CN102453701B - Gene modified CDR3 delta transplanted gamma delta T lymphocyte and its antitumor purpose - Google Patents

Abstract

The invention relates to a Gene modified CDR3 delta transplanted gamma delta T lymphocyte and its antitumor purpose. Retrovirus particles of a gamma delta TCR delta 2 chain and a gamma delta TCR delta 9 chain which contains an OT 3 sequence (FPSHTFHSTVVHT) are respectively packaged, the retrovirus particles are used for infecting normal human PBMC stimulated by an anti-CD3 antibody, the cell membrane surface expresses TCR gamma delta, the gene modified CDR3 delta transplanted gamma 9 delta 2T lymphocyte can be obtained, experiment proves that the cells possess cytotoxic effect on a plurality of tumor cells. The gene modified CDR3 delta transplanted gamma delta T lymphocyte and its antitumor purpose provide a novel method and a strategy for adoptive immunotherapy of the tumor.

Description

Gene modified CDR 3 delta transplanted gamma delta T gamma delta T lymphocytes and but cancer purposes thereof

Technical field

The present invention relates to genetically modified cell and therapeutic field of tumor.Particularly, the present invention relates to T lymphocyte to carry out genetic modification and pass through the field of modified γ 9 δ 2T lymphocyte treatment tumours.

Background technology

The immunotherapy of tumour is placed high hopes by people, and especially tumor specific cytotoxic T lymphocyte adoptive immunotherapy is considered to have the therapeutic strategy of development potentiality.But there is not gratifying curative effect at present experimental or clinical property test.The factors such as this may be low with most of tumor antigenicities extremely faint even disappearance, antigen presentation function defect, surperficial MHC molecule or expression of costimulatory molecules level, tumor mice immunosuppressive condition are closely relevant.In addition, the specific cytotoxic T cell of tumour patient in-vivo tumour (CTL) quantity is few, is difficult in vitro separate and amplification; External long cultivation is difficult to maintain specificity and antineoplastic ability thereof of its tumour identification; The generation of T clone or T cell clone is time-consuming, effort.These factors have all limited the widespread use of T lymphocyte adoptive immunotherapy.

Recently, there is scientist's using gene engineering technique can make the φt cell receptor of α β T Expressions In Lymphocytes tumour-specific, thereby improve the specific recognition ability of T lymphocyte to tumour antigen.Numerous laboratories have all obtained the genetically engineered lymphocyte of virus-specific or tumour-specific, and have proved its antineoplastic ability in vitro with in experimentation on animals.

But the restricted lymphocytic clinical use of α β T that makes genetic modification of MHC of α β T cell recognition tumour antigen is restricted.Tumor cell surface MHC developed by molecule is low or lose, and is difficult to be identified by the α β T lymphocyte of genetic modification, causes tumour immunity to be escaped.And, the α β T lymphocyte of genetic modification should be identified antigen, identify again the MHC molecule of submission antigen simultaneously, therefore, when clinical application, need to separate the T cell clone of the restricted antigen-specific of various different MHC, and determine according to the MHC molecule type of the peptide section of patient's submission can be to the TCR of tumor response.The TCR number of the specific for tumour antigen of finding is at present little, and just counts the tumor related antigen of the cancers such as knurl as melanocyte for minority.Moreover, mainly identify proteantigen for the α β φt cell receptor of transfection, seldom identify the tumour antigen of other two types: carbohydrate and glycolipid antigen.Finally, endogenous and ectogenic α chain and β chain may form and mix TCR, produce unexpected antigen recognition specificity, thereby cause autoimmune disorder.

Another kind of as T cell colony, gamma delta T cells has unique effect in anti-tumor immunotherapy: 1. gamma delta T cells is mainly with MHC non-limiting way identification antigen, and it has overcome the restrictive limitation of MHC of α β T cell antineoplastic immune; 2. antigen recognition spectrum extensively, as peptide class, non-peptide class, alcohols etc., and can be identified the antigen that α β T cell can not be identified, and therefore, can be used as the important supplement of α β T cell mediated immune surveillance in function; 3. tumor-infiltrated gamma delta T lymphocytes is expressed Fc acceptor, in the expression of TCR-CD3 complex body or/and in assembling when defectiveness, still can transmission of signal, this is that α β T cell is not available; 4. bring into play effector function by cytotoxicity and generation cytokine; 5. hematological system tumor and solid tumor are all had to lethal effect.

But the gamma delta T cells preparation preparation being used for the treatment of still exists some problems urgently to be resolved hurrily in method.As immunosuppression in patient's body after middle and advanced stage tumour patient or chemotherapy, cause seed cell quantity to reduce and immunocompetence decline, cause amplification in vitro cell quantity limited, be difficult to reach the cell preparation for the treatment of aequum.Therefore, utilize genetic engineering technique, prepare research cell tumour atopic, nothing/hypotoxic gamma delta T immune effector cell of having of sufficient amount imperative.

The research of gamma delta T cells is engaged in this laboratory for a long time, and the mechanism of gamma delta T CR identification antigen has been carried out to deep discussion.The antigen recognition site of gamma delta T CR is mainly present in the complementary determining region (CDR) of V δ chain, and wherein encoded by germline gene V in CDR1 and CDR2 district, and CDR3 district forms by the somatocyte restructuring of V (D) and J gene.Result of study in the past shows, gamma delta T CR and BCR and antibody are structurally highly similar.CDR3 staple diagram shows, the length variations maximum of heavy chain of antibody (H) and TCR δ chain CDR3, and significantly long compared with light chain (L) and γ chain person.It is the same that the prompting of these architecture basics and heavy chain of antibody CDR3 sequence in antibody recognition Process of Antigen play crucial recognition reaction, and CDR3 δ identifies in antigen and also plays keying action at gamma delta T cells.

Laboratory is the result demonstration to the TCR δ 2-CDR3 genescan in ovarian cancer and normal ovarian tissue and length polymorphism analysis in earlier stage, and the TCR δ 2-CDR3 gene fragment in normal ovarian tissue is polyclone and expresses; By contrast, the TCR δ 2-CDR3 gene fragment in ovarian cancer tissue is few clonal expression.The two fragment length skewness, the latter's length distribution is more extensive, and has more obvious advantage fragment length, and this length distribution difference shows that gamma delta T cells plays an important role in the immune response of ovarian cancer.

Summary of the invention

Contriver analyzes the TCR δ 2-CDR3 sequence drawing according to laboratory early stage from the TIL of 10 routine ovarian cancers, chooses the OT3 sequence (FPSHTFHSTVVHT) that has combination with tumour cell.Total length gamma delta T CR δ 2 chains that packaging contains OT3 sequence respectively and the retroviral particle of gamma delta T CR γ 9 chains, after the infectious titer obtaining, infect the normal pbmc stimulating through anti-cd 3 antibodies, make its surface of cell membrane express TCR γ 9 δ 2, called after γ 9 δ 2 (OT3) T cells.After experimental result shows that γ 9 δ 2 (OT3) T cells are stimulated by kinds of tumor cells albumen, its TNF-α and IFN-γ secretion increase.Meanwhile, kinds of tumor cells is also had to cytotoxicity.And this cytotoxicity can be blocked by the monoclonal antibody of anti-TCR γ δ.These results suggest, γ 9 δ 2 (OT3) T cells can be activated by tumour antigen, and have antitumor action.In order to study the cytotoxic mechanism of action of transfectional cell, contriver uses the inhibitor B FA of Fas/FasL approach and the inhibitor C MA of pore-forming protein/granzyme approach to carry out the cell toxicant blocking experiment of γ 9 δ 2 (OT3) T cells.Found that, BFA only has 20～30% to the maximal percentage inhibition of γ 9 δ 2 (OT3) T cell tumour lethal effects; The killing activity inhibiting rate that CMA kills and wounds the SKOV3 cell of the low expression of Fas to γ 9 δ 2 (OT3) T can reach 56.71%, and the killing activity inhibiting rate of the HO8910 cell to Fas high expression level reaches 33.93%.Combined utilization CMA and BFA reach more than 60% the cytotoxicity inhibiting rate of γ 9 δ 2 (OT3) T cells.Above results suggest, γ 9 δ 2 (OT3) T cells are in the cytotoxicity of tumour, pore-forming protein/granzyme and Fas/FasL approach all play a role, but pore-forming protein/granzyme approach plays a major role, especially even more important in the cytotoxicity of the tumour cell to the low expression of Fas.Contriver is further studied cancer suppressing action in γ 9 δ 2 (OT3) T cell paste.By lotus human ovarian cancer transplanted tumor nude mice model, locally injected into tumor γ 9 δ 2 (OT3) T cells, observe its result for the treatment of to tumour.Result shows, γ 9 δ 2 (OT3) T cells cooperation IL-2 treatments, and the cell infecting with respect to empty carrier coordinates IL-2 treatment group, and the growth of tumour is obviously suppressed, and also extend to some extent the lifetime of nude mice.

Therefore one aspect of the present invention provides a kind of gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes, and the CDR3 district of gamma delta T CR γ 9 chains of described cell and gamma delta T CR δ 2 chains is replaced by OT3 sequence, and wherein OT3 sequence is SEQ ID No:1.

In embodiments of the invention, gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes of the present invention obtain through following step: gamma delta T CR δ 2 chains that 1) packaging contains OT3 sequence total length respectively and the retroviral particle of gamma delta T CR γ 9 chains; 2) use the retroviral particle obtaining to infect the normal pbmc stimulating through anti-cd 3 antibodies, make its surface of cell membrane express TCR γ δ.

Experiment showed, that gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes provided by the invention are after tumour cell albumen stimulates, TNF-α and IFN-γ secretion increase.This shows that gamma delta T lymphocytes of the present invention has antitumor action widely.

Gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes provided by the invention have cytotoxicity to tumour cell.Experiment showed, in cytotoxicity, pore-forming protein/granzyme and Fas/FasL approach all play a role, but pore-forming protein/granzyme approach plays a major role, especially even more important in the cytotoxicity of the tumour cell to the low expression of Fas.

The present invention also provides gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes of the present invention for the preparation of antitumor cell preparation.

Antitumor small cell preparation provided by the invention can be used for treating kinds of tumors, and medicable tumour comprises the tumour of the low expression of Fas.Medicable tumour also comprises ovarian cancer, cervical cancer and B lymphoma etc.

The present invention also provides on the other hand antitumor cell preparation of the present invention is combined to use with IL-2, to obtain better result for the treatment of.

Brief description of the drawings

Fig. 1: the PBLs cell expressing gamma delta T CR of transfection

(A) pcr amplification is transplanted total length δ 2 and γ 9 chains of specific C DR3 sequence.1: γ 9 chains; 2: δ 2.(B) retroviral particle that will contain pMSCVhyg-γ 9 plasmids and pMSCVneo-δ (OT3) plasmid infects the PBMC cell through anti-cd 3 antibodies activation, and the PBMC cell simultaneously empty carrier virion being infected in contrast.RT-PCR amplification TCR γ 9 and TCR δ 2 chain total lengths and CDR3 district separately.The left side: 1: extract the RNA of the PBMC cell infecting from empty carrier virion, 2: the RNA of the PBMC cell that the retroviral particle that contains TCR infects; The right side: use the RNA of the PBMC cell of the retroviral particle infection that contains TCR to carry out RT-PCR (1-5) as template: 1: β-actin; 2: γ 9 total lengths; 3: δ 2 total lengths; 4: the CDR3 sequence of γ 9 chains; 5: the CDR3 sequence that derives from ovarian cancer that δ 2 chains are transplanted; M:marker; Extract the RNA of the PBMC cell infecting from empty carrier virion as template, carry out RT-PCR (6-10) 6: β-actin; 7: γ 9 total lengths; 8: δ 2 total lengths; 9: the CDR3 sequence of γ 9 chains; 10: the CDR3 sequence that derives from ovarian cancer that δ 2 chains are transplanted.(C) Western blotting detects the expression of gamma delta T CR.1: be the cell protein extract (negative control) of transfection empty carrier; 2 is the cell protein extract of TCR virus infection; 3 normal people's gamma delta T cells protein extracts (positive control).(D) collect the cell (γ 9 δ 2 (OT3) T cells) of integrating γ 9 and δ 2 chains, empty carrier control cells, by the expression of Flow cytometry transfectional cell TCR γ δ.The left side is empty carrier control cells; The right is γ 9 δ 2 (OT3) T cells.

The biological function of Fig. 2: γ 9 δ 2 (OT3) T cells

(A) control cells and the Daudi of γ 9 δ 2 (OT3) T cells and empty carrier transfection, Hela, SKOV3, HO8910 and Raji cell protein extract are hatched after 72 hours jointly, collect supernatant ELISA double antibody sandwich method and detect each group of cell IFN-γ secretion.(B) control cells and the Daudi of γ 9 δ 2 (OT3) T cells and empty carrier transfection, Hela, SKOV3, HO8910 and Raji cell protein extract are hatched after 24 hours jointly, collect supernatant ELISA double antibody sandwich method and detect each group of cell TNF-α secretion.*P＜0.05；**P＜0.01。

(C) mtt assay detects the killing activity of γ 9 δ 2 (OT3) T cells to tumour cell.γ 9 δ 2 (OT3) T cells are effector cell, hatch 8 hours at different effect targets with tumour cell (Hela, SKOV3, HO8910) than 37 DEG C, and then every hole adds the MTT that 10 μ L concentration are 5mg/mL, continues to hatch 4 hours.Add DMSO termination reaction.

Fig. 3: γ 9 δ 2 (OT3) T cells to the effect of tumour cell by anti-gamma delta T CR antibody blocking.

(A) γ 9 δ 2 (OT3) T cells are in advance with irrelevant antibody (the mouse IgG1 control antibodies) sealing treatment of anti-gamma delta T CR antibody or isotype 2 hours, then hatch 72 hours from different tumour cell protein extracts, collection supernatant ELISA double antibody sandwich method detects the secretion of each group of IFN-γ.(B, C, D) γ 9 δ 2 (OT3) the T cells of action effect cell are processed 2 hours with anti-gamma delta T CR antibody or the irrelevant antibody (mouse IgG1 control antibodies) of isotype in advance, then from different tumour cell different effect targets than under mixed culture 8 hours, detect the killing activity of γ 9 δ 2 (OT3) T cells to target cell with mtt assay.*P＜0.05；**P＜0.01

Fig. 4: the expression of Flow cytometry tumor cell surface Fas.

The kill mechanism of Fig. 5: γ 9 δ 2 (OT3) T cells.

(A) impact of the cytotoxicity of BFA on γ 9 δ 2 (OT3) T cells.Process after γ 9 δ 2 (OT3) T cell 2h with the BFA of different concns respectively, then mix and do fragmentation test with target cell, establish solvent control simultaneously.(B) impact of the cytotoxicity of CMA on γ 9 δ 2 (OT3) T cells.Process after γ 9 δ 2 (OT3) T cell 2h with the CMA of different concns respectively, then mix and make fragmentation test with target cell, establish solvent control simultaneously.(C) CMA (100nM), after BFA (25 μ M) and combination treatment effector cell, detects the cytotoxicity of γ 9 δ 2 (OT3) T to tumour cell.

Antitumor action in Fig. 6: γ 9 δ 2 (OT3) T cell paste.

After tumor cell inoculation, treat that Subcutaneous tumor tubercle grows to 100mm ³left and right (being about 10 days), is divided into three experimental group by nude mice, treatment group: intratumor injection γ 9 δ 2 (OT3) T cells (1 × 10 at random ⁶cell/50 μ L/, totally 8), empty carrier group: the cell (1 × 10 that intratumor injection empty carrier infects ⁶cell/50 μ L/, totally 8), PBS control group: intratumor injection PBS (50 μ L/, totally 8).Except PBS group, other animals of two groups only give abdominal injection IL-25000U/, every treatment in three days once, carry out altogether four times.Observe and record generalized case and the existence situation of nude mice every day; Measured tumor growth situation and nude mice body weight every 2 days.(A) treatment is after 24 days, lotus human ovarian cancer nude mice profile with and the big or small photo of transplanted tumor.A, B: γ 9 δ 2 (OT3) T; C, D: empty carrier control group; E, F:PBS control group.(B) tumor growth of lotus human ovarian cancer nude mice.(C) growth curve of lotus human ovarian cancer nude mice.(D) body weight change of lotus human ovarian cancer nude mice.

Embodiment

One, materials and methods

1, cell cultures

PBMC is the PBMC of healthy people of fresh separated; RetroPack ^tMpT67 is that the packaging in NIH3T3 source copies retrovirus clone, purchased from Clontech Laboratories, Inc.NIH3T3 is Apoptosis.SKOV ₃, human ovarian's serous papillary cystadenocarcinoma clone; HO8910, people's serous cystadenocarcinoma of ovary clone; Hela, human cervical carcinoma's tumor cell line; Daudi, Human B lymphoma clone; Raji, people Burkitt lymphoma cell line, all can buy from basis institute of Chinese Academy of Medical Sciences cell centre.SKOV3 cell, carries out adherent culture with the McCoy 5A substratum containing 10%FCS.Hela, NIH3T3 and RetroPack PT67 cell, carry out adherent culture with the DMEM high glucose medium containing 10%FCS.HO8910, Raji, and Daudi, carry out adherent or suspension culture with the RPMI-1640 substratum containing 10%FCS.

2, animal

BALB/c nude mouse, 4～6 weeks ages of mouse, body weight 15～20g, female, be purchased from biological products assay institute animal center, in laminar-flow rack under Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's Experimental Animal Center no-special pathogen (specific pathogen free, SPF) condition, raise.

3, bacterial strain and plasmid vector

Bacillus coli DH 5 alpha, purchased from precious biotechnology company limited.Genotype is: supE44 Δ lacU169 ( 80lacZ Δ M15) hsdR17 recA1 end1 gyr96 thi-1 relA1, for amplification and the conversion of plasmid.

PGEM-T or pGEM-T Easy Vector System are for the clone of the PCR product of Taq enzymatic amplification, purchased from Promega company.Express gamma delta T CR pMSCVhyg and pMSCVneo plasmid, purchased from Clontech Laboratories, Inc.Recombinant plasmid and CDR3 district that pcDNA3.1-γ 9 and pcDNA3.1-δ (OT3) are respectively γ 9 full-length genes are the δ of OT3 sequence replacing, built by doctor Xi Xueyan, draw from document X.Xi, Y.Guo, H.Chen, C.Xu, H.Zhang, H.Hu, L.Cui, D.Ba, and W.He, Antigen specificity of gammadelta T cells depends primarily on the flanking sequences of CDR3delta, J.Biol.Chem.284 (2009) 27449-27455.

4, primer (in table 1)

Table 1. is for the γ of the gamma delta T CR that increases and the primer of δ full length gene

Above primer is synthetic by Beijing Qing Ke company.

Experimental technique:

1, the structure of retroviral plasmid

(1) PCR of the γ chain of TCR and δ (OT3) chain gene total length

With pcDNA3.1-γ, pcDNA3.1-δ (OT3) plasmid is template, with pMSCVneoOT3-up, pMSCVneoOT3-down primer amplification goes out to contain TCR δ (OT3) the chain total length of Bgl II and Xho I restriction enzyme site, with pMSCVhyr γ-up, pMSCVhyr γ-down primer amplification goes out to contain the γ chain of Xho I and Bgl II restriction enzyme site.

(2) purifying of PCR product

(3) carrier and PCR product endonuclease reaction

(4) ligation

Total length TCR γ 9 after enzyme is cut is connected with the carrier that TCR δ 2 (OT3) chain is cut with enzyme respectively, is built into recombinant plasmid pMSCVneo-δ (OT3), pMSCVhyr-γ plasmid.

(5) conversion of recombinant plasmid and positive-selecting

(6) a small amount of of plasmid DNA preparation

(7) enzyme of recombinant plasmid is cut qualification

(8) sequential analysis of DNA fragmentation

By enzyme cut assay certificate contain target DNA insert recombinant plasmid carry out determined dna sequence (being completed by Nuo Sai biotech company).With DNAMAN software analysis sequencing result.

2, RetroPack ^tMdetermining of PT67 cells resistance

The each inoculation 1 × 10 in every hole in (1) six orifice plate ⁵individual RetroPack ^tMpT67 cell, adding respectively final concentration is the tetracycline of 0,50,100,200,300,400,500,600,700,800,900,1000 μ g/ml.Cultivate in the backward each hole of 3～4d and supplement corresponding Selective medium, maintain 7～9d.Observe each porocyte survival condition every 24h, determine the suitableeest screening concentration of tetracycline.

The each inoculation 1 × 10 in every hole in (2) six orifice plates ⁵individual RetroPack ^tMpT67 cell, adds respectively G418 to different final concentrations, is respectively 0,100,200,300,400,500,600,700,800,900,1000 and 1100 μ g/ml.Each concentration is established two multiple holes, and cell is cultivated 13 days in 37 DEG C, 5%CO2 incubator, chooses and causes Retro Pack ^tMpT67 cell all dead G418 concentration be after the positive transfection of screening concentration used while cloning.

3, liposome transfection RetroPack ^tMpT67 cell

With the DMEM substratum adjustment RetroPack of serum-free ^tMpT67 cell, to suitable concentration, adds cell suspension 500 μ l in the 24 every holes of orifice plate.By pMSCVhyg and pMSCVneo, pMSCVhyg-γ and pMSCV neo-δ and Lipofectamine ^tM2000 liposomes dilute with the DMEM substratum of 50 μ l serum-frees respectively, juxtaposition equilibrium at room temperature.Within 5 minutes by the vector plasmid having diluted and Lipofectamine ^tM2000 liposomes mix, and continue at room temperature to hatch 20 minutes, and then by above-mentioned mixed solution, (100 μ l) join containing RetroPack ^tMin 24 orifice plates of PT67 cell, vibration mixes gently, puts 37 DEG C, 5%CO ₂in incubator, cultivate.After 24 hours, by RetroPack ^tMthe DMEM perfect medium (being wherein that 800 μ g/ml or Hyg final concentration are 300 μ g/ml containing G418 final concentration) that PT67 cell changes into containing 10% serum carries out positive fine screening.

4, the screening of positive colony

Within every 2 days, change liquid 1 time, screen after 7 days, do not have in a large number the cell of transfection plasmid to occur death, and the RetroPack of transfection plasmid ^tMpT67 cell grows a large amount of cell masses, and cell mass, through trysinization, is proceeded in 96 orifice plates and carried out mono-clonal cultivation by limiting dilution assay, after two weeks, occurs cell clone.Choose mono-clonal to 24 orifice plate enlarged culturing, collect supernatant and measure virus titer.

5, the mensuration of virus titer

NIH3T3 cell is that virus titer is measured, calculated to target cell under Polybrene existence condition.Virus titer (cfu/L)=cell clone mean number × virus liquid extension rate × 10 ³.The viral supernatant liquor of collecting is done respectively to 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4doubly dilution., the virus liquid (8mg/L Polybrene) of drawing respectively 1mL dilution adds in six orifice plates of prior inoculation NIH3T3 cell, make viruses adsorption after 2.5 hours, adding equivalent continues to cultivate 3 days containing the DMEM nutrient solution of 800 μ g/ml G418 or the DMEM nutrient solution of 300 μ g/ml hyg, then change the nutrient solution containing the G418 of 800 μ g/ml, cultivate approximately 14 days, cell clone basically forms.

6, retroviral infection normal people's PBMC

(1) separation of peripheral blood mononuclear cell (PBMC)

(2) retroviral infection process

Through the PBMC of anti-cd 3 antibodies activation, with the RPMI1640 perfect medium that contains retrovirus and 200IU/mlIL-2 resuspended become 1 × 10 ⁶individual/ml, is inoculated into the non-tissue culturing plate that is coated with in advance Retronectin.At 32 DEG C, centrifugal 90 minutes of culture plate 1500 × g, then cell is placed on 37 DEG C, 5%CO ₂incubator is cultivated.The process of this infection repeats twice.Infect the 6th day, collecting cell, then changes the substratum that contains IL-2 and continues to cultivate.At the 8th day that infects, change into and contain respectively geneticin (0.5mg/ml) and hygromycin B (0.1mg/ml) and continue to cultivate after 3 days, change the fresh complete RPMI1640 that contains 200IU/ml IL-2 into and continue cultivation.

7, γ 9 δ 2 (OT3) T transfectional cells kill and wound and kill and wound enclosed experiment to tumour cell

Target cell preparation: the respective target cell that goes down to posterity and cultivate, i.e. SKOV3 cell or other tumour cells, with after corresponding nutrient solution washing 2 times, adjusting cell concn is 1 × 10 ⁵/ ml, adds 96 orifice plates with 100 μ l/ holes, is placed in 37 DEG C, 5%CO ₂cultivate, after cell attachment, can be used for killing experiments.

Effector cell's preparation: effector cell is the PBMC of γ 9 δ 2 (OT3) T cells or transfection empty carrier, adjusts cell concn, makes to imitate target ratio and is respectively 5: 1,10: 1,20: 1, and effector cell adds respectively in each target cell hole, every group of 3 parallel holes.In addition, establish the control wells that only adds target cell or effector cell or only add nutrient solution.

Mtt assay detects killing activity: mixed reaction system is placed in to 37 DEG C, 5%CO ₂cultivate after 4 hours, every hole sucks 100 μ l supernatants, adds 15 μ l MTT, continues to hatch.After 4 hours, every hole adds 100 μ l DMSO, hatches 4 hours, and in measuring wavelength 570nm, reference wavelength 630nm place measures OD value, and calculates killing activity by following formula:

$\frac{T_{\mathrm{OD}}-[{(E+T)}_{\mathrm{OD}}-E_{\mathrm{OD}}}{T_{\mathrm{OD}}}\×100\%$

Wherein, T _oDrefer to the OD value of independent target cell; E _oDfor the OD value of individual effect cell; (E+T) _oDrepresent the OD value of effector cell and target cell mix aperture.In the time killing and wounding enclosed experiment, γ 9 δ 2 (OT3) T cells and anti-gamma delta T CR monoclonal antibody are hatched after 90 minutes in 37 DEG C, measure its cytotoxic activity, to observe the sealing process of monoclonal antibody.

8, analyze the experiment of γ 9 δ 2 (OT3) T cell killing mechanism

(1) expression of Flow cytometry tumor cell surface Fas

Collect cell to be measured, be mixed with 1 × 10 ⁶the cell suspension of/ml.PBS washs 3 times, adds the monoclonal anti-fas antibody 5 μ l of FITC mark after exhaustion supernatant, hatches 60 minutes for 4 DEG C.Centrifugal and with PBS washing 3 times, exhaust the 2% paraformaldehyde stationary liquid that adds 500 μ l after supernatant, on flow cytometer, analyze.Homotype contrast adopts the homotype control antibodies of FITC mark.

(2) cell toxicant suppresses experiment

Mix and make fragmentation test with target cell again after 2 hours with the CMA of different concns or BFA or combination treatment γ 9 δ 2 (OT3) T cells respectively, establish solvent control simultaneously, observe CMA or BFA or combined action on γ 9 impacts of δ 2 (OT3) T cell on tumor cytotoxicity effect, to inquire into the mechanism of lethal effect.

9, antitumor action in γ 9 δ 2 (OT3) T cell paste.

SKOV3 cell, with after 0.25% trysinization counting, with PBS washing 3 times, is suspended from PBS, is seeded in the right shoulder of nude mice back subcutaneous (1 × 10 ⁶, 100 μ l volumes).Treat that Subcutaneous tumor tubercle grows to 0.1mm ³left and right (approximately 10 days), is divided into three experimental group by nude mice, treatment group: intratumor injection γ 9 δ 2 (OT3) T cells (1 × 10 at random ⁶cell/50 μ L/, totally 8), empty carrier group: the cell (1 × 10 that intratumor injection empty carrier infects ⁶cell/50 μ L/, totally 8), PBS control group: intratumor injection PBS (50 μ L/, totally 8).Except PBS group, other animals of two groups only give abdominal injection IL-2 5000U/, every treatment in three days once, carry out altogether four times.Observe and record generalized case and the existence situation of nude mice every day; Measured tumor growth situation and nude mice body weight every 2 days.Its calculation formula is: tumor volume (mm ³)=a × b ²× 0.5.A: knurl body major diameter (mm), b: knurl body minor axis (mm).2 mouse of every group of execution in the 24th day, get tumor nodule after dissection.Remaining continues to observe the lifetime of each group of mouse.

Two, experimental result

Retrovirus infection method is prepared the research of the inside and outside cancer suppressing action of γ 9 δ 2 (OT3) T cell paste

(1) clone and the Sequence Identification of pMSCVhyg-γ 9 and pMSCVneo-δ 2 (OT3) gene

In document, report and contained the total length γ 9 and the δ 2 chain (X.Xi that come from OEC tumor infiltrating lymphocyte (TIL) CDR3 district, Y.Guo, H.Chen, C.Xu, H.Zhang, H.Hu, L.Cui, D.Ba, and W.He, Antigen specificity of gammadelta T cells depends primarily on the flanking sequences of CDR3delta, J Biol Chem.284 (2009) 27449-27455).In brief, according to the TCR δ in GenBank and TCR γ primers, by round pcr, amplify DNA fragmentation and identify through agarose gel electrophoresis.PCR product is connected with pMSCV carrier after reclaiming purifying, product called after pMSCVhyg-γ 9.Connect product and be transformed into after bacillus coli DH 5 alpha, use Bgl II through the positive colony of ammonia benzyl resistance screening, the qualification of Xho I double digestion, the positive colony of getting wherein carries out determined dna sequence.According to sequence comparison, the Insert Fragment in recombinant plasmid is consistent with the sequence of TCR δ 2 (OT3) and TCR γ 9 respectively.Experimental result is shown in Figure 1A.

(2) RT-PCR detects TCR γ 9 and TCR δ 2 chains

The retroviral particle that will contain pMSCVhyg-γ 9 plasmids and pMSCVneo-δ 2 (OT3) plasmid infects the PBMC cell through anti-cd 3 antibodies activation, and the PBMC cell that simultaneously infection is contained to empty carrier virion in contrast.RT-PCR amplification TCR γ 9 and TCR δ 2 chain total lengths and CDR3 district separately, detect the integration of cells infected TCR γ 9 and TCR δ 2 chains.From pMSCVhyg-γ 9 plasmids and pMSCVneo-δ (OT3) plasmid virion cells infected cDNA, amplify TCR γ 9 and TCR δ 2 (OT3) chain total length and CDR3 district separately, prompting TCR γ 9 and TCR δ 2 (OT3) have been incorporated into transfectional cell.Experimental result is shown in Figure 1B.

(3) expression of Flow cytometry transfectional cell TCR γ δ

Collect γ 9 δ 2 (OT3) T cells, turn empty carrier control cells, the expression of Flow cytometry transfectional cell TCR γ δ.As shown in Fig. 1 D, compared with empty carrier transfection control cells, 74% virus transfection cell expressing gamma delta T CR.

(4) Western blotting detects the expression of transfectional cell TCR γ δ

Taking the monoclonal antibody of mouse-anti people γ 9 as primary antibodie, HRP enzyme mark sheep anti-mouse antibody is anti-as two, use chemical luminous substrate develops, and to the control cells protein crude extract of γ 9 δ 2 (OT3) T cells and normal people's gamma delta T cells and transfection empty carrier, row Western blot after SDS-PAGE analyzes.As shown in Figure 1 C, the first swimming lane is the cell (negative control) of transfection empty carrier, and the second swimming lane is γ 9 δ 2 (OT3) T cells, and the 3rd swimming lane is normal people's gamma delta T cells (positive control).Western detected result, proves the expression of transfectional cell TCR γ δ.

(5) biological function of γ 9 δ 2 (OT3) T qualification

1, different tumour cell total proteins and γ 9 δ 2 (OT3) T cells are hatched the expression of rear TNF-α altogether

ELISA double antibody sandwich method detects the secretion of each group of cell TNF-α.Result is as shown in 2A.Using Daudi protein extract as positive control, Raji protein extract is as negative control; Hela, SKOV3, HO8910 protein extract stimulates the amount of γ 9 δ 2 (OT3) T emiocytosis TNF-α higher than the secretion of empty carrier control group, but there is no statistical significance.

2, different tumour total proteins and γ 9 δ 2 (OT3) T cells are hatched the expression of rear IFN-γ altogether

ELISA double antibody sandwich method detects the secretion of each group of cell IFN-γ, and result as shown in Figure 2 B.Using Daudi protein extract as positive control, Raji protein extract is as negative control; Hela, SKOV3, HO8910 protein extract stimulates the amount of γ 9 δ 2 (OT3) T emiocytosis IFN-γ higher than the secretion (p < 0.01, p < 0.01, p < 0.01) of empty carrier control group.，

3, γ 9 δ 2 (OT3) T cells killing and wounding and the enclosed experiment of anti-gamma delta T CR antibody tumour cell

Taking different tumour cells as target cell, γ 9 δ 2 (OT3) T cells are effector cell, and with the killing activity (with kill rate represent) of mtt assay detection γ 9 δ 2 (OT3) T cells to target cell, result as shown in Figure 2 C.γ 9 δ 2 (OT3) T cells all have killing activity to kinds of tumor cells, and along with imitating the raising of target ratio, killing-efficiency also improves.Wherein the strongest to the lethal effect of SKOV3.After monoclonal antibody pre-treatment γ 9 δ 2 (OT3) T cells with anti-TCR γ δ, its killing activity to tumour cell decrease (Fig. 3 A, B, C, D).The above results shows, to tumor cytotoxicity, effect is that TCR relies on to γ 9 δ 2 (OT3) T cells.

(6) the kill mechanism pre-test of γ 9 δ 2 (OT3) T cells

1, the expression of Flow cytometry tumor cell surface Fas

Use the goat anti-mouse IgG of monoclonal anti-fas antibody and FITC mark to carry out immunofluorescence dyeing to kinds of tumor cells such as Hela, SKOV3, HO8910, to determine the expression of these cell surfaces Fas.As shown in Figure 4, flow cytometer detects and shows the result that flow cytometer detects, and SKOV3 is the low express cell of Fas, and its Fas expression rate is 6.46%.HO8910 is Fas high expressing cell, and its Fas expression rate is 70% left and right, and the Fas expression rate of Hela occupy between SKOV3 and HO8910, and its Fas expression rate is in 40% left and right.

2, BFA is to γ 9 δ 2 (OT3) T cell tumour cytotoxic activity restraining effect

After processing γ 9 δ 2 (OT3) T cell 2h with the BFA of different concns respectively, mix and make fragmentation test with target cell again, establish solvent control, to observe the impact of the cytotoxicity of BFA on γ 9 δ 2 (OT3) T cells simultaneously.Result as shown in Figure 5A, BFA part suppresses the lethal effect of γ 9 δ 2 (OT3) T cells to tumour cell, and the increase restraining effect with its concentration also strengthens, when final concentration is 25 μ M, restraining effect reaches peak value, now to HO8910, SKOV3 and Hela tumor cytotoxicity inhibiting rate are respectively 27.91%, 19.01% and 20.56%.Prompting γ 9 δ 2 (OT3) T cells are not only relevant with FasL approach with Fas to the lethal effect of tumour cell, and relate to other approach.

3, CMA is to γ 9 δ 2 (OT3) T cell tumour cytotoxic activity restraining effect

Process γ 9 δ 2 (OT3) T cells with the CMA of different concns respectively and mix and make fragmentation test with target cell again after 2 hours, establish solvent control, to observe the impact of the cytotoxicity of CMA on γ 9 δ 2 (OT3) T simultaneously.As shown in Figure 5 B, CMA has remarkable restraining effect to the tumor cytotoxic activity of γ 9 δ 2 (OT3) T cells to result, and also strengthens with the increase restraining effect of its concentration.Final concentration reaches inhibition peak value while being 100nM, to HO8910, SKOV3 and Hela tumor cytotoxicity inhibiting rate are respectively 33.93%, 56.71% and 46.52%.This prompting γ 9 δ 2 (OT3) T cells mainly depend on pore-forming protein and granzyme approach, the especially tumour cell to the low expression of Fas to the lethal effect of tumour cell.

4, CMA combines 9 δ 2 (OT3) the T cell tumour cytotoxic activity restraining effect to γ with BFA

In order further to determine the lethal effect mechanism of γ 9 δ 2 (OT3) T cells to tumour cell, we have compared CMA (100nM), after BFA (25 μ M) and combined action effector cell, on the impact of γ 9 δ 2 (OT3) T cytotoxicity.Result as shown in Figure 5 C, express high HO8910 cell for Fas, BFA is 24.15% to γ 9 δ 2 (OT3) T cell toxic action inhibiting rates, CMA is that 38.51%, BFA and CMA combined action are 60.95% to the inhibiting rate of cytotoxicity to γ 9 δ 2 (OT3) T cell toxic action inhibiting rates.Prompting is in the kill mechanism of the cell of γ 9 δ 2 (OT3) T cells to Fas high expression level, and Fas and FasL approach and granzyme pore-forming protein approach have all been brought into play effect.For the Hela cell of expressing in Fas, BFA is 18.52% to the inhibiting rate of γ 9 δ 2 (OT3) T cell toxic actions, CMA is that to combine the inhibiting rate of cytotoxicity be 63.27% for 54.96%, BFA and CMA to the cytotoxicity inhibiting rate of γ 9 δ 2 (OT3) T cells.In the kill mechanism of the cell that this prompting is expressed in to Fas at gamma delta T cells, Fas and FasL approach and granzyme pore-forming protein approach have all been brought into play effect, but granzyme pore-forming protein approach has been brought into play Main Function.For the SKOV3 cell of the low expression of Fas, BFA is 21.96% to the inhibiting rate of γ 9 δ 2 (OT3) T cell toxic actions, CMA is that 61.76%, BFA and CMA combined action are 62.08% to the inhibiting rate of cytotoxicity to the inhibiting rate of γ 9 δ 2 (OT3) T cell toxic actions.Prompting gamma delta T cells is in the kill mechanism of tumour cell, and Fas and FasL approach and granzyme pore-forming protein approach have all been brought into play effect, but granzyme/pore-forming protein approach performance Main Function.

(7) antitumor action in γ 9 δ 2 (OT3) T cell paste.

In order to study the in vivo antitumor effect of γ 9 δ 2 (OT3) T cells to human ovarian cancer, we have set up lotus Proliferation of Human Ovarian Cell SKOV3 transplanted tumor nude mice model.After tumor cell inoculation, treat that Subcutaneous tumor tubercle grows to 100mm ³left and right (being about 10 days), is divided into three experimental group by nude mice, treatment group: intratumor injection γ 9 δ 2 (OT3) T cells (1 × 10 at random ⁶cell/50 μ L/, totally 8), empty carrier group: the cell (1 × 10 that intratumor injection empty carrier infects ⁶cell/50 μ L/, totally 8), PBS control group: intratumor injection PBS (50 μ L/, totally 8).Except PBS group, other animals of two groups only give abdominal injection IL-2 5000U/.Fig. 6 A is the profile of the 24th day each treatment group nude mice and the photo of the interior transplanted tumor of mice with tumor body.As can be seen here, treatment group mouse and the tumour of carrying thereof are all significantly less than empty carrier control group or PBS control group.From Fig. 6 B, in knurl, inject γ 9 δ 2 (OT3) T cell tumour tubercle volumes from 0.097 ± 0.088mm ³rise to 0.521 ± 0.332mm ³, and the gross tumor volume of the mouse of the cell that injection empty carrier infects and injection PBS is respectively from 0.104 ± 0.062mm ³with 0.122 ± 0.094mm ³be increased to 1.136 ± 0.492mm ³with 1.272 ± 0.381mm ³.Statistical analysis shows, beginning in the 18th day from treating, and the tumor nodule of injection γ 9 δ 2 (OT3) T cell therapy groups is significantly less than the cell (1 × 10 that empty carrier infects ⁶cell, 8) and PBS control group (P < 0.05).Prompting γ 9 δ 2 (OT3) T genetically engineered cells can killing tumor cell, has therapeutic action.By as shown in 6C, use γ 9 δ 2 (OT3) T cells to treat, with respect to empty carrier and PBS control group, can obviously extend the survival time (P < 0.05) of tumor bearing nude mice.

In whole therapeutic process, the general status of each treatment group mouse is good, feed and movable normal, and body weight is being treated the forward and backward significant difference (Fig. 6 D) that also do not have, and prompting this time treatment does not cause obvious murder by poisoning and damage to nude mice.

Claims (7)

1. gene modified CDR 3 delta transplanted gamma delta T gamma delta T lymphocytes, in the purposes of preparing in antitumor cell preparation, is characterized in that:

δ 2 chains that the gamma delta T CR of described cell is replaced by OT3 sequence by γ 9 chains and CDR3 district form, and wherein OT3 sequence is as shown in SEQ ID No:1;

Wherein said cell obtains through following step:

1) gamma delta T CR δ 2 chains that packaging contains OT3 sequence total length respectively and the retroviral particle of gamma delta T CR γ 9 chains;

2) use the retroviral particle obtaining to infect the PBMC of healthy people stimulating through anti-cd 3 antibodies, make its surface of cell membrane express TCR γ 9 δ 2.

2. gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes according to claim 1, in the purposes of preparing in antitumor cell preparation, is characterized in that described cell is after tumour cell albumen stimulates, and TNF-α and IFN-γ secretion increase.

3. gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes according to claim 1, in the purposes of preparing in antitumor cell preparation, is characterized in that described cell has cytotoxicity to tumour cell.

4. gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes according to claim 3 are in the purposes of preparing in antitumor cell preparation, it is characterized in that described cell mainly brings into play its cytotoxicity to tumour cell by pore-forming protein/granzyme approach.

According to the gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes described in claim 3 or 4 in the purposes of preparing in antitumor cell preparation, it is characterized in that described tumour cell is the tumour cell of the low expression of Fas.

6. gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes according to claim 3 are in the purposes of preparing in antitumor cell preparation, it is characterized in that described cell brings into play its cytotoxicity to tumour cell by Fas/FasL approach.

7. gene modified CDR 3 delta transplanted gamma delta T γ 9 δ 2T lymphocytes according to claim 1 are in the purposes of preparing in antitumor cell preparation, and wherein said tumour is selected from ovarian cancer, cervical cancer, B lymphoma.