Chimeric antigen receptor for expressing membrane-anchored PD-1 antibody and application of chimeric antigen receptor

Abstract

The invention discloses a chimeric antigen receptor for expressing a membrane-anchored PD-1 antibody and application, and relates to the technical field of biological medicines. An MSLN antigen is used as a target spot, an immune checkpoint PD-1 is combined for design, an optimal mode for combining the MSLN antigen and the immune checkpoint PD-1 is found, an MSLN CAR molecule for expressing a membrane anchoring PD-1 antibody is obtained, and a novel CAR-T cell therapy is developed based on the CAR molecule. Specifically, the invention provides a chimeric antigen receptor containing an MSLN targeting structure and a PD-1 antibody anchoring structure, the MSLN targeting structure comprises a coding sequence of the chimeric antigen receptor targeting MSLN, and the PD-1 antibody anchoring structure comprises a coding sequence of a membrane anchored PD-1 antibody. In-vitro and in-vivo experiments show that the treatment effect of the anti PD-1 amplified MSLN CAR-T is superior to that of a second generation MSLN CAR-T and that of a fourth generation MSLN CAR-T secreting a PD-1 antibody, the molecule can enable MSLN and PD-1 to have a synergistic effect, the immune effect of the CAR molecule is greatly improved, and the life cycle of tumor patients is obviously prolonged.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a chimeric antigen receptor expressing a membrane-anchored PD-1 antibody and applications thereof. Background Art

[0002] Chimeric antigen receptor T (CAR-T) cell immunotherapy, as a cell immunotherapy, has achieved remarkable results in a variety of tumors. So far, a variety of CAR-T cell therapies have been approved. Mesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that is widely expressed in tumors. Studies have found that mesothelin is expressed in about 95% of malignant mesotheliomas and 80% to 85% of pancreatic cancers, and is also highly expressed in a higher proportion of cancer types such as ovarian cancer, bile duct cancer, colorectal cancer, and lung cancer. In addition, the selection of targets in CAR-T therapy is very critical. The expression of target antigens on tumor cells needs to be wide enough to ensure that CAR-T cells can effectively recognize and attack most tumor cells. The targets should also be as specific to tumor cells as possible to reduce damage to normal cells, and the target antigens should be stable and not lost during treatment, because tumor cells may escape recognition by the immune system by downregulating or losing surface antigens. As a newly discovered tumor target, MSLN has limited expression in normal tissues and is associated with the effects of multiple tumor-related pathways in the body. It meets the requirements for target selection in the above CAR-T therapy and has therefore become a current research hotspot.

[0003] However, CAR-T faces many challenges in the treatment of solid tumors, such as immunosuppressive tumor microenvironment, and CAR-T therapy based on MSLN targets is no exception. Therefore, how to enhance the therapeutic effect of CAR-T in solid tumors is an important issue that needs to be solved urgently. One of the main reasons for the poor effect of CAR-T cell therapy in solid tumors is the exhaustion of CAR-T cells. It is mainly manifested as poor expansion, reduced activity and non-function. CAR-T cell exhaustion may be related to inhibitory checkpoints such as PD-1, and the formation of immunosuppressive tumor microenvironment is also related to it. Therefore, blocking this signaling pathway may achieve lasting anti-tumor effects and clinical remission. There are currently a variety of strategies for blocking this pathway and thus enhancing CAR-T cell function. These include systemic administration of PD-1 antibodies, using CRISPR / Cas9 technology to knock out PD-1 on CAR-T cells, and constructing CAR-T cells that secrete PD-1 antibodies. However, these strategies all have some defects to a greater or lesser extent. Although systemic administration of PD-1 antibodies can block this pathway, it inevitably brings about systemic toxicity. In addition, the effect of exogenous PD-1 antibodies is short-lived and requires multiple administrations. CRISPR / Cas9 provides a powerful and effective solution for editing the PD-1 gene in CAR-T cells to block immune checkpoints and improve the tumor immune microenvironment. However, its safety and reliability must be considered. Off-target cutting may cause gene mutations and induce cancer. In addition, the activity and proliferation ability of edited T cells may be affected, and PD-1 deficiency also affects the function and phenotype of cells. Studies have shown that CAR-T cells that secrete PD-1 antibodies have good tumor treatment effects, but the PD-1 antibodies produced in this way need to be secreted into the extracellular space first, and then block PD-1 on CAR-T cells. The concentration and stability of PD-1 antibodies cannot be guaranteed. In addition, this is similar to the systemic administration of PD-1 antibodies, which may also produce immune-related adverse reactions. Therefore, it is of great significance to find a method that can enhance the therapeutic effect of MSLN CAR-T therapy without obvious safety issues. Summary of the invention

[0004] To solve the above technical problems, the present invention provides a MSLN CAR-T cell expressing a membrane-anchored PD-1 antibody. We anchor the ScFv of the PD-1 antibody to the cell membrane by connecting it to the CD8 transmembrane region. This cell editing method enables the PD-1 molecules on the surface of the CAR-T cells to be stably self-enclosed. The PD-1 antibody is only expressed on the adoptively transferred CAR-T cells and can synergize with the MSLN tumor target, effectively avoiding the shortcomings of systemic targeting, improving the immune effect while reducing immune-related adverse reactions.

[0005] The first object of the present invention is to provide a chimeric antigen receptor expressing a membrane-anchored PD-1 antibody, wherein the chimeric antigen receptor comprises an MSLN targeting structure and a PD-1 antibody anchoring structure:

[0006] The MSLN targeting structure comprises a coding sequence of a chimeric antigen receptor targeting MSLN;

[0007] The PD-1 antibody anchoring structure comprises a coding sequence of a membrane-anchored PD-1 antibody.

[0008] Furthermore, the chimeric antigen receptor targeting MSLN contains an extracellular region, a transmembrane region 1 and an intracellular region, and the extracellular region contains the MSLN antibody scFv and hinge region 1.

[0009] Furthermore, the amino acid sequence of the MSLN antibody scFv is shown in SEQ ID NO.4, and the hinge region 1 includes the CD8 hinge region (SEQ ID NO.5).

[0010] Furthermore, the transmembrane region 1 includes the CD8 transmembrane region (SEQ ID NO.6).

[0011] Furthermore, the intracellular region contains a co-stimulatory molecule region and a signal transduction molecule region; the co-stimulatory molecule includes 4-1BB (SEQ ID NO.7), and the signal transduction molecule includes CD3Zeta (SEQ ID NO.8).

[0012] Furthermore, in the chimeric antigen receptor, the PD-1 antibody anchoring structure is connected after the MSLN targeting structure, preferably, after the above-mentioned intracellular region.

[0013] Furthermore, the PD-1 antibody anchoring structure is connected to the MSLN targeting structure via a self-cleaving peptide; the self-cleaving peptide includes a 2A peptide, such as a P2A peptide, a T2A peptide, etc., and the most preferred is a P2A peptide (SEQ ID NO.9).

[0014] Further, the membrane-anchored PD-1 antibody comprises an antibody region, a hinge region 2 and a transmembrane region 2, wherein the antibody region comprises a PD-1 antibody scFv. Preferably, the hinge region 2 is located between the antibody region and the transmembrane region 2, and the antibody region is located at one end close to the MSLN targeting structure.

[0015] Furthermore, the amino acid sequence of the PD-1 antibody scFv is shown in SEQ ID NO.3, the hinge region 2 includes the CD8 hinge region (SEQ ID NO.5), and the transmembrane region 2 includes the CD8 transmembrane region (SEQ ID NO.6).

[0016] The second object of the present invention is to provide a nucleic acid molecule encoding the chimeric antigen receptor expressing the membrane-anchored PD-1 antibody.

[0017] The third object of the present invention is to provide a recombinant expression vector containing the nucleic acid molecule.

[0018] Preferably, the different regions of the chimeric antigen receptor expressing the membrane-anchored PD-1 antibody are all located on one plasmid.

[0019] The fourth object of the present invention is to provide a recombinant cell containing the chimeric antigen receptor expressing the membrane-anchored PD-1 antibody.

[0020] 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.

[0021] The fifth object of the present invention is to provide the use of the chimeric antigen receptor, nucleic acid molecule, recombinant expression vector or recombinant cell expressing the membrane-anchored PD-1 antibody in the preparation of tumor therapeutic drugs.

[0022] The sixth object of the present invention is to provide a tumor therapeutic drug containing the chimeric antigen receptor, nucleic acid molecule, recombinant expression vector or recombinant cell expressing the membrane-anchored PD-1 antibody.

[0023] Furthermore, the tumor is a solid tumor, including but not limited to malignant mesothelioma, pancreatic cancer, ovarian cancer, bile duct cancer, colorectal cancer, lung cancer, etc.

[0024] By means of the above scheme, the present invention has at least the following advantages:

[0025] The present invention anchors the ScFv of the PD-1 antibody on the cell membrane by connecting it to the CD8 transmembrane region, which can stably block the PD-1 molecules on the surface of CAR-T cells, block the PD1 / PDL1 pathway, reduce CAR-T cell exhaustion, and significantly enhance the anti-tumor effect of CAR-T cells. In addition, the PD-1 antibody is only expressed on adoptively transferred CAR-T cells, and can synergize with the MSLN tumor target, effectively avoiding the shortcomings of systemic targeting and reducing immune-related adverse reactions. The results show that the MSLN CAR-T expressing the membrane-anchored PD1 antibody has very strong cell-killing toxicity and specificity both in vivo and in vitro, and can secrete high levels of cytokines such as INF-γ. After repeated tumor stimulation in vitro, the cell killing effect is better than that of the second-generation MSLN CAR-T and the fourth-generation MSLN CAR-T cells that secrete PD-1 antibodies, and the cell exhaustion rate is lower, and the same trend is also shown in vivo.

[0026] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention in conjunction with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0028] Figure 1 is a schematic diagram of a chimeric antigen receptor in the present invention;

[0029] Figure 2 This is a comparison of the killing ability of MSLN CAR-T cells expressing membrane-anchored PD1 antibodies on target cells;

[0030] Figure 3 This is a comparison of the IFN-γ secretion ability of MSLN CAR-T cells expressing membrane-anchored PD1 antibodies;

[0031] Figure 4 This is a schematic diagram of the in vivo experiment of MSLN-CAR-T cells expressing membrane-anchored PD1 antibody.

[0032] Figure 5 This is a comparison of the in vivo efficacy of MSLN-CAR-T cells expressing the membrane-anchored PD1 antibody. DETAILED DESCRIPTION

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

[0034] Example 1 Construction of lentiviral expression vector

[0035] The PD-1 antibody scFv region, CD8 hinge region and CD8 transmembrane region were connected in series to form a fusion protein, which was linked to the back of CD3ζ of the MSLN-CAR structure through the P2A peptide and subcloned into the PLVX-EF1a vector (purchased from clontech) to construct PLVX-EF1a-CAR. The combination order of each element in the constructed chimeric antigen receptor lentiviral expression vector is as follows Figure 1 shown.

[0036] The amino acid sequence of the membrane-anchored PD-1 antibody is as follows (SEQ ID NO.1):

[0037] MALPVTALLLPLALLLHAARPQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLS LSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0038] The nucleic acid sequence of the membrane-anchored PD-1 antibody is as follows (SEQ ID NO.2):

[0039]

[0040] The amino acid sequence of the PD-1 antibody is as follows (SEQ ID NO.3):

[0041] MALPVTALLLPLALLLHAARPQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQ GTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK

[0042] The amino acid sequence of the MSLN antibody is as follows (SEQ ID NO.4):

[0043] MALPVTALLLPLALLLHAARPQVQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTT VTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVL

[0044] The amino acid sequence of the CD8 hinge region is as follows (SEQ ID NO.5):

[0045] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0046] The amino acid sequence of the CD8 transmembrane region is as follows (SEQ ID NO.6):

[0047] IYIWAPLAGTCGVLLLSLVITLYC

[0048] The amino acid sequence of the CD137 (4-1BB) intracellular domain is as follows (SEQ ID NO.7):

[0049] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0050] The amino acid sequence of the CD3Zeta is as follows (SEQ ID NO.8):

[0051] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0052] The amino acid sequence of the P2A peptide is as follows (SEQ ID NO.9):

[0053] ATNFSLLKQAGDVEENPGP.

[0054] Example 2 Lentivirus Preparation

[0055] The specific experimental steps are as follows:

[0056] S1, prepare a 15cm dish, inoculate 5*10 6 293T cells (purchased from ATCC) were added with complete culture medium (DMEM high glucose, 10% FBS, double antibody), placed in a 37°C, 5% CO2 incubator, and cultured overnight.

[0057] S2. Take out 100μM PEI and lentiviral packaging plasmids (PLVX-EF1a-CAR, pGP, pVSVG) from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out PBS or HBSS buffer and warm to room temperature. Take 2mL PBS to one well of a 6-well plate, add 10μg PLVX-EF1a-CAR, 4μg pGP, and 2μg pVSVG respectively, pipette up and down to mix thoroughly, add 18μL 100μM PEI, immediately pipette up and down to mix, and let stand at room temperature for 10 minutes.

[0058] S3. Add the above DNA / PEI complex dropwise into a 15 cm culture dish, gently shake the dish and mix thoroughly. Place the dish in a 37°C, 5% CO2 incubator, and after 6 to 8 hours of culture, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium.

[0059] S4. After continuous culture for 48 hours, collect the supernatant of the culture medium containing the virus in the culture dish, filter it with a 0.45μm filter membrane, add 20% volume of 50% PEG6000 solution, incubate at 4℃ for 2h, transfer to a centrifuge tube, balance it, and centrifuge it at 3000xg at 4℃ for 0.5h. After centrifugation, carefully remove the liquid in the centrifuge tube in the biosafety cabinet, add 500μL PBS buffer to resuspend the precipitate, and store the virus at -80℃.

[0060] Example 3 Isolation of primary T cells

[0061] The specific experimental steps are as follows:

[0062] S1. Invert the lymphocyte separation solution upside down several times to mix the Lymphoprep reagent thoroughly.

[0063] 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 blood sample to be separated) and set aside.

[0064] S3. Dilute the blood sample with an equal volume of PBS + 2% FBS.

[0065] S4. Use a pipette to carefully add the diluted blood sample slowly along the tube wall to the upper layer of the separation reagent to avoid mixing the separation reagent and the blood sample.

[0066] S5. Set the centrifuge to 800 x g, set the speed reduction rate to the slowest, and centrifuge at room temperature for 20 minutes.

[0067] S6. After centrifugation, collect the upper light yellow serum into another sterile centrifuge tube and store at -80℃.

[0068] S7. Gently aspirate the mononuclear cell layer at the interface of serum and separation reagent into a new centrifuge tube and wash the cells once with culture medium.

[0069] S8. Adjust the cell density to 1*10 8 cells / mL (total volume not to exceed 2.5 mL) and resuspend in a 5 mL round-bottom tube.

[0070] S9. Add 100 μl / mL antibody cocktail, mix thoroughly, and incubate at room temperature for 15 minutes.

[0071] S10. Take out the magnetic beads and mix them thoroughly by pipetting them up and down at least 5 times.

[0072] S11. Pipette 50 μl of magnetic beads / mL into the above sample, mix thoroughly, and incubate at room temperature for 10 minutes.

[0073] S12. Add complete culture medium to a total volume of 2.5 mL in the tube, insert the tube (with the lid open) into the magnet, and let stand at room temperature for 5 minutes.

[0074] S13. After incubation, keep the tube in the magnetic pole and gently invert it to pour out the cells in the tube.

[0075] 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.

[0076] Example 4 Activation of primary T cells and lentiviral infection

[0077] The specific experimental steps are as follows:

[0078] S1. Adjust the cell density to 1*10 6 cells / mL, cytokine and antibody complexes (final concentration of 300U / mL IL-2, 10ng / mL IL-7, 5ng / mL IL-15, 500ng / mL Anti-CD3 (OKT3), 2ug / mL Anti-CD28) were added and cultured for 48 hours.

[0079] 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.

[0080] S3. After taking out the virus from the -80℃ refrigerator, quickly melt it in a 37℃ water bath. Add the above calculated amount of virus to the six-well plate, add polybrene with a final concentration of 6μg / mL, mix thoroughly, seal the four sides of the six-well plate with sealing film, and centrifuge at 800xg for 1 hour.

[0081] S4. After centrifugation, remove the sealing film and place the six-well plate in a 37°C 5% CO2 incubator for 24 hours.

[0082] S5. Centrifuge at 250 x g for 10 minutes, remove 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 culturing for 3-6 days for use.

[0083] Example 5 Lysis of target cells by anti-PD-1armoured MSLN CAR-T cells prepared by the present invention

[0084] Real-time label-free dynamic cell analysis (RTCA) is a technology that integrates microelectronic cell sensor chips into the bottom of the cell detection plate through a special process to build a cell impedance detection sensing system that can track changes in cell morphology, proliferation and differentiation in real time, dynamically and quantitatively. When cells that grow on the surface of the microelectrode cause changes in the impedance of the adherent electrode interface, this change is correlated with the change in the real-time functional state of the cell. Through real-time dynamic electrode impedance detection, biological information related to cell physiological functions can be obtained, including cell growth, extension, morphological changes, death and adhesion. This method can detect the killing of target cells by CAR-T cells in real time.

[0085] The specific experimental steps are as follows:

[0086] S1. Take the target cells out of the incubator, discard the supernatant, add PBS to rinse once, and discard the PBS.

[0087] S2. Add 0.25% trypsin for digestion. When the cells are basically detached, add complete culture medium to terminate digestion. Transfer to a centrifuge tube and mix well. Take 20 μL of cell suspension and 20 μL of AO / PI dye solution and mix well for counting.

[0088] S3. Take the required amount of cell suspension, centrifuge at 500g for 5 min, discard the supernatant, and resuspend and mix with culture medium.

[0089] S4. Take out the 96W20idf electrode plate and add 100 μL of target cell suspension to each well, 40,000 target cells.

[0090] S5. After the cells are added, the electrode plate is placed on the clean bench and allowed to stand for 15 minutes.

[0091] S6. Put the electrode plate into the Station and fasten it. Click Check to check whether the electrode plate is connected to the workstation and then click Start to start data collection.

[0092] S7. After the cells are added, the electrode plate is placed on the clean bench and allowed to stand for 15 minutes.

[0093] S8. After about 24-36 hours of data collection (depending on the experimental requirements), the target cells are in a stable cell growth plateau phase.

[0094] S9, CAR-T cells and T cells were blown and counted, 20 μL of cell suspension was mixed with 20 μL AO / PI dye solution and counted, the required amount of cell suspension was taken, 500 g, centrifuged for 5 min, the supernatant was discarded, and the culture medium was resuspended to adjust the corresponding concentration.

[0095] S10. Click Pause to remove the electrode plate, aspirate the culture medium, add CAR-T cells and T cell suspension according to the effector-target ratio, and add the corresponding culture medium to the same final volume in each well. Put the electrode plate back into the workstation, click Check, and then click Resume.

[0096] S11. After continuing to collect data for 48 hours (depending on the experimental requirements), click Finish to end the experiment.

[0097] Experimental groups:

[0098] (1) NC group: no effector cells added

[0099] (2) T cell group: T cells were used as effector cells and added at a 1:1 effector-target ratio.

[0100] (3) MSLN CAR-T group: MSLN CAR-T cells were used as effector cells, and effector cells were added at a 1:1 effector-target ratio.

[0101] (4) αPD-1MSLN CAR-T group: MSLN CAR-T cells secreting PD-1 antibodies were used as effector cells, and effector cells were added at a 1:1 effector-target ratio.

[0102] (5) Anti-PD-1armouredMSLN CAR-T group: MSLN CAR-T cells with membrane-anchored PD-1 antibody were used as effector cells, and effector cells were added at an effector-target ratio of 1:1.

[0103] in,

[0104] The preparation of MSLN CAR-T cells was the same as in Example 1-4, except that: compared with anti PD-1armoured MSLNCAR-T cells, the PD-1 antibody scFv region, CD8 hinge region and CD8 transmembrane region were not connected behind CD3ζ of the MSLN-CAR structure;

[0105] The preparation of αPD-1MSLN CAR-T cells is the same as that of Example 1-4, except that compared with anti PD-1armouredMSLN CAR-T cells, there is no CD8 hinge region and CD8 transmembrane region behind the scfv region of the PD-1 antibody.

[0106] Data analysis: Open the analysis software (CP96Analyze) for data analysis, open the file, and select the required data to export.

[0107] Experimental results:

[0108] Different CAR-T cells or T cells were used as effector cells and pancreatic cancer cell lines (ASPC-1, BXPC3, Capan-2, HPAC) were used as target cells to establish a co-culture system, that is, in a 96-well plate, the number of fixed target cells in each well was 40,000, and the co-culture system was cultured in serum-free medium for continuous culture, and data analysis was performed using analysis software (CP96Analyze). The results are shown in Figure 2. Figure 2 As shown, for different pancreatic cancer cell lines, the killing effect of the anti PD-1armouredMSLN CAR-T cells of the present invention is significantly better than that of other experimental groups.

[0109] Example 6 Detection of CAR-T cell factor secretion level

[0110] The specific experimental steps are as follows:

[0111] S1, pancreatic cancer cells Capan-2 were used as target cells and centrifuged at 400 g for 10 min;

[0112] S2. Resuspend the target cells in culture medium and adjust the cell density to 5*10 5 / mL, take a new 96-well plate and inoculate the target cells at 100μL / well. Add 100μL of sterile water to each of the unused wells around the 96-well plate to prevent the water in the middle experimental well from evaporating. Place the plate in a 5% CO2 37℃ incubator and culture overnight.

[0113] S3. Collect the anti PD-1armoured MSLN CAR-T cells or T cells prepared above by centrifugation and resuspend them in serum-free 1640 medium; remove the 96-well plate from the incubator, completely aspirate the medium in the wells, gently wash the cells once with sterile PBS, and then add CAR-T cells or T cells according to the above E / T ratio, and make up the final volume to 100 μL / well; place the well plate in a 5% CO2 37°C incubator and culture for 6 hours. At the same time, set up a control T cell group.

[0114] S4. After the culture is completed, take the well plate out of the incubator, centrifuge the 96-well plate at 1200xg at room temperature for 5 minutes, gently take out the plate, transfer 50 μL of culture supernatant from each well, use ELISA kit to detect the expression of IFN-γ, and use a microplate reader to read the OD value.

[0115] S5. The above acquired data were converted into cytokine concentrations and graphed using GraphPad 6.0.

[0116] Experimental results:

[0117] With anti PD-1armouredMSLN CAR-T cells or T cells as effector cells and pancreatic cancer cell Capan-2 as target cells, a co-culture system was established according to different effector-target ratios, that is, in a 96-well plate, the number of fixed target cells in each well was 40,000, and different numbers of CAR-T cells or T cells were added, respectively. The co-culture system was cultured in serum-free medium. After continuous culture for 8 hours, the well plate was taken out and centrifuged at room temperature at 1200×g for 10 minutes to allow all suspended cells to precipitate to the bottom of the well plate, and then 30 μl of supernatant was taken from each well, and the expression of IFN-γ secreted by CAR-T cells or T cells after being activated by tumor cells in the culture medium supernatant was detected by ELISA; the results are shown in FIG. Figure 3 As shown, T cells cannot effectively kill pancreatic cancer cell lines, while the MLSN CAR-T cells expressing membrane-anchored PD-1 antibodies of the present invention can effectively activate CAR-T cells after binding to target cells, and cause an increase in the secretion and expression of cytokines; when the effector-target ratio is 1:1, after being activated by tumor cells, CAR-T cells can secrete a large amount of IFN-γ, which is significantly higher than that of control T cells.

[0118] Example 7 CAR-T cell in vivo efficacy experiment

[0119] S1. Capan-2 cells in the logarithmic growth phase were collected, the culture medium was removed and the cells were washed twice with PBS before inoculation. Tumor cells were subcutaneously inoculated into NKG mice (male, 6 weeks old, purchased from Saiye Biotechnology Co., Ltd. (Suzhou)), with each mouse inoculated with 5x10 6 cells / 100ul;

[0120] S2. Grouped medication:

[0121] The average tumor volume of mice was 101.61 mm 3 The mice were randomly divided into groups according to the tumor volume. Each group had 6 mice, and there were 4 groups, with a total of 24 mice. The mice were treated with drugs on the day of grouping ( Figure 4 );

[0122] The groups are as follows:

[0123] (1) Model group (NC)

[0124] (2) CAR-T1 group: MSLN CAR-T

[0125] (3) CAR-T2 group: αPD-1MSLN CAR-T

[0126] (4) CAR-T3 group: anti PD-1armoured MSLN CAR-T

[0127] S3. Experimental observation and data collection:

[0128] After cell inoculation, the effect of the tumor on the normal behavior of the animals was routinely monitored weekly; the specific contents included the activity of the experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities;

[0129] After the administration, the tumor size was observed and the mice were weighed. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 );

[0130] S4. 43 days after CAR-T cell treatment, the mice were euthanized and the subcutaneous tumors were isolated, photographed and weighed.

[0131] The experimental results are as follows Figure 5 : MSLN CAR-T cells expressing membrane-anchored PD-1 antibodies have significant efficacy in mouse tumor models and are superior to MSLN CAR-T cells and MSLN CAR-T cells secreting PD-1 antibodies.

[0132] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled 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 methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A chimeric antigen receptor expressing a membrane-anchored PD-1 antibody, characterized in that: The chimeric antigen receptor contains a MSLN targeting structure and a PD-1 antibody anchoring structure: The MSLN targeting structure comprises a coding sequence of a chimeric antigen receptor targeting MSLN; The PD-1 antibody anchoring structure comprises a coding sequence of a membrane-anchored PD-1 antibody.

2. The chimeric antigen receptor expressing membrane-anchored PD-1 antibody according to claim 1, characterized in that: Contain at least one of the following characteristics: (1) The PD-1 antibody anchoring structure is connected behind the MSLN targeting structure; (2) The PD-1 antibody anchoring structure is connected to the MSLN targeting structure via a self-cleaving peptide; wherein the self-cleaving peptide includes a 2A peptide.

3. The chimeric antigen receptor expressing membrane-anchored PD-1 antibody according to claim 1, characterized in that: Contain at least one of the following characteristics: (1) The chimeric antigen receptor targeting MSLN contains an extracellular region, a transmembrane region 1 and an intracellular region, and the extracellular region contains an MSLN antibody scFv and a hinge region 1; (2) The membrane-anchored PD-1 antibody comprises an antibody region, a hinge region 2 and a transmembrane region 2, and the antibody region comprises a PD-1 antibody scFv.

4. The chimeric antigen receptor expressing membrane-anchored PD-1 antibody according to claim 3, characterized in that: Contain at least one of the following characteristics: (1) The transmembrane region 1 is located between the extracellular region and the intracellular region, and the hinge region 1 in the extracellular region is located close to one end of the transmembrane region 1; (2) the hinge region 2 is located between the antibody region and the transmembrane region 2, and the antibody region is located at one end close to the MSLN targeting structure; (3) The intracellular region is connected to the antibody region, and the intracellular region is located in front of the antibody region.

5. The chimeric antigen receptor expressing a membrane-anchored PD-1 antibody according to claim 3 or 4, characterized in that: Contain at least one of the following characteristics: (1) The amino acid sequence of the MSLN antibody scFv is shown in SEQ ID NO.4; (2) the hinge region 1 includes the CD8 hinge region; (3) the transmembrane region 1 includes the CD8 transmembrane region; (4) The intracellular region contains a co-stimulatory molecule region and a signal transduction molecule region, and the co-stimulatory molecule region is located between the transmembrane region 1 and the signal transduction molecule region; wherein the co-stimulatory molecule includes 4-1BB, and the signal transduction molecule includes CD3Zeta; (5) The amino acid sequence of the PD-1 antibody scFv is shown in SEQ ID NO.3; (6) the hinge region 2 includes a CD8 hinge region; (7) The transmembrane region 2 includes the CD8 transmembrane region.

6. A nucleic acid molecule encoding the chimeric antigen receptor expressing the membrane-anchored PD-1 antibody according to any one of claims 1 to 5.

7. A recombinant expression vector containing the nucleic acid molecule of claim 6.

8. A recombinant cell containing the recombinant expression vector according to claim 7.

9. The recombinant cell according to claim 8, characterized in that The starting cells of the recombinant cells include immune cells.

10. A tumor treatment drug, characterized in that: The tumor therapeutic drug contains the chimeric antigen receptor expressing the membrane-anchored PD-1 antibody according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the recombinant expression vector according to claim 7, or the recombinant cell according to claim 8 or 9.

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