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

Abstract

The invention relates to gene-modified CDR3δ transplanted γδT lymphocytes and the tumor suppressing application thereof. The inventors infected normal human PBMCs stimulated with anti-CD3 antibodies by packaging retroviral particles containing the full-length γδ TCRδ2 chain and γδ TCRγ9 chain of the OT3 sequence (FPSHTFHSTVVHT) respectively, so that TCRγδ was expressed on the cell membrane surface, and the genetically modified CDR3δ transplanted type was obtained. γ9δ2 T lymphocytes, experiments have proved that the cells have cytotoxic effect on a variety of tumor cells. The present invention thus provides new methods and strategies for adoptive immunotherapy of tumors.

Description

Gene-modified CDR3δ transplanted γδT lymphocytes and its application for tumor suppression

technical field

The present invention relates to the fields of genetically modified cells and tumor treatment. Specifically, the present invention relates to the field of genetically modifying T lymphocytes and treating tumors with the modified γ9δ2 T lymphocytes.

Background technique

Immunotherapy of tumors has high hopes, especially the adoptive immunotherapy of tumor-specific T lymphocytes is considered to be a potential treatment strategy. However, the current experimental or clinical trials have not shown satisfactory efficacy. This may be closely related to the extremely weak or even absent antigenicity of most tumors, the defect of antigen presentation function, the low expression level of surface MHC molecules or co-stimulatory molecules, and the immunosuppressive state of tumor-bearing organisms. In addition, the number of tumor-specific cytotoxic T lymphocytes (CTL) in cancer patients is small, and it is difficult to isolate and expand in vitro; it is difficult to maintain the specificity of tumor recognition and its anti-tumor ability in long-term culture in vitro; T The generation of cell lines or T cell clones is time-consuming and laborious. These factors limit the wide application of T lymphocyte adoptive immunotherapy.

Recently, some scientists have applied genetic engineering technology to enable αβT lymphocytes to express tumor-specific T cell receptors, thereby improving the specific recognition ability of T lymphocytes to tumor antigens. Many laboratories have obtained virus-specific or tumor-specific genetically engineered lymphocytes, and their anti-tumor ability has been proved in vitro and in animal experiments.

However, the MHC restriction of tumor antigen recognition by αβT cells limits the clinical use of genetically modified αβT lymphocytes. The expression of MHC molecules on the surface of tumor cells is low or lost, which is difficult to be recognized by genetically modified αβT lymphocytes, leading to tumor immune escape. Moreover, the genetically modified αβT lymphocytes need to recognize both the antigen and the MHC molecules presenting the antigen. Therefore, in clinical application, it is necessary to separate a variety of T cell clones specific to different MHC-restricted antigens, and to select specific T cells based on the patient’s presentation. The MHC molecule types of the peptides presented were used to determine the TCRs capable of responding to the tumor. The number of tumor antigen-specific TCRs discovered so far is very small, and they only target a few tumor-associated antigens of cancers such as melanoma. Furthermore, the αβ T cell receptors used for transfection mainly recognize protein antigens and rarely recognize two other types of tumor antigens: carbohydrate and glycolipid antigens. Finally, endogenous and exogenous α and β chains may form hybrid TCRs that generate unintended antigen recognition specificities, thereby triggering autoimmune diseases.

As another type of T cell population, γδT cells have a unique role in anti-tumor immunotherapy: ① γδT cells mainly recognize antigens in an MHC-unrestricted manner, which overcomes the limitation of MHC-restricted anti-tumor immunity of αβ T cells; ② Antigen recognition spectrum is wide, such as peptides, non-peptides, alcohols, etc., and can recognize antigens that cannot be recognized by αβT cells, so it can be used as an important supplement to the immune surveillance of αβT cells in function; ③Tumor-infiltrating γδT lymphocytes express Fc receptors can still transmit signals when the expression or/and assembly of the TCR-CD3 complex is defective, which is not available in αβT cells; ④ exert effector functions through cytotoxicity and cytokine production; ⑤ It has killing effects on hematological tumors and solid tumors.

However, there are still some problems to be solved urgently in the preparation of γδT cell preparations for treatment. For example, immunosuppression in patients with advanced tumors or patients after chemotherapy leads to a decrease in the number of seed cells and a decline in immune activity, resulting in a limited number of expanded cells in vitro, making it difficult to achieve the required amount of cell preparations for treatment. Therefore, it is imperative to prepare a sufficient number of γδT immune effector cells with specific tumor response and no/low toxicity by using genetic engineering technology.

Our laboratory has been engaged in the research of γδT cells for a long time, and has conducted an in-depth discussion on the mechanism of γδTCR antigen recognition. The antigen recognition site of γδTCR mainly exists in the complementarity-determining region (CDR) of Vδ chain, in which CDR1 and CDR2 regions are encoded by germline gene V, while CDR3 region is formed by somatic recombination of V(D) and J genes of. Previous studies have shown that γδTCR is highly similar in structure to BCR and antibodies. The CDR3 length distribution graph shows that the length of the antibody heavy chain (H) and TCRδ chain CDR3 varies the most, and is significantly longer than the light chain (L) and γ chain. These structural basis suggest that, just as the CDR3 sequence of the heavy chain of the antibody plays a key recognition role in the process of antibody recognition of antigen, CDR3δ also plays a key role in the recognition of antigen by γδ T cells.

The preliminary laboratory results of TCRδ2-CDR3 gene scanning and length polymorphism analysis in ovarian cancer and normal ovarian tissue showed that the TCRδ2-CDR3 gene fragment in normal ovarian tissue was polyclonal expression; in contrast, ovarian cancer tissue The TCRδ2-CDR3 gene fragment was oligoclonal expression. The length distribution of the two fragments is uneven, and the length distribution of the latter is more extensive, and has a more obvious dominant fragment length. This difference in length distribution shows that γδT cells play an important role in the immune response of ovarian cancer.

Contents of the invention

The inventor selected the OT3 sequence (FPSHTFHSTVVHT) that binds to tumor cells according to the TCRδ2-CDR3 sequence obtained from the TIL of 10 cases of ovarian cancer in the laboratory. Retroviral particles containing the full-length γδ TCRδ2 chain and γδ TCRγ9 chain containing the OT3 sequence were respectively packaged. After the obtained high-titer virus was obtained, normal human PBMCs stimulated by anti-CD3 antibodies were infected to express TCRγ9δ2 on the cell membrane surface, named γ9δ2 (OT3) T cells. The experimental results showed that the secretion of TNF-α and IFN-γ of γ9δ2 (OT3) T cells increased after being stimulated by various tumor cell proteins. At the same time, it also has cytotoxic effect on a variety of tumor cells. Moreover, this cytotoxic effect can be blocked by monoclonal antibodies against TCRγδ. These results suggest that γ9δ2 (OT3) T cells can be activated by tumor antigens and have anti-tumor effects. In order to study the mechanism of cytotoxicity of transfected cells, the inventors used BFA, an inhibitor of the Fas/FasL pathway, and CMA, an inhibitor of the perforin/granzyme pathway, to conduct cytotoxicity blocking experiments on γ9δ2 (OT3) T cells. The results showed that the maximum inhibitory rate of BFA on the tumor killing effect of γ9δ2(OT3) T cells was only 20-30%; the inhibitory rate of CMA on the killing activity of SKOV3 cells with low expression of Fas by γ9δ2(OT3)T could reach 56.71%. The inhibitory rate of killing activity of HO8910 cells with high Fas expression was 33.93%. The combined application of CMA and BFA can inhibit the cytotoxicity of γ9δ2 (OT3) T cells by more than 60%. The above results suggest that both the perforin/granzyme and Fas/FasL pathways play a role in the cytotoxic effect of γ9δ2(OT3) T cells on tumors, but the perforin/granzyme pathway plays a major role, especially in cells with low expression of Fas. more important in the cytotoxicity of tumor cells. The inventors further studied the tumor suppressor effect of γ9δ2 (OT3) T cells in vivo. Through nude mouse models bearing human ovarian cancer xenografts, γ9δ2 (OT3) T cells were locally injected into the tumor to observe its therapeutic effect on the tumor. The results showed that γ9δ2 (OT3) T cells combined with IL-2 treatment, compared with empty vector infected cells combined with IL-2 treatment group, the tumor growth was significantly inhibited, and the survival period of nude mice was also prolonged.

Therefore, one aspect of the present invention provides a genetically modified CDR3δ transplanted γ9δ2 T lymphocyte, the CDR3 region of the γδ TCRγ9 chain and the γδ TCRδ2 chain of the cell is replaced by an OT3 sequence, wherein the OT3 sequence is SEQ ID No: 1.

In an embodiment of the present invention, the genetically modified CDR3δ transplanted γ9δ2T lymphocytes of the present invention are obtained through the following steps: 1) respectively packaging retroviral particles containing the full-length γδ TCRδ2 chain and γδ TCRγ9 chain of the OT3 sequence; 2) The obtained retroviral particles were used to infect normal human PBMCs stimulated with anti-CD3 antibody to express TCRγδ on the cell membrane surface.

Experiments have proved that the secretion of TNF-α and IFN-γ increases after the gene-modified CDR3δ transplanted γ9δ2T lymphocytes provided by the invention are stimulated by tumor cell proteins. This shows that the γδT lymphocytes of the present invention have extensive anti-tumor effects.

The gene-modified CDR3δ transplanted γ9δ2T lymphocytes provided by the invention have cytotoxic effect on tumor cells. Experiments have shown that both the perforin/granzyme and the Fas/FasL pathway play a role in cytotoxicity, but the perforin/granzyme pathway plays a major role, especially in the cytotoxicity of tumor cells with low Fas expression. important.

The present invention also provides the gene-modified CDR3δ transplanted γ9δ2T lymphocytes of the present invention for preparing anti-tumor cell preparations.

The anti-tumor cell preparation provided by the invention can be used to treat various tumors, and the treatable tumors include tumors with low Fas expression. Treatable tumors also include ovarian cancer, cervical cancer, and B lymphoma.

Another aspect of the present invention also provides the combined use of the anti-tumor cell preparation of the present invention and IL-2 to obtain better therapeutic effect.

Description of drawings

Figure 1: Transfected PBLs express γδTCR

(A) PCR amplification of full-length δ2 and γ9 chains of graft-specific CDR3 sequences. 1: γ9 chain; 2: δ2. (B) PBMC cells activated by anti-CD3 antibody were infected with retroviral particles containing pMSCVhyg-γ9 plasmid and pMSCVneo-δ(OT3) plasmid, and PBMC cells infected with empty vector virus particles were used as control. RT-PCR amplified the full length of TCRγ9 and TCRδ2 chains and their respective CDR3 regions. Left: 1: RNA extracted from PBMC cells infected with empty vector virus particles, 2: RNA of PBMC cells infected with retroviral particles containing TCR; right: RNA extracted from PBMC cells infected with retroviral particles containing TCR as Template for RT-PCR (1-5): 1: β-actin; 2: full-length γ9; 3: full-length δ2; 4: CDR3 sequence of γ9 chain; 5: CDR3 sequence derived from ovarian cancer transplanted with δ2 chain ; M: marker; RNA extracted from PBMC cells infected with empty vector virus particles was used as a template for RT-PCR (6-10) 6: β-actin; 7: γ9 full length; 8: δ2 full length; 9: γ9 10: CDR3 sequence of δ2 chain transplanted from ovarian cancer. (C) Western blotting to detect the expression of γδTCR. 1: Protein extract of cells transfected with empty vector (negative control); 2: Protein extract of cells infected with TCR virus; 3: Protein extract of normal human γδT cells (positive control). (D) Cells integrating γ9 and δ2 chains (γ9δ2(OT3) T cells), empty vector control cells were collected, and the expression of TCRγδ in transfected cells was detected by flow cytometry. On the left are empty vector control cells; on the right are γ9δ2(OT3) T cells.

Figure 2: Biological functions of γ9δ2(OT3) T cells

(A) γ9δ2(OT3) T cells and control cells transfected with empty vector were co-incubated with Daudi, Hela, SKOV3, HO8910 and Raji cell protein extracts for 72 hours, and the supernatant was collected to detect IFN in each group of cells by ELISA double antibody sandwich method -γ secretion. (B) γ9δ2(OT3) T cells and control cells transfected with empty vector were co-incubated with Daudi, Hela, SKOV3, HO8910 and Raji cell protein extracts for 24 hours, and the supernatant was collected to detect TNF in each group by ELISA double antibody sandwich method -alpha secretion. *P<0.05; **P<0.01.

(C) The killing activity of γ9δ2 (OT3) T cells on tumor cells was detected by MTT assay. γ9δ2 (OT3) T cells are effector cells, and were incubated with tumor cells (Hela, SKOV3, HO8910) at different effector-target ratios at 37°C for 8 hours, and then 10 μL of MTT with a concentration of 5 mg/mL was added to each well and incubated for 4 hours. The reaction was terminated by adding DMSO.

Figure 3: The effect of γ9δ2 (OT3) T cells on tumor cells is blocked by anti-γδTCR antibody.

(A) γ9δ2 (OT3) T cells were pre-blocked with anti-γδTCR antibody or isotype-independent antibody (mouse IgG1 control antibody) for 2 hours, then incubated with different tumor cell protein extracts for 72 hours, and the supernatant was collected for ELISA double antibody sandwich The secretion of IFN-γ in each group was detected by method. (B, C, D) γ9δ2 (OT3) T cells as effector cells were pre-treated with anti-γδTCR antibody or isotype-independent antibody (mouse IgG1 control antibody) for 2 hours, and then compared with different tumor cells at different effector-target ratios After mixing and culturing for 8 hours, the killing activity of γ9δ2 (OT3) T cells on target cells was detected by MTT method. *P<0.05; **P<0.01

Figure 4: Fas expression on the surface of tumor cells detected by flow cytometry.

Figure 5: Killing mechanism of γ9δ2 (OT3) T cells.

(A) Effect of BFA on cytotoxicity of γ9δ2(OT3) T cells. The γ9δ2(OT3) T cells were treated with different concentrations of BFA for 2 hours, and then mixed with the target cells for the killing test, and a solvent control was set at the same time. (B) Effect of CMA on cytotoxicity of γ9δ2(OT3) T cells. The γ9δ2(OT3) T cells were treated with different concentrations of CMA for 2 hours, and then mixed with the target cells for the killing test, and a solvent control was set at the same time. (C) After CMA (100 nM), BFA (25 μM) and combined treatment of effector cells, the cytotoxic effect of γ9δ2(OT3)T on tumor cells was detected.

Figure 6: Anti-tumor effect of γ9δ2 (OT3) T cells in vivo.

After inoculation of tumor cells, when the subcutaneous tumor nodules grew to ^about 100 mm3 (about 10 days), the nude mice were randomly divided into three experimental groups. The treatment group: intratumoral injection of γ9δ2 (OT3) T cells (1×10 ⁶ cells /50μL/monkey, 8 rats in total), empty vector group: intratumoral injection of cells infected with empty vector (1×10 ⁶ cells/50 μL/monkey, 8 rats in total), PBS control group: intratumoral injection of PBS (50 μL/carrier , a total of 8). Except for the PBS group, the animals in the other two groups were given intraperitoneal injection of IL-25000U/animal, once every three days, for a total of four times. The general situation and survival of the nude mice were observed and recorded every day; the tumor growth and body weight of the nude mice were measured every 2 days. (A) Photographs of the appearance of nude mice bearing human ovarian cancer and the size of their transplanted tumors after 24 days of treatment. A, B: γ9δ2(OT3)T; C, D: empty vector control group; E, F: PBS control group. (B) Tumor growth in nude mice bearing human ovarian cancer. (C) Growth curves of nude mice bearing human ovarian cancer. (D) Changes in body weight of nude mice bearing human ovarian cancer.

Detailed ways

1. Materials and methods

1. Cell culture

PBMC are freshly isolated normal human peripheral blood mononuclear cells; RetroPack ^TM PT67 is a packaging and replicating retroviral cell line derived from NIH3T3, purchased from Clontech Laboratories, Inc. NIH3T3 is a mouse fibroblast cell line. SKOV ₃ , human ovarian serous papillary cystadenocarcinoma cell line; HO8910, human ovarian serous cystadenocarcinoma cell line; Hela, human cervical cancer tumor cell line; Daudi, human B lymphoma cell line; Raji, human Burkitt lymphoma cell line All tumor cell lines can be purchased from the Cell Center, School of Basic Science, Chinese Academy of Medical Sciences. SKOV3 cells were cultured adherently in McCoy 5A medium containing 10% FCS. Hela, NIH3T3 and RetroPack PT67 cells were cultured adherently in DMEM high glucose medium containing 10% FCS. HO8910, Raji, and Daudi, in RPMI-1640 medium containing 10% FCS for adherent or suspension culture.

2. Animals

BALB/c nude mice, aged 4 to 6 weeks, weighing 15 to 20 g, female, were purchased from the Animal Center of the Institute for the Control of Biological Products, and were free from specific pathogens (SPF) in the Experimental Animal Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. ) in a laminar flow rack.

3. Strains and plasmid vectors

Escherichia coli DH5α was purchased from Bao Biological Engineering Co., Ltd. The genotype is: supE44ΔlacU169 ( 80lacZΔM15) hsdR17 recA1 end1 gyr96 thi-1 relA1, used for amplification and transformation of the plasmid.

pGEM-T or pGEM-T Easy Vector System was used for cloning of PCR products amplified by Taq enzyme, purchased from Promega. The pMSCVhyg and pMSCVneo plasmids for expression of γδ TCR were purchased from Clontech Laboratories, Inc. pcDNA3.1-γ9 and pcDNA3.1-δ(OT3) are the recombinant plasmids of the full-length γ9 gene and the CDR3 region is the δ replaced by the OT3 sequence, constructed by Dr. Xi Xueyan, quoted from literature 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. Primers (see Table 1)

Table 1. Primers used to amplify the full length of the γ and δ genes of the γδ TCR

The above primers were synthesized by Beijing Qingke Company.

experimental method:

1. Construction of retroviral plasmids

(1) PCR of the full-length genes of the gamma chain and delta (OT3) chain of TCR

Use pcDNA3.1-γ, pcDNA3.1-δ(OT3) plasmids as templates, use pMSCVneoOT3-up, pMSCVneoOT3-down primers to amplify the full-length TCRδ(OT3) chain containing BglII and XhoI restriction sites, and use pMSCVhyrγ -up, pMSCVhyrγ-down primer amplifies the γ chain containing XhoⅠ and BglⅡ restriction sites.

(2) Purification of PCR products

(3) Enzyme digestion reaction of vector and PCR product

(4) Connection reaction

The digested full-length TCRγ9 and TCRδ2 (OT3) chains were respectively connected with the digested vectors to construct recombinant plasmids pMSCVneo-δ(OT3) and pMSCVhyr-γ plasmids.

(5) Transformation and positive selection of recombinant plasmids

(6) Small amount preparation of plasmid DNA

(7) Enzyme digestion identification of recombinant plasmids

(8) Sequence analysis of DNA fragments

Recombinant plasmids that were identified by restriction enzyme digestion and contained the target DNA were subjected to DNA sequence determination (completed by Nuosai Biotechnology Co., Ltd.). The sequencing results were analyzed with DNAMAN software.

2. Determination of drug resistance of RetroPack ^TM PT67 cells

(1) Inoculate 1×10 ⁵ RetroPack ^TM PT67 cells in each well of a six-well plate, and add the final concentrations of 0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg/ ml of puromycin. After culturing for 3-4 days, the corresponding selective medium was added to each well and maintained for 7-9 days. The cell viability of each well was observed every 24 hours to determine the optimum screening concentration of puromycin.

(2) Inoculate 1×10 ⁵ RetroPack ^TM PT67 cells in each well of a six-well plate, and add G418 to different final concentrations, which are 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 1100 μg/ml. Two replicate wells were set up for each concentration, and the cells were cultured in a 37°C, 5% CO2 incubator for 13 days. The concentration of G418 that caused the death of all Retro Pack ^PT67 cells was selected as the concentration used for screening positive transfection clones later.

3. Liposome transfection of RetroPack ^TM PT67 cells

After adjusting the RetroPack ^™ PT67 cells to an appropriate concentration with serum-free DMEM medium, add 500 μl of cell suspension to each well of a 24-well plate. Dilute pMSCVhyg and pMSCVneo, pMSCVhyg-γ and pMSCV neo-δ, and Lipofectamine ^™ 2000 liposomes with 50 μl of serum-free DMEM medium, and equilibrate at room temperature. Mix the diluted vector plasmid and Lipofectamine ^TM 2000 liposome within 5 minutes, continue to incubate at room temperature for 20 minutes, then add the above mixture (100 μl) to the 24-well plate containing RetroPack ^TM PT67 cells, lightly Gently oscillate to mix, and culture in a 37°C, 5% CO ₂ incubator. After 24 hours, the RetroPack ^™ PT67 cells were replaced with DMEM complete medium containing 10% serum (in which the final concentration of G418 was 800 μg/ml or the final concentration of Hyg was 300 μg/ml) for positive fine selection.

4. Screening of positive clones

The medium was changed every 2 days. After 7 days of selection, a large number of cells without the transfected plasmid died, while the RetroPack ^TM PT67 cells transfected with the plasmid grew a large number of cell clusters. The cell clusters were digested with trypsin and transferred into Monoclonal culture was carried out in a 96-well plate, and cell clones appeared after two weeks. Single clones were picked out and cultured in 24-well plates, and the supernatant was collected to determine the virus titer.

5. Determination of virus titer

NIH3T3 cells were used as target cells to measure and calculate the virus titer in the presence of Polybrene. Virus titer (cfu/L) = average number of cell clones × dilution factor of virus solution × 10 ³ . Dilute the collected virus supernatant to 10 ^-1 , 10 ^-2 , 10 ^-3 , and 10 ^-4 times respectively. Pipette 1 mL of the diluted virus liquid (8 mg/L Polybrene) into the six wells previously inoculated with NIH3T3 cells In the plate, after the virus was adsorbed for 2.5 hours, an equal amount of DMEM culture solution containing 800 μg/ml G418 or 300 μg/ml hyg was added to continue culturing for 3 days, and then the culture solution containing 800 μg/ml G418 was replaced. After about 14 days, cell clones basically formed.

6. Retrovirus infection of normal human PBMC

(1) Isolation of peripheral blood mononuclear cells (PBMC)

(2) Retrovirus infection process

PBMCs activated by anti-CD3 antibody were resuspended in RPMI1640 complete medium containing retrovirus and 200IU/ml IL-2 to 1×10 ⁶ cells/ml, and inoculated onto non-tissue culture plates coated with Retronectin in advance. At 32°C, the culture plate was centrifuged at 1500×g for 90 minutes, and then the cells were cultured in a 37°C, 5% CO ₂ incubator. This infection process is repeated twice. On the sixth day after infection, the cells were collected, and then the medium containing IL-2 was changed to continue culturing. On the eighth day of infection, replace with geneticin (0.5mg/ml) and hygromycin B (0.1mg/ml) and continue to culture for 3 days, then replace with fresh complete RPMI1640 containing 200IU/ml IL-2 and continue to culture.

7. Killing of tumor cells by γ9δ2(OT3) T transfected cells and killing blocking experiment

Target cell preparation: Subculture the corresponding target cells, namely SKOV3 cells or other tumor cells, wash twice with the corresponding culture medium, adjust the cell concentration to 1×10 ⁵ /ml, add 100 μl/well to a 96-well plate, place Incubate at 37°C, 5% CO ₂ , and use for killing experiments after the cells adhere to the wall.

Preparation of effector cells: the effector cells are γ9δ2 (OT3) T cells or PBMCs transfected with empty vector, adjust the cell concentration so that the effector-target ratio is 5:1, 10:1, 20:1 respectively, and the effector cells are added to each target cell In the hole, there are 3 parallel holes in each group. In addition, set up control wells where only target cells or effector cells or only culture medium are added.

Detection of killing activity by MTT method: place the mixed reaction system at 37° C., 5% CO ₂ and incubate for 4 hours, remove 100 μl of supernatant from each well, add 15 μl of MTT, and continue to incubate. After 4 hours, add 100 μl DMSO to each well, incubate for 4 hours, measure the OD value at the measurement wavelength of 570 nm and the reference wavelength of 630 nm, and calculate the killing activity according to the following formula:

$\frac{{\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; OD</span>}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; [</span>{<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; E.</span>}<span\; class="notranslate">\; +</span>\mathrm{<span\; class="notranslate">\; T</span>}<span\; class="notranslate">\; )</span>}_{\mathrm{<span\; class="notranslate">\; OD</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; E.</span>}}_{\mathrm{<span\; class="notranslate">\; OD</span>}}}{{\mathrm{<span\; class="notranslate">\; T</span>}}_{\mathrm{<span\; class="notranslate">\; OD</span>}}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}<span\; class="notranslate">\; \%</span>$

Among them, T _OD refers to the OD value of target cells alone; E _OD refers to the OD value of effector cells alone; (E+T) _OD represents the OD value of effector cells and target cells mixed wells. In the killing blocking experiment, γ9δ2 (OT3) T cells were incubated with anti-γδTCR monoclonal antibody at 37°C for 90 minutes, and then their cytotoxic activity was measured to observe the blocking effect of the monoclonal antibody.

8. Experiments to analyze the killing mechanism of γ9δ2 (OT3) T cells

(1) Flow cytometry to detect the expression of Fas on the surface of tumor cells

Collect the cells to be tested and prepare a cell suspension of 1×10 ⁶ /ml. Wash with PBS 3 times, absorb the supernatant, add 5 μl of FITC-labeled anti-Fas monoclonal antibody, and incubate at 4°C for 60 minutes. Centrifuge and wash 3 times with PBS, add 500 μl of 2% paraformaldehyde fixative after aspirating the supernatant, and analyze on the flow cytometer. FITC-labeled isotype control antibody was used as the isotype control.

(2) Cytotoxicity inhibition test

γ9δ2(OT3) T cells were treated with different concentrations of CMA or BFA or in combination for 2 hours, and then mixed with target cells for a killing test. At the same time, a solvent control was set up to observe the effects of CMA or BFA or the combination on the effect of γ9δ2(OT3) T cells on tumors. The effect of cell killing to explore the mechanism of killing.

9. Anti-tumor effect of γ9δ2 (OT3) T cells in vivo.

SKOV3 cells were digested and counted with 0.25% trypsin, washed three times with PBS, suspended in PBS, and inoculated subcutaneously on the right shoulder and back of nude mice (1×10 ⁶ , 100 μl volume). When the subcutaneous tumor nodules grew to about 0.1 ^mm3 (about 10 days), the nude mice were randomly divided into three experimental groups. The treatment group: intratumoral injection of γ9δ2 (OT3) T cells (1×10 ⁶ cells/50 μL/mouse, 8 rats in total), empty vector group: intratumoral injection of cells infected with empty vector (1×10 ⁶ cells/50 μL/rat, 8 rats in total), PBS control group: intratumoral injection of PBS (50 μL/rat, 8 rats in total) . Except for the PBS group, the animals in the other two groups were given intraperitoneal injection of IL-2 5000U/animal, once every three days for a total of four times. The general situation and survival of the nude mice were observed and recorded every day; the tumor growth and body weight of the nude mice were measured every 2 days. The calculation formula is: tumor volume (mm ³ )=a×b ² ×0.5. a: tumor long diameter (mm), b: tumor short diameter (mm). On the 24th day, 2 mice in each group were sacrificed, and the tumor nodules were obtained after dissection. The rest continued to observe the survival period of mice in each group.

2. Experimental results

Study on tumor suppressor effect of γ9δ2(OT3) T cells prepared by retrovirus transfection in vitro and in vivo

(1) Cloning and sequence identification of pMSCVhyg-γ9 and pMSCVneo-δ2 (OT3) genes

Full-length γ9 and δ2 chains containing CDR3 regions from OEC tumor-infiltrating lymphocytes (TILs) have been reported in the literature (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). In short, primers were designed according to the TCRδ and TCRγ sequences in GenBank, and the amplified DNA fragments were identified by agarose gel electrophoresis through PCR technology. After recovery and purification, the PCR product was ligated with the pMSCV vector, and the product was named pMSCVhyg-γ9. After the ligation product was transformed into Escherichia coli DH5α, the positive clones screened by ampicillin resistance were identified by double enzyme digestion with BglII and XhoI, and the positive clones were taken for DNA sequence determination. According to sequence comparison, the inserted fragments in the recombinant plasmid were consistent with the sequences of TCRδ2 (OT3) and TCRγ9, respectively. The experimental results are shown in Figure 1A.

(2) Detection of TCRγ9 and TCRδ2 chains by RT-PCR

Retroviral particles containing pMSCVhyg-γ9 plasmid and pMSCVneo-δ2(OT3) plasmid were used to infect PBMC cells activated by anti-CD3 antibody, while PBMC cells containing empty vector virus particles were infected as a control. RT-PCR amplified the full length of TCRγ9 and TCRδ2 chains and their respective CDR3 regions, and detected the integration of TCRγ9 and TCRδ2 chains in infected cells. The full-length TCRγ9 and TCRδ2 (OT3) chains and their respective CDR3 regions were amplified from the cDNA of cells infected with pMSCVhyg-γ9 plasmid and pMSCVneo-δ(OT3) plasmid virus particles, suggesting that TCRγ9 and TCRδ2(OT3) had been integrated into the transfected cells . The experimental results are shown in Figure 1B.

(3) Detection of TCRγδ expression in transfected cells by flow cytometry

γ9δ2 (OT3) T cells were collected, transformed into empty vector control cells, and the expression of TCRγδ in transfected cells was detected by flow cytometry. As shown in Figure 1D, 74% of the virus-transfected cells expressed the γδ TCR compared to the empty vector-transfected control cells.

(4) Detection of TCRγδ expression in transfected cells by Western blotting

Using mouse anti-human γ9 monoclonal antibody as the primary antibody, HRP enzyme-labeled goat anti-mouse antibody as the secondary antibody, using chemiluminescent substrate to develop, the control of γ9δ2 (OT3) T cells and normal human γδT cells and transfection empty vector The crude cell protein extract was analyzed by Western blot after SDS-PAGE. As shown in Figure 1C, the first lane is cells transfected with empty vector (negative control), the second lane is γ9δ2 (OT3) T cells, and the third lane is normal human γδT cells (positive control). The results of Western detection proved the expression of TCRγδ in the transfected cells.

(5) Biological function identification of γ9δ2(OT3)T

1. The expression of TNF-α after the total protein of different tumor cells was co-incubated with γ9δ2 (OT3) T cells

ELISA double antibody sandwich method was used to detect the secretion of TNF-α in each group of cells. The results are shown in 2A. Daudi protein extract was used as a positive control, and Raji protein extract was used as a negative control; Hela, SKOV3, HO8910 protein extracts stimulated γ9δ2 (OT3) T cells to secrete TNF-α higher than that of the empty vector control group, but there was no statistics learning meaning.

2. The expression of IFN-γ after co-incubation of different tumor total proteins and γ9δ2 (OT3) T cells

The ELISA double-antibody sandwich method was used to detect the secretion of IFN-γ in each group of cells, and the results are shown in Figure 2B. Daudi protein extract was used as positive control, Raji protein extract was used as negative control; Hela, SKOV3, HO8910 protein extract stimulated γ9δ2 (OT3) T cells to secrete IFN-γ which was higher than that of empty vector control group (p<0.01 , p<0.01, p<0.01). ,

3. Killing of tumor cells by γ9δ2 (OT3) T cells and blocking experiment of anti-γδTCR antibody

Using different tumor cells as target cells and γ9δ2(OT3) T cells as effector cells, the killing activity of γ9δ2(OT3) T cells on target cells was detected by MTT assay (indicated by killing rate), and the results are shown in Figure 2C. γ9δ2 (OT3) T cells have killing activity on a variety of tumor cells, and with the increase of the effector-target ratio, the killing efficiency also increases. Among them, the killing effect on SKOV3 was the strongest. After pretreatment of γ9δ2(OT3) T cells with anti-TCRγδ monoclonal antibody, their killing activity against tumor cells was reduced (Fig. 3A, B, C, D). The above results indicated that the killing effect of γ9δ2 (OT3) T cells on tumor cells was TCR-dependent.

(6) Preliminary study on the killing mechanism of γ9δ2 (OT3) T cells

1. Flow cytometry to detect the expression of Fas on the surface of tumor cells

Use anti-Fas monoclonal antibody and FITC-labeled goat anti-mouse IgG to perform immunofluorescence staining on Hela, SKOV3, HO8910 and other tumor cells to determine the expression of Fas on the surface of these cells. The result of the flow cytometry test is shown in FIG. 4 . The flow cytometry test showed that SKOV3 was a cell with low Fas expression, and its Fas expression rate was 6.46%. HO8910 is a high Fas expression cell, and its Fas expression rate is about 70%. The Fas expression rate of Hela is between SKOV3 and HO8910, and its Fas expression rate is about 40%.

2. Inhibitory effect of BFA on tumor cytotoxic activity of γ9δ2 (OT3) T cells

The γ9δ2(OT3) T cells were treated with different concentrations of BFA for 2 hours, and then mixed with the target cells for the killing test. At the same time, a solvent control was set up to observe the effect of BFA on the cytotoxicity of the γ9δ2(OT3) T cells. The results are shown in Figure 5A, BFA partially inhibited the killing effect of γ9δ2 (OT3) T cells on tumor cells, and the inhibitory effect was also enhanced with the increase of its concentration, and the inhibitory effect reached the peak when the final concentration was 25 μM. and Hela tumor cell killing inhibition rates were 27.91%, 19.01% and 20.56%. It is suggested that the killing effect of γ9δ2 (OT3) T cells on tumor cells is not only related to Fas and FasL pathways, but also involves another pathway.

3. Inhibitory effect of CMA on tumor cytotoxic activity of γ9δ2 (OT3) T cells

The γ9δ2(OT3)T cells were treated with different concentrations of CMA for 2 hours, and then mixed with the target cells for the killing test. At the same time, a solvent control was set up to observe the effect of CMA on the cytotoxicity of γ9δ2(OT3)T. The results are shown in Figure 5B, CMA has a significant inhibitory effect on the tumor cytotoxic activity of γ9δ2 (OT3) T cells, and the inhibitory effect is also enhanced with the increase of its concentration. When the final concentration is 100nM, the inhibition peak is reached, and the killing inhibition rates of HO8910, SKOV3 and Hela tumor cells are 33.93%, 56.71% and 46.52%, respectively. This suggests that the killing effect of γ9δ2 (OT3) T cells on tumor cells mainly depends on the pathway of perforin and granzyme, especially for tumor cells with low expression of Fas.

4. Inhibitory effect of CMA combined with BFA on tumor cytotoxic activity of γ9δ2 (OT3) T cells

In order to further determine the killing mechanism of γ9δ2(OT3) T cells on tumor cells, we compared the effects of CMA (100 nM), BFA (25 μM) and combined effector cells on the cytotoxicity of γ9δ2(OT3) T cells. The results are shown in Figure 5C. For HO8910 cells with high expression of Fas, the inhibitory rate of BFA on the cytotoxicity of γ9δ2 (OT3) T cells was 24.15%, and the inhibitory rate of CMA on the cytotoxicity of γ9δ2 (OT3) T cells was 38.51%. BFA Combined with CMA, the inhibition rate of cytotoxicity is 60.95%. It suggested that Fas and FasL pathways as well as granzyme perforin pathway played a role in the killing mechanism of γ9δ2(OT3) T cells to cells with high expression of Fas. For Hela cells expressed in Fas, the inhibition rate of BFA on the cytotoxicity of γ9δ2 (OT3) T cells was 18.52%, and the inhibition rate of CMA on the cytotoxicity of γ9δ2 (OT3) T cells was 54.96%. The inhibition rate of toxicity was 63.27%. This suggests that in the killing mechanism of Fas-expressing cells by γδ T cells, both the Fas and FasL pathways and the granzyme perforin pathway play a role, but the granzyme perforin pathway plays a major role. For SKOV3 cells with low expression of Fas, the inhibition rate of BFA on the cytotoxicity of γ9δ2 (OT3) T cells was 21.96%, and the inhibition rate of CMA on the cytotoxicity of γ9δ2 (OT3) T cells was 61.76%. The inhibition rate of cytotoxicity was 62.08%. It suggested that Fas and FasL pathways and granzyme perforin pathway played a role in the killing mechanism of γδT cells to tumor cells, but granzyme/perforin pathway played a major role.

(7) Anti-tumor effect of γ9δ2 (OT3) T cells in vivo.

In order to study the antitumor effect of γ9δ2 (OT3) T cells on human ovarian cancer in vivo, we established a nude mouse model bearing human ovarian cancer cell SKOV3 xenografts. After inoculation of tumor cells, when the subcutaneous tumor nodules grew to ^about 100 mm3 (about 10 days), the nude mice were randomly divided into three experimental groups. The treatment group: intratumoral injection of γ9δ2 (OT3) T cells (1×10 ⁶ cells /50μL/monkey, 8 rats in total), empty vector group: intratumoral injection of cells infected with empty vector (1×10 ⁶ cells/50 μL/monkey, 8 rats in total), PBS control group: intratumoral injection of PBS (50 μL/carrier , a total of 8). Except the PBS group, the animals in the other two groups were given intraperitoneal injection of IL-2 5000U/animal. Fig. 6A is the appearance of the nude mice in each treatment group on the 24th day and the photos of the transplanted tumors in the tumor-bearing mice. It can be seen that the mice in the treatment group and the tumors they carried were significantly smaller than those in the empty vector control group or the PBS control group. It can be seen from Fig. 6B that the volume of tumor nodules increased from 0.097±0.088 mm ³ to 0.521±0.332 mm ³ after intratumoral injection of γ9δ2 (OT3) T cells, while the tumor volumes of mice injected with empty vector-infected cells and mice injected with PBS were respectively Increased from 0.104±0.062mm ³ and 0.122±0.094mm ³ to 1.136±0.492mm ³ and 1.272±0.381mm ³ . Statistical analysis showed that from the 18th day after treatment, the tumor nodules in the γ9δ2 (OT3) T cell-injected treatment group were significantly smaller than those in the empty vector-infected cells (1×10 ⁶ cells, 8 rats) and the PBS control group (P< 0.05). It is suggested that γ9δ2 (OT3) T genetically engineered cells can kill tumor cells and have therapeutic effect. As shown in 6C, treatment with γ9δ2 (OT3) T cells can significantly prolong the survival time of tumor-bearing nude mice compared with the empty vector and PBS control groups (P<0.05).

During the whole treatment process, the general condition of the mice in each treatment group was good, eating and activities were normal, and there was no statistical difference in body weight before and after treatment (Figure 6D), suggesting that the treatment did not cause obvious toxicity and harm to the nude mice. damage.

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.