MSLN CAR-T cell for expressing TRAIL protein and application of MSLN CAR-T cell

Abstract

The invention discloses an MSLN CAR-T cell for expressing TRAIL protein and application of the MSLN CAR-T cell, and relates to the technical field of biological medicine. Spatial epitopes of tumor antigens serve as target spots, anti-tumor factors TRAIL are tried to be combined for design, the optimal combination mode of the tumor antigens and the TRAIL is searched for to play the anti-tumor role to the maximum extent, and finally it is found that when mesothelin (MSLN) serves as the target spot for designing CAR molecules, an MSLN antibody scfv region, a transmembrane region and an intracellular signal region are connected in series, and structural optimization and transformation are conducted on the MSLN antibody scfv region, the transmembrane region and the intracellular signal region; after a TRAIL coding sequence is connected to an intracellular signal region, an MSLN CAR molecule for expressing TRAIL protein is obtained, and a novel CAR-T cell therapy is developed. In-vitro and in-vivo experiments show that the treatment effect of the MSLN-TRAIL CAR-T is superior to that of second-generation MSLN CAR-T and other combination modes, the molecule can enable MSLN and TRAIL to have a synergistic effect, the immune effect of the CAR molecule is greatly improved, and an effective treatment strategy is provided for tumor patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly to an MSLN CAR-T cell expressing TRAIL protein and its application. Background Art

[0002] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily. Because it can selectively induce apoptosis of tumor cells without damaging normal cells, it has attracted wide attention in the field of cancer treatment. Different from other TNF family members such as FasL and TNF-α, TRAIL can specifically induce apoptosis of tumor cells without harming normal cells, making it an extremely attractive candidate drug for cancer treatment. TRAIL exerts its function by binding to its receptors. Currently, five TRAIL receptors are known, including two death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2), and three decoy receptors DcR1 (TRAIL-R3), DcR2 (TRAIL-R4), and soluble receptor OPG. DR4 and DR5 bind to TRAIL and activate downstream signaling pathways to induce apoptosis of tumor cells. This process begins with the formation of the death-inducing signaling complex (DISC), which consists of FADD (Fas-associated death domain protein) and procaspase 8 / 10. In the DISC, procaspase 8 / 10 undergoes autocatalytic cleavage and is activated, and then activates caspase 3, leading to apoptosis. In addition, activated caspase8 / 10 can also promote the activation of the mitochondrial apoptosis pathway by cleaving BID (Bcl-2 interacting protein), thereby enhancing the apoptosis effect. All five receptors of TRAIL have significant homology in their extracellular ligand-binding domains. The intracellular fragments of TRAIL-R1 and TRAIL-R2 contain conserved functional domains, the so-called "death domains", which are responsible for transducing apoptosis signals. TRAIL plays an important role in the immune surveillance of tumor cells.

[0003] Chimeric antigen receptor T (CAR-T) cell immunotherapy, as a type of cell immunotherapy, has achieved remarkable results in various tumors. Up to now, a variety of CAR-T cell therapies have been approved. However, CAR-T faces many challenges in the treatment of solid tumors, such as the immunosuppressive tumor microenvironment. Therefore, how to enhance the therapeutic effect of CAR-T in solid tumors is an important problem that urgently needs to be solved. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a mesothelin (MSLN)-targeted chimeric antigen receptor (CAR) expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. The CAR-T cells prepared based on this CAR molecule can target and kill tumor cells. In particular, constructing the gene sequence expressing TRAIL protein onto the CAR vector can enable CAR-T cells to highly express TRAIL protein, significantly enhancing the anti-tumor effect of CAR-T cells.

[0005] The first object of the present invention is to provide a chimeric antigen receptor, which contains an MSLN-targeting structure and a tumor necrosis factor-related apoptosis-inducing ligand coding region connected in sequence; wherein:

[0006] The MSLN-targeting structure contains a chimeric antigen receptor targeting MSLN, and the chimeric antigen receptor targeting MSLN contains an scfv region of an MSLN antibody, a hinge region, a transmembrane region, and an intracellular signaling region.

[0007] The tumor necrosis factor-related apoptosis-inducing ligand coding region contains a tumor necrosis factor-related apoptosis-inducing ligand coding sequence, and the tumor necrosis factor-related apoptosis-inducing ligand coding region is located after the intracellular signaling region.

[0008] Further, the intracellular signaling region and the tumor necrosis factor-related apoptosis-inducing ligand coding region are connected by a self-cleaving peptide.

[0009] Further, the self-cleaving peptide includes 2A peptides, such as P2A peptide, T2A peptide, etc., and most preferably P2A peptide (SEQ ID NO.8).

[0010] Further, the amino acid sequence of the tumor necrosis factor-related apoptosis-inducing ligand coding sequence is as shown in SEQ ID NO.1, and the nucleotide sequence is as shown in SEQ ID NO.2.

[0011] Further, the amino acid sequence of the scfv region of the MSLN antibody is as shown in SEQ ID NO.3.

[0012] Further, the hinge region includes a CD8 hinge region, and the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO.4.

[0013] Further, the transmembrane region includes a CD8 transmembrane region, and the amino acid sequence of the CD8 transmembrane region is as shown in SEQ ID NO.5.

[0014] Further, the intracellular signaling region contains a co-stimulatory molecule region and / or a signal transduction molecule region; the co-stimulatory molecule region includes a CD137 intracellular signaling region, and the signal transduction molecule region includes a CD3zeta intracellular signaling region.

[0015] Furthermore, the amino acid sequence of the intracellular signaling region of CD137 is as shown in SEQ ID NO.6, and the amino acid sequence of the intracellular signaling region of CD3zeta is as shown in SEQ ID NO.7.

[0016] Furthermore, according to actual needs, an extracellular signal peptide region can be designed in front of the scfv region of the MSLN antibody of the chimeric antigen receptor. Preferably, the signal peptide in the extracellular signal peptide region can be the CD8a signal peptide.

[0017] Furthermore, the scfv region of the MSLN antibody is located before the hinge region, the transmembrane region and the intracellular signaling region are located after the hinge region, and the intracellular signaling region is located at the end of the transmembrane region far from the hinge region.

[0018] The second object of the present invention is to provide a nucleic acid molecule encoding the chimeric antigen receptor.

[0019] The third object of the present invention is to provide an expression vector containing the nucleic acid molecule.

[0020] The fourth object of the present invention is to provide a recombinant cell containing the chimeric antigen receptor.

[0021] Furthermore, the starting cells of the recombinant cells include but are not limited to immune cells, such as T cells, chimeric antigen receptor (CAR)-T cells, T cell receptor (TCR)-T cells, tumor infiltrating lymphocytes (TIL), NK cells, NK-T cells, CAR-NK cells, CAR-NKT cells, dendritic cells, macrophages, CAR-macrophages or any genetically modified tumor-specific immune cells.

[0022] The fifth object of the present invention is to provide a CAR-T cell containing the nucleic acid molecule or the expression vector.

[0023] The sixth object of the present invention is to provide the use of the recombinant cell or the CAR-T cell in the preparation of an anti-tumor drug.

[0024] Furthermore, the anti-tumor drug is used for preventing or treating solid tumors, and the solid tumors include but are not limited to breast cancer, pancreatic cancer, ovarian cancer, cholangiocarcinoma, colorectal cancer, lung cancer, etc.

[0025] The seventh object of the present invention is to provide an anti-tumor drug containing the chimeric antigen receptor, nucleic acid molecule, expression vector, recombinant cell or CAR-T cell.

[0026] By means of the above solution, the present invention has at least the following advantages:

[0027] The present invention innovatively constructs the gene expressing TRAIL protein into the CAR structure, enabling CAR-T cells to highly express TRAIL protein while expressing CAR, significantly enhancing the tumor killing ability of CAR-T cells. Our experimental results show that when the CAR structure is a structure targeting the MSLN target, MSLN CAR-T expressing TRAIL protein has very strong cytotoxicity and specificity both in vitro and in vivo. The synergistic effect of the two exerts excellent anti-tumor effects that cannot be achieved by other structures, and the effect in in vivo experiments is significantly better than that of the second-generation MSLN CAR-T. At the same time, the present invention optimizes the combination mode of TRAIL and MSLN CAR, and its therapeutic effect is significantly better than the combination mode of separately administering MSLN CAR-T and TRAIL, and also better than other drug combination modes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the content of the present invention easier to be clearly understood, the present invention will be further described in detail below according to specific embodiments of the present invention in conjunction with the drawings.

[0029] Figure 1 is a schematic diagram of the chimeric antigen receptor MSLN-TRAIL CAR in the present invention;

[0030] Figure 2 is a comparison result diagram of the killing ability of MSLN CAR-T cells expressing TRAIL protein against target cells;

[0031] Figure 3 is a result diagram of the ability of MSLN CAR-T cells expressing TRAIL protein to secrete IFN-γ;

[0032] Figure 4 is a comparison result diagram of the in vivo drug efficacy of MSLN-CAR-T cells expressing TRAIL protein. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present invention will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0034] Example 1 Construction of Lentiviral Expression Vector

[0035] The nucleic acid sequence encoding TRAIL protein is linked to the back of CD3ζ of the MSLN-CAR structure through P2A peptide and subcloned into the PLVX-EF1a vector (purchased from clontech). The combination order of each element in the constructed chimeric antigen receptor lentiviral expression vector is as Figure 1 shown:

[0036] The amino acid sequence of the TRAIL protein is as follows (SEQ ID NO.1):

[0037] MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG

[0038] The nucleic acid sequence of the TRAIL protein is as follows (SEQ ID NO.2):

[0039] ATGGCTATGA TGGAAGTGCA GGGGGGGCCA AGCCTCGGCC

[0040] AGACATGTGT TCTGATAGTCATCTTCACCG

[0041] TGCTGTTGCA GTCACTTTGC GTGGCCGTGA CCTATGTGTA

[0042] CTTTACCAAT GAGCTGAAGC AGATGCAGGA

[0043] TAAGTACTCC AAGTCTGGTA TTGCTTGCTT TCTCAAGGAA

[0044] GACGATAGTTATTGGGATCC TAACGACGAA

[0045] GAGAGCATGA ATTCCCCGTG TTGGCAAGTG AAGTGGCAAC

[0046] TGCGCCAGCT CGTTCGGAAAATGATCCTGA

[0047] GAACCAGCGA AGAGACTATT AGCACAGTGC AGGAAAAACA

[0048] GCAGAATATC TCTCCTCTGG TGAGGGAAAG

[0049] AGGACCACAG AGAGTGGCTG CACATATTAC GGGAACAAGA

[0050] GGCCGCTCTAACACCCTTAG CTCTCCTAAC

[0051] TCAAAAAACG AAAAGGCTCT GGGTCGCAAA ATTAACAGCT

[0052] GGGAAAGTTC TCGCTCAGGC CATTCCTTCT

[0053] TGTCCAACCT TCATCTGCGG AATGGTGAAC TCGTAATCCA

[0054] CGAAAAGGGT TTCTATTATATATATAGCCA

[0055] GACCTACTTC AGATTTCAGG AGGAGATCAA AGAAAATACT

[0056] AAGAACGACAAACAGATGGT CCAATACATC

[0057] TACAAGTACA CCAGCTATCC GGATCCCATT CTCCTCATGA

[0058] AATCCGCACG CAACTCCTGC TGGAGCAAAG

[0059] ATGCAGAATA TGGCCTTTAT TCAATCTATC AGGGCGGGAT

[0060] TTTTGAGCTGAAAGAGAACGACAGAATTTT

[0061] TGTATCCGTC ACTAATGAGC ATCTGATTGA TATGGACCAC

[0062] GAAGCATCCT TTTTTGGAGC CTTCTTGGTG

[0063] GGTTGA

[0064] The amino acid sequence of the MSLN antibody (Anti-MSLN ScFv) is as follows (SEQ ID NO.3):

[0065] MALPVTALLLPLALLLHAARPQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVL

[0066] The amino acid sequence of the CD8 hinge region (CD8Hing) is as follows (SEQ ID NO.4):

[0067] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0068] The amino acid sequence of the CD8 transmembrane region (CD8TM) is as follows (SEQ ID NO.5)

[0069] IYIWAPLAGTCGVLLLSLVITLYC

[0070] The amino acid sequence of the CD137 (4-1BB) intracellular domain is as follows (SEQ ID NO.6):

[0071] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEGGCEL

[0072] The amino acid sequence of the CD3Zeta is as follows (SEQ ID NO.7):

[0073] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0074] The amino acid sequence of the P2A peptide is as follows (SEQ ID NO.8):

[0075] ATNFSLLKQAGDVEENPGP。

[0076] Example 2 Lentivirus Preparation

[0077] The specific experimental steps are as follows:

[0078] S1. Prepare a 15-cm dish, inoculate 5×10 6 293T cells (purchased from ATCC), add complete medium (DMEM high glucose, 10% FBS, double antibiotics), and place it in a 37°C, 5% CO2 incubator for overnight culture.

[0079] S2. Take out 100 μM PEI and lentivirus packaging plasmids (PLVX-EF1a-CAR, pGP, pVSVG) from the refrigerator. After thawing at room temperature, mix them thoroughly by pipetting up and down. Take out PBS or HBSS buffer and warm it to room temperature. Take 2 mL of PBS into one well of a 6-well plate, add 10 μg of PLVX-EF1a-CAR, 4 μg of pGP, and 2 μg of pVSVG respectively. After mixing thoroughly by pipetting up and down, add 18 μL of 100 μM PEI, and immediately mix by pipetting up and down. Let it stand at room temperature for 10 minutes.

[0080] S3. Dropwise add the above DNA / PEI complex into the 15-cm culture dish, gently shake the culture dish to mix thoroughly. Place the culture dish in a 37°C, 5% CO2 incubator. After culturing for 6 - 8 hours, remove the medium containing the transfection reagent and replace it with fresh complete medium.

[0081] After continuous culture for 48 hours, collect the supernatant of the medium containing the virus in the culture dish, filter it through a 0.45-μm filter membrane, add a 20% volume of 50% PEG6000 solution, incubate at 4°C for 2 h, transfer it to a centrifuge tube, balance it, and centrifuge at 3000 xg at 4°C for 0.5 hour. After centrifugation, in a biosafety cabinet, carefully aspirate the liquid in the centrifuge tube, add 500 μL of PBS buffer to resuspend the precipitate, and store the virus at -80°C.

[0082] Example 3 Isolation of Primary T Cells

[0083] The specific experimental steps are as follows:

[0084] S1. Invert the lymphocyte separation solution several times up and down to thoroughly mix the Lymphoprep reagent.

[0085] S2. In a biosafety cabinet, add 15 mL of Lymphoprep reagent to a 50-mL centrifuge tube (or a 15-mL centrifuge tube, depending on the volume of the separated blood sample) for standby.

[0086] S3. Dilute the blood sample with an equal volume of PBS + 2% FBS.

[0087] S4. Carefully add the diluted blood sample dropwise along the tube wall to the upper layer of the separation reagent using a pipette, avoiding mixing of the separation reagent and the blood sample.

[0088] S5. Set the centrifuge to 800 x g, with the slowest speed of descent, and centrifuge at room temperature for 20 minutes.

[0089] S6. After centrifugation, collect the upper light yellow serum into another sterile centrifuge tube and store it at -80 °C.

[0090] S7. Gently aspirate the monocyte layer at the serum-separation reagent interface into a new centrifuge tube and wash the cells once with the medium.

[0091] S8. Adjust the cell density to 1×10 8 cells / mL (total volume not exceeding 2.5 mL), and resuspend in a 5 mL round-bottom tube.

[0092] S9. Add 100 μl / mL of antibody cocktail, mix well, and incubate at room temperature for 15 minutes.

[0093] S10. Take out the magnetic beads and pipette up and down at least 5 times to mix well.

[0094] S11. Aspirate 50 μl of magnetic beads / mL into the above sample, mix well, and incubate at room temperature for 10 minutes.

[0095] S12. Add complete medium to a total volume of 2.5 mL in the tube, insert the tube (with the cap open) into the magnetic pole, and let it stand at room temperature for 5 minutes.

[0096] S13. After incubation, keep the tube in the magnetic pole, gently invert it, and pour out the cells in the tube.

[0097] S14. Resuspend the cells in X-vivo 15 medium and add 10% FBS, 300 U / mL IL-2, 5 ng / mL IL-15, and 10 ng / mL IL-7.

[0098] Example 4 Activation and Lentiviral Infection of Primary T Cells

[0099] The specific experimental steps are as follows:

[0100] S1. Adjust the cell density to 1×10 6Cells / mL, add cytokine and antibody complex (IL-2 at a final concentration of 300 U / mL, IL-7 at 10 ng / mL, IL-15 at 5 ng / mL, Anti-CD3 (OKT3) at 500 ng / mL, Anti-CD28 at 2 μg / mL), and culture continuously for 48 hours.

[0101] S2. Calculate the required amount of virus according to MOI = 20. The calculation formula is as follows: Required amount of virus (mL) = (MOI * number of cells) / virus titer.

[0102] S3. After taking out the virus from the -80 °C refrigerator, quickly melt it in a 37 °C water bath. Add the calculated amount of virus obtained above to a six-well plate, add polybrene at a final concentration of 6 μg / mL, mix well, seal the four sides of the six-well plate with sealing film, and centrifuge at 800 x g for 1 hour.

[0103] S4. After centrifugation, tear off the sealing film, place the six-well plate in an incubator at 37 °C and 5% CO2, and continue to culture for 24 hours.

[0104] S5. Centrifuge at 250 x g for 10 minutes, discard the culture medium supernatant containing the virus, resuspend the cell pellet with fresh culture medium, transfer the cells to a new six-well plate, and continue to culture for 3 - 6 days for standby.

[0105] Example 5 Lysis of target cells by CAR-T cells

[0106] Real Time Cellular Analysis (RTCA) is a cell impedance detection sensing system that integrates a microelectronic cell sensor chip into the bottom of a cell detection plate through a special process to construct a real-time, dynamic, and quantitative tracking system for changes in cell morphology, proliferation, and differentiation. When cells growing adherently on the surface of the microelectrode cause a change in the impedance of the adherent electrode interface, this change is correlated with the real-time functional state change of the cells. Biological information related to cell physiological functions, including cell growth, spreading, morphological changes, death, and adhesion, can be obtained through real-time dynamic electrode impedance detection. This method can be used to detect the killing of target cells by CAR-T cells in real time.

[0107] The specific experimental steps are as follows:

[0108] S1. Take out the ovarian cancer cells from the incubator, discard the supernatant, add PBS to wash once, and discard the PBS.

[0109] S2. Add 0.25% trypsin for digestion. When the cells are basically detached, add complete culture medium to terminate the digestion, transfer to a centrifuge tube and mix well. Take 20 μL of the cell suspension and mix it with 20 μL of AO / PI staining solution for counting.

[0110] S3. Take the required amount of cell suspension, centrifuge at 500 g for 5 min, discard the supernatant, and resuspend and mix well with the medium.

[0111] S4. Take out the 96W20idf electrode plate, add 100 μL of target cell suspension (40,000 target cells) to each well.

[0112] S5. After adding the cells, place the electrode plate in the laminar flow hood and let it stand for 15 min.

[0113] S6. Place the electrode plate in the Station and fasten it. Click Check to check if the electrode plate is well connected to the workstation, and then click Start to start data acquisition.

[0114] S7. After adding the cells, place the electrode plate in the laminar flow hood and let it stand for 15 min.

[0115] S8. After about 24 - 36 h of data acquisition (determined according to the experimental requirements), the target cells are in a stable cell growth plateau phase.

[0116] S9. Resuspend and count the CAR-T cells or T cells. Take 20 μL of cell suspension and mix it with 20 μL of AO / PI staining solution for counting. Take the required amount of cell suspension, centrifuge at 500 g for 5 min, discard the supernatant, and resuspend with the medium to adjust the corresponding concentration.

[0117] S10. Click Pause, take out the electrode plate, aspirate the medium, add the CAR-T cells or T cell suspension according to the effector-to-target ratio, and supplement the corresponding medium to make the final volume in each well the same. Put the electrode plate back into the workstation, click Check, and then click Resume.

[0118] S11. After continuing data acquisition for 48 h (determined according to the experimental requirements), click Finish to end the experiment.

[0119] Experimental groups:

[0120] (1) Tumor cell group: No effector cells were added.

[0121] (2) T cell group: Primary T cells were used as effector cells, and effector cells were added at an effector-to-target ratio of 1:1.

[0122] (3) MSLN CAR-T group: MSLN CAR-T cells were used as effector cells, and effector cells were added at an effector-to-target ratio of 1:1.

[0123] (4) MSLN CAR-T + TRAIL group: The prepared MSLN-TRAIL CAR-T cells were used as effector cells, and effector cells were added at an effector-to-target ratio of 1:1.

[0124] Among them,

[0125] The preparation of MSLN CAR-T cells is the same as that in Examples 1-4, except that: compared with MSLN-TRAIL CAR-T cells, the P2A peptide and TRAIL coding sequence are not connected behind CD3ζ of the MSLN-CAR structure.

[0126] Data analysis: Open the analysis software (CP96Analyze) for data analysis, open the file, and select the required data for export.

[0127] Experimental results:

[0128] Using CAR-T cells or T cells as effector cells and ovarian cancer cell lines as target cells, a co-culture system was established according to the same effector-to-target ratio, that is, in a 96-well plate, the number of target cells fixed in each well was 20,000. CAR-T cells or T cells were added respectively, and the co-culture system was cultured with serum-free medium. After continuous culture, data analysis was performed using the analysis software (CP96Analyze). The results are as Figure 2 shown. Compared with the T cell group, the chimeric antigen receptor targeting MLSN can effectively kill ovarian cancer cell lines, and the MSLN CAR-T cells expressing TRAIL protein have a better killing effect.

[0129] Example 6 Detection of CAR-T Cell Cytokine Secretion Level

[0130] The specific experimental steps are as follows:

[0131] S1. Use ovarian cancer cells OVCAR3 as target cells and centrifuge at 400g for 10 min;

[0132] S2. Resuspend the target cells in the medium, adjust the cell density to 5×10 5 cells / mL. Take a new 96-well plate and inoculate the target cells at a volume of 100 μL / well. For the unused wells around the 96-well plate, add 100 μL of sterile water to each well to prevent water evaporation in the middle experimental wells. Place the plate in a 5% CO2, 37 °C incubator and culture overnight.

[0133] S3. Centrifuge and collect the above-prepared MSLN-TRAIL CAR-T cells and resuspend them in serum-free 1640 medium; Take out the 96-well plate from the incubator, completely aspirate the medium in the wells, gently wash the cells once with sterile PBS, then add MSLN-TRAIL CAR-T cells according to the above E / T ratio, and make up the final volume to 100 μL / well; Place the plate in a 5% CO2, 37 °C incubator and culture for 6 hours. At the same time, set up a control T cell group.

[0134] S4. After the culture is completed, take out the well plate from the incubator, centrifuge the 96-well plate at 1200 x g at room temperature for 5 minutes, gently take out the plate, transfer 50 μL of the culture medium supernatant from each well, detect the IFN-γ expression using an ELISA kit, and read the OD value using an enzyme-linked immunosorbent assay reader.

[0135] S5. Plot the data obtained above using GraphPad 6.0.

[0136] Experimental results:

[0137] Using MSLN-TRAIL CAR-T cells as effector cells and ovarian cancer cells OVCAR3 as target cells, a co-culture system was established according to different effector-to-target ratios. That is, in a 96-well plate, the number of target cells in each well was fixed at 20,000, and different numbers of CAR-T cells were added respectively. The co-culture system was cultured with serum-free medium. After continuous culture for 8 hours, take out the well plate, centrifuge at 1200 x g at room temperature for 10 minutes to precipitate all the suspended cells to the bottom of the well plate. Then, take out 30 μL of the supernatant from each well and detect the expression level of IFN-γ secreted by CAR-T cells after being activated by tumor cells in the culture medium supernatant by ELISA; the results are as Figure 3 shown. After the MLSN CAR-T cells expressing the TRAIL protein bind to the target tumor cells, they can effectively activate the CAR-T cells and cause an increase in the secretion and expression level of cytokines. When the effector-to-target ratio is 0.25:1, after the CAR-T cells are activated by tumor cells, they can secrete a large amount of IFN-γ, which is significantly higher than that of control T cells.

[0138] Example 7 In vivo efficacy experiment of CAR-T cells

[0139] S1. Collect OVCAR3 cells in the logarithmic growth phase, remove the culture medium, wash twice with PBS, and then inoculate; the tumor cells are subcutaneously inoculated into NKG mice (male, 6 weeks old) purchased from Cyagen Biosciences Inc. (Suzhou), and each mouse is inoculated with 5 x 10 6 cells / 100 μL;

[0140] S2. Group and administer drugs:

[0141] The average tumor volume of the mice is 100.06 mm 3 , and they are randomly grouped according to the tumor volume. There are 6 mice in each group, 3 groups, a total of 18 mice. The day of grouping is defined as day D0, and drug treatment is administered on the day of grouping;

[0142] Experimental grouping:

[0143] (1) Solvent group: Inject 100 μL of PBS;

[0144] (2) MSLN CAR-T group: Inject 1E06 MSLN CAR-T cells;

[0145] (3) MSLN CAR-T + TRAIL group: Inject 1E06 MSLN-TRAIL CAR-T cells;

[0146] S3. Experimental observation and data collection:

[0147] After cell inoculation, regularly monitor the impact of the tumor on the normal behavior of the animals every week; the specific contents include the activity of the experimental animals, food intake and water consumption, weight gain or loss, eyes, hair, and other abnormal conditions;

[0148] After starting the administration, observe the tumor size and weigh the mice. The calculation method of the tumor volume is: Tumor volume (mm 3 ) = 0.5 × (long diameter of the tumor × short diameter of the tumor 2 );

[0149] S4. Euthanize the mice 35 days after CAR-T cell treatment, isolate the subcutaneous tumors, take pictures, and weigh them.

[0150] Experimental results: As Figure 4 shown, MSLN CAR-T cells expressing TRAIL protein have significant efficacy in a mouse tumor model and are superior to general MSLN CAR-T cells.

[0151] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor contains a MSLN targeting structure and a tumor necrosis factor-related apoptosis-inducing ligand coding region which are connected; The MSLN targeting structure comprises a chimeric antigen receptor targeting MSLN, and the chimeric antigen receptor targeting MSLN contains an MSLN antibody scfv region, a hinge region, a transmembrane region and an intracellular signaling region; The tumor necrosis factor-related apoptosis-inducing ligand coding region contains a tumor necrosis factor-related apoptosis-inducing ligand coding sequence, and the tumor necrosis factor-related apoptosis-inducing ligand coding region is located after the intracellular signaling region.

2. The chimeric antigen receptor according to claim 1, wherein It contains at least one of the following features: (1) The intracellular signaling region is connected to the tumor necrosis factor-related apoptosis-inducing ligand coding region through a self-cleaving peptide; (2) The MSLN antibody scfv region is located before the hinge region, the transmembrane region and the intracellular signaling region are located after the hinge region, and the intracellular signaling region is located at the end of the transmembrane region far from the hinge region; (3) An extracellular signal peptide region is further provided in front of the MSLN antibody scfv region of the chimeric antigen receptor.

3. The chimeric antigen receptor according to claim 2, wherein It contains at least one of the following features: (1) The self-cleaving peptide includes 2A peptide; (2) The self-cleaving peptide includes P2A peptide and / or T2A peptide; the amino acid sequence of the P2A peptide is as shown in SEQ ID NO.8; (3) The signal peptide in the extracellular signal peptide region includes CD8a signal peptide.

4. The chimeric antigen receptor according to claim 1, wherein It contains at least one of the following features: (1) The amino acid sequence of the tumor necrosis factor-related apoptosis-inducing ligand is as shown in SEQ ID NO.1; (2) The amino acid sequence of the MSLN antibody scfv region is as shown in SEQ ID NO.3; (3) The hinge region includes a CD8 hinge region; the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO.4; (4) The transmembrane region includes a CD8 transmembrane region; the amino acid sequence of the CD8 transmembrane region is as shown in SEQ ID NO.5; (5) The intracellular signaling region contains a co-stimulatory molecule region and / or a signal transduction molecule region; the co-stimulatory molecule region includes a CD137 intracellular signaling region, and the signal transduction molecule region is a CD3zeta intracellular signaling region.

5. A nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1-4.

6. An expression vector containing the nucleic acid molecule according to claim 5.

7. A recombinant T cell containing the nucleic acid molecule according to claim 5.

8. A CAR-T cell, characterized in that, The CAR-T cell contains the nucleic acid molecule according to claim 5 or the expression vector according to claim 6.

9. Use of the chimeric antigen receptor according to any one of claims 1-4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the recombinant cell according to claim 7 or the CAR-T cell according to claim 8 in the preparation of an anti-tumor drug.

10. An anti-tumor drug, characterized in that, Containing the chimeric antigen receptor according to any one of claims 1-4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the recombinant cell according to claim 7 or the CAR-T cell according to claim 8.

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