Universal CAR-T cell preparation technology and its use

Abstract

A method for preparing universal CAR-T cells and its use. The RNP complex when preparing universal CAR-T includes an RNP complex consisting of Cas9 protein and sgRNA. The sgRNA is a CD4 sgRNA and / or a CD8 sgRNA. The CD4 gene and / or CD8 gene of the universal CAR-T cells or T cells are knocked out and / or inactivated. The CAR-T cells express CD44 and / or CD62L can enhance the activity of CAR-T cells, improve the efficacy of CAR-T, improve the phenotype of CAR-T cells, reduce the expression of exhaustion, and improve the killing of CAR-T cells against tumor target cells, which can be applied to tumor targeted therapy and allogeneic CAR-T therapy.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims priority to Chinese Patent Application No. 202111652734.4 (Title: Preparation Technology of Universal CAR-T Cells and Its Use) filed on December 30, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention claims priority to Chinese Patent Application No. 202111652757.5 (Title: Preparation Technology of Universal CAR-T Cells and Its Use) filed on December 30, 2021, the entire contents of which are incorporated herein by reference.

[0003] The present invention claims priority to Chinese Patent Application No. 202111657691.9 (Title: Preparation Method and Use of Universal CAR-T Cells) filed on December 30, 2021, the entire contents of which are incorporated herein by reference.

[0004] The present invention claims priority to Chinese Patent Application No. 202111657717.X (Title: Preparation Method and Use of Universal CAR-T Cells) filed on December 30, 2021, the entire contents of which are incorporated herein by reference. [Technical field]

[0005] The present invention relates to the technical fields of cell biology, molecular biology and drug discovery, and specifically to a method for preparing universal CAR-T cells and their use. [Background technology]

[0006] CAR-T immunotherapy uses genetic engineering to modify a patient's own T lymphocytes to express a chimeric antigen receptor, which can effectively overcome immune evasion, such as downregulating the expression of MHC molecules and reducing antigen presentation, so that tumor cells cannot escape. Currently, two types of T cell products with chimeric antigen receptors (CARs) approved in China are both derived from autologous T cells, and these autologous CAR-T cells approved for clinical use must be produced on a customized basis. However, due to the high production costs and long production process, such autologous T cell production platforms are still important limiting factors for large-scale clinical application, and there are also risks such as production failure. At the same time, the production process of personalized, customized autologous CAR-T cells has also limited the wide application in different tumor types. Therefore, it is necessary to use universal allogeneic T cells to prepare universal CAR-T cells, and these cells can be used for large-scale clinical application as "off-the-shelf" ready-to-use therapeutic agents. Universal CAR-T cells can significantly shorten the treatment cycle and reduce costs, and the problems of graft rejection-related reactions and therapeutic efficacy can be gradually solved through strategies such as gene editing technology and stem cell transplant donor-derived T cells, giving them a greater advantage in future market competition.

[0007] Universal CAR-T cells are an allogeneic live cell transplant drug that uses T cells extracted from the peripheral blood of a healthy donor as a starting sample, resolves the issue of immune rejection between allogeneic and allogeneic cells through gene editing technology, and is then equipped with a CAR structure that specifically targets cancer cells. The T cells are then finally returned to the body of an allogeneic cancer patient to specifically target tumor cells.

[0008] Genome editing technologies, including ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease) and CRISPR-related technologies, are used to prepare universal T cells. At present, the common operating methods in the industry are roughly the same, and all of them use gene editing to knock out TCR molecules and / or MHC-related molecules that cause strong immune rejection in allogeneic T cells, thereby solving the problem of immune rejection. In addition, many mechanisms use such gene editing to further knock out PD-1, CTLA-4, CD52, or introduce CAR suicide gene switches, etc., in order to improve therapeutic efficacy or safety.

[0009] Although this type of engineering is theoretically feasible, there is currently no relevant literature that provides a detailed analysis of the effects on cell survival and function of T cells after depletion of TRC or MHC molecules, which is also one of the reasons why the industry is currently facing great challenges.

[0010] At present, the major challenge facing the industry is that the produced universal CAR-T cells have a relatively poor in vivo persistence, and even if the reinfusion dose or reinfusion frequency is increased, it is difficult to achieve the ideal therapeutic effect, whereas autologous CAR-T cells have a relatively low reinfusion dose and a higher relief rate. There are many possible reasons for this result. As a key gene that enables T cells to exert various immune-related functions, there is no relevant research that clearly points out whether TCR also plays an important or crucial auxiliary role in other functions of T cells. However, the advantages of ready-to-use, ready-made, low treatment cost, etc. that can be achieved by universal CAR-T cells are not realized by autologous CAR-T cells. Therefore, it is necessary to prepare a universal CAR-T with a long persistence in vivo. Summary of the Invention

[0011] In view of the above, the present invention provides a technology for preparing universal CAR-T cells and its use.

[0012] In order to achieve the above object, the present invention provides the following technical means.

[0013] A first object of the present invention is to provide an RNP complex that can be used to knock out a target site in a T cell.

[0014] In order to achieve the above object, the technical means of the present invention are as follows.

[0015] An RNP complex comprising a Cas9 protein and an sgRNA, wherein the sgRNA is a CD4 sgRNA and / or a CD8 sgRNA.

[0016] Furthermore, the RNP complex is a CRISPR / Cas9 intracellular operating form.Extracellular delivery methods include plasmid delivery methods, virus delivery methods, mRNA (Cas9) and gRNA delivery methods, mRNA (Cas9) and plasmid (gRNA) delivery methods, virus (Cas9) and gRNA delivery methods, virus (Cas9) and plasmid (gRNA) delivery methods, plasmid (Cas9) and virus (gRNA) delivery methods, and plasmid (Cas9) and gRNA delivery methods.

[0017] Furthermore, in the RNP complex, the CD4 gene is the CD4 gene on human chromosome 12: 6,789,528-6,820,799 (located on the forward strand), and the CD8 gene is the CD8A gene on human chromosome 2: 86,784,610-86,808,396 (located on the reverse strand) and the CD8B gene on human chromosome 2: 86,815,339-86,861,924 (located on the reverse strand).

[0018] Furthermore, the sequence of the CD4 sgRNA is set forth in any one of SEQ ID NOs: 1-6 or 14-24, and the sequence of the CD8 sgRNA is set forth in any one of SEQ ID NOs: 7-11 or 25-33.

[0019] A second object of the present invention is to provide T cells with long in vivo persistence that can be used for the further preparation of CAR-T cells.

[0020] In order to achieve the above object, the technical means of the present invention are as follows.

[0021] T cells in which the CD4 and / or CD8 genes have been knocked out and / or inactivated.

[0022] Some of the target sequences used in the present invention are shown in SEQ ID NO: 1-11. The sequences are not limited to the sequences listed here, and include CD4 gene on human chromosome 12: 6,789,528-6,820,799 (located on the forward strand), CD8A gene on human chromosome 2: 86,784,610-86,808,396 (located on the reverse strand), and CD8B gene on human chromosome 2: 86,815,339-86,861,924 (located on the reverse strand). Table 1, the location of gene editing markers, shows the target sequences that can cause deletions in the gene itself within any one of the included regions.

[0023] Furthermore, the T cells are obtained by gene editing.

[0024] Moreover, the T cells are obtained by natural isolation.

[0025] Additionally, the T cells have genes for β2-microglobulin, and / or common subunit of HLA class I molecules, and / or CIITA that are rendered inoperative or deleted.

[0026] Furthermore, said T cells are allogeneic T cells.

[0027] In addition, the T cells also express HLA-E and / or HLA-G and / or HLA-F proteins.

[0028] In addition, the T cells express CD44.

[0029] Moreover, preferably, said T cells also express the CD62L gene.

[0030] Furthermore, in said T cells, the amino acid sequence of the CD62L gene is shown in SEQ ID NO: 13-17.

[0031] Furthermore, in the T cells, the amino acid sequence of the CD44 is set forth in SEQ ID NO:12, and the amino acid sequence of the CD62L is set forth in SEQ ID NO:13.

[0032] Further, in the T cells, the CD4 gene and / or CD8 gene of the T cells are knocked out and / or inactivated. The amino acid sequence of the CD44 gene is shown in SEQ ID NO: 12. Some of the target sequences of the CD4 gene and CD8 gene are shown in SEQ ID NO: 1-11, including but not limited to the sequences listed herein, CD4 gene on human chromosome 12: 6,789,528-6,820,799 (located on the forward strand), CD8A gene on human chromosome 2: 86,784,610-86,808,396 (located on the reverse strand), and CD8B gene on human chromosome 2: 86,815,339-86,861,924 (located on the reverse strand). Locations of gene editing targets Table 1, Table 3-Table 5 show target sequences that can cause deletions in the gene itself in any one of the regions included. Furthermore, to improve knockout efficiency, a single guanine G can be introduced before any sequence in which the first base in the target sequence is not G.

[0033] The CD62L native amino acid sequence is shown in SEQ ID NO:13.

[0034] Since CD62L expressed natively or in its native sequence has a mode of degradation or hydrolysis due to autocleavage that occurs under certain physiological conditions (when the degree of activation is relatively high), its persistence can be improved by mutating it to the non-degradation mode sequence described below by amino acid substitution, deletion, or other shortening.

[0035] [Table 1]

[0036] [Table 2]

[0037] The third object of the present invention is to provide a method for preparing said T cells, which provides a preparation idea for providing cells with long persistence.

[0038] In order to achieve the above object, the technical means of the present invention are as follows.

[0039] The RNP complex is used to knock out and / or inactivate the CD4 gene and / or the CD8 gene in T lymphocytes by gene editing technology.

[0040] In the method for preparing T cells, the target cells are obtained by any one of the following methods.

[0041] (1) Using fresh or frozen T cells, knock out or inactivate the CD4 and CD8 genes by gene editing. The CD4 and CD8 genes may be knocked out or inactivated simultaneously, or knock out or inactivate CD4 or CD8 before knocking out or inactivating CD8 or CD4. Finally, obtain target cells. (2) Select CD4-positive T cells, knock out or inactivate the CD4 gene by gene editing to obtain target cells. (3) Select CD8-positive T cells, knock out or inactivate the CD8 gene by gene editing to obtain target cells. (4) Select CD4-positive T cells, knock out or inactivate the CD4 gene by gene editing, select CD8-positive T cells, knock out or inactivate the CD8 gene by gene editing, and finally mix the T cells whose CD4 gene has been knocked out or inactivated by gene editing with the T cells whose CD8 gene has been knocked out or inactivated by gene editing to obtain target cells.

[0042] Furthermore, said T cells may be activated and / or non-activated T lymphocytes.

[0043] Furthermore, the timing of knocking out or inactivating the CD4 and / or CD8 genes in the T cells is after T cell expansion or before T cell expansion.

[0044] Furthermore, the gene editing technology involves remodeling T cells by electroporation or non-electroporation methods.

[0045] Furthermore, the gene editing techniques include CRISPR / (Cas9 or Cpf1), ZFN, and TALEN.

[0046] A fourth object of the present invention is to provide universal CAR-T cells that are more persistent in vivo and are characterized by being capable of expressing CD44 and / or CD62L.

[0047] The protein encoded by the CD44 gene is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. It is a receptor for hyaluronic acid (HA) and can also interact with other ligands (e.g., osteopontin, collagen, matrix metalloproteinases (MMPs)). This protein is involved in various cellular functions including lymphocyte activation, recirculation and homing, hematopoiesis and tumor metastasis. Currently, research on CD44 is mainly focused on its role in cancer stem cells and the role of its various variants in cell adhesion, with very few studies targeting CD44 for CAR-T cell therapy and no related reports yet on its direct use in CAR-T treatment.

[0048] The L-selectin (CD62L) gene encodes a cell surface adhesion molecule belonging to the adhesion / homing receptor family, and its expression on cells continues to be downgraded by the activation of the self-splicing mechanism during continuous activation of T cells as a marker of T cell activation. Studies have shown that, even with the downgrading phenomenon, the antitumor ability of cytotoxic T lymphocytes (CTLs) expressing CD62L is also significantly higher than that of CTLs that do not express CD62L (doi:10.1172 / JCI24480doi:10.1073 / pnas.0503726102). This is thought to be due to the homing ability of CD62L. However, some studies have also shown that L-selectin is not related to homing but is associated with the activation of therapeutic T cells in tumors (doi:10.3389 / fimmu.2019.01321). In the study (doi:10.3389 / fimmu.2019.01321), a mouse model of adoptive immunotherapy was used to investigate the effect of wild-type mouse CD8 + Using T as a control, a mutant CD62L gene-modified mouse was generated by deleting a portion of the CD62L membrane-proximal hydrolytic cleavage site. This mutant gene-modified mouse was characterized by the expression of mouse CD8 + Increased expression of L-selectin on T cells. As shown by the results of the study, mutant CD62L genetically engineered mice showed increased expression of L-selectin on CD8 +Although it improves the ability of T cells to control the growth of solid tumors, the ultimate effect and specific mechanism achieved by overexpressing CD62L on T cells or CAR-T cells in conventional techniques remain unclear.

[0049] The fifth object of the present invention is to provide CAR-T cells with high long-term viability and a method for preparing the same, which are commercially applicable and the method is highly reproducible.

[0050] In order to achieve the above object, the technical means of the present invention are as follows.

[0051] CAR-T cells prepared from the T cells.

[0052] Furthermore, the CAR in a CAR-T cell comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

[0053] Furthermore, the CAR-T cells are allogeneic CAR-T cells.

[0054] In addition, the CAR-T cells also express membrane-bound IL15.

[0055] In addition, the CAR-T cells also express secreted cytokines, including IL2, IL7, IL12, IL21, and IL15.

[0056] In addition, the CAR-T cells can also express and recognize fusion proteins of molecules in the tumor microenvironment, the extracellular structures of which recognize PD1, PDL1, SIRPγ, SIRPδ, and TIGIT.

[0057] Furthermore, the CAR structure in the CAR-T cell can be activated or inactivated by control. The controlled activation or inactivation structures include peptide neoepitope (PNE), fluorescein (FITC), 10 amino acids (5B9 tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv, leucine ZipFv bound to antibody, and streptavidin 2 (mSA2) biotin binding domain. These molecules can be recognized by the antibody or ZipCAR structure bound to the CAR, which can induce and activate the signal pathway of the CAR-T cell. Such a system controls the activity of the CAR-T cell by changing the number of switch molecules bound to the antibody.

[0058] Furthermore, the CAR-T cells can recognize solid tumors and blood tumor cells / tissues. The antigen recognition region of the CAR-T cells can recognize antigens including CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, or TAG-72.

[0059] The sixth object of the present invention is to provide a method for preparing the CAR-T cells, which comprises obtaining target cells by any one of the following two methods:

[0060] (1) harvesting T cells, infecting them with a virus containing CD44 and a CAR structure, or infecting them with a virus containing CD62 and a CAR structure, and then introducing RNP into the T cells infected with the CAR structure by electroporation to obtain target cells; or (2) T cells are collected and obtained, and RNP is introduced into the T cells by electroporation, and then the T cells are infected with a virus containing CD44 and a CAR structure, or with a virus containing CD62 and a CAR structure to obtain target cells.

[0061] Furthermore, the preparation method obtains target cells by either one of the following two methods.

[0062] (1) Activating T cells in which the CD4 and / or CD8 genes have been knocked out or inactivated, infecting them with a virus containing CD62L, CD44 and a CAR structure, and then introducing RNP into the T cells infected with the CAR structure by electroporation to obtain target cells; or

[0063] (2) T cells in which the CD4 and / or CD8 genes have been knocked out or inactivated are activated, and then RNP is introduced into the T cells by electroporation. The T cells are then infected with a virus containing CD62L, CD44, and a CAR structure to obtain target cells.

[0064] Furthermore, in the preparation method, the T cells are modified by CRISPR / Cas9 gene editing technology when the CD4 and / or CD8 genes are knocked out or inactivated. The RNP complex related to the CRISPR / Cas9 gene editing technology is specifically an RNP complex containing CD4sgRNA, CD8sgRNA and Cas9 protein.

[0065] Furthermore, in the preparation method, the molar ratio of the Cas9 protein to the sgRNA is 1:2.

[0066] Preparation of CAR-T cells 1) constructing a viral vector containing a target gene fragment (including a CAR structural sequence for a specific tumor target and a CD62L gene fragment driven by different promoters); 2) infecting T cells with the viral vector to obtain target cells; Includes.

[0067] Furthermore, in the above method, in step 2), expression of the CD62L gene is enhanced by lentivirus infection, gene targeting, lipofection, electroporation, or the like.

[0068] A seventh object of the present invention is to provide a cell composition, which is capable of collecting a large number of functional cells, and which has a therapeutic effect.

[0069] In order to achieve the above object, the technical means of the present invention are as follows.

[0070] A cell composition comprising the T cells and / or the CAR-T cells.

[0071] Furthermore, the proportion of the T cells or the CAR-T cells contained in the cell composition is 90%, 80%, 70%, or 60% or more by number.

[0072] Furthermore, the proportion of the T cells or the CAR-T cells contained in the cell composition is 60% or more by number.

[0073] Furthermore, the proportion of the T cells or the CAR-T cells contained in the cell composition is 70% or more by number.

[0074] Furthermore, the proportion of the T cells or the CAR-T cells contained in the cell composition is 80% or more by number.

[0075] Furthermore, the proportion of the T cells or the CAR-T cells contained in the cell composition is 90% or more by number.

[0076] Furthermore, the cell composition may be activation-induced CIK cells or NKT cells.

[0077] Furthermore, the T lymphocytes in the cell composition in which the CD4 and CD8 genes have been inactivated express a chimeric antigen receptor.

[0078] An eighth object of the present invention is to provide a use of the T cell, the recombinant vector, the CAR-T cell, or the cell composition, which provides an excellent therapeutic idea for clinical use.

[0079] In order to achieve the above object, the technical means of the present invention are as follows.

[0080] Use of the T cells, the CAR-T cells, the recombinant vector, and / or the cell composition in the preparation of a cellular therapeutic drug.

[0081] Further, the use is the use of the T cell, the CAR-T cell, the recombinant vector, and / or the cell composition in a medicament for treating acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer, and / or skin cancer.

[0082] The use of the RNPs according to the invention in cell therapy drugs used in tumor treatment.

[0083] Use of CD4 gene inhibitors, knockout agents and / or CD4 gene inhibitors, knockout agents as cell proliferation agents, activity enhancers, phenotype distribution improvers, and CD4 gene inhibitors, knockout agents as exhaustion marker expression inhibitors in the preparation of therapeutic T-cells or CAR-T cells.

[0084] Use of CD62L and / or CD44 in the preparation of a CAR-T cell activity enhancer.

[0085] Furthermore, the use of a combination of CD44 and CD62L in the preparation of a CAR-T cell activity enhancer.

[0086] The use of CD62L and / or CD44 in the preparation of an inhibitor of PD-1 and LAG-3 factors secreted by CAR-T cells.

[0087] Terminology Use of CD62L and / or CD44 genes as tumor cell killing promoters in the preparation of CAR-T cells, said CAR-T cells being normal CAR-T cells, CAR-T cells not expressing CD4 and / or CD8 genes, or universal CAR-T cells.

[0088] The CAR structures described in the present invention may be conventional first, second or third generation CARs, or may be novel CAR structures such as improved dual CARs, tunable CAR structures (e.g., FRB / FKBP12 tuned), etc.

[0089] The CAR constructs described in the present invention contain one or more components of natural killer cell receptors (NKRs), thus forming NKR-CARs. NKR components include killer cell immunoglobulin-like receptors (KIRs) (e.g., KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1), natural cytotoxicity receptors (NCRs) (e.g., NKp30, NKp44, NKp4 6), the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors (e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD2F-10); Fc receptors (FcRs) (e.g., CD16 and CD64), and Ly49 receptors (e.g., LY49A, LY49C). The NKR-CAR molecule may interact with an adaptor molecule or an intracellular signaling domain (e.g., DAP12).

[0090] The lentiviral vector according to the present invention is essentially selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV).

[0091] The CAR-T of the present invention is disclosed in patent applications 201710305078.8, 201710613317.6, 201710618293.3, 201510304389.3, 201510648252.X, 201810207761.2, 201810934637.6, 201811125906.0, 2019 10407001.0, 202010257762.5, 202010259458.4, 202010260805.5, 202010260849.8, 202010559366.8, 202110556147.9 and other published CAR-T structures can be used. The use of such double negative universal CAR-T cell therapy can be applied to any CAR structure or target, and is not limited by CAR target, structure, and T cells that do not express CD4 and CD8 genes do not affect the expression of any CAR structure.

[0092] The gene editing technology used in the present invention includes but is not limited to CRISPR series technology (Cas9, Cpf1), ZFN, TALEN. The double negative universal T cell described herein can be obtained by any gene editing technology.

[0093] The allogeneic T cells of the present invention refer to T cells whose CD4 and CD8 genes have been inactivated by gene editing technology and which can be used allogeneically to treat specific indications.

[0094] The pharmaceutical composition of the present invention can be used in combination with one or more of chemotherapy drugs, other tumor-targeting monoclonal antibody drugs, antibody-drug coupling conjugates (ADCs), immune checkpoint inhibitor drugs, and small molecule drugs.

[0095] The hinge region sequence in the CAR-T construct according to the present invention can be derived from IgG, CD8, CD7, CD4.

[0096] The transmembrane domain sequences in the CAR-T constructs according to the invention can be derived from CD8, CD28, CD3ε, CD4, CD16, CD137, CD80 and CD86.

[0097] The intracellular signaling regions in the CAR-T constructs described in the present invention can be derived from CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B7-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2C, CD83, CD86, and CD127.

[0098] The antigen recognition region according to the present invention may be a ligand / receptor capable of recognizing a target antigen. The ScFv may be a mouse antibody, a fully human antibody, or a human-mouse chimeric antibody ScFv, or may be a single domain antibody such as a shark, alpaca, or camel antibody, or may be a bispecific antibody, or may be combined with an artificially designed target-specific fibronectin type III (FN3) domain that recognizes a specific target.

[0099] The CAR-T in the present invention is disclosed in patent applications 201710305078.8, 201710613317.6, 201710618293.3, 201510304389.3, 201510648252.X, 201810207761.2, 201810934637.6, 201811125906.0, 201811125906.0, 201810207761.2, 201810934637.6, 201811125906.0, 201810207761.2 ... 1910407001.0, 202010257762.5, 202010259458.4, 202010260805.5, 202010260849.8, 202010559366.8, 202110556147.9 and other published CAR-T structures can be used. The use of such double negative universal CAR-T cell therapy can be applied to any one CAR except for the CD44 target, and is not limited by other targets or structures of the CAR.

[0100] In the preparation method disclosed in the present invention, activated T cells may be infected with the CAR construct via a virus or a transposon system may be used.

[0101] In the present invention, DU-CAR-T refers to universal CAR-T cells in which the CD4 and / or CD8 genes have been knocked out or inactivated.

[0102] In the present invention, 44-19CAR refers to CD44-expressing CAR-T cells prepared by autoimmune cells.

[0103] In the present invention, L-19CAR refers to CD62-expressing CAR-T cells prepared by autoimmune cells.

[0104] In the preparation method disclosed in the present invention, activated T cells may be infected with the CAR construct via a virus or a transposon system may be used.

[0105] In the present invention, the conventional universal CAR-T expressing CD44 prepared using allogeneic cells is CU-44-CAR-T, and the universal CAR-T expressing CD44 prepared using allogeneic cells is DU-44-CAR-T.

[0106] In the present invention, the conventional universal CAR-T expressing CD62L prepared using allogeneic cells is CU-62-CAR-T, and the universal CAR-T expressing CD62 prepared using allogeneic cells is DU-62-CAR-T.

[0107] Beneficial Effects of the Invention 1) The double negative universal CAR-T cells provided in the present invention can not only be effectively expressed in T lymphocytes, but also enhance the activity of CAR-T cells, improve the efficacy of CAR-T, improve the phenotype of CAR-T cells, reduce the expression of exhaustion, and improve the killing of CAR-T cells against tumor target cells, and can be applied to tumor targeted therapy. 2) The present invention provides selectable universal CAR-T cell candidate cells that can not only effectively kill tumor target cells in vivo and in vitro, but also can be used for tumor targeted therapy, and have a higher anti-tumor effect than traditional autologous CAR-T cells and traditional universal CAR-T cells. 3) The universal CAR-T cells provided by the present invention, like conventional universal CAR-T cells, only induce a low GVH response in vitro and can be applied to allogeneic CAR-T therapy, which has important guiding significance for the clinical application of CAR-T and the development of new strategies for combination tumor treatment. 4) DU-CAR-T prepared by the method provided by the present invention achieves the same level of low GVH reaction as conventional universal CAR-T cells, and the 44-CAR, L-CAR and universal DU-CAR of the present invention are superior or not inferior to autologous CAR cells in all aspects. 5) The DU-CAR-T cells prepared by the method provided by the present invention have stronger proliferation ability, higher activity, better phenotype distribution, lower exhaustion marker expression, and better in vitro efficacy than natural DN-T or DN-CAR-T cells prepared using it, with highly significant statistical differences. In addition, the cells of the present invention are more accessible, and even with the same starting amount of PBMC, the effective cells finally obtained are much more than DN-CAR-T cells. This greatly reduces the production cost and provides more possibilities for its clinical application. [Brief description of the drawings]

[0108] [Figure 1] The detection results of the knockout rate are shown. [Diagram 2] The detection results of CAR positivity rate are shown. [Diagram 3] 1 shows the results of in vitro pharmacodynamics detection. [Figure 4] The phenotype detection results are shown. [Diagram 5] The results of exhaustion marker detection are shown. [Figure 6] The results of vitality monitoring are shown. [Figure 7] The results of GVH intensity detection are shown. [Figure 8] A comparison of the in vivo anti-tumor detection results of 19CAR, DU-19CAR, and CU-19CAR is shown. [Figure 9] A comparison of the in vivo anti-tumor detection results of 19CAR and 44-19CAR is shown. [Figure 10] 1 shows a comparison of the in vivo anti-tumor detection results of DU-44-19CAR and 19CAR. [Figure 11] The detection results of CAR positivity rate are shown. [Figure 12] 1 shows the results of in vitro pharmacodynamics detection. [Figure 13] The detection results of a flow cytometer are shown. [Figure 14] The results of detection of fatigue markers are shown. [Figure 15] The results of GVH intensity detection are shown. [Figure 16] The results of flow cytometry detection are shown. [Figure 17] The results of flow cytometry detection are shown. [Figure 18] 1 shows the results of in vitro pharmacodynamics detection. [Figure 19] The detection results of the factors are shown. [Figure 20] The results of proliferation and activity detection are shown. [Figure 21] The phenotypic detection results are shown. [Figure 22] The results of detection of fatigue markers are shown. [Diagram 23] The results of detection of activation markers are shown. [Figure 24] The detection results of a flow cytometer are shown. [Diagram 25] 4 shows the results of an in vitro pharmacodynamic evaluation. [Figure 26] The phenotypic detection results are shown. [Figure 27] The results of fatigue detection are shown. [Figure 28] The results of proliferation and activity detection are shown. [Figure 29] The results of GVH intensity detection are shown. [Diagram 30] The results of in vivo drug efficacy detection are shown. [Diagram 31] The results of RT-PCR of CAR-T copy number in mouse blood are shown. [Diagram 32] FIG. 1. Efficiency of gene knockout. [Diagram 33] In vitro killing diagram of DU-19CAR, CD4 single negative 4N-19CAR, and CD8 single negative 8N-19CAR. [Diagram 34] The GVH intensity of mixed cells of CD4 single negative and CD8 single negative cells mixed in equal proportions is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] The present invention discloses a technique for preparing universal CAR-T cells and its use. Those skilled in the art can appropriately modify the process parameters based on the contents of this specification to realize the present invention. It should be noted that all substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and use of the present invention are described by preferred embodiments. Those skilled in the art can realize and apply the technology of the present invention by modifying or appropriately changing and combining the method and use described herein without departing from the content, spirit and scope of the present invention.

[0110] The double negative T cells protected by the present invention can be combined with any target and structure of chimeric antigen receptor (CAR) to obtain the technical effect disclosed in the present invention. Here, a chimeric antigen receptor targeting CD19 is specifically described as an example. Similarly, the vector is any one of a lentivirus expression vector, a reverse transcription virus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector, and a plasmid. Here, a lentivirus vector is described as an example. The preparation scheme can use an electroporation method and a non-electroporation method, but in this embodiment, the electroporation method is described as an example.

[0111] In the present invention, magnetic bead selection is performed by using biotin-labeled CD4 and CD8 antibodies and antibiotic-bound microbeads, or by using CD4 and CD8 positive magnetic beads and passing them through a column. In addition, selection may be performed using a flow sorter or by a method that can obtain the required part, and the required part may be selected at the end. The antibodies used to detect knockout efficiency and selection purity by flow cytometry are CD3: PE-CY7, CD4: FITC, and CD8: BV510. In the present invention, the method for preparing the double negative CAR-T cells is as follows: 1. CD4 and / or CD8 single positive T cells are obtained, and infected with a virus containing a CAR and / or CD44 and / or CD62L structure after or without activation for several hours, and cultured for several hours, and then RNP electroporation is performed on the T cells infected with the CAR structure, followed by culture, and selection of the target cells. 2. CD4 and / or CD8 single positive T cells are obtained, and RNP is introduced into the T cells by electroporation after activation for several hours or without activation, and then the cells are cultured for several hours, infected with a virus containing a CAR and / or CD44 and / or CD62L structure, and then cultured to select the target cells. 3. Cryopreserved or fresh mononuclear cells are obtained, and RNP is introduced into the cell nucleus by electroporation after activation for several hours or without activation, and CD4 and / or CD8 single positive T cells are selected and obtained, and RNP is further introduced into the cell nucleus by electroporation, and CD4 and CD8 double negative T cells are selected and obtained. 4. Cryopreserved or fresh mononuclear cells are obtained, and RNP is introduced into the cell nucleus by electroporation after activation for several hours or without activation, and CD4 and CD8 double negative T cells are selected and obtained, and then infected with a virus containing a CAR structure and / or CD44 and / or CD62L, and then cultured to obtain the target cells.

[0112] The structure of the CAR used in the present invention is extracellular recognition region (anti-CD19ScFv)-CD8-derived hinge-CD8-derived transmembrane region-intracellular signaling region (CD137-CD3), and the amino acid sequence is shown in SEQ ID NO:12.

[0113] [ka]

[0114] The preparation technique of universal CAR-T cells provided by the present invention and the raw materials and reagents used in the use thereof are all commercially available. The present invention will be further described below with reference to examples.

[0115] The present invention will now be further described with reference to examples.

[0116] Example 1: Results of knockout of CD4 and CD8 genes in T cells

[0117] Using a Lonza electroporator, select an electroporation program suitable for the introduction of T cells by electroporation. The molar ratio of Cas9 protein:sgRNA can be 1:2 or other suitable ratios. The RNP needs to be incubated in vitro for 5-15 minutes. Then, it is mixed evenly with an appropriate amount of T cells resuspended in electroporation buffer, transferred to an electroporation cup, and electroporated. After electroporation is completed, it is transferred to a culture flask containing an appropriate amount of medium for continued culture. 72 h after electroporation, CD4 or CD8 flow cytometry antibodies with detectable fluorescent labels can be incubated to detect knockout efficiency. Figure 32 shows the knockout efficiency of some of the targets used in Table 6. Under different conditions, other targets can also show relatively high knockout effect or higher knockout effect. Any target sequence that causes deletion of the gene itself in the gene region where CD4 and CD8 are knocked out can be used.

[0118] In the present invention, the positions of gene editing targets are shown in Tables 1 and 3-5. Target sequences that can cause deletion in the gene itself in any one of the regions included in the table are shown. The results are shown in FIG. 1. Some of the target sequences used may be shown in SEQ ID NOs: 14-33. The sequences are not limited to the sequences shown here, and may be any target sequences that cause deletion in the gene itself in the gene region where CD4 and CD8 are knocked out, and include the CD4 gene on human chromosome 12: 6,789,528-6,820,799 (located on the forward strand), the CD8A gene on human chromosome 2: 86,784,610-86,808,396 (located on the reverse strand), and the CD8B gene on human chromosome 2: 86,815,339-86,861,924 (located on the reverse strand). Gene editing target positions are shown in Tables 6 and 7. The target sequences that induce deletions in the gene itself in any included region are shown below.

[0119] [Table 3]

[0120] [Table 4]

[0121] [Table 5]

[0122] [Table 6]

[0123] [Table 7]

[0124] Example 2: Preparation of DU-19CAR Method for obtaining DU-19CAR prepared by the technical method of the present invention The starting sample was peripheral blood from healthy individuals, and CD4 / CD8 single positive T cells were selected by magnetic beads or flow cytometry, respectively. They were continuously stimulated with Dynabeads containing CD3 and CD28 antibodies to obtain the normal negative control group CT / CD4, the single positive control group 4P-CT / CD8, and the single positive control group 8P-CT. They were infected with lentivirus containing CAR to obtain the normal CAR-T / CD4 single positive 4P-CAR-T / CD8 single positive 8P-CAR-T.

[0125] Alternatively, the RNP complex of sgRNA targeting CD4 and / or CD8 and Cas9 protein may be introduced by electroporation (see Example 1 for a specific embodiment), and after electroporation, CD4 / CD8 double negative DU-CAR-T and / or CD4 / CD8 single negative 4N-CAR-T / 8N-CAR-T cells may be selected by flow cytometry or magnetic beads, and appropriate amounts of factors or any combination of medium and cytokines suitable for T cell culture may be added to the culture medium. Here, other steps in the CAR-T cell preparation process follow the steps described in our previous patent applications.

[0126] Example 3: Evaluation of CD4 and CD8 double-negative DU-19CAR 1. Method for detecting CAR positivity PL antibody labeled with PE. As can be seen from Figure 11, gene editing was performed on CD4 and CD8 on 19CAR, and high-purity double-negative CAR-T cells were selected by magnetic beads. The percentages of CD4 and CD8 in the non-gene-edited group were 53.46% and 43.43%, respectively. After gene knockout, the percentage of DT reached 89.93%, and after selection by magnetic beads, high-purity double-negative CAR-T cells with a purity of 99.94% were obtained. Such manipulation did not affect the expression of the CAR structure on T cells. The positive expression levels of the DU-19CAR group and the 19CAR group were both about 85%, while the expression level of the conventional universal CU-19CAR was 76%, which was slightly lower than the other two groups.

[0127] 2. In vitro efficacy and killing evaluation method The cells were plated at a specific effector-to-target ratio (E:T = CAR-T:tumor cells) and cultured for a given time in 1640 complete medium, and the viability of the target cells was detected by luciferase lysis method to calculate the cytotoxicity efficiency. The secretion level of interferon-gamma factor can be detected by using a kit applicable to this factor level. For details, please refer to the kit instructions of each manufacturer.

[0128] As can be seen from the in vitro pharmacodynamic evaluation results shown in Figure 12, the in vitro killing of DU-19CAR corresponds to that of the conventional universal CU-19CAR, and when the ratio is 1:4 and the killing time is 24h, the killing efficiency of both reaches about 90%, whereas the factor secretion level of the new universal DU-19CAR group of the present invention is twice that of the conventional universal CU-19CAR group, which indicates that the method of the present invention is advantageous in improving its tumor killing ability and attacking tumor cells more effectively.

[0129] 3. Phenotype detection method Flow cytometer detection Antibodies: CCR7:APC, CD45RA:BV421, CD45RO:Percp5.5 Phenotypic Classification Standards T memory stem cells: TSCM (CD45RA+CD45RO-CCR7+), similar T memory stem cells: TSCM-Like (CD45RA+CD45RO+CCR7+), effector memory T: TEM (CD45RA-CD45RO+CCR7-), central memory T: TCM (CD45RA-CD45RO+CCR7+), effector T: TE (CD45RA+CD45RO-CCR7-), others (100%-TSCM-TSCM-Like-TEM-TCM-TE) As shown in Figure 13, the phenotypic detection results show that compared with the conventional autologous 19CAR, the universal DU-19CAR groups have a relatively close subtype distribution, a slightly lower TSCM, but a slightly higher TE, which is advantageous for the instantaneous killing of tumor cells.

[0130] 4. How to detect fatigue markers Flow cytometer detection Antibodies: PD-1:APC, LAG-3:BV421, TIM-3:Percp5.5 As shown in Figure 14, the expression data results of fatigue marker detection show that neither of the two groups of CARs expresses TIM-3, but compared with the traditional autologous 19CAR, the expression of PD-1 and LAG-3 on the universal DU-19CAR of the present invention is lower, which indicates that the universal DU-19CAR of the present invention has longer persistence and is favorable for improving anti-tumor ability.

[0131] 5. Measurement method of GVH strength The mixed lymphocyte reaction (MLR) was measured by the method of microsphere absolute counting, and a mixture of donor cells (CAR-T): recipient cells (donor cells and healthy PBMCs of different HLA subtypes) was prepared at a specific ratio and cultured in 1640 basal medium for 8 days. The absolute cell count was detected by flow cytometry on days 0 and 8, respectively, and the relative GVH intensity was calculated based on the change in cell number.

[0132] As can be seen from the detection results shown in Figure 15, the intensity of the GVH reaction caused by the double negative DU-19CAR of the present invention is equivalent to that of the conventional universal CAR-T cells CU-19CAR, and both are lower than the control groups of autologous CAR-T cells 19CAR and CT groups. This result indicates that the DU-19CAR prepared by the method of the present invention can be applied to allogeneic CAR-T immunotherapy in the same way as the conventional universal CAR-T cells.

[0133] 6. In vivo pharmacodynamics evaluation method Mice were introduced on day 11, and after a series of tests such as quarantine, they were allowed to participate in the study. On day -3, 5e5 Nalm6-Luc-GFP target cells were injected into the tail vein, and on day 0, the mice were randomly divided into groups, and CAR-T cells were reinjected so that the number of CAR-T effective cells was 2e6. After that, bioimaging was performed every 7 days to observe the in vivo tumor elimination status of the mice.

[0134] As can be seen from the in vivo efficacy results shown in Figure 8, the 19CAR group, the DU-19CAR group and the CU-19CAR group had a significant tumor suppression effect in vivo compared with the Control T group. As can be seen from the fluorescence value curve data analysis of the DU-19CAR group and the CU-19CAR group, the CU-19CAR group has a significantly better in vivo efficacy compared with the DU-19CAR group, and the in vivo efficacy of the DU-19CAR of the present invention is slightly better than that of the autologous group 19CAR, which indicates that the in vivo anti-tumor effect of the new universal CAR-T cells of the present invention is much higher than that of the traditional universal CAR-T cells, and is also higher than that of the traditional autologous CAR-T cells. This is also one of the unexpected discoveries of the present invention.

[0135] In summary, the in vitro and in vivo pharmacodynamic evaluation, phenotype detection, and exhaustion marker detection of the DU-19CAR designed in the present invention showed that the DU-19CAR of the present invention is superior to conventional universal CAR-T cells in all aspects. The mixed lymphocyte reaction (MLR) was measured by the microsphere absolute counting method, and the degree of GVH reaction induced by the new universal DU-19CAR designed in the present invention was equivalent to that of conventional universal CAR-T cells (CU-19CAR), and it can be used for allogeneic reinfusion therapy.

[0136] Example 4: Evaluation of DU-19CAR in CD4 single negative, CD8 single negative, and a combination of CD4 single negative and CD8 single negative

[0137] 1. In vitro pharmacodynamics evaluation As can be seen from the in vitro pharmacodynamic evaluation results shown in Figure 33, the in vitro antitumor capabilities of DU-19CAR, CD4 single negative 4N-19CAR, and CD8 single negative 8N-19CAR are comparable to the control group autologous 19CAR, and the killing efficiency of each reaches 80% or more. The non-specific killing shown by K562-Luc-GFP is a normal phenomenon. This indicates that the in vitro antitumor capabilities of DU-19CAR of the present invention or DU-19CAR consisting of CD4 negative and CD8 negative 19CAR are not reduced by gene editing.

[0138] 2. Measurement of GVH strength The mixed lymphocyte reaction (MLR) and flow cytometry detection methods were used to measure and prepare cells at a specific ratio. Specifically, recipient cells (PBMCs from healthy donors with a different HLA subtype from donor cells) were first treated with mitomycin C to suppress their growth and eliminate interference, and then donor cells and / or recipient cells were stained with different colored dyes (CTV and / or CFSE) to distinguish them, and donor cells (activated T cells): recipient cells were mixed at a specific ratio. Here, in DU-CT and CT, where donor cells consist of CD4 positive / negative and CD8 positive / negative T cells, the component ratios of cells in each group are shown in Table 8. Immediately after mixing, the cells were detected by flow cytometry to distinguish their respective ratios, and cultured in 1640 basal medium. The changes in the respective ratios were detected by flow cytometry at specified times, and the relative strength of GVH was calculated based on the changes in the cell ratios.

[0139] In Figure 34, after 4 days of co-culture, the cell proliferation ratio due to the rejection of donor cells to host PBMCs was significantly lower in the DU-CT group than in the control CT group. These results indicate that the double negative CAR-T cells prepared by the method of the present invention can be used for allogeneic CAR-T immunotherapy.

[0140] [Table 8]

[0141] Example 5: Natural DNT or DN-CAR-T prepared using it (DN-19CAR: natural CD4 - CD8 - T cells) and the universal DU-CAR-T (DU-19CAR: CD4 T cells obtained by gene editing) prepared by the technology of the present invention. - CD8 - T cell)

[0142] Natural DNT refers to double negative T cells in human peripheral blood that do not express natural CD4 and CD8. Such cells have an antigen presentation pathway that is not restricted by MHC, and the probability of causing allo-rejection in allogeneic use is extremely low (1-5% of the total lymphocytes in peripheral blood), but the proportion of these cells in human peripheral blood is extremely low, and in vitro amplification is very difficult, resulting in poor therapeutic effects for indications. The universal double negative cells prepared by the technology of the present invention are initially derived from CD4 single positive or CD8 single positive T cells in human peripheral blood (40-80% of the total lymphocytes in peripheral blood), and such cells have an antigen presentation process that is restricted by MHC, cannot be directly used for allogeneic use, and require further manipulation to avoid allo-rejection. The present invention uses gene editing technology to inactivate CD4 and CD8 to inactivate CD4 - CD8 - We obtained double-negative T cells. In the present invention, we demonstrated that such cells have the necessary conditions to be applicable to allogeneic therapy, similar to conventional universal T cells (see data related to GVH).

[0143] 1. How to obtain a natural DNT Starting sample: Peripheral blood from healthy individuals (1) CD4 / CD8SPT cells (CD4 or CD8 cells) were removed using magnetic beads, and then stimulated with antibodies against CD3 and CD28 for 24-48 hours. Then, the cells were enriched in a medium containing IL2 and IL4 (and / or) IL7 (and / or) IL15 (and / or) IL21. (2) CD3 + CD4 - / CD8 - T cells were selected and stimulated with antibodies against CD3, CD28, and other antigens for 24-48 hours, and then enriched in medium containing IL2 and IL4 (and / or) IL7 (and / or) IL15 (and / or) IL21.

[0144] Method for obtaining DN-19CAR: Natural DNTCs were obtained, cultured for 24 h, and infected with CAR-containing lentivirus.

[0145] Six batches of peripheral blood from healthy individuals from different sources were collected, their PBMCs were separated, and the three batches with the highest DNT content were selected by detection (selection index: CD3 + CD4 - CD8 - The starting cells that should be used for the preparation of DU-19CAR were SPT (selection index: CD3 + , CD4 + / CD8 + The results are shown in Table 9.

[0146] [Table 9]

[0147] The statistics of the number of CAR-T cells obtained on the 6th day of cell activation, calculated with a starting amount of 1e8, are shown in Table 10. As can be seen from the data in the table, for the same starting amount of PBMC, after selecting a batch with a relatively high DNT rate, the obtained DU-19CAR cells were at least 3 times that of DN-19CAR cells, and even up to 35 times more than DN-19CAR cells. For the proliferation data that began to be monitored on the 6th day of activation, as shown in FIG. 23, in the late proliferation, DN-19CAR cells were much lower than DU-19CAR cells. As can be seen from the above results, the application range of DU-19CAR cells is wider, and it can meet the needs of various applications, especially fields with high requirements for cell amount such as clinical applications. As can be seen from FIG. 16, the CAR positive rate of DU-CAR-T cells is more than 60%, which is much higher than 25.4% of N-DN-CAR-T cells, that is, the rate of effective cells is higher, which is more favorable for killing tumor cells.

[0148] [Table 10]

[0149] 3. How to get the DU-19CAR The preparation was the same as before, and the cells were selected by magnetic beads, and then sorted by column using biotin-labeled CD4 and CD8 antibodies and antibiotic-bound microbeads. As shown in Figure 17, the negative portion can be selected. The antibodies used to select knockout efficiency and selection purity by flow cytometry were CD3: PE-CY7, CD4: FITC, and CD8: BV510.

[0150] As can be seen from Figure 17, the purity of DU-19CAR cells reached 100%, the selection purity was high, and the DNT ratio of DN-CAR-T cells was 96.6%. This indicates that the purity of DNT obtained by enrichment is relatively high, but slightly lower than that of the method of the present invention. This indicates that in terms of safety, the DU-CAR-T cells obtained by the method of the present invention have a greater advantage.

[0151] 4. In vitro pharmacodynamics evaluation As shown in Figure 18, when the effector to target ratio is 1:8, the killing results show a statistically significant difference between the DU-19CAR of the present invention and the conventional autologous 19CAR, and a highly significant statistically significant difference between the DU-19CAR of the present invention and the DN-19CAR with natural DNT, which indicates that the killing ability of the DU-CAR-T cells of the present invention is significantly higher than that of the DN-CAR-T cells and slightly better than that of the autologous CAR-T cell group.

[0152] 5. Factor detection results As can be seen from Figure 19, any kit that can be used to detect interferon gamma can be used to detect the supernatant medium after killing, and the method can be referred to the instructions for each reagent. As can be seen from the results, the DU-19CAR of the present invention has a significant statistical difference with the conventional autologous 19CAR, and the DU-19CAR of the present invention has a very significant statistical difference with the DN-19CAR by natural DNT. This indicates that the double negative universal CAR-T cells of the present invention have a higher in vitro efficacy from the level of factor secretion.

[0153] 6. Monitoring growth and activity Monitoring began when high-purity double-negative DU-19CAR was selected by magnetic beads (day 5 or day 6 after activation). Regarding the monitoring method, a predetermined starting amount (e.g., 1e6) of cells was taken from each group and monitored, and then monitored every 2-3 days. When it was observed that the cells of a certain group no longer proliferated and their activity began to decrease, monitoring was stopped. Cells should not be discarded throughout the monitoring process. As shown in FIG. 20, the proliferation ability and cell activity of the universal DU-CAR-T of the present invention are significantly superior to those of the natural double-negative DN-Control T or the double-negative DN-19CAR cells derived therefrom. This is because the universal DU-19CAR cells obtained by the preparation method of the present invention have both unaffected proliferation and activity and have unexpected effects, whereas the natural DNT has poor proliferation, is difficult to rapidly meet the amount required for treatment, and the production cost is significantly higher, making it difficult to promote clinical application.

[0154] 7. Phenotype, exhaustion markers, and activation marker detection 1) Phenotype detection results: As shown in Figure 21, the TE ratio of the DU-19CAR group of the present invention was significantly higher than that of the DN-19CAR group. Since the duration of action of CAR-T is short and instantaneous killing ability is an important indicator of drug efficacy, the killing results of this example indicate that the killing ability of the DU-19CAR of the present invention is higher.

[0155] 2) Detection results of exhaustion markers: As shown in Figure 22, the expression levels of three kinds of exhaustion molecules in the DU-19CAR of the present invention were all lower than those of the DN-19CAR. As can be seen from the results, the DU-19CAR of the present invention has higher persistence and a slower rate of apoptosis, so it can better exert its antitumor efficacy.

[0156] 3) Detection method of activation markers: Flow cytometer detection Antibodies: CD25:FITC, CD44:BV421, CD95:BV4711 As can be seen from the detection results of activation markers shown in FIG. 23, except for a small difference in CD25, CD95 and CD44 both showed similar expression.

[0157] Example 6: Preparation of CD44-expressing T cells and 44-19 CAR (chimeric antigen receptor T cells) cells CD44 can be combined with any target and structure of chimeric antigen receptor (CAR) to obtain the technical effect disclosed in the present invention. Here, a chimeric antigen receptor targeting CD19 is specifically described as an example. Similarly, the vector may be any one of a lentivirus expression vector, a reverse transcription virus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector, and a plasmid. Here, a lentivirus vector is described as an example.

[0158] 1) Construction of CD44 expression vector The fragment of interest and the vector fragment were ligated with T4 ligase (purchased from Promega) to obtain a lentiviral vector expressing a chimeric antigen receptor, such as the gene fragment corresponding to the CD44 amino acid sequence shown in SEQ ID NO: 12. The plasmid was extracted using a plasmid extraction kit (Invitrogen) (see the instruction manual for the specific method). The vector may be any one of lentiviral expression vectors, reverse transcription virus expression vectors, adenovirus expression vectors, adeno-associated virus expression vectors, DNA vectors, RNA vectors, and plasmids. To explain the specific preparation method, the lentiviral vector is taken as an example.

[0159] The gene sequence encoding the obtained CD44 molecule was double-digested with restriction enzymes NheI and XhoI (purchased from Thermo), and the lentivirus expression vector pRRLSIN.cPPT.PGK-GFP.WPRE plasmid was enzymatically digested with restriction enzymes NheI and XhoI (http: / / www.addgene.org / 12252 / ), and the pRRLSIN.cPPT.PGK-GFP.WPRE plasmid was simultaneously enzymatically digested with restriction enzymes NheI and XhoI (see the instructions for the enzyme digestion reaction). The enzyme digest was separated by agarose gel electrophoresis, and the DNA fragment was recovered using an agarose gel DNA fragment recovery kit. Then, the synthesized CD44 sequence fragment having the NheI and XhoI restriction enzyme sites was simultaneously enzymatically digested with the restriction enzymes NheI and XhoI, and then ligated to the above-mentioned DNA fragment obtained by the enzyme digestion to obtain a CD44 expression vector.

[0160] 2) Construction of a vector that expresses CD44 and specifically recognizes the CAR gene of tumors A chimeric antigen receptor sequence was synthesized that contained a single-chain antibody ScFv containing an anti-human CD19 antigen, a hinge region, a transmembrane region, and an intracellular signaling segment.

[0161] Then, PCR amplification was performed using the chimeric antigen receptor sequence and the CD44 molecule as templates. The reaction system was injected according to the instructions for KOD FX NEO DNA polymerase (purchased from TOYOBO), and the PCR reaction conditions were 95°C, 5 min pre-denaturation; 95°C, 10 s denaturation; 55°C, 15 s annealing; 68°C, 30 s extension; 30 cycles. The amplified product was identified on a 1% agarose gel (w / v). Then, DNA fragments were recovered using a recovery kit (Promega) (see the instructions for specific methods). The chimeric antigen receptor and CD44 molecule fragments were recovered and the DNA recovered fragments were sequenced.

[0162] (2) Construction of a lentiviral vector expressing a chimeric antigen receptor:CD19CAR-binding peptide (polycistronic structure)-targeting CD44.

[0163] The CAR molecule and the CD44 molecule are linked by a polycistronic structure. The polycistronic structure is a self-cleaving peptide or an internal ribosome entry site (IRES). The self-cleaving peptide is T2A, P2A, E2A or F2A. Here, P2A is taken as an example to obtain an expression vector targeting CD19CAR-P2A-CD44.

[0164] The enzyme digestion reaction was carried out according to the instructions. After separating the enzyme digest by agarose gel electrophoresis, the DNA fragments were recovered using an agarose gel DNA fragment recovery kit, and then the target fragment and the vector fragment were ligated with T4 ligase (purchased from Promega) to obtain a lentiviral vector expressing a chimeric antigen receptor and a CD44 molecule. 5 μL of the lentiviral vector was taken and transformed into E. coli TOP10, and after culturing at 30 ° C for 16 h, a monoclonal antibody was selected, and the selected monoclonal antibody was cultured under conditions of 30 ° C for 12 h, and the plasmid was extracted using a plasmid extraction kit (Invitrogen) (see the instructions for specific methods). The extracted plasmid was enzymatically digested with the restriction enzyme HindIII and identified by electrophoresis.

[0165] 3) Obtain vectors in steps 2) and 3) and prepare lentivirus (1) Lentivirus packaging In this embodiment, the lentivirus was packaged using the calcium phosphate method. The specific steps are as follows: 293T cells were cultured in DMEM medium containing 10% FBS (w / v) to an appropriate condition, and then 293T cells were cultured at 1×10 5 / cm 2 density of one 75cm 2The cells were transferred to a culture flask and cultured for 22 h to ensure that the cell confluence was 70-80% at the time of transfection. In addition, the culture medium was replaced with pre-warmed DMEM medium containing 2% FBS (w / v) and cultured for 2 h as a backup. 680 μL of ddH2O, 20 μg lentivirus vector (vectors described in steps 1) and 2), and 100 μL of 2.5 mM CaCl2 were placed in a 15 mL centrifuge tube and mixed uniformly, and then 2×HBS was dropped into the mixture using a pipette, and mixed uniformly by pipetting with a 10 mL pipette, and then left to stand at room temperature for 15 min. Next, the mixture was dropped onto the prepared cells, and then cultured for 12-16 h, after which the culture medium was replaced with DMEM medium containing 10% FBS (w / v). After the cells were cultured for 48 h and 72 h, the cell supernatants were collected and used for virus purification, respectively.

[0166] (2) Purification of lentivirus The virus supernatant was collected in a 50mL centrifuge tube, centrifuged at 3000r / min for 10min, filtered through a 0.45μm filter membrane, and the filtrate was centrifuged at 3000r / min for 10min, then transferred to a new 50mL centrifuge tube. Depending on the amount of virus supernatant, 50% PEG6000 (w / v) and 4M NaCl were added, and the total volume was adjusted to 35mL (final concentration of PEG6000 8.5%, final concentration of NaCl 0.3M) with medical saline, left in a 4℃ refrigerator for 90min, mixed by inversion for 30min / time, centrifuged the sample at 4℃ and 5000r / min for 30min, discarded the entire supernatant, resuspended the virus in 200μL of DMEM medium containing 10% FBS, dispensed into 1.5mL EP tubes at 40μL / tube, and stored at -80℃ as a backup.

[0167] (3) Measurement of lentivirus titer A, Virus-infected 293T cells 12h ago, 293T cells were plated in a 24-well plate containing 10% FBS (w / v) DMEM culture medium to ensure that the cell confluence before virus infection was 40%-70% and the cell condition was good. 1μL of 6g / mL polybrene solution was added to each well, and then 1μL of purified lentivirus vector was taken and diluted 10 times with medical saline to prepare the working solution and added to the corresponding wells. After 24h, the culture medium was replaced with DMEM medium containing 10% FBS (w / v). After 72h infection, the cells were collected by centrifugation at 1000r / min for 5min and the gene cluster was extracted.

[0168] B. Genome extraction The collected cells were resuspended in PBS, centrifuged at 1000r / min for 5 min, the supernatant was completely discarded, and the process was repeated once. The cells were resuspended in 200μL of PBS, 20μL of protease K and 200μL of AL lysis solution were added, mixed uniformly by pipetting, and incubated at 56℃ for 10 min. Then, 200μL of pre-cooled ethanol was added, mixed by inversion, the solution was transferred to a filtration column, left at room temperature for 1 min, centrifuged at 8000r / min for 1 min, the supernatant was completely discarded, 700μL of AW1 solution was added, centrifuged at 8000r / min for 1 min, the supernatant was completely discarded, 500μL of AW2 solution was added, centrifuged at 8000r / min for 1 min, the supernatant was completely discarded, and the filtration column was transferred to a new 1.5mL EP tube and left for 1 min with the lid open to evaporate the ethanol. 50 μL of sterilized ddH2O (preheated at 60° C.) was added, the mixture was left to stand for 2 minutes, and centrifuged at 12,000 r / min for 1 minute (the genome extraction kit was the QIAamp DNA Blood Mini Kit purchased from Qiagen).

[0169] C. Measurement of virus titer by qRT-PCR The reaction system is SYBR (R) Premix Ex Taq TMThe mixture consisted of 10 μL of GAG II (2×), 1 μL of upstream primer (GAG up), 1 μL of downstream primer (GAG dn), 1 μL of extracted genome, and 7 μL of RNase-Free dH2O. At least three duplicate wells were prepared for each sample and standard. Then, amplification was performed with the following process: 95°C, 30s pre-denaturation; 95°C, 5s denaturation; 60°C, 30s annealing; 72°C, 30s extension. After the reaction was completed, the data was analyzed with analysis software, and the virus titer was calculated by the standard curve. The results are shown in TU / mL.

[0170] 4) Lentivirus-infected T cells (1) Isolation of human peripheral blood mononuclear cells Approximately 60 mL of normal human peripheral blood was collected in a blood collection tube containing anticoagulant, and dispensed into two 50 mL centrifuge tubes (30 mL / tube), and diluted with 7.5 mL of hydroxyethyl starch. The centrifuge tube was left at room temperature for approximately 30 min to allow natural sedimentation, and the upper layer plasma was collected, which was then centrifuged at 1400 r / min for 15 min, and then resuspended in saline to allow sedimentation. The cell suspension was carefully added to the lymphocyte separation solution in a volume ratio of 1:1, and gradient centrifugation was performed at 400 g for 15 min, with the centrifuge deceleration rate DEC set to 5. After centrifugation, the centrifuge tube was divided into four layers from top to bottom: plasma layer, circular milky peripheral blood lymphocyte (PBMC) layer, clear separation solution layer, and red blood cell layer. The second layer of PBMC was carefully aspirated into a new centrifuge tube and washed twice with saline, centrifuged once at 400g for 10 min and once at 1100r / min for 5 min. The cells were then resuspended in saline, the PBMC cell suspension was aspirated with a pipette and transferred to a culture flask, and cultured in RPMI 1640 complete medium containing 10% FBS (w / v).

[0171] (2) Lentiviral vector-infected T lymphocytes Freshly prepared mononuclear cells (PBMCs) were cultured in RPMI 1640 complete medium containing 10% FBS, activated by adding anti-CD3 monoclonal antibody on day 1, and lentivirus infection was performed for the previous 3 days. The lentivirus vectors described in 1) and 2) were added at a multiplicity of infection (MOI) of 2, respectively, and uninfected peripheral blood lymphocytes (PBMCs) were used as blank controls. After 24 h, the medium was replaced with RPMI 1640 complete medium containing 500 IU / mL recombinant human IL-2, and the cells were subsequently cultured for 10-20 days.

[0172] In some examples, T cells co-expressing CD44 and CAR can also be prepared by the following scheme. A. CD44-expressing T cells were prepared using the scheme in step 1) above, and CD44-expressing T cells were obtained. The CD44 T cells were then transduced with a CAR gene to obtain T cells that simultaneously express CD44 and CAR. B. CAR-expressing T cells (CAR-T cells) were obtained by a similar step to step 2) above. The difference is that the CAR expression vector does not contain the CD44 gene, and the CD44 gene was further transduced based on the obtained CAR-T cells to obtain T cells (44-19CAR) that simultaneously express CD44 and CAR. The expression of CD44 and CAR is shown in Figure 2. Both CD44 and CAR can be normally expressed on T cells.

[0173] Example 7: Preparation of DU-44-19CAR 1. Gene knockout experiments Referring to Example 1, DU-T cells in which CD4 and CD8 were simultaneously knocked out were prepared.

[0174] 2. How to get the DU-44-19CAR The starting sample was peripheral blood from healthy donors. CD4 / CD8 single positive T cells were selected by magnetic beads or flow cytometry, continuously stimulated with Dynabeads containing CD3 and CD28 antibodies, infected with lentivirus containing CAR, transfected with RNP mixture of sgRNA targeting CD4 and CD8 and Cas9 protein by electroporation, and after electroporation, DU-CAR-T cells were selected by flow cytometry or magnetic beads, and the appropriate amount of factors or any combination of medium and cytokines suitable for the culture of T cells were added to the culture medium.

[0175] How to obtain the DU-44-19CAR (both of the following two methods are available): 1. CD4 and / or CD8 single positive T cells were obtained and activated for several hours, then infected with a virus containing CD44 and a CAR structure. After culturing for several hours, RNP was introduced into the infected T cells by electroporation, and the cells were subsequently cultured to select the target cells. 2. CD4 and / or CD8 single positive T cells were obtained and activated for several hours, after which RNP was introduced into the T cells by electroporation. After culturing for several hours, the T cells were infected with a virus containing CD44 and CAR structure, and then cultured to select the target cells.

[0176] Preparation sequence for obtaining 44-19CAR: CD4 and / or CD8 single positive T cells are obtained and activated for several hours, then infected with a virus containing CD44 and CAR structures, and subsequently cultured.

[0177] Example 8: Evaluation of DN-44-19CAR and 44-19CAR 1. CAR expression Gene editing was performed on CD4 and CD8 on 44-19CAR cells, and high-purity double-negative CAR-T cells were selected using magnetic beads. Such manipulation does not affect the expression of the CAR structure on T cells. As can be seen from Figure 2, the CAR expression levels on 44-19CAR and DU-44-19CAR cells were both above 55%. After DU-19CAR selection, the proportion of double-negative CAR-T was high, at over 99%.

[0178] 2. In vitro killing The tumor cells were plated at a specific effector-to-target ratio E:T = CAR-T:tumor cells and cultured in 1640 complete medium for a given time, and the viability of the target cells was detected by luciferase lysis method, and the cytotoxicity efficiency was calculated. The secretion level of interferon-gamma factor can be detected by using a kit that can be used to detect this factor level. For detailed operations, please refer to the instructions of the kit provided by each manufacturer.

[0179] As shown in FIG. 3 and Tables 11 to 14, the in vitro pharmacodynamic evaluation results showed that the in vitro efficacy of 44-19CAR was comparable to that of the autologous 19CAR, and when the effector-to-target ratio was 1:4 and the killing time was 24 h, the killing efficiency reached more than 90%, and similarly, DU-44-19CAR had comparable killing ability compared to 19CAR. As a result of factor secretion, both 44-19CAR and DU-44-19CAR were lower than 19CAR. This indicates that in the case of equivalent killing ability, the 44-19CAR and DU-44-19CAR of the present invention did not affect their killing ability even if they secreted fewer factors. This reduces the risk of cytokine storm and improves the safety of CAR-T therapy. Regarding IFN-γ, IFN-γ is a TH1 molecule, and currently CAR-T cells exert specific functions, but if IFN-γ secretion is too high, it is related to the side effect cytokine "storm" (CRS) that occurs in CAR-T treatment, so high secretion of IFN-γ has both advantages and disadvantages. The data corresponding to Figure 3 is shown below.

[0180] Table 11: 44-19CAR Kill Data [Table 11]

[0181] Table 12: Kill data for the DU-44-19CAR [Table 12]

[0182] Table 13: IFN-γ (pg / ml) secretion data for 44-19CAR [Table 13]

[0183] Table 14: IFN-γ (pg / ml) secretion data for DU-44-19CAR [Table 14]

[0184] 3. Phenotypic outcomes and fatigue markers Detection by flow cytometer; Antibodies: CCR7: APC, CD45RA: BV421, CD45RO: Percp5.5; Phenotypic classification standard: T memory stem cells: TSCM (CD45RA+CD45RO-CCR7+), like T memory stem cells: TSCM-Like (CD45RA+CD45RO+CCR7+), effector memory T: TEM (CD45RA-CD45RO+CCR7-), central memory T: TCM (CD45RA-CD45RO+CCR7+), effector T: TE (CD45RA+CD45RO-CCR7-), others (100%-TSCM-TSCM-Like-TEM-TCM-TE). Detection method for exhaustion markers: detection by flow cytometer; antibodies: PD-1: APC, LAG-3: BV421, TIM-3: Percp5.5.

[0185] As shown in Figure 4 and Tables 15-16, the phenotype detection results showed that compared with the autologous 19CAR, the 44-19CAR and universal DU-44-19CAR had a relatively close subtype distribution in each group, a slightly higher TSCM, and a slightly lower TE, but the comparison of the killing results did not affect the tumor cell killing ability. In addition, as can be seen from Figure 5 and Tables 17-18, as can be seen from the data results of the exhaustion marker detection expression amount, none of the three groups of CARs expresses TIM-3, but compared with the autologous 19CAR, the expression of PD-1 and LAG-3 on the 44-19CAR and universal DU-44-19CAR of the present invention are all lower, which also suggests that the universal 44-19CAR and DU-44-19CAR of the present invention have a longer persistence, which is advantageous for improving anti-tumor ability. The data corresponding to Figure 4 is shown below.

[0186] Table 15: Phenotypic data for 44-19CAR [Table 15]

[0187] Table 16: Phenotypic data of DU-44-19CAR [Table 16]

[0188] The data corresponding to Figure 5 is as follows: Table 17: 44-19CAR Exhaustion Markers [Table 17] Table 18: DU-44-19CAR exhaustion markers [Table 18]

[0189] 4. Liveness detection After RNP is introduced by electroporation, the target cells need to be purified by selection. This time point is generally performed 5-7 days after activation. Cell activity begins to be monitored on the day of selection. The main embodiment is to obtain cell activity at a given time point by counting with a cell counter. In order to maintain experimental consistency, 19CAR and 44-19CAR do not need to be selected, but both are monitored at the same time point as DU-44-19CAR.

[0190] As shown in Figure 6 and Tables 19 to 20, the activity of the 44-19CAR and universal DU-44-19CAR of the present invention was comparable to that of the 19CAR in the first few days after selection. However, after 7 days, the activity of the CAR-T cells of the present invention was still maintained at 80% or more, whereas the activity of the autologous 19CAR group was significantly decreased. The data corresponding to Figure 6 is shown below.

[0191] Table 19: Activity values ​​of 44-19CAR [Table 19]

[0192] Table 20: Activity values ​​of DU-44-19CAR [Table 20]

[0193] 5. Detection of GVH intensity The mixed lymphocyte reaction (MLR) was measured by the method of microsphere absolute counting. A mixture of donor cells (CAR-T): recipient cells (donor cells and healthy PBMCs of different HLA subtypes) was prepared at a specific ratio and cultured in 1640 basal medium for 8 days, and the absolute cell count was detected by flow cytometry on days 0 and 8, respectively, and the relative intensity of GVH was calculated based on the change in cell number.

[0194] The detection results of GVH intensity are shown in Figure 7 and Table 21. The GVH reaction intensity induced by the universal DU-44-19CAR of the present invention on day 8 was equivalent to that of the conventional universal CAR-T cells CU-44-19CAR, and was weaker than the control groups autologous 19CAR, 44-19CAR and CT groups. As can be seen from these results, the DU-44-19CAR prepared by the method of the present invention can be used in allogeneic CAR-T immunotherapy, similar to the conventional universal CAR-T cells. The data corresponding to Figure 7 are shown below.

[0195] Table 21: Anti-GVH values ​​for DU-44-19CAR [Table 21]

[0196] 7. In vivo efficacy experiment 2 (comparison between 19CAR and 44-19CAR, DU-44-19CAR and 19CAR) Mice were introduced on day -11, and were quarantined and subjected to a series of tests before being included in the experiment. On day -3, 5e5 Nalm6-Luc-GFP target cells were injected into the tail vein. On day 0, the mice were randomly divided into groups, and CAR-T cells were reinjected so that the number of CAR-T effective cells was 2e6. After that, bioimaging was performed every 7 days to observe the in vivo tumor elimination status of the mice.

[0197] As shown in Figure 9, the results of in vivo efficacy show that the 19CAR group and the 44-19CAR group exhibited significant tumor suppression effects in vivo compared with the Control T group. The data of in vivo antitumor effect shows that the 19CAR group is equivalent to the 44-19CAR group. However, as can be seen from the CAR-T survival results, the survival of 44-19CAR is greater, which indicates that the 44-19CAR of the present invention can improve the survival of CAR-T cells in vivo, and thus can kill tumors sustainably.

[0198] Analysis of the fluorescence curve data of the DU-44-19CAR group and the 19CAR group showed that the antitumor effect of DN-44-19CAR was significantly better than that of the 19CAR group, as shown in Figure 9. This indicates that the universal CAR-T cells DU-44-19CAR of the present invention not only achieve universality, but also have higher efficacy than the conventional autologous CAR-T cells.

[0199] In summary, in vitro pharmacodynamic evaluation, GVH strength detection, and in vivo antitumor detection were performed on the DU-44-19 CAR-T designed in the present invention, and the results showed that it is superior to conventional CAR-T in terms of reducing the risk of cytokine storm, improving in vivo survival, and antitumor effect.

[0200] Example 9: Preparation of CD62L-expressing T cells and L-19CAR (chimeric antigen receptor T cells) cells

[0201] The CD62L molecule can be combined with any target and structure of chimeric antigen receptor (CAR) to obtain the technical effect disclosed in the present invention. Here, the specific application of the chimeric antigen receptor targeting CD19 is described as an example. Similarly, the vector may be any one of a lentivirus expression vector, a reverse transcription virus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector, and a plasmid. Here, the lentivirus vector is described as an example. For specific preparation steps, see Example 1.

[0202] The gene fragment corresponding to the amino acid sequence of CD62L is shown in SEQ ID NO:13.

[0203] In some examples, T cells co-expressing CD62L and CAR can also be prepared by the method described in Example 6. CD62L can be used instead of CD44.

[0204] The expression of CD62L and CAR is shown in Figure 24. Both CD62L and CAR could be normally expressed in T cells.

[0205] Example 10: Preparation of DU-L-19CAR 1. Gene knockout experiments Referring to Example 1, DU-T cells in which CD4 and CD8 were simultaneously knocked out were obtained.

[0206] 2. How to get the DU-L-19CAR The starting sample was peripheral blood from a healthy individual. CD4 / CD8 single positive T cells were selected by magnetic beads or flow cytometry, and continuously stimulated for 48 h with Dynabeads containing antibodies to CD3 and CD28, infected with lentivirus containing CAR for 24 h, and RNP mixture of sgRNA targeting CD4 and CD8 and Cas9 protein was introduced by electroporation for 48 h. DU-CAR-T cells were selected by flow cytometry or magnetic beads 72 h after electroporation, and the culture medium was supplemented with appropriate amounts of factors (IL2 and IL4 (and / or) IL7 (and / or) IL15 (and / or) IL21) at 1640, or any combination of medium and cell factors suitable for culturing T cells.

[0207] How to get the DU-L-19CAR 1. CD4 and / or CD8 single positive T cells were obtained and activated for several hours, then infected with a virus containing CD62L and a CAR structure. After culturing for several hours, the infected T cells were subjected to RNP electroporation, and then cultured to select the target cells. 2. CD4 and / or CD8 single positive T cells were obtained and activated for several hours, after which RNP was introduced into the T cells by electroporation. After culturing for several hours, the T cells were infected with a virus containing CD62L and CAR structure, and then cultured to select the target cells.

[0208] Example 11: Evaluation of DN-L-19CAR and L-19CAR 1. CAR expression Gene editing was performed on CD4 and CD8 on L-19CAR cells, and high-purity DU-19CAR cells were selected using magnetic beads. This operation did not affect the expression of the CAR structure on T cells. As can be seen from Figure 24, the expression levels of CAR were all higher than 70%, and after DU-L-19CAR selection, the proportion of double-negative CAR-T cells reached 100%.

[0209] 2. In vitro killing

[0210] Specific effector to target ratios were plated, see each manufacturer's kit instructions for details.

[0211] The results of in vitro pharmacodynamic evaluation are shown in Figure 25 and Tables 22 to 25. The in vitro efficacy of L-19CAR and DU-L-19CAR is equivalent to that of the autologous 19CAR, and when the effector-to-target ratio is 1:4 and the killing time is 24 h, the killing efficiency reaches more than 90%, and the detection results of factor secretion show no difference. The data for Figure 25 are shown in the table below.

[0212] Table 22: L-19CAR Kill Data [Table 22]

[0213] Table 23: DU-L-19CAR Kill Data [Table 23]

[0214] Table 24: Secretion data of IFN-γ (pg / ml) of L-19CAR [Table 24]

[0215] Table 25: Secretion data of IFN-γ (pg / ml) of DU-L-19CAR [Table 25]

[0216] 3. Phenotypic results Flow cytometer detection The phenotypic detection results are shown in Figure 26 and Tables 26 to 27. Compared with the autologous 19CAR, the L-19CAR and universal DU-L-19CAR groups had relatively close distributions of their respective subtypes, slightly lower TSCM, but slightly higher TE. This is favorable for improving the instantaneous killing ability of CAR-T. The data for Figure 26 are shown in the table below.

[0217] Table 26: Phenotypic data of L-19CAR [Table 26]

[0218] Table 27: Phenotypic data for DU-L-19CAR [Table 27]

[0219] 4. Fatigue marker Detection method for fatigue markers: Flow cytometer detection Antibodies: PD-1:APC, LAG-3:BV421, TIM-3:Percp5.5

[0220] As for the results of the detection data of the expression level of exhaustion markers, as can be seen from Figure 27, none of the CARs in each group expresses TIM-3, but the expression levels of LAG-3 and PD-1 on the L-19CAR and universal DU-L-19CAR of the present invention are lower than those on the autologous 19CAR. This also indicates that the L-19CAR and universal DU-L-19CAR of the present invention have a longer persistence and a longer time to apoptosis. This is advantageous for improving the sustained anti-tumor ability of CAR-T cells.

[0221] 5. Detection of proliferation and activity After RNP is introduced by electroporation, it is necessary to purify the target cells by sorting. This time point is generally 5-7 days after activation. Cell activity starts to be monitored on the day of sorting. The main embodiment is to obtain cell activity at a specific time point by counting with a cell counter. In order to keep the consistency of the experiment, it is not necessary to sort 19CAR and L-19CAR, but to monitor them at the same time point as DU-L-19CAR.

[0222] The monitoring results of proliferation and activity are shown in FIG. 28 and Tables 28 to 29. As can be seen from the results, the universal DU-L-19CAR of the present invention has better proliferation ability and cell activity than the autologous 19CAR. Among them, in terms of proliferation fold, the DU-L-19CAR of the present invention had a large difference with the other two groups, the proliferation rate was significantly better than the autologous 19CAR group, and the cell activity was also higher than the autologous 19CAR group. This indicates that the preparation method of the present invention has the effect of significantly improving the proliferation ability of CAR-T cells. Therefore, this method can be further used for therapeutic purposes.

[0223] The data for Figure 28 is shown in the table below. Table 28: Activity values ​​of DU-L-19CAR [Table 28]

[0224] Table 29: DU-L-19CAR proliferation numbers [Table 29]

[0225] 6. GVH strength detection The results of GVH intensity detection are shown in Figure 29 and Table 30. The intensity of the GVH reaction caused by the double negative DU-L-19CAR of the present invention was lower than that of the conventional universal CAR-T cells CU-L-19CAR, and significantly lower than the control group autologous CAR-T cells L-19CAR, 19CAR group and CT group. This result indicates that the DU-L-19CAR prepared by the method of the present invention can be applied to immunotherapy using allogeneic CAR-T, similar to the conventional universal CAR-T cells.

[0226] The data for Figure 29 is shown in the table below. Table 30: Anti-GVH values ​​for DU-L-19CAR [Table 30]

[0227] 7. In vivo experiment (comparison of DU-L-19CAR, L-19CAR and 19CAR)

[0228] Mice were introduced on day 11, and were quarantined and subjected to a series of tests before being included in the study. On day -3, 5e5 Nalm6-Luc-GFP target cells were injected into the tail vein, and on day 0, the mice were randomly divided into groups and re-injected with CAR-T cells so that the effective cell count of CAR-T was 2e6. After that, bioimaging was performed every 7 days to observe the in vivo tumor elimination status of the mice.

[0229] Regarding the in vivo efficacy results, as shown in FIG. 30, the 19CAR group, the DU-L-19CAR group, and the L-19CAR group showed a significant tumor suppression effect in vivo compared with the Control T group. Analysis of the fluorescence value curve data of the DU-L-19CAR group, the L-19CAR group, and the 19CAR group showed that the DU-L-19CAR group and the L-19CAR group have higher in vivo efficacy compared with the 19CAR group. This shows that the L-19CAR group prepared by the method of the present invention has higher efficacy. The universal DU-L-19CAR of the present invention not only realizes universality, but also has higher efficacy compared with the conventional autologous CAR-T cells. This has a strong enlightening effect on the therapeutic field of universal CRA-T cells.

[0230] Furthermore, the CAR-T copy number in mouse blood was detected by RT-PCR, and as shown in Figure 31, the L-19CAR of the present invention had more than twice as many CAR-T cells persisting in vivo as the conventional autologous 19CAR group. This is sufficient to demonstrate that the method of the present invention improves the persistence of CAR-T in vivo, thereby further improving the sustained anti-tumor ability of CAR-T in vivo.

[0231] In summary, the results of in vitro pharmacodynamic evaluation, proliferation and activity monitoring, and GVH strength detection of the DU-L-19 CAR-T designed in the present invention show that it is superior to conventional CAR-T in all aspects, including antitumor effect, cell proliferation ability, cell activity, and in vivo survival.

[0232] The above is a detailed description of the preparation technology of universal CAR-T cells provided by the present invention and its use. In this specification, the principle and embodiment of the present invention are described by specific examples, but the description of the above examples is only for understanding the method and the purpose of the present invention. It should be noted that those skilled in the art can make some improvements and modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications are also included in the scope of the claims of the present invention.

Claims

1. An RNP complex comprising an RNP complex comprising a Cas9 protein and an sgRNA, The RNP complex, wherein the sgRNA is a CD4 sgRNA and / or a CD8 sgRNA.

2. The RNP complex according to claim 1, characterized in that the RNP complex is a CRISPR / Cas9 intracellular operation format, and the extracellular delivery techniques include a plasmid delivery technique, a viral delivery technique, a protein (Cas9) and (RNA) gRNA delivery technique, an mRNA (Cas9) and gRNA delivery technique, an mRNA (Cas9) and plasmid (gRNA) delivery technique, a viral (Cas9) and gRNA delivery technique, a viral (Cas9) and plasmid (gRNA) delivery technique, a plasmid (Cas9) and virus (gRNA) delivery technique, a plasmid (Cas9) and gRNA delivery technique, and combinations thereof.

3. The RNP complex according to claim 1 or 2, characterized in that the CD4 gene is the CD4 gene on human chromosome 12: 6,789,528-6,820,799 (located on the forward strand), and the CD8 gene is the CD8A gene on human chromosome 2: 86,784,610-86,808,396 (located on the reverse strand) and the CD8B gene on human chromosome 2: 86,815,339-86,861,924 (located on the reverse strand).

4. The sequence of the CD4 sgRNA is shown in any one of SEQ ID NOs: 1-6 or 14-24, and the sequence of the CD8 sgRNA is shown in any one of SEQ ID NOs: 7-11 or 25-33. The RNP complex according to claim 1 or 2.

5. Use of the RNP complex according to any one of claims 1 to 4 as an agent for promoting the secretion level of interferon-γ factor in allogeneic CAR-T cells.

6. Use of a CD4 gene inhibitor, knockout agent and / or a CD8 gene inhibitor, knockout agent as a cell proliferation agent, activity enhancer, phenotype distribution improving agent, or exhaustion marker expression inhibitor in the production of therapeutic T-cells or CAR-T cells.

7. A T cell in which a target site has been knocked out using the RNP complex according to any one of claims 1 to 4.

8. The T cell according to claim 7, characterized in that the CD4 gene and / or the CD8 gene of the T cell have been knocked out and / or inactivated.

9. The T cell according to claim 7 or 8, characterized in that the T cell expresses CD44 and / or CD62L.

10. The T cell according to any one of claims 7 to 9, characterized in that the amino acid sequence of the CD44 is shown in SEQ ID NO: 12 and the amino acid sequence of the CD62L is shown in SEQ ID NO:

13.

11. The T cell according to any one of claims 7 to 10, characterized in that the T cell is obtained by gene editing.

12. The T cell according to any one of claims 7 to 11, characterized in that the T cell is obtained by natural isolation.

13. The T cell according to any one of claims 7 to 12, characterized in that the genes for β2-microglobulin, and / or common subunit of HLA class I molecules, and / or CIITA of the T cell are non-functional or deleted.

14. The T cell according to any one of claims 7 to 13, characterized in that the T cell is an allogeneic T cell.

15. The T cell according to any one of claims 7 to 14, characterized in that the T cell also expresses HLA-E and / or HLA-G and / or HLA-F.

16. A method for preparing T cells according to any one of claims 7 to 15, comprising: A preparation method, comprising knocking out and / or inactivating the CD4 gene and / or the CD8 gene in a T lymphocyte by gene editing technology using the RNP complex according to any one of claims 1 to 4.

17. (1) Using fresh or cryopreserved T cells, knock out or inactivate the CD4 and CD8 genes by gene editing, either simultaneously knock out or inactivate the CD4 and CD8 genes, or knock out or inactivate CD4 or CD8, and then knock out or inactivate CD8 or CD4, and finally obtain the target cells; (2) A method of selecting CD4-positive T cells and obtaining target cells by knocking out or inactivating the CD4 gene by gene editing; (3) A method for selecting CD8-positive T cells and obtaining target cells by knocking out or inactivating the CD8 gene by gene editing; and (4) A method of selecting CD4-positive T cells, knocking out or inactivating the CD4 gene by gene editing, selecting CD8-positive T cells, knocking out or inactivating the CD8 gene by gene editing, and finally mixing the T cells in which the CD4 gene has been knocked out or inactivated by gene editing with the T cells in which the CD8 gene has been knocked out or inactivated by gene editing to obtain target cells; The preparation method according to claim 16, characterized in that the target cells are obtained by any one of the methods above.

18. The preparation method according to claim 16 or 17, characterized in that the T cells are activated and / or non-activated T lymphocytes.

19. The preparation method according to any one of claims 16 to 18, characterized in that the CD4 gene and / or CD8 gene in the T cells is knocked out or inactivated after expansion of the T cells or before expansion of the T cells.

20. The preparation method according to any one of claims 16 to 19, characterized in that the gene editing technology is to remodel T cells by electroporation or non-electroporation.

21. The preparation method according to any one of claims 16 to 20, characterized in that the gene editing technology comprises one or more of CRISPR, ZFN or TALEN.

22. T cells obtained by the preparation method according to any one of claims 16 to 21.

23. A CAR-T cell prepared using a T cell according to any one of claims 7 to 15 or claim 22.

24. The CAR-T cell according to claim 23, wherein the CAR in the CAR-T cell comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

25. The CAR-T cell according to claim 23 or 24, wherein the CAR-T cell is an allogeneic CAR-T cell.

26. The CAR-T cell according to any one of claims 23 to 25, further expressing membrane-bound IL15.

27. The CAR-T cell according to any one of claims 23 to 26, wherein the CAR-T cell further expresses secreted cytokines, the cytokines including one or more of IL2, IL7, IL12, IL21, or IL15.

28. The CAR-T cell according to any one of claims 23 to 27, characterized in that the CAR-T cell can further express and recognize a fusion protein of a molecule in a tumor microenvironment, and the extracellular structure of the fusion protein recognizes one or more of PD1, PDL1, SIRPγ, SIRPδ, or TIGIT.

29. The CAR-T cell according to any one of claims 23 to 28, characterized in that the CAR structure can be activated or inactivated by control, and the structure controlling activation or inactivation is one or more of peptide neoepitope (PNE), fluorescein (FITC), 10 amino acids (5B9 tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv, leucine ZipFv bound to an antibody, or streptavidin 2 (mSA2) biotin-binding domain.

30. The CAR-T cell can recognize solid tumor and blood tumor cells / tissues, and the antigen recognition region of the CAR-T cell is an antigen: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Tr The CAR-T cell according to any one of claims 23 to 29, which is capable of recognizing one or more of op2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, or TAG-72.

31. (1) A method of collecting and obtaining T cells, infecting them with a virus having a CAR structure, and introducing RNPs into the T cells infected with the CAR structure by electroporation to obtain target cells; or (2) A method of collecting and obtaining T cells, introducing RNPs into the T cells by electroporation, and then infecting the T cells with a virus containing a CAR structure to obtain target cells; The method for preparing a CAR-T cell according to any one of claims 23 to 30, wherein the target cell is obtained by any one of the methods described above.

32. (1) A method in which T cells are collected and infected with a virus containing CD62 and a CAR structure, or infected with a virus containing CD44 and a CAR structure, and RNP is introduced into the T cells infected with the CAR structure by electroporation to obtain target cells; or (2) A method of collecting and obtaining T cells, introducing RNPs into the T cells by electroporation, and then infecting the T cells with a virus containing CD44 and a CAR structure, or with a virus containing CD62 and a CAR structure, to obtain target cells; The preparation method according to claim 31, characterized in that the target cells are obtained by any one of the methods above.

33. (1) A method in which T cells are collected and then infected with a virus containing CD62L, CD44 and a CAR structure, and RNP is introduced into the T cells infected with the CAR structure by electroporation to obtain target cells; or (2) A method in which T cells are collected and obtained, RNPs are introduced into the T cells by electroporation, and the T cells are then infected with a virus containing CD62L, CD44, and a CAR structure to obtain target cells; The preparation method according to claim 32, characterized in that the target cells are obtained by any one of the methods above.

34. A preparation method according to any one of claims 31 to 33, characterized in that in the process of introducing RNP by electroporation, the RNP complex is the RNP complex described in claim 1.

35. The preparation method according to any one of claims 31 to 34, characterized in that the molar ratio of the Cas9 protein to the sgRNA is 1:

2.

36. A recombinant vector infected into a CAR-T cell according to any one of claims 23 to 30 or the CAR-T cell prepared by the preparation method according to any one of claims 31 to 35, The recombinant vector comprises a CAR structure.

37. The recombinant vector according to claim 36, comprising a functional fragment of a CD44 gene and / or a functional fragment of a CD62L gene.

38. The recombinant vector comprises: (I) CD44 and CAR structures; (II) CD62L and a CAR structure, or (III) CD62L, CD44 and CAR structure 38. A recombinant vector according to claim 36 or 37, comprising:

39. The recombinant vector according to any one of claims 36 to 38, characterized in that the expression vector used in the recombinant vector is one or more of a lentivirus expression vector, a reverse transcription virus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector, and a plasmid.

40. A cell composition comprising a T cell according to any one of claims 7 to 15, or claim 22, and / or a CAR-T cell according to any one of claims 23 to 30, or the CAR-T cell prepared by the preparation method according to any one of claims 31 to 35.

41. The cell composition of claim 40, wherein the proportion of the T cells or the CAR-T cells contained in the cell composition is 90%, 80%, 70%, or 60% or more by number.

42. The cell composition according to claim 40 or 41, characterized in that the cell composition is an activation-induced CIK cell or NKT cell.

43. Use of the T cell according to any one of claims 7 to 15, or claim 22 and / or the CAR-T cell according to any one of claims 23-30, the CAR-T cell prepared by the preparation method according to any one of claims 31-35, and / or the recombinant vector according to any one of claims 36-39, and / or the cell composition according to any one of claims 40-42, in the preparation of a cell therapeutic drug.

44. The use according to claim 43, characterized in that the use is use of the T cell according to any one of claims 7 to 15 or claim 22 and / or the CAR-T cell according to any one of claims 23-30, or the CAR-T cell prepared by the preparation method according to any one of claims 31-35 and / or the recombinant vector according to any one of claims 36-39 and / or the cell composition according to any one of claims 40-42 in the preparation of a medicament for treating acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer and / or skin cancer.

45. Use of CD62L and / or CD44 in the preparation of an enhancer of CAR-T cell activity.

46. Use of a combination of CD44 and CD62L in the preparation of an enhancer of CAR-T cell activity.

47. Use of CD62L and / or CD44 in the preparation of an inhibitor of PD-1 and LAG-3 factors secreted by CAR-T cells.

48. Use of CD62L and / or CD44 genes as promoters of tumor cell killing in the preparation of CAR-T cells, The CAR-T cell is a normal CAR-T cell, a CAR-T cell that does not express CD4 and / or CD8 genes, or a universal CAR-T cell.

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