NY-ESO-1 specific TCR-T receptor and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to an NY-ESO-1 specific TCR-T receptor and application thereof. The genome of the NY-ESO-1 specific TCR-T receptor comprises an alpha-chain constant region gene and a beta-chain constant region gene of a human T cell receptor alpha95: LY subjected to murine transformation, and the amino acid sequences of the alpha-chain constant region gene and the beta-chain constant region gene are respectively shown as SEQ ID NO.8 and SEQ ID NO.10. The NY-ESO-1 specific TCR-T receptor comprises an alpha chain as shown in an amino acid sequence of SEQ ID NO: 4 and a beta chain as shown in an amino acid sequence of SEQ ID NO: 6. The TCR expression efficiency and stability of the NY-ESO-1 specific TCR-T receptor are greatly improved, the effectiveness and safety of in-vivo application of the NY-ESO-1 specific TCR-T receptor are promoted, and the NY-ESO-1 specific TCR-T receptor is simple and easy to design, has high reference value for TCR-T preparation and application and is suitable for popularization and application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a NY-ESO-1 specific TCR-T receptor and an application thereof. Background Art

[0002] In recent years, adoptive cell therapy for solid tumors has developed rapidly, especially TCR-T therapy. TCR-T therapy uses gene editing technology to transfect genes encoding T cell receptors (TCR) into T cells derived from autologous or allogeneic donors of tumor patients, so that they express TCR structures to specifically recognize tumor cell membrane (endo) antigen epitopes and exert anti-tumor effects. Among them, the stable and efficient expression of TCR on the surface of T cells is crucial for the efficacy of TCR-T. TCR is a heterodimer structure, including an α peptide chain and a β peptide chain, each peptide chain contains a variable region (V region) and a constant region (C region). In the process of constructing TCR-T using traditional TCR gene sequences, the phenomenon of mispairing of exogenously introduced TCRα / β chains and endogenously expressed TCRα / β in T cells often occurs, resulting in problems such as low expression efficiency of exogenous TCR and cross-reactive toxicity, which seriously affects the efficacy and safety of TCR-T. Therefore, by optimizing the TCR gene sequence and improving the pairing priority of exogenous TCR, it is expected to construct TCR-T that stably and efficiently expresses TCR, and promote the efficacy and safety of TCR-T against solid tumors.

[0003] NY-ESO-1 is a tumor-testis antigen, which is specifically expressed in a variety of solid tumor cells, such as synovial sarcoma (82%), melanoma (45%), liver cancer (23%), esophageal squamous cell carcinoma (19%), lung large cell carcinoma (17.8%), triple-negative breast cancer (16%), etc. Its expression in normal tissues is limited to the immune-privileged organ, the testis. Therefore, it is a relatively ideal antigen target at this stage. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a NY-ESO-1 specific TCR-T receptor that can efficiently target tumor tissues, has low toxicity and a broad anti-tumor spectrum in view of the deficiencies in the prior art.

[0005] The technical problem that the present invention needs to solve is to provide the application of the above-mentioned NY-ESO-1 specific TCR-T receptor.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A NY-ESO-1 specific TCR-T receptor, characterized in that the genome of the NY-ESO-1 specific TCR-T receptor includes an α chain constant region gene and a β chain constant region gene of a murinized human T cell receptor α95:LY;

[0008] Among them, the amino acid sequences of the α chain constant region gene and the β chain constant region gene of the murinized human T cell receptor α95:LY are shown in SEQ ID NO.8 and SEQ ID NO.10, respectively.

[0009] Among them, the human T cell receptor α95:LY is a variant of the human 1G4 TCR.

[0010] Among them, the NY-ESO-1 specific TCR-T receptor has a genome comprising a human variable region of the α chain and a human variable region of the β chain.

[0011] Specifically, the human variable region of the α chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO: 11, CDR2 with an amino acid sequence as shown in SEQ ID NO: 12, and CDR3 with an amino acid sequence as shown in SEQ ID NO: 13; the human variable region of the β chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO: 14, CDR2 with an amino acid sequence as shown in SEQ ID NO: 15, and CDR3 with an amino acid sequence as shown in SEQ ID NO: 16.

[0012] The NY-ESO-1 specific TCR-T receptor comprises an α chain as shown in the amino acid sequence of SEQ ID NO: 4, and a β chain as shown in the amino acid sequence of SEQ ID NO: 6.

[0013] Specifically, the α chain and the β chain are connected by a P2A sequence;

[0014] Furthermore, the P2A sequence is: ATNFSLLKQAGDVEENPGP.

[0015] The nucleic acid molecule encoding the NY-ESO-1 specific TCR-T receptor is also within the scope of protection of the present invention.

[0016] The expression vector comprising the nucleic acid molecule is also within the scope of protection of the present invention.

[0017] Recombinant cells comprising the nucleic acid molecule or the expression vector are also within the scope of protection of the present invention.

[0018] The pharmaceutical composition comprising the nucleic acid molecule or the expression vector or recombinant cell is also within the scope of protection of the present invention.

[0019] The use of the NY-ESO-1 specific TCR-T receptor, the nucleic acid molecule, the recombinant cell, or the pharmaceutical composition in the preparation of drugs for detecting, treating or preventing diseases is also within the scope of protection of the present invention.

[0020] The disease is a tumor disease with high expression of NY-ESO-1, such as melanoma and liver cancer.

[0021] Beneficial Effects

[0022] (1) The present invention improves the pairing priority of exogenous TCR by modifying the α chain constant region gene and the β chain constant region gene of the human 1G4 TCR variant α95:LY, constructs a TCR-T that stably and efficiently expresses TCR, and greatly improves the TCR expression efficiency and stability of NY-ESO-1-specific TCR-T.

[0023] (2) The NY-ESO-1-specific TCR-T cells constructed by the present invention efficiently and stably express TCR, exhibit strong NY-ESO-1-positive tumor cell-specific killing effects in vivo and in vitro, and have in vivo biosafety.

[0024] (3) The NY-ESO-1 specific TCR-T constructed by the present invention can efficiently target tumor tissues, has low toxicity and a broad anti-tumor spectrum, and can be developed in the direction of controllable clinical universal therapy to further explore the anti-tumor application of optimized TCR-T. At the same time, the design of the present invention is simple and easy to implement, has a high reference value for the preparation and application of TCR-T, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0026] Figure 1 This is a structural diagram of the "Sleeping Beauty" transposon system encoding the NY-ESO-1 specific TCR gene. The TCR gene includes the human NY-ESO-1 TCR α chain gene and β chain gene, and the two gene chains are connected by the P2A sequence (ATNFSLLKQAGDVEENPGP). The constant regions of the TCR α chain gene and β chain gene have been mouse-derived.

[0027] Figure 2Figure 2 is the flow cytometry analysis result of NY-ESO-1TCR expression efficiency. A is the cross-circle gate result of T cells stained with PC7-coupled anti-human CD8 antibody and PE-coupled HLA-A*02:01NY-ESO-1 tetramer on the 3rd day after PBLs were transfected with the plasmids before and after the modification. The data in the upper right quadrant are CD8 expressing NY-ESO-1TCR. + The percentage of T lymphocytes; B is the CD8 measured by flow cytometry on the third day after PBLs were transfected with the plasmids before and after the modification. + Expression efficiency of NY-ESO-1-specific TCR in T lymphocytes.

[0028] Figure 3 The results of IFN-γ secretion after 24 hours of co-incubation of PBLs before and after transfection with human melanoma cell line A375 that highly expresses NY-ESO-1 antigen and human liver cancer cell line SK-HEP-1 that lowly expresses NY-ESO-1 antigen. Among them, the untransfected PBLs are before transfection, and the NY-ESO-1TCR-T is after transfection.

[0029] Figure 4 The results of CD28 and CD137 activation phenotype expression after PBLs were co-incubated with human melanoma cell line A375 with high expression of NY-ESO-1 antigen and human liver cancer cell line SK-HEP-1 with low expression of NY-ESO-1 antigen before and after transfection for 24 hours. A shows the CD28 molecules on T cells labeled with PE-conjugated anti-human CD28 antibody, and the peak graph shows the expression of CD28 molecules in each group of cells; B shows the mean fluorescence intensity (MFI, Mean fluorescence intensity) of CD28 molecules coupled with fluorescence (PE-CD28) on the surface of cells in each group using a bar graph; C shows the CD137 molecules on T cells labeled with APC-conjugated anti-human CD137 antibody, and the peak graph shows the expression of CD137 molecules in each group of cells; D shows the mean fluorescence intensity (MFI, Mean fluorescence intensity) of CD137 molecules coupled with fluorescence (APC-CD137) on the surface of cells in each group using a bar graph.

[0030] Figure 5 To use bar graphs to statistically analyze the killing results of NY-ESO-1TCR-T against human melanoma cell line A375 with high expression of NY-ESO-1 antigen and human liver cancer cell line SK-HEP-1 with low expression of NY-ESO-1 antigen at different effector-target ratios, as well as the killing results of untransfected PBLs against A375.

[0031] Figure 6The figure shows the tumor inhibition effect of NY-ESO-1TCR-T in mice bearing A375 subcutaneous tumors. A is the tumor growth curve of each group of mice during the cell reinfusion treatment process; B is the statistical graph of tumor volume of each group of mice at the end of cell reinfusion treatment; C is the statistical graph of tumor weight of each group of mice at the end of cell reinfusion treatment.

[0032] Figure 7 The safety evaluation of NY-ESO-1TCR-T in mice bearing A375 subcutaneous tumors. A is the weight change curve of each group of mice during the treatment period; B is the important organ tissue morphology of each group of mice at the end of the treatment. DETAILED DESCRIPTION

[0033] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0034] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0035] Example 1: Modification of target TCR gene sequence

[0036] First, the constant regions of the TCRα chain gene sequence (the sequence is shown in SEQ ID NO.1) and the TCRβ chain gene sequence (the sequence is shown in SEQ ID NO.2) of the human 1G4 TCR variant α95:LY (the variant has been disclosed in the article Single and dual aminoacid substitutions in TCR CDRs can enhance antigen-specific T cell functions, Doi:10.4049 / jimmunol.180.9.6116) were murinized. Specifically, the nucleotide sites at positions 388 to 846 of the TCRα chain gene of the human 1G4 TCR variant α95:LY were replaced with the constant region gene of mouse origin; at the same time, the nucleotide sites at positions 397 to 936 of the TCRβ chain gene of the human 1G4 TCR variant α95:LY were replaced with the constant region gene of mouse origin.

[0037] Then, the TCRα chain (the nucleotide sequence of the TCRα chain is shown in SEQ ID NO.3) and the TCRβ chain (the nucleotide sequence of the TCRβ chain is shown in SEQ ID NO.5) containing the human variable region sequence and the mouse constant region sequence are connected through the P2A sequence (SEQ ID NO.17: ATNFSLLKQAGDVEENPGP) to obtain the target TCR gene sequence.

[0038] Specifically, the sequence information of the target TCR gene is shown in Table 1.

[0039] Table 1 Sequence information of target TCR gene

[0040]

[0041]

[0042] Example 2: Construction of TCR plasmid

[0043] The target gene sequence was loaded into the Sleeping Beauty transposon (purchased from Suzhou Aikonde Biotechnology Co., Ltd.) to obtain the TCR plasmid.

[0044] The specific construction process is as follows:

[0045] 1. Target TCR gene synthesis: Based on the above gene modification, Suzhou Aikonde Biotechnology Co., Ltd. was commissioned to synthesize the target TCR gene.

[0046] 2. Enzyme digestion: Use NcoI and NotI restriction endonucleases to digest the "Sleeping Beauty" transposon and target TCR gene sequence to produce sticky ends.

[0047] 3. Connect the transposon vector and the target gene sequence: Use DNA ligase to connect the "Sleeping Beauty" transposon after enzyme digestion and the target TCR gene sequence to obtain the target plasmid.

[0048] 4. Transformation of the target plasmid: Thaw the competent bacteria DH5α stored in a -80°C refrigerator on ice; Add 1 μg of the target plasmid to 50 μL of competent bacteria and gently mix the two, and let them stand on ice for 30 minutes; Heat-shock the mixing system in a 42°C water bath for 90 seconds (strictly control the time), and then quickly transfer to ice and let it stand for 2 minutes (do not shake the system); Add the above system to 500 μL of LB liquid culture medium without ampicillin (preheated in a 37°C water bath), and culture in a shaker at 37°C, 220rpm for 45 minutes, and set up a pure LB culture medium group as a blank control; Pipette 100-200 μL of the above bacterial solution and evenly spread it on an LB solid culture medium plate containing ampicillin; After the bacterial solution on the plate is absorbed, invert the plate and incubate it in a 37°C constant temperature incubator for about 16 hours.

[0049] Note: The formula of LB liquid culture medium is: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, and dilute to 1000mL with ddH2O; add 5mol / L NaOH (about 0.2mL) solution to adjust the pH value to 7.2. Among them, LB solid culture medium is added with agar powder: 15-20g / L. After the preparation is completed, the mixed system is placed in a high temperature of 121℃ and autoclaved for 30min. After sterilization, the system is cooled to about 50-60℃, and a 100mg / mL concentration of ampicillin solution is added at a volume ratio of 1:1000.

[0050] 5. Amplification of target plasmid: Observe the growth of colonies on the LB plate the day after plasmid transformation, take the coated plate with normal colony morphology and appropriate distribution density, and pick up the monoclonal colony with a sterile 10μL pipette tip; inject the monoclonal colony into 3mL LB liquid culture medium containing ampicillin with the pipette tip, and culture it on a shaker at 37°C and 220rpm for 6h; take the bacterial solution with appropriate turbidity, inoculate it into 100mL LB liquid culture medium containing ampicillin at a volume ratio of 1:200-500, and mix it to form a bacterial culture system; place the system in a shaker at 37°C and 220rpm and continue to culture for 12h (the remaining bacterial solution should be frozen in a -80°C refrigerator for seed preservation or sent for sequencing for sequence verification).

[0051] 6. Use QIAGEN's endotoxin-free plasmid extraction kit to extract the target plasmid.

[0052] (1) Preparation before extraction: Mix RNAase A solution at a final concentration of 100 μg / mL into Buffer P1 solution and store at 4°C; mix LyseBlue solution at a ratio of 1:1000 into Buffer P1 solution.

[0053] (2) Check the state of SDS in Buffer P2 to prevent crystallization. Preheat to 37°C if necessary; precool Buffer P3 solution at 4°C; prepare isopropanol reagent; add 40 mL of 96-100% ethanol solution to the bottle of endotoxin-free water provided in the kit; prepare the necessary experimental utensils free of endotoxins and pathogens.

[0054] (3) Centrifuge the amplified bacterial solution at 4°C, 6000g, and 15 min to obtain precipitated bacterial cells.

[0055] (4) Discard the supernatant, resuspend the cells in 10 mL of Buffer P1 and thoroughly vortex to mix the cells.

[0056] (5) Add 10 mL of Buffer P2 solution, invert 4 to 6 times to mix, and let stand at room temperature for 5 min.

[0057] (6) During the incubation process, screw the QIA filter cartridge cap onto the outlet nozzle of the QIA filter cartridge, and then gently insert the QIA filter cartridge into a sterile 50 mL centrifuge tube.

[0058] (7) Add 10 mL of Buffer P3 solution to the mixture in step (5). Immediately invert gently 4 to 6 times to mix thoroughly. Pour the mixture into the filter tube of the QIA filter and let it stand at room temperature for 10 min.

[0059] (8) Remove the cap from the QIA filter cartridge outlet nozzle, gently insert the plunger into the QIA filter cartridge, and squeeze the cell lysate into a 50 mL sterile centrifuge tube.

[0060] (9) Add 2.5 mL of Buffer ER to the lysate filtrate, invert the mixture about 10 times, and incubate on ice for 30 min.

[0061] (10) While the system is standing, place the QIAGEN-tip 500 column on the receiving tube and slowly add 10 mL of Buffer QBT solution on top of the column until the Buffer QBT solution soaks the entire resin column under the action of gravity and flows into the receiving tube to balance the QIAGEN-tip 500 column.

[0062] (11) After the incubation of the mixed system in step (9) is completed, it is transferred to a balanced QIAGEN-tip 500 column, and the mixed system is allowed to flow naturally through the column under the action of gravity, and the DNA component is purified into the resin column.

[0063] (12) Discard the column filtrate and add 30 mL of Buffer QC solution to the column. Allow it to flow through the column naturally under the action of gravity and rinse twice.

[0064] (13) Discard the column filtrate and add 15 mL of Buffer QN solution to the column to elute the DNA into a 50 mL sterile centrifuge tube at the bottom of the column to obtain a DNA suspension.

[0065] (14) Add 10.5 mL of isopropanol to the DNA suspension and mix quickly and thoroughly to precipitate the DNA.

[0066] (15) Centrifuge the system containing the target DNA at 4°C, 15,000 g, for 30 min, and gently remove the supernatant to obtain a DNA precipitate.

[0067] (16) Add 5 mL of endotoxin-free 70% ethanol to the DNA precipitate to elute the DNA. Centrifuge at 15,000 g for 10 min at 4 °C and gently aspirate the supernatant, avoiding touching the DNA precipitate.

[0068] (17) Place the DNA precipitate in an open centrifuge tube and allow it to dry naturally for 5–10 min. Wait for the remaining supernatant to evaporate naturally and dissolve the DNA precipitate with an appropriate volume of ddH2O.

[0069] (18) Use NanoDrop spectrophotometer to detect the purity of the extracted target plasmid and ensure OD 260 / 280 The value is between 1.8 and 2.0; at the same time, the concentration of the target plasmid is detected and recorded. According to subsequent experiments, the target plasmid solution is packaged and stored in a -20℃ refrigerator.

[0070] Example 3: TCR target plasmid transfection

[0071] 1. Use the "Sleeping Beauty" transposition system to electroporate the TCR target plasmid into peripheral blood lymphocytes (PBLs). Figure 1 This is a structural diagram of the "Sleeping Beauty" transposon system encoding the NY-ESO-1-specific TCR gene.

[0072] 1. Extraction of peripheral blood lymphocytes (PBLs)

[0073] (1) Peripheral blood of healthy volunteers in blood collection tubes was diluted with normal saline at a ratio of 1:1, and the diluted peripheral blood was transferred to a 50 mL centrifuge tube.

[0074] (2) Take another 50 mL centrifuge tube and add 10 mL of human lymphocyte separation solution (Ficoll solution) to each tube.

[0075] (3) Use a Pasteur pipette to draw the diluted peripheral blood and slowly add it to the Ficoll liquid surface along the wall of the centrifuge tube (do not break the liquid surface during the process) so that the volume ratio of Ficoll solution, peripheral blood and physiological saline in the final system is 1:1:1.

[0076] (4) Density gradient centrifugation was performed with the following parameters: three rises and three falls, 800 g, 25 min, and 20°C.

[0077] (5) After centrifugation, the mixed system in the centrifuge tube can be seen to be divided into layers (the upper layer is plasma, the lower layer is mainly red blood cells and granulocytes, the middle layer is Ficoll solution, and there is a white film layer mainly composed of PBLs at the interface between the upper and middle layers). Use a pipette to insert into the white film layer to absorb PBLs and add them to the serum-free and antibiotic-free X-VIVO 15 culture medium.

[0078] (6) Wash the PBLs by centrifugation at 350 g and 4 °C for 10 min with nine steps of rotation and nine steps of rotation. Discard the supernatant.

[0079] (7) Resuspend the PBL pellet in 10 mL of sterile red blood cell lysis buffer and let it stand for 2 to 3 minutes.

[0080] (8) Centrifuge the solution at 350 g and 4 °C for 3 min with a nine-fold increase and decrease, and discard the supernatant to obtain the PBLs precipitate.

[0081] (9) Resuspend the pellet in 10 mL of serum-free and antibiotic-free X-VIVO 15 medium and mix well.

[0082] (10) Centrifuge the solution at 350 g and 4 °C for 10 min with a nine-fold increase and decrease, discard the supernatant, and continue washing the PBLs.

[0083] (10) After washing twice, the PBLs precipitate was resuspended in complete culture medium (X-VIVO 15 + 10% FBS) to obtain a culture system containing PBLs.

[0084] 2. Activation of peripheral blood lymphocytes (PBLs)

[0085] (1) The culture system containing PBLs was transferred to a 6-well plate and incubated in a cell culture incubator at 37° C. and 5% CO 2 for 2 h until the monocytes adhered to the bottom wall of the 6-well plate.

[0086] (2) Aspirate the suspended cell suspension consisting mainly of T lymphocytes and centrifuge at 350 g for 10 min.

[0087] (3) Discard the supernatant, resuspend the T lymphocyte pellet, resuspend the PBLs pellet in complete culture medium (X-VIVO 15 + 10% FBS), and add 0.1 μg / mL OKT-3 monoclonal antibody and 300 U / mL recombinant human IL-2 to stimulate T lymphocyte proliferation and activation.

[0088] (4) Depending on the condition of the cells, consider replacing half of the medium or expanding the volume. After 48 to 72 hours of cytokine stimulation, activated T lymphocytes are obtained and await transfection.

[0089] 3. Gene transfection of T lymphocytes based on the “Sleeping Beauty” transposon system

[0090] (1) Observe the activation of T lymphocytes. Collect well-activated T lymphocytes into a sterile 15 mL centrifuge tube 48 hours or 72 hours after cytokine induction.

[0091] (2) Wash the cells with PBS solution preheated in a 37°C water bath, centrifuge slowly at 800 rpm for 5 min, and discard the supernatant (to avoid liquid residue that affects transfection efficiency).

[0092] (3) Prepare a blank 24-well plate, set up different groups, add 500 μL of X-VIVO 15 medium to each well of each group, and place it in a 37°C, 5% CO2 cell culture incubator for prewarming.

[0093] (4) Preheat the electroporation buffer and the "Sleeping Beauty" transposon system plasmid to room temperature, and then mix the two evenly.

[0094] (5) Resuspend the washed T lymphocytes with a transfection reagent containing the plasmid and transfer them to an electroporation cup (avoid the generation of bubbles in this step to reduce the transfection efficiency). Select an appropriate electroporation program for electroporation.

[0095] (6) After the electroporation is completed, 500 μL of preheated culture medium is immediately pipetted into the electroporation cup, and the electroporation cup is placed in a 37°C, 5% CO2 cell culture incubator for 5 to 10 minutes.

[0096] (7) After electroporation, the cells were aspirated into a 24-well plate containing pre-warmed X-VIVO 15 medium (this step requires gentle operation) and continued to be cultured in a cell culture incubator at 37°C and 5% CO2.

[0097] (8) 24 h after transfection, replace the culture medium with fresh complete medium (X-VIVO 15 + 10% FBS) to resuspend the cells, and add 100 U / mL recombinant human IL-2, 10 ng / mL recombinant human IL-7 and 10 ng / mL recombinant human IL-15 to amplify the transfected T lymphocytes.

[0098] (9) After 48 hours of transfection, cells from each group were aspirated for flow cytometry analysis after flow antibody / tetramer staining to verify the transfection efficiency.

[0099] (10) Observe the cell growth status and replenish fresh immune cell-specific complete culture medium approximately every 2 to 3 days, add cytokines, and expand the culture volume if necessary.

[0100] 2. Double staining of transfected PBLs with HLA-A*02:01NY-ESO-1Tetramer SLLMWITQC-PE (MBL, TB-M011-1) and CD8-PC7 (Biolegend) fluorescent antibodies.

[0101] 1. Prepare cell suspension: Set up a plasmid transfection group before modification (before modification) and a plasmid transfection group after modification (after modification), centrifuge each group of cells, resuspend the cells in PBS solution to a density of 10^7 / mL, and pipette each group of cells into a 1.5mL Eppendorf tube for staining.

[0102] 2. Flow cytometry antibody staining: Mix the antibody and cells with 10 μL PE-HLA-A*02:01SLLMWITQC Tetramer / 50 μL cell suspension, and incubate at room temperature for 20-30 min. After incubation, add APC-anti-human-CD8 antibody to each tube and incubate at 4°C for 30 min. Add PBS solution to wash non-specific staining, centrifuge at 300 g for 5 min, discard the supernatant, and keep the precipitate. Resuspend the cell precipitate with 200 μL PBS solution, put it on the flow cytometer, adjust the voltage and compensation parameters, and circle the positive cell population.

[0103] 3. Flow cytometry was used to analyze the ratio of double positive cells and compare the ratio of tetramer positive ("Tetramers(+)") cells in CD8 positive ("CD8(+)") cells before and after the modification. The results are as follows: Figure 2 shown.

[0104] from Figure 2 It can be seen that after plasmid modification, CD8 expressing NY-ESO-1TCR + The percentage of T lymphocytes increased from 1.67% to 17.0% ( Figure 2 A). Among them, in the modified group, CD8 + In T lymphocytes, the expression efficiency of NY-ESO-1 specific TCR can reach 26.5% ( Figure 2 B).

[0105] Example 4: Study on the specific IFN-γ secretion ability of NY-ESO-1TCR-T, the expression of immune activation molecules CD28 and CD137, and cytotoxicity

[0106] NY-ESO-1TCR-T was co-incubated with the melanoma cell line A375 (preserved by the laboratory of the Affiliated Cancer Center of the Medical School of Nanjing University) with high expression of NY-ESO-1 and the liver cancer cell line SK-HEP-1 (preserved by the laboratory of the Affiliated Cancer Center of the Medical School of Nanjing University) with low expression of NY-ESO-1 to study the specific IFN-γ secretion ability of NY-ESO-1TCR-T, the expression of immune activation molecules CD28 and CD137, and the cytotoxicity.

[0107] 1. Cytokine microsphere assay to evaluate IFN-γ secretion level of NY-ESO-1TCR-T

[0108] (1) Prepare standard samples with different concentration gradients: Use 4 mL of PBS solution to dissolve the IFN-γ standard in the CBA kit, blow the system thoroughly to make it uniform, and let it stand at room temperature for 15 min to equilibrate, and obtain the TOP concentration standard; then, mark the TOP, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, and 1 / 256 concentration gradient centrifuge tubes, add the TOP concentration standard solution to the TOP tube, and dilute the remaining concentration gradient tubes with PBS solution according to the proportional value, blow the diluted system thoroughly to make it uniform, and wait for detection.

[0109] (2) Preparation of samples to be tested: A375 cells and SK-HEP-1 cells in the logarithmic growth phase were collected and resuspended in complete tumor cell culture medium (1640 + 10% FBS) to 10 5 The cell suspension was prepared at a concentration of 10 / mL. Both cells were set up as untransfected PBLs group and NY-ESO-1TCR-T group. The cell suspension was inoculated in a round-bottom 96-well plate at 100 μL / well, and 3 or more duplicate wells were set up for each group. The number of untransfected T cells in the untransfected PBLs group and the number of NY-ESO-1TCR-T cells in the NY-ESO-1TCR-T group were counted, and the cells of the two groups were resuspended to 10 in complete culture medium (1640 + 10% FBS). 6 Each group of T cells was added to a round-bottom 96-well plate inoculated with tumor cells at a concentration of 100 μL / well. The final volume of each well was 200 μL. The co-culture system was placed in a 37°C, 5% CO2 cell culture incubator for 22 to 24 hours. After the incubation was completed, the samples to be tested were prepared, the supernatant of each well was aspirated, and the samples were tested.

[0110] (3) Preparation of suspension of cytokine detection microspheres and fluorescent antibodies: Take the microsphere solution and antibody solution in the CBA kit, gently oscillate for 30 seconds using a vortexer to resuspend the microspheres, then use PBS solution to dilute the microsphere and antibody solution 100 times at a volume ratio of 1:1, prepare an appropriate working concentration of microsphere-antibody suspension according to the number of samples to be tested, and oscillate the diluted system to mix well.

[0111] (4) Incubate the microsphere antibody suspension with the standard and the sample to be tested: Take 20-40 μL of the standard sample solution and the supernatant sample to be tested in each group, add the microsphere antibody suspension with the same volume as the sample to be tested (the microsphere antibody suspension should be fully shaken for 30 seconds before use), mix the mixture thoroughly, and incubate it at room temperature in the dark for 2-3 hours.

[0112] (5) Detection: After the incubation, wash each group of samples with 100 μL washing buffer, centrifuge at 300 g for 5 min, discard the supernatant, resuspend the sample with 200 μL PBS solution, and use flow cytometry to detect the fluorescence intensity of different IFN-γ concentration gradients and the supernatant samples of each group.

[0113] (6) Draw a standard curve corresponding to IFN-γ concentration and flow cytometry fluorescence intensity: use the fluorescence intensity as the ordinate and the concentration gradient of the IFN-γ standard sample as the abscissa, draw a curve using Excel software, and then perform fitting to obtain the standard curve.

[0114] (7) According to the standard curve, the IFN-γ concentration corresponding to the fluorescence intensity of the supernatant in each group is obtained.

[0115] The results are as follows Figure 3 As shown in the figure, by co-incubation with A375 cells that highly express NY-ESO-1 antigen, the IFN-γ secretion level of NY-ESO-1TCR-T was significantly higher than that of untransfected PBLs, and this difference was statistically significant (**, P = 0.0083); however, when NY-ESO-1TCR-T and untransfected PBLs were co-incubated with SK-HEP-1 cells that lowly express NY-ESO-1 antigen, there was no statistical difference in the IFN-γ secretion level between the two groups. The above results indicate that NY-ESO-1TCR-T can specifically recognize NY-ESO-1 antigen epitopes and exert NY-ESO-1 antigen-specific immune effects represented by IFN-γ secretion levels.

[0116] 2. Detection of the immune activation phenotype of NY-ESO-1TCR-T by flow cytometry antibody staining

[0117] (1) A375 cells and SK-HEP-1 cells in the logarithmic growth phase were collected and resuspended in complete tumor cell culture medium (1640 + 10% FBS) to 10 6 The two types of cells were set up as untransfected PBLs group and NY-ESO-1TCR-T group. The cell suspension was inoculated in a 24-well plate at 500 μL / well, and 3 or more duplicate wells were set up for each group.

[0118] (2) Count the number of untransfected T cells in the untransfected PBLs group and the number of NY-ESO-1TCR-T cells in the NY-ESO-1TCR-T group, and resuspend the cells in both groups to 10 μl in complete culture medium (1640 + 10% FBS). 6 / mL cell density, each group of T cells was added to the 24-well plate inoculated with tumor cells at 500 μL / well, and the final volume of each well was 1 mL.

[0119] (3) The co-culture system was placed in a 37°C, 5% CO2 cell culture incubator and incubated for 24 h.

[0120] (4) After incubation, the lymphocytes in the 24-well plate were transferred to a flow cytometry tube and labeled with fluorescent antibodies APC-anti-human CD137 and PE-anti-human CD28 targeting the immunoreactivity phenotype of T cells. A negative control (Blank) group and a single-stained flow cytometry antibody group were set up and incubated at 4°C in the dark for 30 min.

[0121] (5) After staining, add 1 mL of PBS solution to each tube to resuspend and wash the cells, centrifuge at 300 g for 5 min, discard the supernatant, resuspend the cell pellet with 200 μL of PBS solution, and detect the lymphocyte phenotype by flow cytometry.

[0122] The results are as follows Figure 4 As shown, when the SK-HEP-1 cell line with low expression of NY-ESO-1 antigen was co-cultured and stimulated, the CD28 molecule on NY-ESO-1TCR-T ( Figure 4 A&B) and CD137 molecules ( Figure 4 The expression of C&D in the NY-ESO-1TCR-T cells was not significantly different from that in the untransfected PBLs group. Only when the A375 cell line with high expression of NY-ESO-1 antigen was co-cultured and stimulated, the CD28 molecule ( Figure 4 A&B) and CD137 molecules ( Figure 4 The expression of C&D in the PBLs group was significantly higher than that in the untransfected PBLs group. The above results show that NY-ESO-1TCR-T has NY-ESO-1 antigen-specific immune response ability.

[0123] 3. Evaluation of the killing function of NY-ESO-1TCR-T in vitro

[0124] (1) Target cell preparation: Human solid tumor cell lines with HLA-A*02:01 typing, including human melanoma cells A375 with high expression of NY-ESO-1 antigen and human liver cancer cells SK-HEP-1 with low expression of NY-ESO-1 antigen, were used as target cells. When the cells grew to the logarithmic growth phase, the cells were digested with trypsin and collected in a centrifuge tube. The cells were centrifuged at 1000 rpm for 5 min to obtain a cell pellet. The cells were washed once with saline. After counting the cells, the cells were resuspended in saline to a final concentration of 10 610 μM / mL, add CFSE dye solution with a working concentration of 4 μM and mix with tumor cells, place in a 37°C, 5% CO2 cell culture incubator in the dark for 10 min; after the incubation, use 5 times the volume of normal saline to terminate the staining reaction, centrifuge at 1000 rpm for 5 min, discard the supernatant, keep the cell pellet, wash the cells twice with normal saline; resuspend the cells with complete culture medium (1640+10% FBS) to a final concentration of 10 6 The cell suspension was added into the flow tube at 100 μL / well and placed in a cell culture incubator at 37°C and 5% CO2 for later use.

[0125] (2) Effector cell preparation: Set up an untransfected PBLs group and a NY-ESO-1TCR-T group, with 3 or more replicate wells in each group; resuspend each group of T cells in complete culture medium (1640 + 10% FBS) to a final concentration of 4×10 7 The cells were diluted in different tubes according to the predetermined effector-target ratio (1:1, 5:1, 10:1, 20:1, 30:1, 40:1), and the effector cells and CFSE-labeled target cells were mixed in different flow cytometry tubes with 100 μL of cell suspension per well and incubated in a cell culture incubator at 37°C and 5% CO2 for 6 h.

[0126] (3) PI staining: The incubated cells were collected by centrifugation at 300 g for 5 min. The cells of each group were resuspended in 100 μL of normal saline, and PI was added to a final concentration of 50 μg / mL. The cells were incubated at 4°C in the dark for 15 min. The stained cells were collected by centrifugation, washed once, and resuspended in 200 μL of normal saline. The results were analyzed by flow cytometry and the killing graph was drawn.

[0127] The results are as follows Figure 5 As shown in the figure, when the effect-target ratio was 5, the killing ratio of NY-ESO-1TCR-T against A375 cell line began to be significantly higher than that of untransfected PBLs, and this difference became more and more significant as the effect-target ratio increased, indicating that compared with untransfected PBLs, NY-ESO-1TCR-T has the ability to kill tumor cells in vitro. When the effect-target ratio was 30 and 40, the killing ratio of NY-ESO-1TCR-T against A375 cell line began to be significantly higher than that against SK-HEP-1 cell line. The above results show that NY-ESO-1TCR-T can specifically kill tumor cells with high expression of NY-ESO-1 antigen in vitro, and the killing effect increases with the increase of the effect-target ratio.

[0128] Example 5: In vivo anti-tumor effect and biosafety evaluation of NY-ESO-1TCR-T

[0129] The NY-ESO-1TCR-T cells were infused back into the nude mouse model of melanoma A375 subcutaneous tumors by tail vein to determine the in vivo anti-tumor effect and biosafety of NY-ESO-1TCR-T cells. The specific steps are as follows:

[0130] (1) Model construction: The cell suspension of human melanoma cell line A375 cultured to the logarithmic growth phase was collected by trypsin digestion, centrifuged at 1000 rpm for 5 min at 4°C, and the supernatant was discarded. The cells were washed three times with PBS solution and centrifuged again to obtain a cell pellet. The cells were counted and A375 cells were resuspended to 5×10 7 A sufficient amount of A375 cells was placed on ice for subcutaneous tumor inoculation at a cell density of 10 / mL. An appropriate number of 4-6 week-old, 15-20 g SPF-grade BALB / cnude male mice were selected as tumor carriers. 10 / mL of each mouse was injected with a 1 mL sterile syringe. 7 The A375 tumor cells were inoculated in the subcutaneous area of ​​the upper right groin of the nude mice (the needle was gently inserted into the dorsal skin of the mouse to reach the subcutaneous pocket, that is, the area where there is no resistance when pushing the syringe, avoiding penetrating into the underlying muscle layer). The skin of the nude mice was disinfected with iodine before and after inoculation. After the tumor cell inoculation, the survival and tumor growth of the nude mice were monitored every day, and the maximum long diameter (L) and the maximum transverse diameter (W) perpendicular to the long diameter of the tumor were measured. The tumor volume change was calculated and recorded according to "tumor volume (V) = (L×W×W) / 2". When the tumor volume reached 60-70mm 3 Follow-up experiments were performed around 2 pm.

[0131] (2) Experimental groups: The tumor-bearing mice were evenly divided into a PBS group, a non-transfected PBLs group, and a NY-ESO-1TCR-T group.

[0132] (3) Cell preparation: Each tumor-bearing mouse was infused with 10 7 Cells were collected and resuspended in 10 8 The cells were placed in an ice box and waited for reinfusion.

[0133] (4) Treatment mode: Each group of cells was gently vortexed and resuspended, and 100 μL of each group of cells / mouse was infused into the tail vein. The first treatment date was recorded as Day 0, and then the drug was administered once every 8 days for a total of 3 treatments. On the second day after each cell treatment, 40,000 U / mL recombinant human IL-2 was injected intraperitoneally for a total of 3 days.

[0134] (5) Data recording: Starting from Day 0, the survival of nude mice and the growth of tumors were monitored every 2 days using a vernier caliper. The maximum long diameter (L) and the maximum transverse diameter (W) perpendicular to the long diameter of the tumor were measured. The changes in tumor volume were calculated and recorded according to "tumor volume (V) = (L × W × W) / 2". The weight of tumor-bearing mice was weighed and recorded at the same time as the tumor diameter was measured.

[0135] (6) H&E staining: After the treatment of each group of mice, 3 mice / group were selected, and the heart, liver, spleen, lung, and kidney tissues of the mice were fixed in 4% paraformaldehyde. The hematoxylin-eosin staining (H&E staining) method was used to observe the changes in tissue morphology of multiple tissues and to evaluate the in vivo safety of the treatment model. The steps of H&E staining are as follows:

[0136] A. Tissue fixation: Use 4% paraformaldehyde to fix the tissue. Soak the tissue in enough 4% paraformaldehyde and fix it at 4°C for 24 to 48 hours. Then discard the fixative and wash it with PBS solution for 3 times, 3 minutes each time.

[0137] B. Tissue gradient dehydration: Tissue gradient dehydration was performed by soaking in 70% ethanol for 20 min, 80% ethanol for 1 h, 90% ethanol for 1 h, 95% ethanol (first time) for 2 h, 95% ethanol (second time) for 2 h, anhydrous ethanol (first time) for 15 min, anhydrous ethanol (second time) for 15 min, anhydrous ethanol (third time) for 15 min, a 1:1 mixture of xylene and ethanol for 15 min, xylene (first time) for 25 min, xylene (second time) for 25 min, and xylene (third time) for 15 min.

[0138] C. Paraffin-embedded tissue: Embed tissue in paraffin at 60°C for 1 hour each time, for a total of 3 times. After the paraffin solidifies and embeds, store the tissue at 4°C.

[0139] D. Paraffin tissue sectioning: Take out the paraffin-embedded tissue and slice it with a blade (be careful to use a sharp blade and try to avoid wrinkles and knife marks when slicing). Then spread the thin slices in a 50°C water bath for about 1 minute (adding an appropriate amount of ethanol to the toe slices can avoid failure in spreading). After the slices are fully spread, lay the slide flat and stick the slices in the center.

[0140] E. Baking: Baking at 65℃ for 30min;

[0141] F. Dewaxing and hydration: Dewaxing was performed in the following order: xylene (first time) soaking for 5 min, xylene (second time) soaking for 5 min, anhydrous ethanol (first time) soaking for 2 min, anhydrous ethanol (second time) soaking for 2 min, 95% ethanol (first time) soaking for 1 min, 95% ethanol (second time) soaking for 1 min, 80% ethanol soaking for 1 min, and then the sections on the slides were rinsed with naturally running water for 30 min;

[0142] G. Removal of endogenous enzyme activity: Soak tissue sections in 3% H2O2 for 15 minutes to remove endogenous peroxidase activity, and then rinse with natural running water for 30 minutes;

[0143] HH&E staining: Place the tissue sections in hematoxylin staining solution for 5 minutes, then rinse the tissue sections with natural running water for 3 minutes, differentiate with 1% hydrochloric acid ethanol for 30 seconds, rinse the tissue sections with natural running water for 1 minute, soak the tissue sections in dilute ammonia water for a few seconds to turn blue, rinse with natural running water for 1 minute, then place the tissue sections in eosin staining solution for 2 minutes, and rinse with natural running water for 3 minutes;

[0144] I. Dehydration and permeabilization of tissue sections: Dehydrate and transparentize the sections by soaking in 80% ethanol for 1 min, 95% ethanol (first time) for 2 min, 95% ethanol (second time) for 2 min, anhydrous ethanol (first time) for 3 min, anhydrous ethanol (second time) for 3 min, 3:1 xylene carbolic acid for 1 min, xylene (first time) for 5 min, and xylene (second time) for 5 min.

[0145] J. Covering microscopy: Remove the slides from xylene, wait for the tissue sections to dry, then drop neutral resin on top of the sections, gently place a cover slip and press lightly to expel bubbles, air-dry the sections and store at room temperature or directly observe and collect images under an optical microscope.

[0146] The experimental results of evaluating the anti-tumor effect of NY-ESO-1TCR-T in vivo are as follows Figure 6 As shown in the figure, it can be seen that the tumor growth of mice in the NY-ESO-1TCR-T group was the slowest ( Figure 6 A in Figure 3 ); At the end point of cell reinfusion therapy, the tumor volume and weight of mice in the NY-ESO-1TCR-T group were significantly smaller than those in the non-transfected PBLs group (*, P = 0.0221; *, P = 0.0130) and the blank control group infused with PBS (**, P = 0.0048; *, P = 0.0198), indicating that NY-ESO-1TCR-T exerts an in vivo anti-tumor effect against tumor tissues that highly express NY-ESO-1 antigens, which is consistent with the results of the in vitro cell killing experiment ( Figure 6 B & C in.

[0147] The experimental results of biosafety evaluation are as follows: Figure 7 As shown in the figure, it can be seen that there is no significant difference in the weight changes of mice in each group during the cell reinfusion treatment ( Figure 7 A in the figure); at the end of cell reinfusion treatment, no obvious lesions were found in the important organs of mice in each group ( Figure 7 The above results indicate that the NY-ESO-1TCR-T prepared by plasmid modification is relatively safe for use in mice bearing A375 subcutaneous tumors.

[0148] The present invention provides a NY-ESO-1 specific TCR-T receptor and its application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A NY-ESO-1 specific TCR-T receptor, characterized in that: The NY-ESO-1 specific TCR-T receptor, whose genome includes the α chain constant region gene and β chain constant region gene of the murinized human T cell receptor α95:LY; Among them, the amino acid sequences of the α chain constant region gene and the β chain constant region gene of the murinized human T cell receptor α95:LY are shown in SEQ ID NO.8 and SEQ ID NO.10, respectively.

2. The NY-ESO-1 specific TCR-T receptor according to claim 1, characterized in that The genome of the NY-ESO-1 specific TCR-T receptor includes a human variable region of the α chain and a human variable region of the β chain.

3. The NY-ESO-1 specific TCR-T receptor according to claim 2, characterized in that The human variable region of the α chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO: 11, CDR2 with an amino acid sequence as shown in SEQ ID NO: 12, and CDR3 with an amino acid sequence as shown in SEQ ID NO: 13; the human variable region of the β chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO: 14, CDR2 with an amino acid sequence as shown in SEQ ID NO: 15, and CDR3 with an amino acid sequence as shown in SEQ ID NO:

16.

4. The NY-ESO-1 specific TCR-T receptor according to claim 1, characterized in that The NY-ESO-1 specific TCR-T receptor comprises an α chain as shown in the amino acid sequence of SEQ ID NO: 4, and a β chain as shown in the amino acid sequence of SEQ ID NO:

6.

5. The NY-ESO-1 specific TCR-T receptor according to claim 4, characterized in that The α chain and the β chain are connected by a P2A sequence, wherein the P2A sequence is: ATNFSLLKQAGDVEENPGP.

6. A nucleic acid molecule encoding the NY-ESO-1 specific TCR-T receptor according to any one of claims 1 to 5.

7. An expression vector comprising the nucleic acid molecule of claim 6.

8. A recombinant cell comprising the nucleic acid molecule of claim 6 or the expression vector of claim 7.

9. A pharmaceutical composition comprising the nucleic acid molecule of claim 6, the expression vector of claim 7, or the recombinant cell of claim 8.

10. Use of the NY-ESO-1 specific TCR-T receptor according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the recombinant cell according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of a drug for detecting, treating or preventing a disease.

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