CAR-T cell of targeted GPC-3 autocrine CiTEs fusion protein as well as preparation method and application of CAR-T cell

Abstract

The invention relates to a CAR-T (Chimeric Antigen Receptor-T) cell of a targeted GPC-3 autosecretion CiTEs fusion protein as well as a preparation method and application of the CAR-T cell. The fusion protein comprises a chimeric antigen receptor fragment and a bifunctional checkpoint inhibitory T cell adapter fragment, the chimeric antigen receptor fragment and the bifunctional checkpoint inhibitory T cell adapter fragment are connected through 2A peptide, and the chimeric antigen receptor fragment comprises a GPC3 single-chain antibody region, a transmembrane region and an intracellular signal transduction region; the bifunctional checkpoint inhibitory T cell adapter fragment comprises a GPC3 single-chain antibody region, a checkpoint inhibitory functional domain and a CD3 single-chain antibody region. The fusion protein is separated into CAR and CiTEs by the 2A peptide through a self-cleavage mechanism. CAR endows T cells with new specific recognition and killing capabilities, and CiTEs limit the risk of adverse events related to immunity.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid detection technology, and in particular to a CAR-T cell targeting GPC-3 autocrine CiTEs fusion protein, a preparation method and an application thereof. Background Art

[0002] According to statistics, there are about 905,700 new cases of liver cancer and 830,200 deaths worldwide each year, and its mortality rate ranks third among all malignant tumors. Liver cancer has an insidious onset and progresses rapidly. Most patients have lost the opportunity for radical treatment when they seek medical treatment and are in urgent need of systemic drug treatment. Chemotherapy is the main means of systemic treatment for advanced liver cancer, but its efficacy is limited and its adverse reactions are obvious. In recent years, targeted therapy and immunotherapy have developed rapidly, providing more options for the first-line treatment of advanced liver cancer.

[0003] Liver cancer, or primary liver cancer, mainly includes three different pathological types: hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). The three types differ greatly in pathogenesis, biological behavior, pathological histology, treatment methods, and prognosis, with HCC accounting for 75% to 85% and ICC accounting for 10% to 15%.

[0004] There are many treatments for HCC, but few are effective. Surgical resection has always been the main treatment for early HCC. Early surgical treatment can indeed achieve radical cure, but most patients with HCC are already in the middle and late stages when diagnosed. Most patients cannot undergo early surgery due to huge tumors, multiple metastases inside and outside the liver, anatomical limitations of intrahepatic tumors, or poor liver function. This leads to a very poor prognosis for HCC. According to relevant statistics, the 5-year overall survival (OS) of HCC patients is only 12.5%. Since the beginning of this century, the treatment of hepatocellular carcinoma has been constantly explored and innovated, and the treatment system has indeed been effectively supplemented. For example, the emergence of treatment methods such as transarterial chemoembolization (TACE), radiofrequency ablation (RFA), and stereotactic body radiotherapy (SBRT) has enriched the systemic treatment of liver cancer. Although new treatment methods have been widely used in clinical practice, the benefits of treatment for patients with advanced hepatocellular carcinoma are far from meeting the expectations of HCC patients. In 2008, the multi-target kinase inhibitor sorafenib was officially included in the first-line treatment of advanced HCC by the National Comprehensive Cancer Network (NCCN) guidelines for its good therapeutic effect and safety for patients with advanced HCC; however, from the application of sorafenib in clinical practice to the current long-term observation study, its clinical effect has been limited.

[0005] Chronic inflammation is also a factor that cannot be ignored in the progression of tumor cells. Chronic inflammation is usually considered to be the continuous expression of different cytokines and the recruitment of immune cells to problem areas. HBV virus infection causes immunosuppression. With the development of chronic infection, peripheral immune tolerance is generated and eventually tumors are produced. In chronic viral hepatitis, immune suppression checkpoints, including PD-1 / PD-L1, CTLA-4 and T cell immunoglobulin and mucin domain-3 (TIM-3), play an important role in immunosuppression by downregulating the response of T cells.

[0006] Tumor microenvironment (TME) refers to the internal environment of the tumor, which is mainly composed of tumor cells, stromal cells (such as tumor-associated fibroblasts), immune cells and their secreted factors, vascular endothelial cells and extracellular matrix (ECM). It is a complex integrated system. These immune cells and related matrix components recruited and activated by tumor cells form a tumor-suppressing inflammatory microenvironment to hinder the occurrence and development of tumors during tumor colonization or early growth. After continuous tumor antigen stimulation and immune activation response, the related effector cells in the microenvironment are in a state of exhaustion or remodeling, unable to perform normal functions or even promote the malignant characteristics of tumors, thereby producing an immunosuppressive microenvironment.

[0007] Liver cancer can induce tumor immune tolerance through various mechanisms, evade the body's immune killing, and eventually continue to progress and metastasize. Therefore, exploring the mechanism of liver cancer inducing immune tolerance and formulating targeted immunotherapy strategies are new development directions in the field of liver cancer treatment in the future. Summary of the invention

[0008] The purpose of the present invention is to disclose a CAR-T cell targeting GPC-3 autocrine CiTEs fusion protein and a preparation method and application thereof, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0009] To achieve the above object, the present invention is implemented through the following technical solutions: The first aspect of the present invention is to provide a fusion protein having both PD-1 / PDL-1 immune checkpoint inhibition function and GPC-3 chimeric antigen receptor targeting function.

[0010] The second aspect of the present invention is to provide a nucleotide molecule encoding the fusion protein described in the first aspect of the present invention.

[0011] The third aspect of the present invention is to provide a recombinant vector containing the nucleotide molecule described in the second aspect of the present invention.

[0012] The fourth aspect of the present invention is to provide a recombinant cell containing the recombinant vector described in the third aspect of the present invention.

[0013] The fusion protein of the first aspect of the present invention includes a chimeric antigen receptor fragment and a bifunctional checkpoint inhibitory T cell engager fragment, wherein the chimeric antigen receptor fragment and the bifunctional checkpoint inhibitory T cell engager fragment are connected by a 2A peptide, and the chimeric antigen receptor fragment includes a GPC3 single-chain antibody region, a transmembrane region and an intracellular signal transduction region; the bifunctional checkpoint inhibitory T cell engager fragment includes a GPC3 single-chain antibody region, a checkpoint inhibition domain and a CD3 single-chain antibody region.

[0014] The 2A peptide separates the fusion protein into a chimeric antigen receptor (CAR) and a dual-function checkpoint inhibitory T cell engager (CiTEs) through a self-cleavage mechanism. CAR combines a specific antigen recognition domain with the signal transduction part of T cells, thereby endowing T cells with new specific recognition and killing capabilities, while CiTEs confine the inhibitory function of PD-1 / PD-L1 to the local redirected T cells that interact with targeted tumor-associated antigens (TAA), while avoiding the systemic activation of T cells associated with anti-PD-1 or anti-PD-L1 immune checkpoint inhibitors, thereby limiting the risk of immune-related adverse events.

[0015] In some embodiments of the first aspect of the present invention, the amino acid sequence of the GPC3 single-chain antibody region is shown in SEQ ID No: 1. The GPC3 single-chain antibody region is a single-chain variable fragment (scFv) formed by connecting the heavy chain variable region (Viarable Heavy chain, VH) and the light chain variable region (Viarable light chain, VL) that specifically recognize and bind to the target protein in the GPC3-specific antibody GC33 with reference to the GPC3-specific antibody GC33. The GPC3 single-chain antibody region is the part that confers specificity to the CAR target antigen, so that the T cells containing the CAR can target GPC3-positive cells.

[0016] In some embodiments of the first aspect of the present invention, the amino acid sequence of the checkpoint inhibitory domain is as shown in SEQ ID No: 2. The checkpoint inhibitory domain is derived from the extracellular structure of the PD-1 protein, has low affinity to PD-L1 expressed on tumor cells, cannot directly target cells expressing PD-L1, and cannot non-specifically block the interaction of PD-1 / PD-L1, but can compete with PD-L1 binding to the tumor to neutralize the inhibitory effect of the immune checkpoint.

[0017] In some embodiments of the first aspect of the present invention, the amino acid sequence of the CD3 single-chain antibody region is shown in SEQ ID No: 3. The CD3 single-chain antibody region is a scFv formed by connecting the VH and VL that specifically recognize and bind to the target protein in the CD3-specific antibody OKT-3 with a flexible linker. The CD3 single-chain antibody region is the part that gives CiTEs T cell specificity.

[0018] In some embodiments of the first aspect of the present invention, the amino acid sequence of the chimeric antigen receptor fragment is shown in SEQ ID No: 4. The chimeric antigen receptor fragment shown in SEQ ID No: 4 comprises a GPC3 single-chain antibody region, a first flexible linker, a CD8 transmembrane region, a CD28 co-stimulatory intracellular domain, and a CD3ζ intracellular signaling region.

[0019] In some embodiments of the first aspect of the present invention, the amino acid sequence of the bifunctional checkpoint inhibitory T cell engager fragment is shown in SEQ ID No: 5. The bifunctional checkpoint inhibitory T cell engager fragment shown in SEQ ID No: 5 comprises a GPC3 single-chain antibody region, a second flexible connection, a checkpoint inhibitory domain, a third flexible connection and a CD3 single-chain antibody region.

[0020] The nucleotide molecule of the second aspect of the present invention is a nucleotide sequence encoding any of the fusion proteins described above. For example, the nucleotide sequence encoding the GPC3 single-chain antibody region is shown in SEQ ID No: 6; the nucleotide sequence encoding the checkpoint inhibition domain is shown in SEQ ID No: 7; the nucleotide sequence encoding the CD3 single-chain antibody region is shown in SEQ ID No: 8; the nucleotide sequence encoding the fusion protein is shown in SEQ ID No: 9.

[0021] The recombinant vector described in the third aspect of the present invention comprises the nucleotide molecule described in the second aspect of the present invention.

[0022] In some embodiments of the third aspect of the present invention, the recombinant vector is a transduction plasmid pCDH-EF1, and its nucleotide sequence is shown in SEQ ID No:12.

[0023] The recombinant cell according to the fourth aspect of the present invention comprises the recombinant vector according to the third aspect of the present invention.

[0024] In some embodiments of the fourth aspect of the present invention, the recombinant cell is a lentiviral expression vector.

[0025] In some embodiments of the fourth aspect of the present invention, a lentiviral expression vector is constructed by transduction plasmid pCDH-EF1, packaging plasmid psPAX2, film plasmid pMD2.G and HEK293T cell line. The nucleotide sequence of psPAX2 is shown in SEQ ID No: 13, and the nucleotide sequence of pMD2.G is shown in SEQ ID No: 14.

[0026] The lentiviral expression vector can express recombinant lentiviral products, and T lymphocytes transfected with the products will become autocrine CiTEs CAR-T cells. The CiTEs secreted by the autocrine CiTEs CAR-T cells at the tumor lesions can inhibit or neutralize the immunosuppression regulated by the PD-1 / PDL-1 pathway at the lesions, allowing the autocrine CiTEs CAR-T cells to proliferate and remain at the lesions and play a therapeutic role - solving the immune escape phenomenon caused by the immunosuppression regulated by the PD-1 / PD-L1 immune checkpoint pathway of tumor cells, and enhancing the survival and killing effect of the autocrine CiTEs CAR-T cells at the lesions.

[0027] Taking PD-L1 expressed on tumor cells as the intervention target, CAR-T cell therapy technology is combined with CiTEs technology. The modified autocrine CiTEs CAR-T cells reverse the negative regulatory effects on immune cells in the tumor microenvironment by secreting CiTEs. The original fusion protein structure is designed. This fusion protein can bind to the PD-L1 protein expressed by tumor cells with reference to the extracellular structure of the PD-1 protein on the surface of immune cells, neutralizing the inhibitory effect of tumor cells on immune cells. By co-expressing CiTEs, the inhibition of CAR-T cells is relieved or neutralized, maximizing the therapeutic benefits of CAR-T cells.

[0028] The modified autocrine CiTEs CAR-T cells break through or enhance the redirection of immune cells in the tumor microenvironment by secreting CiTEs. The CiTEs design the scFv structure with reference to the specific recognition structure of the GPC3-targeted antibody GC33, which enables CiTEs to specifically recognize and bind to tumor cells expressing GPC3. At the same time, CiTEs design the scFv structure with reference to the specific recognition structure of the CD3-targeted antibody OKT-3, which enables CiTEs to specifically recognize and bind to T lymphocytes expressing CD3. A large number of studies have reported the use of anti-human CD3 monoclonal antibodies to recognize and interact with the ε chain of the CD3 molecule on the TCR-CD3 complex, thereby enhancing the activation and proliferation of T lymphocytes. Therefore, in addition to connecting T lymphocytes, the anti-CD3scFv in CiTEs can also give one of the activation signals to resting T lymphocytes in the tumor microenvironment.

[0029] Ultimately, CiTEs connect T lymphocytes and target tumor cells, bypassing TCR-MHC-I interactions. Therefore, regardless of antigen specificity, CiTEs as T cell binders trigger T cell activation and produce cytotoxic molecules including perforin and granzyme to kill tumor cells. Therefore, during the period when the autocrine CiTEs CAR-T cells enter the body and persist, the continuous secretion of CiTEs in the tumor microenvironment can enable T lymphocytes in hot tumors to have the ability to surround cancer cells, enhance the in vivo T cell-mediated cytotoxic response and CAR-T cells to jointly exert anti-tumor effects.

[0030] In addition to targeting PD-L1 on the tumor surface, the CiTEs also target the GPC3 protein on the surface of liver cancer cells, ultimately confining the inhibitory function of PD-1 / PD-L1 to the local redirected T cells that target TAA interactions while avoiding the systemic activation of T cells associated with anti-PD-1 or anti-PD-L1 ICIs, thereby limiting the risk of immune-related adverse events. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a schematic diagram of the structure of the fusion protein described in Example 1; Figure 2 is the electrophoresis diagram of the nucleotide molecule cleavage fragments prepared in Example 2; Figure 3 is the electrophoresis diagram of the pCDH-EF1 restriction fragment prepared in Example 2; Figure 4 is the electrophoresis diagram of the recombinant vector prepared in Example 2; Figure 5 is an amplification curve diagram of the autocrine CiTEs CAR-T cells prepared in vitro in Example 5; Figure 6 is a graph showing changes in the viability of the autocrine CiTEs CAR-T cells prepared in vitro in Example 5; Figure 7 It is the original scatter plot of the purity flow cytometry detection of autocrine CiTEs CAR-T cells described in Example 5; Figure 8 is the IC50 standard curve of the anti-PD-L1 neutralizing antibody in Example 5; Fig. 9 is a bar graph of the GPC3 protein expression rates of different target cells in Example 6; Fig.10 It is a bar graph of the killing activity of the autocrine CiTEs CAR-T cells against different target cells at different effector-target ratios described in Example 6; Fig.11is the ratio of activated T lymphocyte subsets after co-culture of different functional cells in Example 7; Fig.12 The effect of the cell culture supernatants from different functional cell sources in Example 8 on the killing of target cells by T lymphocytes; Fig.13 It is the original data diagram of the scatter plot of the T lymphocyte proliferation regulation flow cytometry in Example 8; Fig.14 It is a comparison of the regulatory effects of co-culture of different functional cells on T lymphocyte resistance to the immune checkpoint protein PD-L1 in Example 8; Fig.15 is a graph showing changes in body weight of tumor-bearing mice after administration of autocrine CiTEs CAR-T cells in Example 9; Fig.16 This is a comparison of the number of surviving tumor-bearing mice at different time points after administration of the autocrine CiTEs CAR-T cells in Example 9. DETAILED DESCRIPTION

[0032] The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the Molecular Cloning Experiment Guide (3rd edition) or in accordance with the methods and product instructions; the biological materials described in the methods, unless otherwise specified, can be obtained from commercial channels.

[0033] Example 1: Design of fusion protein structure.

[0034] Design a fusion protein that can simultaneously express CAR targeting GPC3 and CiTEs that competitively bind to PD-L1, the structure of which is as follows Figure 1 As shown, including: (1) Signal peptide: encoding CD8α signal peptide (CD8α Leader); (2) GPC3 single-chain antibody region: Refer to the single-chain variable fragment (Anti GPC3 scFv) in the GPC3-specific antibody GC33 that specifically recognizes and binds to the target protein, which provides the target antigen specificity for CAR; (3) The first flexible linker: a linker fragment (Linker-1) used to connect the scFv and the transmembrane junction region, giving the scFv the required flexibility; (4) CD8 transmembrane region: derived from the transmembrane domain (CD8 TM) in the amino acid sequence structure of CD8α protein; (5) CD28 co-stimulatory intracellular domain: The intracellular fragment of the CD28 protein molecule (CD28 Cyto), which is involved in the reception and transmission of the second signal required for T cell activation; (6) CD3ζ intracellular signal transduction region: CD3ζ chain (CD3ζ Cyto), the intracellular structure of CD3 molecules, provides the first signal for T cell activation in CAR; (7) 2A peptide: a self-cleaved 2A peptide (P2A) that allows the amino acid sequence of subsequent translation to be cleaved intracellularly and then released to the extracellular space; (8) GPC3 single-chain antibody region: Refer to the single-chain variable fragment (Anti GPC3 scFv) in the GPC3-specific antibody GC33 that specifically recognizes and binds to the target protein, which provides the target antigen specificity for CiTEs; (9) Second flexible linker: provides the connection function between Anti GPC3 scFv and PD-1-ex components, giving CiTEs the required flexibility (Linker-2); (10) Checkpoint inhibitory domain: a fragment derived from the extracellular structure of the PD-1 protein that achieves immune checkpoint inhibition by competing for and binding to the tumor's PD-L1 (PD-1-ex); (11) The third flexible linker: a linker fragment (Linker-3) that provides the connection function between PD-1-ex and Anti CD3 scFv components and gives CiTEs the required flexibility; (12) CD3 single-chain antibody region: Refer to the single-chain variable fragment (Anti CD3 scFv) in the CD3-specific antibody OKT-3 that specifically recognizes and binds to the target protein, which provides the target T cell specificity for CiTEs.

[0035] When the recombinant T cells are able to express the above-mentioned fusion protein, the CiTEs protein expressed in the tumor microenvironment as the CAR-T cells proliferate can enhance the binding of anti-CPC3 CAR-T cells to target tumor cells and provide stimulation signals in vivo; the immune checkpoint inhibition of the PD-1 / PD-L1 signaling axis in the tumor microenvironment mediated by GPC3-positive tumor cells in vivo is specifically inhibited by the CiTE protein, which can enable the immune cells that play a role in tumor treatment in the body, including the body's own immune cells and CAR-T cells, to restore normal function in the tumor microenvironment and remain for a long time, thereby reducing immune rejection.

[0036] The nucleotide sequence of the nucleotide molecule encoding the fusion protein after adding a suitable restriction site is shown in SEQ ID No: 1.

[0037] Example 2: Construction of lentiviral vector.

[0038] The nucleotide molecule encoding the fusion protein constructed in Example 1 (SEQ ID No: 1) and the corresponding upstream primer (SEQ ID No: 10) and downstream primer (SEQ ID No: 11) were commissioned to be synthesized by Yiming (Suzhou) Cell Biotechnology Co., Ltd. After synthesis, the DNA solution was delivered by ultra-low temperature freezing. Before use, the delivered primers were diluted with nuclease-free water to a DNA concentration of 12.5 μmol / L, and stored in aliquots at -80°C.

[0039] The following plasmids were selected to construct lentiviral vectors: pCDH-EF1 with a nucleotide sequence as shown in SEQ ID No: 12, psPAX2 with a nucleotide sequence as shown in SEQ ID No: 13, and pMD2.G with a nucleotide sequence as shown in SEQ ID No: 14, and they were commissioned to be synthesized by GenScript Biotech Co., Ltd. After synthesis, the company transduced them into TransStbl3 Chemically Competent Cells (Stbl3 competent cells) for delivery.

[0040] (1) Resuscitation, expansion and cryopreservation of competent cells: (1-1) Prepare LB Agar solid culture medium (preferably 2% agarose concentration) and LB liquid culture medium. Sterilize the prepared culture medium with moist heat before use.

[0041] (1-2) After the transduced competent cells are delivered, they are rewarmed until they are dissolved in liquid culture medium. After the inoculation recovery is complete, they are cultured in an inverted state at 37°C for 16 to 20 hours. The culture medium becomes turbid when observed.

[0042] (1-3) Aseptically transfer the cultured bacterial suspension and inoculate it into LB Agar solid medium. Incubate the medium at 37°C for 16-20 hours. After observing small colonies, store the medium at 4°C.

[0043] (1-4) Aseptically add ampicillin to the LB liquid medium prepared above at a volume ratio of 1:1000 and mix well to form the LB screening medium.

[0044] (1-5) Aseptically select a single clone from the solid culture medium and inoculate it into liquid LB screening medium. Incubate the culture medium at 37°C in a constant temperature shaker at 225 rpm for 5 hours until the medium becomes turbid.

[0045] (1-6) The confirmed positive bacterial solution was centrifuged at 12,000 rpm for 3 minutes for subsequent construction of recombinant plasmids; or the precipitate was resuspended in 50% glycerol and frozen for long-term storage at -80°C for future use.

[0046] (2) Plasmid extraction and purification: The three plasmids were extracted in large quantities using the TIANGEN Endotoxin-free Plasmid Extraction Kit (Enhanced, DP120). The steps are as follows: (2-1) Prepare the working solution by adding RNase A (10 mg / mL) to solution P1.

[0047] (2-2) The bacterial pellet of the engineered plasmid was thoroughly centrifuged to remove all the supernatant. Solution P1 was added to the pellet and vortexed to mix well. Then, an equal volume of solution P2 was added.

[0048] (2-3) Invert thoroughly to allow the bacteria to fully lyse and place at room temperature for 5 minutes. Then add solution P2 and invert thoroughly to mix until a white flocculent precipitate appears.

[0049] (2-4) The plasmid suspension was placed at room temperature for 10 minutes, and centrifuged at 8228×g for 10 minutes to precipitate the white flocs. The plasmid suspension was poured into filter CS1 to recover the filtrate.

[0050] (2-5) After adding equilibrium solution BL to adsorption column CP6, centrifuge at 8228×g for 10 min and discard the effluent.

[0051] (2-6) Add endotoxin-free solution ER to the plasmid suspension and mix until it turns transparent yellow. Add 0.2 times the volume of isopropanol, mix well and transfer to the equilibrated adsorption column CP6. Centrifuge at room temperature at 8228×g for 2 minutes and discard the effluent.

[0052] (2-7) Add buffer ED to the loaded adsorption column CP6 and centrifuge at room temperature at 8228×g for 2 minutes. Discard the flow-through and repeat the above steps twice.

[0053] (2-8) Add rinse solution PW to the adsorption column CP6 loaded with sample and centrifuge at room temperature at 8228×g for 2 minutes. Discard the flow-through and repeat the above operation twice.

[0054] (2-9) Centrifuge the adsorption column CP6 at 8228×g for 2 minutes at room temperature, discard the effluent, open the lid and let it dry; drip elution buffer TB onto the adsorption membrane in mid-air and let it stand at room temperature for 5 minutes.

[0055] (2-10) The extracted plasmid solution was sampled and the concentration and purity of DNA were detected using the Qubit 4 Fluorometer Nucleic Acid Protein Fluorescence Quantification System and the Qubit dsDNA High Sensitivity (HS) Kit (32851) was used for detection.

[0056] (2-11) Centrifuge the adsorption column at 8228×g for 5 minutes at room temperature to collect the eluate, transfer it to -20℃ for storage or directly use it to construct the recombinant target plasmid or for lentiviral transfection vector.

[0057] (3) PCR amplification of the nucleotide molecule: The nucleotide molecules were dissolved in nuclease-free water to a DNA concentration of 1 μg / μL. The PCR reaction system was prepared according to the 25 μL system. The reaction system was constructed using the technical solution of TIANGEN 2×Pfu PCR premix reagent (KP201) (as shown in Table 1).

[0058]

[0059] The constructed reaction was subjected to PCR reaction cycles according to Table 2.

[0060]

[0061] After the reaction is completed, the recovered product is stored at -80°C for the next production.

[0062] (4) Purification and recovery of the nucleotide molecules: The amplified product of the nucleotide molecule is recovered using the TIANGEN Universal DNA Purification Recovery Kit (DP214). The operation steps are as follows: (4-1) Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column CB2, centrifuge at 13400 × g for 1 min, and discard the waste liquid.

[0063] (4-2) Add an equal volume of solution PC according to the volume of the PCR product and mix well.

[0064] (4-3) Add the mixed product solution to the equilibrated adsorption column CB2 and place it at room temperature for 2 minutes; centrifuge at 13400×g for 1 minute and discard the waste liquid.

[0065] (4-4) Add 600 μL of rinse solution PW (with anhydrous ethanol added) to the adsorption column CB2 and centrifuge at 13400×g for 1 min. Discard the waste liquid.

[0066] (4-5) Add 600 μL of rinse solution PW (with anhydrous ethanol added) to the adsorption column CB2 again, centrifuge at 13400×g for 1 min, and discard the waste liquid.

[0067] (4-6) Centrifuge the loaded adsorption column CB2 at 13400×g for 2 minutes, discard the waste liquid, and dry it at room temperature for 10 minutes.

[0068] (4-7) Add the same volume of elution buffer EB as the PCR product solution (not less than 30 μL) to the adsorption column CB2, let it stand at room temperature for 2 minutes, centrifuge at 13400×g for 2 minutes, and collect the effluent DNA solution aseptically.

[0069] (4-8) The recovered DNA solution was tested for DNA concentration and purity using the Qubit 4 Fluorometer Nucleic Acid Protein Fluorescence Quantification System and the Qubit dsDNA High Sensitivity (HS) Kit (32851) was used for detection.

[0070] (4-9) The recovered product was stored at -20 °C for the next step of production.

[0071] (5) Construction of transduction plasmid: (5-1) Enzymatic cleavage and recovery of the nucleic acid molecule.

[0072] The amplified product of the nucleic acid molecule has been cut with 5' restriction site (BamI) and 3' restriction site (NotI) at both ends. Therefore, BamHI (10 U / µL) restriction endonuclease (Thermo Scientific TM , Catalog No.: ER0051) and NotI (10 U / µL) restriction endonuclease (Thermo Scientific TM , Catalog No.: ER0592) for double enzyme digestion.

[0073] The amplified product was dissolved in nuclease-free water until the DNA concentration was greater than 1 μg / μL. A double enzyme digestion reaction system was prepared according to the system per 20 μL (as shown in Table 3).

[0074]

[0075] After the reaction system is fully mixed, the reaction is incubated at 37°C in a PCR instrument for 16 hours. After the enzyme digestion is completed, 80% of the reaction tube is incubated for 20 minutes to inactivate the reaction enzyme.

[0076] The double enzyme digestion products of the nucleic acid molecules were recovered by gel excision using the TIANGEN Universal DNA Purification Recovery Kit (DP214) combined with 0.5% agarose gel electrophoresis. The operation steps are as follows: (5-1-1) Weigh 0.5 g of agarose and add it to a 100 mol / L TAE solution. Microwave until the solid particles are dissolved. Add 4 μL of EB solution and shake evenly.

[0077] (5-1-2) When the gel is cooled to 50°C, place it in the gel tank and insert the sample comb. After the gel is formed, remove the sample comb and transfer it to the electrophoresis tank filled with TAE solution.

[0078] (5-1-3) Use a pipette to load the reaction product into the gel loading well and add a control DNA marker to indicate the corresponding base length.

[0079] (5-1-4) Start electrophoresis at a voltage of 110 V until only the dye moves to the bottom of the agarose gel. Take out the gel and place it on a UV transilluminator to observe the electrophoresis results. Figure 2 shown.

[0080] (5-1-5) Use a gel cutter to cut the agarose gel from the target fragment position (about 3679 bp), weigh it, add an equal volume of solution PC according to the weight of the gel block, mix well, and place in a 50℃ water bath for 10 minutes until the gel block is fully dissolved.

[0081] (5-1-6) Add the mixed gel cutting product solution to the equilibrated adsorption column CB2 and place it at room temperature for 2 minutes. Centrifuge at 13400×g for 1 minute and discard the waste liquid.

[0082] (5-1-7) Add 600 μL of rinse solution PW (with anhydrous ethanol added) to the adsorption column CB2 and centrifuge at 13,400 × g for 1 minute. Discard the waste liquid.

[0083] (5-1-8) Add 600 μL of rinse solution PW (with anhydrous ethanol added) to the adsorption column CB2 again. Let it stand for 5 minutes and then centrifuge at 13400×g for 1 minute. Discard the waste liquid.

[0084] (5-1-9) Centrifuge the loaded adsorption column CB2 at 13400×g for 2 minutes, discard the waste liquid, and dry it at room temperature for 10 minutes.

[0085] (5-1-10) Add the same volume of elution buffer EB as solution PC (not less than 30 μL) to the adsorption column CB2, and leave it at room temperature for 2 minutes. Centrifuge at 13400×g for 2 minutes and aseptically collect the effluent DNA solution.

[0086] (5-1-11) The recovered DNA solution was tested for DNA concentration and purity using the Qubit 4 Fluorometer Nucleic Acid Protein Fluorescence Quantification System and the Qubit dsDNA High Sensitivity (HS) Kit (32851) was used for detection.

[0087] (5-1-12) Recover the complete double-enzyme digestion product and store it at -20℃ for the next production step.

[0088] (5-2) Double restriction enzyme digestion of pCDH-EF1.

[0089] The pCDH-EF1 was designed to have 5' restriction site (BamI) and 3' restriction site (NotI) added to both ends of the structure carrying the target exogenous gene. Therefore, BamHI (10 U / µL) restriction endonuclease (ThermoScientific TM, Catalog No.: ER0051) and NotI (10 U / µL) restriction enzymes (Thermo Scientific TM , Catalog No.: ER0592) for double enzyme digestion.

[0090] The plasmid purified in step (2) was dissolved in nuclease-free water until the DNA concentration was greater than 1 μg / μL. A double restriction enzyme digestion reaction system was prepared according to the 20 μL system (as shown in Table 4).

[0091]

[0092] After the reaction system is fully mixed, the reaction is incubated at 37°C in a PCR instrument for 16 hours. After the enzyme digestion is completed, 80% of the reaction tube is incubated for 20 minutes to inactivate the reaction enzyme.

[0093] (5-3) Recovery of pCDH-EF1.

[0094] The pCDH-EF1 double-digestion product was recovered by gel excision using the TIANGEN Universal DNA Purification Recovery Kit (DP214) combined with 0.5% agarose gel electrophoresis. The steps are as follows: (5-3-1) Weigh 0.5 g of agarose and add it to 100 mol / L TAE solution. Microwave until the solid particles are dissolved. Add 4 μL of EB solution and shake evenly.

[0095] (5-3-2) When the gel is cooled to 50°C, place it in the gel tank and insert the sample comb. After the gel is formed, remove the sample comb and transfer it to the electrophoresis tank filled with TAE solution.

[0096] (5-3-3) Use a pipette to load the reaction product into the gel loading well and add a control DNA marker to indicate the corresponding base length.

[0097] (5-3-4) Start electrophoresis at a voltage of 110 V until only the dye moves to the bottom of the agarose gel. Take out the gel and place it on a UV transilluminator to observe the electrophoresis results. Figure 3 shown.

[0098] (5-3-5) Use a gel cutter to cut the agarose gel from the target fragment position (about 7128 bp), weigh it, add an equal volume of solution PC according to the weight of the gel block, mix well, and place in a 50℃ water bath for 10 minutes until the gel block is fully dissolved.

[0099] (5-3-6) Add the mixed gel cutting product solution to the equilibrated adsorption column CB2 and place it at room temperature for 2 minutes. Centrifuge at 13400×g for 1 minute and discard the waste liquid.

[0100] (5-3-7) Add 600 μL of rinse solution PW (with anhydrous ethanol added) to the adsorption column CB2 and centrifuge at 13400×g for 1 minute. Discard the waste liquid.

[0101] (5-3-8) Add 600 μL of rinse solution PW (with anhydrous ethanol added) to the adsorption column CB2 again. Let it stand for 5 minutes and then centrifuge at 13400×g for 1 minute. Discard the waste liquid.

[0102] (5-3-9) Centrifuge the loaded adsorption column CB2 at 13400×g for 2 minutes, discard the waste liquid, and dry it at room temperature for 10 minutes.

[0103] (5-3-10) Add the same volume of elution buffer EB as solution PC (not less than 30 μL) to the adsorption column CB2, and leave it at room temperature for 2 minutes. Centrifuge at 13400×g for 2 minutes, and collect the effluent DNA solution aseptically.

[0104] (5-3-11) The recovered DNA solution was tested for DNA concentration and purity using the Qubit 4 Fluorometer Nucleic Acid Protein Fluorescence Quantification System and the Qubit dsDNA High Sensitivity (HS) Kit (32851) was used for detection.

[0105] (5-3-12) Recover the pCDH-EF1 double-digestion product and store it at -20℃ for the next production.

[0106] (5-4) The nucleotide molecule is connected to pCDH-EF1.

[0107] The purified nucleotide molecule double digestion product was recovered and connected with the pCDH-EF1 double digestion product to construct a transduction plasmid for subsequent lentiviral vector production. Because the cuts at both ends of the double digestion product were produced by BamHI and NotI restriction enzymes, T4 DNA ligase (5 U / μL) (Brand: Invitrogen TM , catalog number: 15224041) to connect the two sticky ends.

[0108] The corresponding technical solution is to adjust the nucleotide molecule double digestion product and the pCDH-EF1 double digestion product to 10 pmol / mL with nuclease-free water according to the nucleic acid concentration test results. Prepare the T4 DNA ligase reaction system according to each 20 μL system (as shown in Table 5).

[0109]

[0110] After the reaction system was fully mixed, the reaction was incubated in a PCR instrument at 26°C for 1 hour.

[0111] The constructed transduction plasmid was recovered by gel excision using the TIANGEN Universal DNA Purification Recovery Kit (DP214) combined with 0.5% agarose gel electrophoresis. The steps are as follows: (5-4-1) Weigh 0.5 g agarose and add it to 100 mol / L TAE solution. Microwave until the solid particles are dissolved. Add 4 μL EB solution and shake evenly.

[0112] (5-4-2) When the gel is cooled to 50°C, place it in the gel tank and insert the sample comb. After the gel is formed, remove the sample comb and transfer it to the electrophoresis tank filled with TAE solution.

[0113] (5-4-3) Use a pipette to load the reaction product into the gel loading well and add a control DNA marker to indicate the corresponding base length.

[0114] (5-4-4) Start electrophoresis at a voltage of 110 V until only the dye moves to the bottom of the agarose gel. Take out the gel and place it on a UV transilluminator to observe the electrophoresis results. Figure 4 shown.

[0115] (5-4-5) Use a gel cutter to cut the agarose gel from the target fragment position (about 10807 bp), weigh it, add an equal volume of solution PC according to the weight of the gel block, mix well, and place in a 50℃ water bath for 10 minutes until the gel block is fully dissolved.

[0116] (5-4-6) Add the mixed gel cutting product solution to the equilibrated adsorption column CB2 and place it at room temperature for 2 minutes. Centrifuge at 13400×g for 1 minute and discard the waste liquid.

[0117] (5-4-7) Add 600 μL of rinse solution PW (with anhydrous ethanol added) to the adsorption column CB2 and centrifuge at 13,400 × g for 1 minute. Discard the waste liquid.

[0118] (5-4-8) Add 600 μL of rinse solution PW (with anhydrous ethanol added) to the adsorption column CB2 again. Let it stand for 5 minutes and then centrifuge at 13400×g for 1 minute. Discard the waste liquid.

[0119] (5-4-9) Centrifuge the loaded adsorption column CB2 at 13400×g for 2 minutes, discard the waste liquid, and dry it at room temperature for 10 minutes.

[0120] (5-4-10) Add the same volume of elution buffer EB as solution PC (not less than 30 μL) to the adsorption column CB2, and leave it at room temperature for 2 minutes. Centrifuge at 13400×g for 2 minutes, and collect the effluent DNA solution aseptically.

[0121] (5-4-11) The recovered DNA solution was tested for DNA concentration and purity using the Qubit 4 Fluorometer Nucleic Acid Protein Fluorescence Quantification System and the Qubit dsDNA High Sensitivity (HS) Kit (32851) was used for detection.

[0122] (5-4-12) Recover the purified transduction plasmid and store it at -20℃ for the next production step.

[0123] (6) Virus packaging and purification: (6-1) HEK293T cells were selected as packaging cells. The corresponding production cells were purchased from ATCC, and the corresponding working cell bank was established within the company and stored at ultra-low temperature before use.

[0124] Prepare complete culture medium for HEK293T cells: DMEM high glucose medium (Gibco TM , Catalog No.: 11965092) + 10% fetal bovine serum (Gibco TM , Catalog No.: 10100147C) + 4 mmoul / L L-glutamine (Invitrogen, Catalog No.: 11360-070) + 1 mmoul / L MEM sodium pyruvate and trypsin-EDTA (0.25%), containing phenol red (Gibco TM , item number: 25200072).

[0125] The frozen cells were resuspended in HEK293T complete cell culture medium after thawing in a 37°C water bath, and washed twice by centrifugation at 300×g for 6 minutes. The cell pellet was recovered and resuspended in complete culture medium and counted according to 1.5×10 4 cells / cm 2 Inoculate into cell culture flasks or multi-layer cell factory culture vessels.

[0126] HEK293T cells were cultured at 37°C and 5% CO2 until the confluence rate was greater than 90%. The complete medium residue was removed by brushing and digested with trypsin-EDTA (0.25%). The cell suspension recovered by digestion was terminated with complete medium and centrifuged at 300×g for 6 minutes and washed twice. The recovered cell pellet was resuspended in complete medium and counted according to 6×10 4 cells / cm 2 Inoculate into cell culture flasks or multi-layer cell factory culture vessels.

[0127] When HEK293T cells are cultured at 37°C, 5% CO2 for 24 hours until the fusion rate reaches 80%, they can be used for subsequent lentiviral packaging.

[0128] (6-2) Configuration of plasmid transfection system.

[0129] The plasmid transfection scheme is based on the common principle of polyethyleneimine (PEI) cationic polymer transfection. PEI40000 (also known as PEIMAX) was selected for plasmid co-transfection. The corresponding transfection system is Polyethylenimine Linear (PEI) MW40000 (rapid lysis) Linear PEI transfection reagent (rapid lysis) MW40000 (brand: YEASEN, item number: 40816ES02 / 40816ES03). Follow the instructions for use before use. Gibco TM Opti-MEM TM Dissolve in reduced serum medium to prepare a 1 mg / mL stock solution. 2 Add 1 mL of transfection basic mix. The corresponding system is shown in Table 6.

[0130]

[0131] After dissolving the corresponding plasmid DNA product in the above mixed system using reduced serum medium, add 38 μL PEI transfection reagent MW40000 (1 mg / mL) per 1 mL, vortex mix, and let stand at room temperature for 30 minutes to obtain transfection mix.

[0132] (6-3) Transfection of 293T cells.

[0133] The HEK293T cells used for co-transfection were thoroughly washed with PBS buffer to remove residual FBS and then added with Opti-MEM TM The cells were washed once with serum-reduced medium and cultured for 2 hours.

[0134] Remove all liquid and add transfection mix, according to the ratio of 75 cm 2 Add 1 mL of transfection mix solution and mix well, then culture at 37°C, 5% CO2 for 8 hours.

[0135] 8 hours after transfection, pour out the transfection mix and place it on each 75 cm 2 Add 10 mL PBS to wash the mixture and discard. Supplement HEK293T cells with complete culture medium and incubate at 37°C, 5% CO2 for 72 hours.

[0136] According to every 75 cm 2 15 mL of complete culture medium was added to collect all the culture supernatant, and after centrifugation at 300×g for 6 minutes to remove the detached cells, all the supernatant was recovered to produce the viral vector.

[0137] (6-4) Viral vector purification and storage.

[0138] The recovered virus solution can be temporarily stored at -70℃ for short-term storage before production. The collected virus solution is centrifuged to remove the detached cells and then filtered and sterilized with a 0.22 μm PES filter. After sterilization, it can be temporarily stored at -70℃ for short-term storage.

[0139] (6-4-1) Add the filtered virus solution into Merck Centricon Plus-70 100 kDa ultracentrifuge tubes and centrifuge at 25,000 rpm for 1.5 hours at 4°C to recover the precipitate at the bottom.

[0140] (6-4-2) Resuspend the virus precipitate in 50 mL of sterile PBS and add it to a Centricon Plus-70 centrifugal filter (Millipore, Catalog No.: UFC700308, Specification: 3 kDa cutoff, 15 mL to 70 mL volume). Seal and perform ultrafiltration.

[0141] (6-4-3) Ultrafiltration conditions are 4°C, 3500×g centrifugation for 20 minutes to the predetermined concentrated volume. After the centrifugation, remove the centrifuge device, and fully resuspend and wash the lentiviral product on the retention membrane with the corresponding volume of lentiviral vector cryopreservation protection solution (provided by Jiangsu Yiming Biotechnology Co., Ltd.)

[0142] (6-4-4) The lentiviral suspension obtained was packaged and stored at -80°C, and samples were taken for virus titer detection.

[0143] (6-4-5) The expected virus MOI is greater than 20 and the virus titer is greater than 5.22×10 7 / mL, and prepare the lentivirus working solution for transfection after aliquoting.

[0144] (6-5) Virus titer detection: The designed lentivirus product is used to infect T lymphocytes, so the target cells used for infection titer detection are Jukart cells. The corresponding cell lines were purchased from ATCC, and the corresponding working cell bank was established within the company and stored at ultra-low temperature before use.

[0145] (6-5-1) Prepare Jukart cell complete culture medium: RPMI 1640 culture medium, GlutaMAX TM Additive, HEPES (Gibco TM Product number: 72400047) + 10% fetal bovine serum (Gibco TM , item number: 10100147C)+IL-2 100 IU / mL (recombinant human interleukin-2 for injection (Yuancexin), brand: Beijing Yuance Pharmaceutical Co., Ltd. National Medicine Standard No. S10980069).

[0146] (6-5-2) The frozen cells were revived in a 37°C water bath and resuspended in complete cell culture medium. The cells were centrifuged at 300×g for 6 minutes and washed twice. The cell pellet was recovered and resuspended in complete culture medium and counted according to 1×10 6 cells / mL were inoculated into cell culture flasks.

[0147] (6-5-3) After the cells were cultured at 37°C and 5% CO2 for 48 hours, they were collected and centrifuged at 300×g for 6 minutes and washed twice with PBS. The collected cell pellet was resuspended in complete medium and counted at 2×10 5 cells / mL were inoculated into a 24-well cell culture plate, with 500 μL in each well, for a total of 24 wells.

[0148] (6-5-4) The sampled virus product solution was diluted in a 10-fold gradient, and the corresponding volume of lentivirus product solution was added to each well, respectively 1 μL, 1×10 -1 μL, 1×10 -2 μL, 1×10 -3 μL, 1×10 -4 μL, with 3 replicate wells for each concentration gradient.

[0149] (6-5-5) After adding the virus solution, culture the cells at 37°C, 5% CO2 for 48 hours. Then, add 500 μL of fresh Jukart cell complete medium and culture the cells for another 96 hours.

[0150] (6-5-6) After transfection, the cultured cells were recovered by centrifugation at 300 × g and washed twice with PBS. The cell pellets were added with Trizol reagent (Invitrogen TM , catalog number: 15596026) to extract total RNA.

[0151] (6-5-7) RNA extracted with chloroform was transcribed using the RevertAid RT reverse transcription kit (ThermoScientific TM , Catalog Number: K1691) were used to obtain the cDNA products of each experimental group.

[0152] (6-5-8) qRT-PCR detection was performed using the cDNA products of each group as templates, and actin was used as the internal reference gene in each group. That is, 3 qRT-PCRs were set up in each replicate well of each experimental group, and the upstream primer and the downstream primer were added for amplification. 3 replicate wells of actin internal reference were set up in each virus dilution gradient.

[0153] (6-5-9) The titer value was determined based on the difference in Ct values ​​between the internal reference actin well and each dilution ratio experimental group. The titer of the corresponding lentiviral product solution was greater than 5.22×10 7 / mL. Generally, a Ct value difference of 2 or more is considered to be significant. The corresponding titer is calculated based on the lowest dilution ratio at which a difference in Ct value is detected, and it is considered that at least one infectious virus particle exists at this dilution ratio.

[0154] Example 3: Construction of autocrine CiTEs CAR-T cells.

[0155] Prepare T lymphocyte activation complete medium and sorting buffer according to the following scheme: The formula of the complete medium for T lymphocyte activation is shown in Table 7. After preparation, it is temporarily stored at 4°C.

[0156]

[0157] The preparation scheme of sorting buffer is as follows: take 2 mL of MACS BSA Stock Solution in a 50 mL centrifuge tube, and then take 40 mL of autoMACS Rinsing Solution and mix them, that is, dilute them at a ratio of 1:20. After dilution, filter and sterilize using a 0.22 μm filter, collect the filtrate, and store it at 4°C before use after preparation.

[0158] The formula of the complete medium for T lymphocyte expansion is shown in Table 8. After preparation, it is temporarily stored at 4°C.

[0159]

[0160] (1) Initial white blood cell collection: After the hepatocellular carcinoma patients who are suitable for treatment with this technology are confirmed by epidemiological tests, heparin anticoagulated peripheral blood is drawn for subsequent autocrine CiTEs CAR-T cell production, or a medical institution with leukocyte component apheresis qualifications performs apheresis on the patients. The volume of the collected leukocyte suspension is greater than 100 mL, and the anticoagulant during collection is a blood preservation solution containing sodium citrate, and the transportation temperature is between 2 and 24°C. The peripheral blood and apheresis products do not undergo a frozen environment during transportation.

[0161] (2) T lymphocyte sorting and activation: (2-1) Transfer the collected autologous peripheral blood or single-collected leukocyte product from the patient into a sterile centrifuge tube under Class A cleanliness conditions in accordance with aseptic operation requirements.

[0162] (2-2) Centrifuge the cells at 1800 rpm for 10 minutes (acceleration: 9, deceleration: 5). After centrifugation, separate and discard the yellow plasma on the top layer. Add 1.5 times the original volume of physiological saline to the cell pellet and dilute and mix.

[0163] (2-3) After dilution, the cells were evenly added to the human peripheral blood lymphocyte separation solution (prepared by FICOLL) (TBD Tianjin Haoyang, catalog number: LTS1077). The cell suspension was added to the upper layer at a volume ratio of cell suspension: human lymphocyte separation solution = 25:15.

[0164] (2-4) Perform density gradient centrifugation on the cells. The centrifugation conditions are: 20°C, 800×g horizontal gradient centrifugation for 20 minutes (acceleration 1, deceleration 0). Observe the cell stratification, aspirate the cells in the buffy coat layer, add sufficient physiological saline to wash twice, centrifuge at 1500 rpm for 10 minutes (acceleration 9, deceleration 5), and discard the supernatant.

[0165] (2-5) Blow the cell pellet evenly with 100 mL of normal saline and take a 200 μL sample to count the cell density and viability.

[0166] (2-6) The total amount of recovery is greater than 1.8×10 8 The cells were centrifuged at 1500 rpm for 10 minutes (acceleration 9, deceleration 5), the supernatant was discarded, the suspension was resuspended with T lymphocyte activation complete medium, and then inoculated into a cell culture flask, and cultured in a 37°C, 5% CO2 incubator for 1 hour.

[0167] (2-7) After the cells adhere to the wall, gently flip the culture flask to recover the suspended cells and take samples to count them. Confirm that the total number of recovered cells is greater than 8×10 7 cells.

[0168] (2-8) Pipette the corresponding volume of CTS according to 3 times the number of cells TM (Cell Therapy System) Dynabeads TM CD3 / CD28 magnetic bead suspension (Brand: Gibco TM , Catalog No.: 40203D). The magnetic beads were diluted with sorting buffer and then DynaMag TM -15 magnet (Brand: Invitrogen TM , Product No.: 12301D) adsorption recovery.

[0169] (2-9) Centrifuge the recovered cells at 1500 rpm for 6 minutes (acceleration 9, deceleration 9), discard the supernatant and resuspend in sorting buffer precooled to 4°C, and rinse the cell suspension with CD3 / CD28 magnetic beads.

[0170] (2-10) The mixed magnetic beads were transferred to a small volume aeration tube and placed in a refrigerator for incubation for 30 minutes. The mixture of magnetic beads and cells was recovered and diluted with sorting buffer and washed and combined into the same centrifuge tube.

[0171] (2-11) Using DynaMag TM-15 magnet to recover the sorted positive cells, and gently wash with sorting buffer to remove negative cells. Recovered positive cells are resuspended in T lymphocyte activation complete medium and inoculated into cell culture bottles, and placed in a 37°C, 5% carbon dioxide incubator for 24-48 hours.

[0172] (3) Lentiviral transfection: (3-1) After washing and recovery of the sorted and cultured T lymphocytes, 100 μL of the samples were diluted and combined for counting, and 3×10 6 T cells as control T cells (Mock-T group) were inoculated into culture flasks and cultured with complete T lymphocyte activation medium for quality inspection.

[0173] (3-2) The remaining cells were centrifuged at 1500 rpm for 6 minutes (acceleration 9, deceleration 9), and the supernatant was discarded. The cell pellet was resuspended to 3×10 in T lymphocyte activation complete medium according to the cell count results. 6 cells / mL density.

[0174] (3-3) According to the number of cells and the virus titer, the transfection lentivirus prepared from the lentiviral vector described in Example 2 was taken out from the ultra-low temperature refrigerator and reconstituted at 4°C.

[0175] (3-4) Take Polybrene (hexadimethrine bromide) (10 mg / mL) (brand: YEASEN, catalog number: 40804ES76) and mix it with T lymphocyte activation complete medium at a volume ratio of 1:500 to prepare Polybrene working solution.

[0176] (3-5) According to the requirement of 50 μL of anti GPC3-CAR-CiTE transfection lentivirus solution for every 400 μL of cell suspension, the above-prepared Polybrene working solution and anti GPC3-CAR-CiTE transfection lentivirus solution are first mixed and then gently added to the surface of the cell suspension.

[0177] (3-6) After adding polybrene to the mixture of virus and cells, reduce shaking and place it in a 37°C, 5% CO2 incubator for 24 to 48 hours.

[0178] (3-7) Add the same volume of T lymphocyte activation complete medium to mix and dilute the virus solution. After dilution, place the cells in a 37°C, 5% CO2 incubator for 24-48 hours to obtain autocrine CiTEs CAR-T cells.

[0179] (4) Control T cell expansion: The control T cells sorted above do not need to be transfected with lentivirus and continue to be cultured with rehydration. Observe the growth under a microscope every 48 hours and gently shake to disperse the cell aggregates. Take 30 μL of the sample to count the cell number and viability.

[0180] After each count, evenly blow away the cell aggregates, add T lymphocyte activation complete medium and resuspend the cells to 0.5×10 6 cells / mL to 1×10 6 The cell suspension was inoculated into a cell culture flask and placed in a 37°C, 5% CO2 incubator for 48-72 hours. The total culture period was 11 to 16 days.

[0181] (5) Autocrine CiTEs CAR-T cell expansion: (5-1) After the lentiviral transfection is completed, the cells are evenly blown and recovered into a centrifuge tube. 30 μL of the sample is taken to count the number of cells. The recovered cells are centrifuged at 1500 rpm for 6 minutes (acceleration 9, deceleration 9). After discarding the supernatant, T lymphocyte expansion complete medium is added to resuspend the cells to 1×10 6 The cell suspension was inoculated into a cell culture flask and placed in a 37°C, 5% CO2 incubator for 48-72 hours.

[0182] (5-2) After the medium is replaced, the autocrine CiTEs CAR-T cells and control T cells are cultured in a breathable cell culture flask or cell culture bag and placed in a 37°C, 5% CO2 incubator. The growth is observed under a microscope every 48 hours, and the cell aggregates are gently shaken to disperse. 30 μL of the sample is taken to count the number of cells and the purity of the target cells is detected by flow cytometry.

[0183] After each count, evenly blow away the cell aggregates, add T lymphocyte expansion complete medium and resuspend the cells to 0.5×10 6 cells / mL to 1×10 6 cells / mL density.

[0184] The cell suspension was inoculated into a cell culture flask and cultured in a 37°C, 5% CO2 incubator for 48 to 72 hours, for a total of 11 to 16 days.

[0185] Example 4: Method for using autocrine CiTEs CAR-T cells.

[0186] (1) Cell centrifugation enrichment: Culture until the total number of cells is greater than 5×10 8After the cells were separated, all cells were recovered and combined. The recovered cells were centrifuged at 1500 rpm for 6 minutes (acceleration 9, deceleration 9), the supernatant was discarded and resuspended in Bomeili A compound electrolyte injection containing 1% human albumin for injection. The cell suspension was centrifuged again at 1500 rpm for 6 minutes (acceleration 9, deceleration 9), the supernatant was discarded and resuspended in Bomeili A compound electrolyte injection containing 1% human albumin for injection to a small volume.

[0187] (2) Removal of magnetic beads: The autocrine CiTEs CAR-T cell suspension was homogenized and loaded into a 50 mL centrifuge tube. The centrifuge tube containing the cell suspension was placed into the DynaMag TM -50 Magnet (Brand: Invitrogen TM , Catalog No.: 12302D), recover all the cell suspension that has been adsorbed. After washing the adsorbed magnetic beads, recover all the cells in the cell suspension and combine them. Centrifuge the recovered cells at 1500 rpm for 6 minutes (acceleration 9, deceleration 9), discard the supernatant and resuspend to 45 mL with Bomeili A compound electrolyte injection containing 1% human albumin for injection.

[0188] After blowing again, load into 50 mL centrifuge tube and use DynaMag TM -50 magnet adsorption to remove magnetic beads and recover all unadsorbed cell suspension.

[0189] (3) Cell cryopreservation: 30 μL of the recovered autocrine CiTEs CAR-T cell suspension was sampled for cell number counting and flow cytometry detection of target cell purity. The recovered cells were centrifuged at 1500 rpm for 6 minutes (acceleration 9, deceleration 9), the supernatant was discarded, and the cells were resuspended to 40 mL with cell freezing solution.

[0190] The corresponding cell freezing solution preparation scheme is shown in Table 9, and it is stored at 4°C in the dark after preparation.

[0191]

[0192] The washed cells were centrifuged again at 1500 rpm for 6 minutes (acceleration 9, deceleration 9), the supernatant was discarded and the cells were resuspended in cell freezing solution to 30 mL. After retaining 2 mL of the sample for sterility testing, all the remaining cell suspension was drawn with a syringe and filled into the cell freezing bag CryoMACS Freezing Bag 50 (brand: Miltenyi, Germany, item number: 200-074-400).

[0193] The cells were sealed using Thermo Scientific TMCryoMed TM The sample is programmed to cool to -80°C in a controlled rate freezer and then temporarily stored in a meteorological liquid nitrogen tank for ultra-low temperature storage until use for treatment.

[0194] (4) Patient pretreatment: Patients with confirmed hepatocellular carcinoma approved by the institutional review board of the participating institution, who are not suitable for surgery or local treatment, and have failed or cannot tolerate standard systemic treatment, and have no effective treatment at the time of enrollment. At the same time, the patient's tumor tissue sample is GPC3 positive (immunohistochemistry test), and their T lymphocytes are obtained to prepare the autocrine CiTEs CAR-T cells shown in the above technical scheme.

[0195] Lymphodepleting chemotherapy: Fludarabine (30 mg / m 2 body surface area, 4 days) and cyclophosphamide (500 mg / m 2 The autocrine CiTEs CAR-T cells were intravenously infused within 3 days after lymphocyte pretreatment.

[0196] (5) Medication treatment: Autocrine CiTEs CAR-T cell input dose on the day of treatment: 2~5×10 6 CAR-T / kg body weight, the proportion of autocrine CiTEs CAR-T cells is not less than 10%, and the proportion of live cells in autocrine CiTEs CAR-T cells is not less than 90%. The administration method is intravenous infusion: the infusion volume is 50~200 mL, and the speed is 10 mL / minute.

[0197] After the autocrine CiTEs CAR-T cell preparation product prepared above was rewarmed in a 37°C water bath until there was no visible solid matter in the cryopreservation bag, the product liquid was drawn into the bagged sodium chloride injection solution by syringe, and then washed and combined into the same bag. An intravenous channel was established to infuse all the cell suspension.

[0198] Example 5: Detection and verification of autocrine CiTEs CAR-T cells.

[0199] Taking a cell sample collected from a patient with advanced hepatocellular carcinoma as an example, nucleated cells were separated from the patient's leukocyte apheresis product by density gradient centrifugation, and autocrine CiTEs CAR-T cells used for subsequent treatment were prepared according to the method provided in Example 3.

[0200] (1) In vitro preparation efficiency of autocrine CiTEs CAR-T cells: During the isolation and culture process, a Cellometer K2 automatic cell counter was used to count the number of cell proliferation and viability. Figure 5 As shown, the cell viability changes as Figure 6 As shown. The autocrine CiTEs CAR-T cells can achieve 594.5-fold expansion in vitro, and can produce corresponding cells for disease treatment to achieve the goal of technical design.

[0201] (2) Positive expression rate of autocrine CiTEs CAR-T cells: After lentiviral transfection, i.e., on the 7th and 15th days of in vitro culture of autocrine CiTEs CAR-T cells, 1×10 6 The cells suspension is analyzed by flow cytometry to detect the proportion of CD3+T lymphocytes and the proportion of cells expressing the nucleotide molecule.

[0202] The corresponding detection method is to collect the autocrine CiTEs CAR-T cell suspension and the control T cell suspension, each greater than 1×10 6 cells. After being dispersed into a cell suspension, PBS was added for dilution and centrifuged at 1500 rpm for 6 minutes (acceleration 9, deceleration 9). The complete medium components were removed and resuspended in PBS. Each type of cell was evenly divided into 2 tubes and centrifuged again. Take one tube of centrifuged cell sediment from each group and add the following flow cytometry working solution to incubate at 4℃ for 30 minutes for staining. The staining solution formula is shown in Table 10.

[0203]

[0204] After all staining is completed, the cell suspension is diluted with PBS and centrifuged at 1500 rpm for 6 minutes (acceleration 9, deceleration 9) to remove the staining solution. Each tube of cells in each group is resuspended in PBS to 100 μL and tested using a flow cytometer. The test results are shown in the figure. Figure 7 The test results showed that the GPC3 CAR positive rate of the Mock-T group was 0.03%, and the CD3+ positive rate was 98.78%; the GPC3 CAR positive rate of the autocrine CiTEsCAR-T cell group was 65.67%, and the CD3+ positive rate was 99.12%.

[0205] (3) Autocrine CiTEs CAR-T cells express and secrete CiTEs, which specifically reduce the binding activity of PD-1 and PD-L1: The PD-1:PD-L1 [Biotinylated] Inhibitor Screening ELISA Kit (Cat. No.: EP-133) from Biopsy Group was used to detect the inhibitory effect of CiTE protein in the culture supernatant on the binding activity of PD-1 and PD-L1. The corresponding cell culture supernatant was filtered at 0.22 μm to remove cells and cell debris before reconstitution and loading. The corresponding detection method can be briefly described as follows: (3-1) Dissolve the human PD-L1 protein, biotinylated PD-1 protein, anti-PD-L1 neutralizing antibody, streptavidin-HRP reagent and chromogenic substrate provided in the kit to prepare a working solution, wherein the anti-PD-L1 neutralizing antibody is diluted in a concentration gradient as a standard curve group; (3-2) Coating the bound PD-L1 protein on a high affinity plate and blocking it; (3-3) After washing the well plate, add the cell culture supernatant to be tested (autocrine CiTEs CAR-T cell group and Mock-T group) and the standard curve group - anti-PD-L1 neutralizing antibody working solution, incubate and bind, and then elute; (3-4) Add biotinylated PD-1 protein to bind to PD-L1 coated on the bottom of the well plate, incubate and then elute; (3-5) Add streptavidin-HRP, followed by TMB or other colorimetric HRP substrate; (3-6) The half-maximal inhibitory concentration of the compound in the test sample for binding to PD-1:PD-L1 will be determined by comparing the 450 nm absorbance readings between different experimental groups.

[0206] The results of the specificity test of CiTE in reducing the binding activity of PD-1 and PD-L1 are shown in Table 11 and Figure 8 shown.

[0207]

[0208] The statistical data of the autocrine CiTEs CAR-T cell culture supernatant containing CiTEs interfering with the PD-1:PD-L1 binding activity are shown in Table 12.

[0209]

[0210] From the test results, it can be seen that the culture supernatant of the autocrine CiTEs CAR-T cells contains biologically active substances that can inhibit the binding activity of the immune checkpoint PD-1 / PD-L1, and the corresponding neutralization inhibition rate is 25.86%. This indicates that the cells can secrete the corresponding protein after modification to specifically bind to the PD-L1 coated in the detection kit, proving that the CiTEs designed in this technology can be secreted into the culture supernatant by the prepared autocrine CiTEs CAR-T cells, and specifically inhibit PD-1 / PD-L1-mediated immune checkpoint inhibition.

[0211] Example 6: Verification of the ability of autocrine CiTEs CAR-T cells to specifically kill liver cancer tumor cells with high expression of GPC3.

[0212] The autocrine CiTEs CAR-T cells prepared in Example 5 were selected to set up an autocrine CiTEs CAR-T cell group and a Mock-T group, respectively. The sampled cells were subsequently co-cultured with target cells for specific killing ability detection.

[0213] Studies have reported that HepG2, Huh7, and LM3 liver cancer cell lines express GPC3, while the normal liver cell line HL7702 does not express GPC3. There are differences in the expression of GPC3 in HepG2, Huh7, and LM3 liver cancer cell lines. Therefore, standard cell lines collected by ATCC were purchased from suppliers. Complete culture medium was prepared with high-glucose DMEM + 10% fetal bovine serum, and the adherent culture method was adopted. The inoculation density was 0.5×10 6 cells / mL, and HepG2, Huh7, LM3 and HL7702 cells were passaged and expanded respectively and used as target cells for subsequent specific killing ability detection.

[0214] When the cells were cultured to the target number at 37°C and 5% CO2, they were washed and then lysed with trypsin-EDTA (0.25%) containing phenol red (brand: Gibco TM , Catalog No.: 25200056) to digest adherent cells. After centrifugation and washing, the recovered suspension was labeled with Glypican 3 Recombinant Rabbit Monoclonal Antibody (024), APC (Brand: Invitrogen, Catalog No.: MA5-40986) and flow cytometry was used to detect the expression of GPC3 protein in different cell lines.

[0215] The target cells identified by flow cytometry were recovered, digested, washed, resuspended and counted, and the cell count confirmed that the viability was greater than 95%. Each target cell was cultured at 2×10 4 Each well of a 96-well cell culture plate was inoculated with 50 μL of the corresponding complete medium. Then 2×10 4 cells, 1×10 5 cells and 2×10 5 The autocrine CiTEs CAR-T cells of cells are inoculated into the corresponding target cell wells. That is, the ratio of the number of effector cells to the number of target cells in the co-culture during the killing ability detection is 1:1, 5:1 and 10:1 respectively.

[0216] The above cells were cultured at 37°C, 5% CO2 concentration in the dark for 4 hours, and then stained and detected using the lactate dehydrogenase (LDH) release method LDH Assay Kit (Cytotoxicity) (Brand: abcam, Catalog No.: ab65393). The corresponding target cell control group was treated with lysate. The 96-well cell culture plate was centrifuged, and the corresponding cell culture supernatant was transferred to another 96-well plate, and the LDH reaction substrate and reaction working solution were added to react at room temperature. The optical density (OD value) of each well was measured on an enzyme-linked detector, the detection wavelength was 492 nm, and the reference wavelength was 650 nm, and the specific killing activity of the autocrine CiTEs CAR-T cells was calculated.

[0217] Test results such as Fig. 9 and Fig.10 As shown, the expression of GPC3 protein in different target cells in the killing ability detection experiment is different. The liver cancer cell lines HepG2 and LM3 liver cancer cell lines express GPC3 with higher abundance than Huh7, and the normal liver cell line HL7702 does not express it. The autocrine CiTEs CAR-T cells were co-cultured with different target cells to detect their killing effects. It was found that the normal liver cell line HL7702 that did not express GPC3 did not express no killing or low killing. It has a strong killing effect on the liver cancer cell lines HepG2 and LM3 that highly express GPC3, and the killing effect increases with the increase in the expression abundance of GPC3 and the number of effector cells.

[0218] The results showed that the autocrine CiTEs CAR-T cells could specifically kill the liver cancer cell lines expressing GPC3, and had no obvious toxicity to normal liver cells, thus meeting the purpose of technical design and having a certain degree of safety.

[0219] Example 7: Autocrine CiTEs CAR-T cells express and secrete CiTEs to activate resting T cells.

[0220] It was confirmed that the liver cancer cell line HepG2 expressed the target protein GPC3, while the normal liver cell line HL7702 did not express GPC3. The above cells were mixed and co-cultured according to Table 13. The inoculation number of HepG2 and HL7702 was 2×10 5 cells / well in a 12-well cell culture plate, and inoculate with the original complete medium 24 hours before co-culture until the cells fully adhere to the bottom of the plate.

[0221]

[0222] According to the technical implementation scheme of the present invention, PBMCs were separated from the patient's frozen leukocyte apheresis product by Ficoll density gradient centrifugation, and the concentration of PBMCs was greater than 1×10 6cells / mL were suspended and cultured in complete T lymphocyte activation medium for 24 hours at 37°C and 5% CO2. The cells were washed by centrifugation for experimental detection. The cell count confirmed that the viability was greater than 90% and adjusted to 4×10 6 cells / mL, so that the co-culture was detected at 2×10 6 cells / well.

[0223] 48 hours before the start of co-culture, the functional cells were sampled at 2×10 5 cells / well were seeded in Transwell inserts (with polyester membrane 6.5 mm, pore size: 0.4 μm) (Brand: CORNING, Cat. No.: 3470). This insert is known to allow cell secretions from internal and external culture conditions to pass through but not cells and cell debris to pass directly.

[0224] 1.1 mL / well of complete medium for T lymphocyte activation was used for inoculation and culture. Before inoculation of the responder cells, the original cell culture supernatant in the well was recovered and used to resuspend the responder cells and PBMC.

[0225] Before co-culture inoculation, the bottom of the culture plate inoculated with target cells was washed with physiological saline to remove the culture supernatant, and autologous PBMC cells resuspended with functional cell culture supernatant were added to the outside of the Transwell nest for co-culture inoculation with target cells. Use sterile tweezers to gently pick up the corresponding Transwell nest inoculated with functional cells and place it in the culture well of the experimental group. A total of 1 mL of cell culture supernatant recovered from each well was used to resuspend all cells. After co-culture inoculation, the cells were cultured at 37°C and 5% CO2 for 8, 36, and 72 hours, respectively.

[0226] The corresponding test was flow cytometry to detect the activation level of T lymphocytes in PBMCs of each group. The changes in the proportion of CD45+ / CD3+ / CD69+ cells were detected after 8, 36 and 72 hours of culture.

[0227] Each time the test was performed, the Transwell nest and the cells inside were removed aseptically. All the suspended cells at the bottom of the culture well were recovered and dispersed into a cell suspension, then washed with PBS, centrifuged at 1500 rpm for 6 minutes (acceleration 9, deceleration 9), the complete culture medium components were removed and resuspended in PBS, and various cells were evenly divided into 3 tubes and centrifuged again. The centrifuged cell pellets of each experimental group were added with 100 μL of the following flow cytometry working solution, incubated at 4°C for 30 minutes, and stained. The dye formula is shown in Table 14.

[0228]

[0229] After all staining is completed, the cell suspension is diluted with PBS and centrifuged at 1500 rpm for 6 minutes (acceleration 9, deceleration 9) to remove the staining solution. Each tube of cells in each group is resuspended in PBS to 100 μL and detected by flow cytometry.

[0230] Test results such as Fig.11 As shown, the autocrine CiTEs CAR-T cells can secrete fusion proteins containing CiTEs to specifically activate T lymphocytes in tumor lesions.

[0231] The autocrine CiTEs CAR-T cells in this embodiment are inoculated in the Transwell nesting and cannot directly contact and kill the target cells, and can only regulate the activation of the reactive cells by secreting CiTEs. It is known that the liver cancer tumor cell line HepG2 expresses the target GPC3 which can be specifically bound by CiTEs, and the normal liver cell line HL7702 does not express the target protein and therefore does not bind to CiTEs. The corresponding reactive cells are autologous PBMCs, which are mixed immune cell populations, in which CD45+CD3+T lymphocytes can be activated as killer T lymphocytes through the pathway of classic APC antigen presentation-TCR receptor specific recognition and activation. The expression of various molecules can be used to indicate T cell function. CD69 and CD25 are upregulated through TCR signal transduction, but have different kinetic characteristics. CD69 can be detected within a few hours after TCR connection, while CD25 increases afterwards. In this way, the activation process of infiltrating T lymphocytes at the tumor lesion is simulated, and the activation index is detected to verify the function of CiTEs.

[0232] According to the experimental design of this project, the co-culture system of experimental group A contains CiTEs that specifically bind to target cells, and there are tumor cells that express the target protein GPC3 in the culture system. Experimental group B is normal liver cells that do not express the target protein. Experimental group C is an experimental control group without CiTEs, while experimental group D is a blank group without CiTEs and specific target cells. According to the flow cytometry results, the T lymphocytes recovered in experimental group A are activated faster than the control group in an environment containing CiTEs and target cells, and the activation effect is target specific. At the same time, the activated T lymphocytes are rapidly transformed into a subpopulation with killing ability. It is suggested that the technology of this project can improve the activation of T lymphocytes infiltrating tumor lesions, enhance their killing effect on tumors, and this enhancement effect is specific to the target. It meets the design goals of this technology.

[0233] Example 8: Autocrine CiTEs CAR-T cells express and secrete CiTEs to enhance the killing effect of T cells on target cells.

[0234] The number of HepG2 and HL7702 cells to be inoculated was 2×10 5cells / well in a 96-well cell culture plate. 24 hours before co-culture, the cells were inoculated with the original complete medium until they were fully adhered to the bottom of the cell culture plate.

[0235] The above cells were mixed and co-cultured according to Table 15. The number of HepG2 and HL7702 cells inoculated was 2×10 5 cells / well in a 12-well cell culture plate, and inoculate with the original complete medium 24 hours before co-culture until the cells fully adhere to the bottom of the plate.

[0236]

[0237] According to the technical implementation scheme of the present invention, PBMCs were separated from the patient's frozen leukocyte apheresis product by Ficoll density gradient centrifugation, and the concentration of PBMCs was greater than 1×10 6 cells / mL were suspended and cultured in complete T lymphocyte activation medium for 24 hours at 37°C and 5% CO2. The cells were washed by centrifugation for experimental detection. Cell counting confirmed that the viability was greater than 90%, and the culture supernatant of the sampled autocrine CiTEs CAR-T cells and the control T cell culture supernatant were resuspended to 1×10 6 cells / mL, so that the co-culture was detected at 1×10 5 cells / well Before co-culture inoculation, the bottom of the culture plate inoculated with target cells was washed with physiological saline to remove the culture supernatant, and the corresponding PBMC cell suspension was added at a volume of 100 μL per well. After co-culture inoculation, the cells were cultured at 37°C and 5% CO2 for 5 consecutive days.

[0238] Cell Counting Kit-8 (Brand: MedChemExpress, Catalog No.: HY-K0301) was used to detect the number of adherent cells every 24 hours to evaluate the effect of the components in the sampled cell culture supernatant on the killing effect of immune cells such as T lymphocytes in PBMC on target cells. Before each test, the culture supernatant was aspirated and discarded after gentle horizontal shaking. 100 μL PBS was added to each well and gently shaken horizontally, and the supernatant was aspirated again and discarded. The plate was centrifuged, and all the supernatant was removed upside down to retain the cell pellet. 90 μL of PBS and 10 μL of CCK-8 working solution were added to the test wells. After addition, it was placed at 37°C and incubated at a concentration of 5% CO2 for 4 hours. The absorbance at 450 nm was measured with an enzyme reader, and the absorbance values ​​were continuously measured for 5 days to draw the growth and proliferation curve of the bottom-adherent tumor cells.

[0239] Test results such as Fig.12As shown. It is verified that the autocrine CiTEs CAR-T cells can secrete CiTEs to specifically enhance the killing effect of immune cells in tumor lesions on liver cancer cells.

[0240] The CAR-T cell culture supernatant containing CiTEs was recovered. It is known that the liver cancer tumor cell line HepG2 expresses the target GPC3 which can be specifically bound by CiTEs, while the normal liver cell line HL7702 does not express the target protein and therefore does not bind to CiTEs. The corresponding reactive cells are autologous PBMCs, which are mixed immune cell populations, among which CD45+CD3+T lymphocytes can be activated into killer T lymphocytes through the pathway of classical APC antigen presentation-TCR receptor specific recognition and activation.

[0241] The cell viability test kit (CCK-8) can be used for simple and accurate cell proliferation and toxicity analysis. The cell lines HepG2 and HL7702 in this project are both mammalian cells that grow on the wall. After being killed by immune cells, they lose their complete cell structure and lyse and fall off. Therefore, after removing the culture supernatant in continuous culture, the OD of CCK8 staining is detected. 450 The absorbance can be used to evaluate the number and proliferation of target cells remaining at the bottom of the culture plate.

[0242] The co-culture system of experimental group A contained CiTEs that specifically bound to target cells, and there were tumor cells that expressed the target protein GPC3 in the culture system. Experimental group B contained normal liver cells that did not express the target protein. Experimental group C was an experimental control group without CiTEs, and experimental group D was a blank group without CiTEs and specific target cells.

[0243] According to the flow cytometry results, the immune cells recovered in experimental group A killed tumor cells HepG2 faster than the control group in an environment containing CiTEs and target cells, and the activation was target specific, that is, normal liver cells did not show rapid killing. This suggests that the autocrine CiTEs CAR-T cells can increase the activation of immune cells infiltrating tumor lesions, especially T lymphocytes, and enhance their killing effect on tumors, and this enhancement is specific to the target. It is in line with the design goals of this technology.

[0244] Example 9: Autocrine CiTEs CAR-T cells express and secrete CiTEs to reduce the inhibitory regulatory effect of tumor cells on the immune checkpoints of T cells.

[0245] The autocrine CiTEs CAR-T cells, control T cells and autologous PBMC cells were sampled for co-culture detection. After the recovered cells were washed to remove the complete medium components, the cell count confirmed that the viability was greater than 95%, and each 1×10 7Resuspend cells in 3.5 mL of PBS. 7 Add 1 μL of 5 mmol / L CFSE solution to cells and mix thoroughly (CFSE Cell Division Tracker Kit, brand: Biolegend, catalog number: 423801). Incubate at 37°C for 10 minutes. Then add T lymphocyte activation complete medium to terminate labeling. The CFSE-labeled autologous PBMC cells were mixed with the corresponding volume of CFSE solution as described in this technical solution. TM (Cell Therapy System) Dynabeads TM CD3 / CD28 magnetic bead suspension (Brand: Gibco TM , Catalog No.: 40203D) for T lymphocyte sorting, and the sorted T lymphocytes are labeled. 6 cells / well to prepare the corresponding cells and recover the corresponding magnetic beads.

[0246] The functional cells sampled during co-culture inoculation were 1×10 6 cells / well were seeded in Transwell inserts (with polyester membrane 6.5 mm, pore size 0.4 μm, brand: CORNING, catalog number: 3470). It is known that this insert allows cell secretions from internal and external culture conditions to pass through, but cells and cell debris cannot pass directly. The experimental groups are shown in Table 16. According to 1×10 per well 6 cells / well inoculated with autologous T lymphocytes in the Transwell insert, and the medium for co-culture of cells was fresh T lymphocyte activation complete medium. After inoculation, PD-L1 protein, Human (HEK293) (Brand: MedChemExpress, Catalog No.: HY-P73361) was added to the co-culture system to the target final concentration of the system - corresponding to IC 50 =0.2191 μg / mL.

[0247]

[0248] The cells were cultured at 37°C and 5% CO2 in the dark for 4 days to simulate the immune checkpoint inhibitory effect of PD-L1-expressing tumor cells on the infiltrating autocrine CiTEs CAR-T cells and T lymphocytes in the tumor microenvironment. The suspended cells inside and outside the Transwell chamber of each group were recovered, washed by centrifugation with PBS, and the corresponding proliferation inhibition rate was detected by flow cytometry. This proves the neutralizing effect of the autocrine CiTEs CAR-T cells on immune checkpoint inhibition.

[0249] Test results such as Fig.13 and Fig.14 As shown, after adding PD-L1 protein to the half-inhibitory concentration in the co-culture system, the proliferation coefficient of autocrine CiTEs CAR-T cells and autologous T lymphocytes (group A vs group B) decreased, but the decrease was lower than that without adding autocrine CiTEs CAR-T cells (group A vs group C). This indicates that in the presence of autocrine CiTEs CAR-T cells, the regulatory effect of PD-L1 protein on immune cells is inhibited through the immune checkpoint mechanism. This indicates that the autocrine CiTEs CAR-T cells can secrete CiTEs to neutralize the inhibitory effect of PD-L1 on T lymphocytes and CAR-T cells.

[0250] Example 9: Effect of autocrine CiTEs CAR-T cells on the survival of tumor-bearing mice in vitro.

[0251] A total of 45 female NOD / SCID mice aged 14 to 20 weeks were selected. The HepG2 cells cultured above were counted to ensure that the viability was above 90%. The density was adjusted to 6×10 6 1×10 cells / mL was injected subcutaneously on the back of mice 6 HepG2 cells. 14 days after inoculation, subcutaneous tissue samples were collected from the injection site to confirm the presence of hepatocellular carcinoma tissue in the mice. All surviving mice were then randomly divided into two treatment groups, with 22 mice in each group. The experimental group (autocrine CiTEs CAR-T cell therapy) and the control group (control T cell therapy) were intravenously injected with 1×10 7 The weight and overall survival rate of the mice were recorded. Fig.15 and Fig.16 shown.

[0252] Compare the animal weight and survival rate of the subcutaneous transplanted liver cancer animal model after treatment with autocrine CiTEs CAR-T cells. The comparison of the number of long-term surviving animals proves that the autocrine CiTEs CAR-T cells can have a therapeutic effect in vivo and can treat or improve the condition of hepatocellular carcinoma tumor-bearing animals and prolong their disease-bearing survival. At the same time, the weight changes of surviving animals indicate that the condition is relatively stable after treatment with this technology, and there are fewer complications such as tumor growth and hepatic ascites. It is suggested that the autocrine CiTEs CAR-T cells have development prospects in the treatment of liver cancer, and the therapeutic effect in vivo can achieve the purpose of technical design.

[0253] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A fusion protein, characterized in that It comprises a chimeric antigen receptor fragment and a bifunctional checkpoint inhibitory T cell engager fragment, wherein the chimeric antigen receptor fragment and the bifunctional checkpoint inhibitory T cell engager fragment are connected by a 2A peptide, the chimeric antigen receptor fragment comprises a GPC3 single-chain antibody region, a transmembrane region and an intracellular signal transduction region; the bifunctional checkpoint inhibitory T cell engager fragment comprises a GPC3 single-chain antibody region, a checkpoint inhibition functional domain and a CD3 single-chain antibody region.

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of the GPC3 single-chain antibody region is shown in SEQ ID No:

1.

3. The fusion protein according to claim 1, characterized in that The amino acid sequence of the checkpoint inhibition domain is shown in SEQ ID No:

2.

4. The fusion protein according to claim 1, characterized in that The amino acid sequence of the CD3 single-chain antibody region is shown in SEQ ID No:

3.

5. The fusion protein according to claim 1, characterized in that: The amino acid sequence of the chimeric antigen receptor fragment is shown in SEQ ID No:

4.

6. The fusion protein according to claim 1, characterized in that: The amino acid sequence of the dual-functional checkpoint inhibitory T cell engager fragment is shown in SEQ ID No:

5.

7. A nucleotide molecule, characterized in that It encodes the fusion protein according to any one of claims 1 to 6.

8. A recombinant vector, characterized in that: Comprising the nucleotide molecule according to claim 7.

9. The recombinant vector according to claim 8, characterized in that: The recombinant vector is pCDH-EF1, and the nucleotide sequence is shown in SEQ ID No:

12.

10. A recombinant cell, characterized in that Comprising the recombinant vector according to claim 8 or 9.

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