies, Minneapolis, MN, USA) counts IFN-γ spot formation cell (SFC).

RNA is extracted and RT-PCR

RNA extractions and RT-PCR are carried out by [30] reported before.In short, with 1mLTrizol reagents (Invitrogen；Carlsbad, CA, USA) total serum IgE is extracted from prostate gland cancer cell.By 3 microgram RNA in 30 μ l volumes Reverse transcription is cDNA, and the PCR that every kind of cDNA1 μ l are used for then with PSGR specific primers pair reacts：Primer 1：5’- GAAGATCTATGAGTTCCTGCAACTTC-3’(SEQ ID NO:22), primer 2：5’-CCCAAGCTT TCACTTGCCTCCCACAG-3’(SEQ ID NO:23).Beta-actin is used as loading control：Primer 1：5’- CATGATGGAGTTGAAGGTAGTTTCG-3’(SEQ ID NO:24)；Primer 2：5’-CAGACTATGCTGTCCCTGTACGC- 3’(SEQ ID NO:25).PCR reactions are carried out under the following conditions：94 DEG C 2 minutes, 94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 1 point 20 seconds, it is total 35 circulation, 72 DEG C 10 minutes, and beta-actin carry out 25 circulation.The PCR primer of subsequent loading equivalent and with Detected through gel electrophoresis.

Cytotoxicity detects

(Promega is detected by lactic dehydrogenase (LDH)；Madison, WI, USA) test PSGR derivative peptide specifics Cytotoxicity of the T cell to PC3 and LNCaP.Detection illustrates to carry out according to manufacturer.LDH releases are calculated based on following equation：

Cytotoxicity (%)=(experiment-effector is spontaneous-the spontaneous LDH releases of target)/(the spontaneous LDH releases of target maximum-target) x100

Spontaneous release, and use and LDH reagents are determined using single target cell or single effector cell conditioned medium Contained cracked solution is incubated together in box target cell supernatant determines maximum release.Identify whether it is HLA-I for measure T cell Limitation, cultivate originate when by anti-HLA-I, anti-HLA-II, or anti-CD19 monoclonal antibodies (be all from ATCC, Manassas, VA, USA) in adding hole.

Intracellular IFN-γ cell factor dyeing

PSGR derives peptide-specific T-cell (0.5-1x10⁶) by and 0.5x10⁶Load is with and without peptide (5 μ g/mL) 37 DEG C of cultures 4 are small on 48 orifice plates in the presence of GolgiStop (BD Pharmingen, San Diego, CA, USA) for T2 cells When.Cell is dyed with anti-CD8 and anti-IFN-γ, and uses FACScalibur Instrumental Analysis.

Statistics

Using Student t check analyses ELISA and ELISPOT detect in experimental port with compare between quantitative differences.P <0.05 is considered as notable.

As a result

PSGR derived peptides specific CTL s induction in healthy donors

To determine that we are from 10 HLA-A2+ healthy donors with the presence or absence of the T cell precursor of PSGR reactions in health objects In obtain PBMCs, and it is stimulated with every kind of in 21 kinds of PSGR derived peptides (table 1) of the binding motif containing HLA-A2 in vitro.2 After all peptides stimulates, the supernatant of T cell is stimulated from peptide by ELISA detection and analysis, is with or without pair with detecting response load The IFN-γ of the T2 cells of peptide is answered to discharge.As shown in table 2,13 kinds of PSGR derived peptides can be at least 1 in 10 health objects Peptide-specific T-cell response is induced on position.Importantly, PSGR3, PSGR4 and PSGR14 can be in 7 in 10 health objects Inducing T cell response on position, show that this 3 kinds of peptides are immune prototypes and to be potentially able to the expansion of antigen in health objects special Property T cell.

2. 10 HLA-A2 of table⁺Health objects PBMCs peptide-specific T-cell induction

Note：Value represents IFN-γ concentration (pg/ml) in supernatant；- do not complete

PSGR derived peptides specific CTL s presence in patients with prostate cancer

Due to being found that anti-PSGR3, PSGR4 and PSGR14 peptide-specific T-cell in the health objects more than 70%, It is presumed that identify that the CTL precursors of this 3 kinds of peptides may be also high in patients with prostate cancer PBMCs.For detection ours it is assumed that I Have detected this 3 kinds of peptide candidates can be from HLA-A2⁺Induction peptide specific CTL s in patients with prostate cancer PBMCs.Before coming from The PBMCs of row adenocarcinoma patients is collected and stimulated in vitro with PSGR3, PSGR4 or PSGR14 peptides.As shown in Figure 3, PSGR3, PSGR4 and PSGR14 is really induction of peptide specific CTLs from patients with prostate cancer PBMCs.

Table 3.HLA-A2⁺Patients with prostate cancer PBMCs peptide-specific T-cell induction

Note：Value represents IFN-γ concentration (pg/ml) in supernatant

PSGR derives identification of the peptide specific to prostate cancer cell line

To obtain a large amount of PSGR peptide-specific T-cells further to analyze, we have expanded the PSGR identified in table 2 and 3 Peptide-specific T-cell.To determine effective peptide concentration of the load T2 cells of T cell identification, We conducted peptide titration experiments.Such as Shown in Figure 1A, for all 3 kinds of peptides, 5 μ g/ml peptide concentration is enough HLA-A2 points on the T2 cells of saturation T cell identification Sub- binding site.Therefore, we use the peptide concentration preload T2 cells always in ELISA and/or ELISPOT detections.Expand The T cell of increasing keeps antigentic specificity, and the IFN-γ of significant quantity is secreted after the T2 cytositimulations to have loaded corresponding peptide, but Control peptide is not so (Figure 1A, B, C, D).ELISPOT detects PSGR peptide specifics T in the T cell that further demonstrate amplification The presence (Fig. 1 E, F, G) of cell.

Whether can be identified for determination PSGR derivative peptide-specific T-cells and kill HLA-A2⁺The prostate cancer of PSGR expression Cell, we use HLA-A2 feminine gender PC3 cell lines and HLA-A2 positive LNCaP prostate cancer cell lines.Examined by RT-PCR The PSGR expression surveyed in both cell lines.[31] consistent with report before, PSGR high expression in LNCaP, but in PC3, In DU145 or normal prostate cell system PNT1A not so (Fig. 2A).As shown in Figure 2 B, from healthy donors and patient PSGR3, PSGR4, or PSGR14 specific T-cells can identify and kill the HLA-A2 positives, and the LNCaP of PSGR expression is but right PC3 cells negative HLA-A2 can not.These results show inside PSGR peptide-specific T-cells identification prostate tumor cells The t cell epitope managed and shown.

T cell identifies PSGR derived peptides with HLA-I restrictive ones

Whether the response for deriving inducing peptide for test PSGR depends on CD8⁺T cell, we are by PSGR derived peptide specificity Ts Cell with load with and without corresponding peptides T2 cells in the presence of GolgiStop 3 co-incubation 4 hours on 48 orifice plates. Then carry out CD8 molecules and the dyeing of intracellular IFN-γ.It was found that only CD8⁺T cell response is loaded with the T2 cells production of corresponding peptides Raw IFN-γ, and CD4⁺T cell does not produce IFN-γ (data are not shown).

To determine that PSGR derives peptide-specific T-cell identifies whether it is HLA-I restricted to LNCaP cells, we By LNCaP cells and PSGR derive peptide-specific T-cell anti-HLA-I monoclonal antibodies (W6/32) or control antibodies (HLA-II monoclonal antibodies or Anti- CD19 monoclonal antibodies) in the presence of co-incubation.As shown in Figure 3 B, the cytotoxicity of these peptide-specific T-cells is resisted completely The addition of HLA-I monoclonal antibodies is suppressed, but is not suppressed by anti-HLA-II (HLA-DR) or control monoclonal antibody (anti-CD19), and this shows It is HLA-I restricted that PSGR, which derives identification of the peptide-specific T-cell to LNCaP cells,.

While the invention has been described with reference to an exemplary embodiment, it should be understood that can occur for a person skilled in the art Change and modifications.Therefore, the limitation outside being limited in claim should not be added in the present invention.

The disclosure that all documents quoted in the application are described by reference integrally includes the present invention.

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Claims

Any of 1.PSGR3, PSGR4 and PSGR14 peptide or its two or more combination are preparing the medicine for the treatment of prostate cancer In purposes, wherein PSGR3 is by SEQ ID NO：3 amino acid sequence is formed, and PSGR4 is by SEQ ID NO：4 amino acid sequence Row are formed, and PSGR14 is by SEQ ID NO：11 amino acid sequence is formed.
2. the purposes described in claim 1, peptide more than two of which is co-administered.
3. the purposes described in claim 1, peptide more than two of which is administered simultaneously.
4. the purposes described in claim 1, peptide carry out order administration more than two of which.
5. the purposes described in claim 1, wherein peptide carry out intradermal administration.
6. the purposes described in claim 1, wherein peptide are carried out herein in connection with granulocyte macrophage colony stimulating factor (GM-CSF) Using.
7. the purposes described in claim 6, wherein peptide and GM-CSF are co-administered in multiple injection.
8. the purposes described in claim 6, wherein peptide and GM-CSF are administered simultaneously.
9. the purposes described in claim 6, wherein peptide and GM-CSF are sequentially applied.
10. the purposes described in claim 1 or claim 6, wherein herein in connection with the amount for being effectively increased T cell immune response TLR9 activators, CTLA4 inhibitor or PD-1 inhibitor are administered.
11. the purposes described in claim 10, wherein TLR9 activators are CpG- oligodeoxynucleotides (CpG-ODN).
12. the purposes described in claim 10, wherein CTLA4 inhibitor are monoclonal antibody.
13. the purposes described in claim 10, wherein PD-1 inhibitor are monoclonal antibody.
14. the purposes described in claim 11, wherein peptide were applied at 1,4 and 10 week, and were then applied to 4 years within every 6 months.
15. the purposes described in claim 10, wherein peptide were applied at 1,4 and 10 week, and were then applied to 4 years within every 6 months, and Wherein CTLA4 inhibitor was applied at 1,4 and 10 week, and was then applied to 52 weeks within every 8 weeks.
16. the purposes described in claim 10, wherein peptide were applied at 1,4 and 10 week, and were then applied to 4 years within every 6 months, and Wherein PD-1 inhibitor was applied at 1,4 and 10 week, and was then applied to 52 weeks within every 8 weeks.
Any of 17.PSGR3, PSGR4 and PSGR14 peptide or its two or more combination are preparing the epidemic disease of prevention prostate cancer Purposes in seedling, wherein PSGR3 are by SEQ ID NO：3 amino acid sequence is formed, and PSGR4 is by SEQ ID NO：4 amino acid Sequence composition, PSGR14 is by SEQ ID NO：11 amino acid sequence is formed.