Modified immune effector cells and methods for preparing same

Abstract

The present invention provides modified immune effector cells, in which the expression and / or activity of the TRAC gene and the HLA-A gene is down-regulated, the expression and / or activity of the B2M gene is not down-regulated, and the expression and / or activity of the CIITA gene is not down-regulated, compared with the expression and / or activity of the corresponding genes in unmodified corresponding cells, and a method for preparing the modified immune effector cells is also provided.

Description

[Technical field]

[0001] The present application relates to biopharmaceuticals, and more particularly to modified immune effector cells and methods for preparing same. [Background technology]

[0002] Antitumor immunotherapy has shown broad potential to induce durable and robust responses in a variety of malignancies and can be used to treat many different types of cancer. Current antitumor immunotherapy mainly includes two types of immunotherapy: immune cell-targeted monoclonal antibody (mAb) therapy and adoptive cell therapy (ACT). ACT refers to the return of autologous or heterologous lymphocytes stimulated and expanded in vitro to the human body to achieve antitumor effects, but this therapy is said to have relatively good therapeutic effects only in patients with matching MHC polymorphism. CAR-T is a new and effective MHC-independent adoptive cell therapy, where CAR is also called chimeric antigen receptor, an artificial receptor that mimics the function of TCR, and specifically recognizes antigens on the surface of tumor cells and targets tumor cells. However, the timely and successful production and infusion of autologous CAR T cells is the biggest obstacle to implementing effective CAR T cell therapy. For example, if a patient has previously been treated with a chemotherapy regimen, the chemotherapy drug may cause the patient's T cells to have difficulty in proliferating in vitro or to have a reduced function, resulting in insufficient quantities of CAR-T cell products to be produced, or the quality of the products to be relatively poor. In addition, currently, autologous CAR-T is often used in ALL or CLL patients, and its application in solid tumor patients faces great challenges, such as the heterogeneity of tumor antigens, which limits the application of autologous CAR-T. In order to increase the flexibility of CAR and expand the scope of antigen recognition, universal CAR-T has emerged. The main design principle of universal CAR T cells is to generate tumor antigen-specific T cells from allogeneic healthy donors. Although the proliferation efficiency and survival rate of CAR T cells obtained by this method are improved, the endogenous TCR of allogeneic T cells recognizes the allogeneic antigen of the receptor, which leads to graft-versus-host disease (GVHD). In addition, the expression of HLA on the surface of allogeneic T cells also induces the host immune system's rapid rejection (HVGR). Therefore, the problem of immune rejection by allogeneic cell therapy requires further investigation. DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0003] The present application provides modified immune effector cells, in which the expression and / or activity of the TRAC gene and the HLA-A gene is down-regulated, the expression and / or activity of the B2M gene is not down-regulated, and the expression and / or activity of the CIITA gene is not down-regulated, compared to the expression and / or activity of corresponding genes in corresponding unmodified cells. [Means for solving the problem]

[0004] In some embodiments, the modification downregulates the expression and / or activity of two genes, the two genes consisting of the TRAC gene and the HLA-A gene.

[0005] In some embodiments, the expression and / or activity of the TRAC gene and the HLA-A gene is downregulated, the expression and / or activity of the B2M gene is not downregulated, and the expression and / or activity of the CIITA gene is not downregulated compared to a corresponding wild-type cell.

[0006] In some embodiments, the expression and / or activity of two genes is downregulated compared to a corresponding wild-type cell, said two genes consisting of the TRAC gene and the HLA-A gene.

[0007] In some embodiments, the immune effector cells comprise T cells.

[0008] In some embodiments, downregulating the expression level and / or activity of the gene comprises downregulating the expression and / or activity of a nucleic acid molecule encoding the gene; and / or downregulating the expression and / or activity of a protein product encoded by the gene.

[0009] In some embodiments, the modification comprises gene mutation and / or gene silencing.

[0010] In some embodiments, the modification comprises administering to the immune effector cells one or more agents selected from the group consisting of antisense RNA, siRNA, shRNA, and a CRISPR / Cas9 system.

[0011] In some embodiments, the modification comprises administration of a CRISPR / Cas9 system to immune effector cells.

[0012] In some embodiments, the alleles of the HLA-A gene are selected from the group consisting of: A*02, A*11, A*24, A*30, A*33, A*03, A*01, and A*26.

[0013] In some embodiments, the expression level and / or activity of up to two alleles within said HLA-A gene is downregulated.

[0014] In some embodiments, the expression level and / or activity of one allele of the HLA-A gene is downregulated.

[0015] In some embodiments, the modification comprises administering to the immune effector cells an sgRNA that targets an exon portion of the HLA-A gene.

[0016] In some embodiments, the sgRNA targeting an exon portion of the HLA-A gene comprises a nucleotide sequence set forth in any of SEQ ID NOs. 16-54, 91-92.

[0017] In some embodiments, the modification also includes administering to the immune effector cells an sgRNA that targets an exon portion of the TRAC gene.

[0018] In some embodiments, the sgRNA targeting an exon portion of the TRAC gene comprises a nucleotide sequence set forth in any of SEQ ID NOs. 1-15.

[0019] In some embodiments, the antisense RNA comprises a nucleotide sequence set forth in any of SEQ ID Nos. 93-96.

[0020] In some embodiments, the modification further comprises administration of a Cas protein to the cell.

[0021] In some embodiments, the Cas protein comprises a Cas9 protein.

[0022] In some embodiments, the immune effector cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory structure, and a primary signaling domain.

[0023] In some embodiments, the antigen binding domain specifically binds to a tumor antigen.

[0024] In some embodiments, the tumor antigen is selected from the group consisting of CD19, CD133, CD123, CD22, CD30, CD171, CA125, C-met, L1CAM, EC, DLL3, CD99, CS1, 5T4, CD138, CS-1 (also known as CD2 subclass 1, CRACC, SLAMF7, CD319, 19A24), C-type lectin-like molecule-1 (CD133 or CD123), CD33, epidermal growth factor receptor variant III (CD171), ganglioside G2 (GD2), ganglioside GD3, TNF receptor phagocytosis (TNF-α) and TNF-α. Myri B-cell maturation antigen (CA125), Tn antigen (e.g., C-met, L1CAMα-CD138), prostate-specific membrane antigen (CRACC); receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72) (EPCAM), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), mesothelin, insulin Interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (TAG72), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (CEAβ), stage-specific embryonic antigen-4 (e.g., proteasome, megalin factor), CD20, folate receptor alpha, receptor tyrosine kinase CD276 (CD117) (LMP2), cell surface-associated mucin 1 (MUC1), epidermal growth factor receptor (IL-13Ra2), neural cell adhesion molecule (CD213A2), IL-11Ra, prostatic acid phosphatase tase (PSCA), mutant elongation factor 2 (TAG72), liver protein B2, fibroblast activation protein alpha (CD44v6), insulin-like growth factor 1 receptor (CEA receptor), carbonylase IX (EPCAM), cell surface associated mucin 1 (CD276), oxidoglycoprotein 9, prostatic acid phosphatase, mutant 100 glycoprotein 9, oncogene fusion protein consisting of breakout cluster (CD276) and IL-13Ra2 murine leukemia viral oncogene homolog 1 (CD117) (IL-13Ra2 or CD213A2), tyrosinase,Liver protein type A receptor 2 (IL-11Ra), fucosyl-GM1; sialyl-PSCA adhesion molecule (PSCA), transglutaminase 5 (TAG72), macromolecule-melanoma-associated antigen (CEAβ), o-acetyl-GD2 ganglioside (TAG72), folate receptor β, tumor endothelial marker 1 (CEA), tumor endothelial marker 7-associated (EPCAM), tight junction protein 6 (CD276), thyroid-stimulating hormone receptor (CD117), G protein-coupled receptor class C group 5 member D (IL-13Ra2 or CD213A2), chromosome X open reading frame 6 1 (IL-11Ra), CD97, IL-11Ra, anaplastic lymphoma kinase (CD44v6), polysialic acid, placenta specific 1 (CEA receptor), CD44v6 hexosamine moiety (EPCAM), mammary differentiation antigen (EPCAM), urothelial differentiation specific glycoprotein (CD117) (LMP2) 2 (MUC1), hepatitis A virus cellular receptor 1 (IL-13Ra2), adrenergic receptor beta 3 (CD213A2), ubiquitin (PSCA) 3 (TAG72), G protein-coupled receptor 20 (sLe), lymphocyte antigen 6 complex locus K9 (TGS5), Olfactory receptor 51 (TAG72), TCR gamma variable protein, E2 (e.g., proteasome, megalin factor), tumor variable protein E2m, cancer / testis antigen 1 (EPCAM), cancer / testis antigen 2 (CD276), melanoma-associated antigen 1 (CD117), ETS translocation mutant gene 6 on chromosome 12p (IL-13Ra2 or CD213A2), sperm protein 17 (IL-11Ra), X antigen family 1A (PSCA), angiopoietin-binding cell surface receptor 2 (TAG72), melanoma cancer testis antigen-1 (CEAβ), melanoma cancer testis antigen-2 (TAG72), F os-associated antigen 1, p53, p53 mutants, prostate-specific protein (CEA), prostate cancer tumor antigen-1 (MUC1), T cell-recognized melanoma antigen 1 (IL-13Ra2) rat sarcoma (CD117) mutants, human telomerase reverse transcriptase (IL-13Ra2 or CD213A2), sarcoma translocation breakpoints, melanoma apoptosis inhibitor protein (IL-11Ra), ERG (PSCA), N-acetylglucosaminyltransferase V (TAG72), paired frame protein IL-11Ra (CEAβ), androgen receptor, cyclin B1,PSCA avian myelocytosis viral oncogene neuroblastoma-derived homolog (TAG72), Ras homolog family member C (CEA), tyrosinase-related protein 2 (EPCAM), cytochrome CEA (CD276), T-cell-discriminating squamous cell carcinoma antigen 3 (CD213A2), paired frame protein CD117 (PSCA), precursor protein-binding protein sp32 (TAG72), lymphocyte-specific protein tyrosine kinase (CD44v6), A-kinase anchor protein 4 (CEA receptor), synovial sarcoma X breakpoint 2 (EPCAM), receptor for advanced glycation end products (EPCAM), pea pod protein (CD117), human papillomavirus E6 (MUC1), human papillomavirus E7 (IL-13Ra2), endopeptidase E7, carboxylase Her2 / n eu, intestinal binding protein sp, lymphocyte-specific protein (Abl), synovial protein anchor protein CD276, synovial sarcoma X breakpoint 72, synovial sarcoma protein, leukocyte-associated immunoglobulin-like receptor 1 (IL-13Ra2 or CD213A2), Fc fragment of IgA receptor (IL-11Ra), leukocyte immunoglobulin-like receptor subfamily A member 2 (PSCA), PSCA molecule-like family member f (TAG72), C-type lectin domain family 12 member A (CEAβ), bone marrow stromal cell antigen 2 (TAG72), mucin-like hormone receptor 2 containing an EGF-like module (CEA), lymphocyte antigen 75 (EPCAM), phosphatidylinositol proteoglycan-3 (CD276), Fc receptor-like 5 (CD117) and / or immunoglobulin lambda-like polypeptide 1.

[0025] In some embodiments, the antigen-binding domain is selected from the group consisting of: a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a single domain antibody, and an antigen-binding fragment thereof.

[0026] In some embodiments, the antigen binding domain targets a solid tumor.

[0027] In some embodiments, the solid tumor is selected from the group consisting of lung cancer, breast cancer, colon cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, vulvar cancer, childhood solid tumor, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma.

[0028] In some embodiments, the antigen binding domain targets a non-solid tumor.

[0029] In some embodiments, the non-solid tumor is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, polyhilar cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic syndrome, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid cell neoplasm, B lymphocytoma, Waldenstrom's macroglobulinemia.

[0030] In some embodiments, the transmembrane domain comprises a protein selected from CD28, CD3e, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGAD, CD11d, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, CD11a, TNFR2.

[0031] In some embodiments, the costimulatory domain comprises a costimulatory domain selected from the following proteins: D137, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, CD160 (BY55), LIGHT, NKG2C, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, L FA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 , PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(S LAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and / or NKG2D.

[0032] In some embodiments, the primary intracellular signaling domain comprises a functional signaling domain selected from the following proteins: CD3zeta, FcRgamma (FCER1G), FcgammaRIIa, FcRbeta (FcεR1b), CD3gamma, CD3delta, CD3ε, CD79a, CD79b, DAP10, and / or DAP12.

[0033] In some embodiments, the hinge region connects the antigen binding domain and the transmembrane domain, and the hinge region comprises a hinge region selected from the following proteins: a human Ig (immunoglobulin) hinge region, a GS connector, a KIR2DS2 hinge region, or a CD8a hinge region.

[0034] The present application also provides a method of producing a modified immune effector cell as described herein, comprising the steps of: down-regulating the expression and / or activity of the TRAC gene and the HLA-A gene in said immune effector cell relative to the expression and / or activity of the corresponding genes in said non-modified corresponding cell, wherein the expression and / or activity of the B2M gene is not down-regulated, and wherein the expression and / or activity of the CIITA gene is not down-regulated.

[0035] In some embodiments, the modification downregulates the expression and / or activity of two genes, the two genes consisting of the TRAC gene and the HLA-A gene.

[0036] In some embodiments, the expression and / or activity of the TRAC gene and the HLA-A gene is downregulated, the expression and / or activity of the B2M gene is not downregulated, and the expression and / or activity of the CIITA gene is not downregulated compared to a corresponding wild-type cell.

[0037] In some embodiments, the expression and / or activity of two genes is downregulated compared to a corresponding wild-type cell, said two genes consisting of the TRAC gene and the HLA-A gene.

[0038] In some embodiments, the expression level and / or activity of the downregulated gene comprises downregulating the expression and / or activity of a nucleic acid molecule encoding the gene; and / or downregulating the expression and / or activity of a protein product encoded by the gene.

[0039] In some embodiments, the modification comprises gene mutation and / or gene silencing.

[0040] In some embodiments, the modification comprises administering to the immune effector cells one or more agents selected from the group consisting of antisense RNA, siRNA, shRNA, and a CRISPR / Cas9 system.

[0041] In some embodiments, the modification comprises administration of a CRISPR / Cas9 system to immune effector cells.

[0042] In some embodiments, the modification comprises administering to the immune effector cells an sgRNA that targets an exon portion of the HLA-A gene.

[0043] In some embodiments, the sgRNA targeting an exon portion of the HLA-A gene comprises a nucleotide sequence set forth in any of SEQ ID NOs. 16-54, 91-92.

[0044] In some embodiments, the modification comprises administering to the immune effector cells an sgRNA that targets an exon portion of the TRAC gene.

[0045] In some embodiments, the sgRNA targeting an exon portion of the TRAC gene comprises a nucleotide sequence set forth in any of SEQ ID NOs. 1-15.

[0046] In some embodiments, the antisense RNA comprises a nucleotide sequence set forth in any of SEQ ID Nos. 93-96.

[0047] In some embodiments, the modification further comprises administration of a Cas protein to the cell.

[0048] In some embodiments, the Cas protein comprises a Cas9 protein.

[0049] The present application also provides compositions, which include the modified immune effector cells described herein and a pharma- ceutically acceptable carrier.

[0050] In some embodiments, the composition comprises a population of cells that comprises the modified immune effector cells described herein.

[0051] The present application also provides an application of the modified immune effector cells of the present application in the preparation of CAR-T cells.

[0052] The present application also provides the application of the modified immune effector cells of the present application in the preparation of a drug, the drug comprising Homeopathic Therapy Used for.

[0053] The present application also provides a method for treating a patient or subject comprising administering to the patient or subject the modified immune effector cells described herein. Homeopathic Therapy The present invention provides a method for:

[0054] The present application also provides: Homeopathic Therapy The present invention provides modified immune effector cells as described herein for

[0055] The present application also provides an application of the modified immune effector cells of the present application in preparing a drug, the drug being used in tumor treatment.

[0056] The present application also provides a method of treating a tumor, the method comprising administering to a patient or subject the modified immune effector cells of the present application.

[0057] The present application also provides the modified immune effector cells, which are used in the treatment of tumors.

[0058] In some embodiments, the tumors include solid tumors and non-solid tumors.

[0059] In some embodiments, the tumor is selected from the group consisting of lung cancer, breast cancer, colon cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, vulvar cancer, solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma.

[0060] Those skilled in the art will readily ascertain other aspects and advantages of the present application from the following detailed description. Effect of the Invention

[0061] In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the present application allows those skilled in the art to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the present invention. Therefore, the accompanying drawings and description of the present application are merely illustrative and not restrictive. [Brief description of the drawings]

[0062] Specific features of the invention according to the present application are set forth in the appended claims. A better understanding of the features and advantages of the invention according to the present application can be obtained by reference to the exemplary embodiments described in detail herein and the accompanying drawings, the brief description of which is as follows:

[0063] [Figure 1] 1 shows the results of Sanger sequencing after Sg9 RNA editing of the TRAC gene. [Diagram 2] The results of TA cloning in which the TRAC gene was edited with Sg9RNA are shown. [Diagram 3] 1 shows the results of flow cytometry in which the TRAC gene was edited with Sg9RNA. [Figure 4] 1 shows the results of Sanger sequencing after Sg2 RNA editing of the HLA-A02 gene. [Diagram 5] 1 shows the results of Sanger sequencing after Sg5RNA editing of the HLA-A02 gene. [Figure 6] 1 shows the results of Sanger sequencing after Sg10-3 RNA editing of the HLA-A11 gene. [Figure 7] 1 shows the results of Sanger sequencing after Sg21 RNA editing of the HLA-A11 gene. [Figure 8] 1 shows the results of simultaneous knockout of HLA-A02 and TRAC in modified immune effector cells. [Figure 9] HLA-A02 and TRAC protein levels in modified immune effector cells are shown. [Figure 10] The mRNA levels of TRAC, HLA-A, B2M and CIITA in modified immune effector cells are shown. [Figure 11] 1 shows protein levels of B2M and CIITA in modified immune effector cells. [Figure 12] Protein levels of TRAC, HLA-A, B2M and CIITA in modified immune effector cells are shown. [Figure 13] 1 shows knockout of TRAC and HLA-A mRNA levels in modified immune effector cells. [Figure 14] CD69 and CD137 protein levels in modified immune effector cells are shown. [Figure 15] Co-culture of modified immune effector cells with NK cells. [Figure 16] IFN-γ expression levels of modified immune effector cells are shown. [Figure 17] Protein levels of TRAC, HLA-A, B2M and CIITA in modified immune effector cells are shown. [Figure 18] 1 shows the efficiency of infection of modified immune effector cells with CAR. [Figure 19] Fold expansion of modified immune effector cells is shown. [Figure 20] The killing effect of modified immune effector cells on CD19-positive target cells is shown. [Figure 21] 1 shows a schedule for administering modified immune effector cells. [Figure 22] 1 shows the killing effect of modified immune effector cells against tumors in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] In the following, embodiments of the present invention will be described using certain specific embodiments, but those familiar with this technology can easily understand other advantages and effects of the present invention from the disclosure in this application.

[0065] The present application is further described below: In the present invention, unless otherwise specified, the scientific and technical terms used in the present application have the meanings that can be commonly understood by those skilled in the art. Furthermore, the terms and experimental procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in the respective fields. In addition, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0066] As used herein, the term "immune effector cell" refers to an immune cell that is generally involved in an immune response and that exerts an effector function. For example, exerting the effector function may include removing foreign antigens or promoting an immunoeffector response. Immune response cells include plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0067] In the present application, the term "modify" generally refers to a change that changes the state or structure of a cell and / or a change that changes the state or structure of a cell. The modification is typically a change in the state or structure of a cell compared to a corresponding cell without the modification, and the modification may include, for example, a change in the expression level or function of an endogenous gene by genetic engineering means in which the expression level of an endogenous gene of a cell is downregulated, upregulated or not expressed, and the genetic engineering means may include gene editing by homologous recombination, CRISPR / CAS9 system, etc. The change may further include a change in the expression, structure or function of a cellular protein, for example, a change in the expression, structure or function of a corresponding protein achieved by a change in the expression level or function of the endogenous gene, for example, a change in protein expression, structure or function achieved by regulation of protein translation, post-translational modification. The modification may also include the introduction of an exogenous gene, the expression of an exogenous protein, etc.

[0068] In this application, the term "TRAC" generally refers to the T cell receptor alpha constant region. T cell receptors (TCRs) are specific receptors that are typically present on the surface of T cells and recognize antigens bound to major histocompatibility complex (MHC) molecules. TCRs are typically composed of two different protein chains (heterodimers). In humans, the TCRs of the majority of T cells consist of one alpha and one beta chain (encoding TRA and TRB, respectively), and such T cells are called αβ T cells, while a minority of T cells consist of one gamma and one delta chain (encoding TRG and TRD, respectively), and such T cells are called γδ T cells. Typically, αβ T cells account for approximately 95% of the total number of T cells, and γδ T cells account for approximately 5% of the total number of T cells, but this ratio changes during individual development and pathological conditions (e.g., leukemia) and also varies between species. Each chain that constitutes the TCR contains a variable region and a constant region. In humans, the gene encoding the α chain (TRA, for example, the information shown in HGNC:12027) is located on chromosome 14 and is composed of a multigene segment, including a variable region (V), a joining region (J) and a constant region (C). The TRAC gene usually refers to the gene sequence encoding the T cell receptor α chain constant region (C) (for example, the information shown in HGNC:12029), which is located on chromosome 14 (14 q11.2;14:22, 547, 505-22, 552, 131). Usually, one of the variable region (V) genes that encodes the antigen recognition region of the N fragment rearranges with one of the joining regions (J) to produce a functional V region exon, which is transcribed and joined to the constant region (C) by splicing, thereby forming the T cell receptor α chain coding sequence.

[0069] In this application, the term "HLA-A" generally refers to the human leukocyte antigens polypeptide chains encoded by the HLA-A gene (e.g., information shown in HGNC:4931) located on human chromosome 6p21.3. HLA-A is one of the three major polypeptide types that constitute human cell surface class I MHC molecules, the others being HLA-B and HLA-C. A heterodimer consisting of an α chain encoded by the HLA-A gene and a β chain (β2-microglobulin) encoded by the B 2 M gene is an HLA-A class MHC I molecule. The α chain encoded by the HLA-A gene can include an α1 domain, an α2 domain, an α3 domain, a transmembrane region, and a cytoplasmic region, of which the α1 domain and the α2 domain bind to peptide fragments to present the peptide fragments from the MHC I molecule (e.g., HLA-A class) to immune system cells. In humans, as in most mammals, the α-chain of the MHC I molecule is polymorphic with many variations in its primary structure, and 2432 HLA-A alleles encoding 1740 activating proteins and 117 inactivating proteins have been identified as of December 2013. In this application, HLA-A alleles may include sequence information of different HLA-A alleles named by the WHO HLA Factor Naming Committee, as contained in the IMGT / HLA database version 3.38.0 (https: / / www.ebi.ac.uk / ipd / imgt / hla / ).

[0070] In this application, the term "B2M" refers to β2-microglobulin, which is usually part of a class MHC I molecule. β2-microglobulin (also called the β chain) can form a class MHC I molecule with the HLA-encoded α chain. B2M is normally expressed in all nucleated cells. In humans, β2-microglobulin is encoded by the B2M gene located at 15 q21.1 (e.g., information shown in HGNC:914).

[0071] In this application, the term "CIITA" generally refers to the transactivator of the class II major histocompatibility complex (MHCII). The transactivator is a protein with an acidic transcriptional activation domain, four LRRs (leucine-rich repeats) and a GTP-binding domain. The protein can be located in the cell nucleus and, as a positive regulator of the transcription of class II major histocompatibility complex (MHCII) genes, is called the "master regulator" of their expression. The protein can also bind GTP and is transported into the cell nucleus using this binding; there it usually acts like a coactivator through its acetyltransferase (AT) activity. In humans, the protein is encoded by a gene located at 16 p13.13 (e.g. information given in HGNC:7067) and can give rise to several transcript variants encoding different isoforms.

[0072] As used herein, the term "wild-type cell" refers to a cell that normally occurs in or is derived from nature.

[0073] As used herein, the term "T cells" generally refers to thymus-derived cells that are involved in a variety of cell-mediated immune responses.

[0074] In this application, the term "nucleic acid", "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term may include nucleic acids containing analogs of naturally occurring nucleotides, which have similar binding properties as those of the reference nucleic acid (e.g., sequence information is provided) and are metabolized in a manner similar to those of naturally occurring nucleotides. Unless otherwise specified, the sequence of the nucleic acid may include conservatively modified variants thereof, such as truncated codon substitutions, alleles, orthologs, SNPs, complementary sequences, and sequences as specified.

[0075] In this application, the term "expression" generally refers to the transcription and / or translation of a particular nucleotide sequence.

[0076] As used herein, the term "gene mutation" refers to a change in the composition or sequence of base pairs that typically occurs structurally in a gene, such as a point mutation resulting from a single base change, or multiple base deletions, duplications, and insertions.

[0077] In this application, the term "gene silencing" generally refers to blocking the expression of a particular gene by a regulatory mechanism. It mainly includes two types: one is transcriptional gene silencing (TGS) at the transcriptional level by factors such as DNA methylation, heterochromatinization, and position effect, and the other is post-transcriptional gene silencing (PTGS), i.e., affecting gene expression by specific interference with target RNA at the post-transcriptional level. Usually, when a gene is silenced, the expression of the corresponding gene is downregulated / reduced. When a gene is knocked out, it usually appears not to be expressed, for example, when all alleles of a particular gene are knocked out, the expression of the gene appears to disappear. Gene silencing is generally considered as a gene knockdown mechanism, and a commonly used method for gene silencing is RNAi, etc.

[0078] As used herein, the term "endogenous" refers to any substance that is produced by or within an organism, cell, tissue, or system.

[0079] As used herein, the term "exogenous" refers to a substance introduced from or produced outside a biological, cellular, histological, or system.

[0080] In this application, the term "antisense RNA" generally refers to a single-stranded RNA that is complementary to the transcript mRNA (messenger RNA). Antisense RNA binds to mRNA to suppress gene expression. For example, after binding to a target mRNA, antisense RNA increases the sensitivity of this double-stranded RNA molecule to RNA enzyme III, causing it to be degraded. For example, antisense RNA binds to the upstream non-coding region of an mRNA and directly inhibits the translation of the target mRNA.

[0081] In this application, the term "siRNA" generally refers to the abbreviation of small interfering RNA or short interfering RNA. siRNA is a type of double-stranded non-coding RNA molecule, about 18-28 base pairs in length, which causes mRNA degradation through complementary binding with mRNA, thereby disrupting the expression of a specific gene. In some embodiments, siRNA may be the product of treating long double-stranded RNA or shRNA with Dicer enzyme. In some implementations, siRNA enters cells and forms an RNA-induced silencing complex (RISC) with other proteins, where the meaningful strand causes degradation and the anti-meaning strand can bind to the complementary target sequence, thereby achieving gene silencing.

[0082] In this application, the term "shRNA" is usually an abbreviation of short hairpin RNA, i.e., "short hairpin RNA". shRNA usually contains two short inverted repeats, separated by a stem loop sequence that forms a hairpin structure. It may contain 5 to 6 T bases as a transcription terminator for RNA polymerase III. In some embodiments, shRNA enters cells via a viral vector or plasmid, transcribes under the action of polymerase II or polymerase III, and the transcript is exported from the cell nucleus (usually Exportin 5) and then transported to RISC via Dicer processing, where the sense strand is degraded and the antisense strand binds to a complementary target sequence, thereby achieving gene silencing.

[0083] As used herein, the term "CRISPR / Cas system" generally refers to a group of molecules including RNA-guided nucleases or other effector molecules and gRNA molecules that can direct and effect RNA-guided nucleases or other effector molecules to a target sequence, e.g., to cause degradation of the target sequence. In some embodiments, the CRISPR system includes a gRNA and a Cas protein, e.g., a Cas9 protein. A system that includes Cas9 or a functional variant thereof is referred to herein as a "Cas9 system" or a "CRISPR / Cas9 system." In some embodiments, the gRNA molecule and the Cas molecule can complex to form a ribonucleoprotein (RNP) complex.

[0084] As used herein, the terms "gRNA molecule" or "guide RNA", "guide RNA", "guide RNA", "guide RNA molecule", "gRNA" and the like are used interchangeably and generally refer to a nucleic acid molecule that specifically guides an RNA-directed nuclease or other effector molecule (typically complexed with a gRNA molecule) to a target sequence. In some embodiments, this guidance is achieved by hybridizing a portion of the gRNA to DNA (e.g., via a gRNA guide domain) and binding a portion of the gRNA molecule to an RNA-guided nuclease or other effector molecule (e.g., at least gRNAtracr). In some embodiments, the gRNA molecule is comprised of a single contiguous polynucleotide molecule, and is referred to herein as a "single guide RNA" or "sgRNA". In other embodiments, the gRNA molecule is comprised of multiple (e.g., two) polynucleotide molecules that can associate (typically hybridize) with themselves, and is referred to herein as a "dual guide RNA" or "dgRNA".

[0085] In this application, the term "Cas protein" generally refers to an enzyme responsible for DNA cleavage in the CRISPR / Cas system. It may include enzymes of the CRISPR / Cas system type I, II, and III, such as "Cas3", "Cas9", and "Cas10".

[0086] In this application, the term "Cas9 protein" generally refers to the enzyme from a bacterial type II CRISPR / Cas system that is responsible for cleaving DNA. Cas9 can include the wild-type protein and functional mutants thereof.

[0087] As used herein, an "allele" generally refers to a potentially different form of variation of a gene sequence at a locus. A locus, also called a genetic locus or genetic site, refers to a fixed location on a chromosome, such as where a particular gene is located. The location of a locus within a genome is called a genetic map.

[0088] In the present application, the term "chimeric antigen receptor (CAR)" generally refers to an antigen receptor formed by fusing the antigen-binding domain of an antibody that recognizes a tumor-associated antigen (TAA) or a binding fragment of another target molecule with an intracellular signal domain "immunoreceptor tyrosine-based activation motifs (ITAM, usually CD3ζ or FcεRIγ)". For example, the basic structure of a CAR may include one tumor-associated antigen (TAA) or other target molecule antigen-binding domain (usually scFv derived from a monoclonal antibody antigen-binding domain), one extracellular hinge region, one transmembrane region, and one intracellular immunoreceptor tyrosine-based activation motif (ITAM).

[0089] In the present application, the term "binding domain" generally refers to a domain that binds (specifically) to a given target epitope or a given target site on a target molecule (e.g. an antigen), or that interacts with said given target epitope or given target site, or that recognizes said given target epitope or given target site.

[0090] In this application, the term "specific binding" refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that allows the presence of the target to be determined in the presence of a heterogeneous population of molecules, typically including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is one that binds to that target with greater affinity, affinity, ease, and / or persistence than it binds to other targets. In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

[0091] In this application, the term "transmembrane domain" refers to a polypeptide or protein that is usually encoded by at least one exon at the DNA level, which includes an extracellular region, a transmembrane region, and an intracellular region. A transmembrane domain generally includes three distinct structural regions: an extracellular region at the N-terminus, a conserved transmembrane stretch region in the middle, and a cytoplasmic region at the C-terminus. A transmembrane domain may also include an intracellular or cytoplasmic domain.

[0092] In this application, the term "hinge region" refers to the region that is usually located between the binding domain and the transmembrane domain in the CAR structure. Hinge regions are usually derived from the IgG family, such as IgG1 and IgG4, and some are derived from IgD and CD8, but usually have some flexibility, which affects the spatial constraint between the CAR molecule and its specific target, and thus the contact between the CAR T cell and the tumor cell.

[0093] In this application, the term "costimulation" refers to the activation source of the second signaling by lymphocytes, which usually occurs by the interaction of a costimulatory molecule with its receptor on the surface of immune cells involved in adaptive immunity (T cell / B cell or antigen presenting cell / T cell). For example, the full activation of T cells depends on the action of dual signaling and cytokines. The first signal of T cell activation comes from the specific binding of its receptor TCR with an antigen, i.e., T cell antigen recognition; the second signal of T cell activation comes from the interaction of a costimulatory molecule on an antigen presenting cell with the corresponding receptor on the surface of the T cell.

[0094] In this application, the term "costimulatory domain" generally refers to the intracellular portion of the corresponding receptor of a costimulatory molecule that can transmit a costimulatory signal (also called a second signal). For example, in CAR-T cells, the costimulatory domain from CD137 (or other costimulatory molecule receptor) is activated after the extracellular binding domain in the CAR structure binds to the corresponding antigen, and transmits a costimulatory signal.

[0095] In this application, the term "primary signaling domain" generally refers to an intracellular amino acid sequence capable of generating a signal that promotes immune effector function of a cell comprising a CAR, such as a CAR-T cell. For example, examples of immune effector function in a CAR-T cell may include auxiliary activities including cytolytic activity and secretion of cytokines. In some embodiments, the primary signaling domain transmits an effector function signal and directs the cell to a specialized function. While it is possible to use the entire primary signaling domain, it is often not necessary to use the entire chain. As long as a truncated portion of the primary signaling domain is used, such a truncated portion can be used in place of the complete chain, provided that it is capable of transmitting the effector function signal. The term primary signaling domain refers to including any short portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0096] In the present application, the term "tumor antigen" refers to a molecule (protein, sugar, lipid, etc.) that is typically expressed on the surface of tumor cells, either intact or as a fragment, and that can be used to bias agents to tumor cells. In some embodiments, the tumor antigen may be a marker of expression on both normal and cancer cells, such as a lineage marker, such as CD 19 on B cells. In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed in tumor cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold or more overexpression compared to normal cells. In some embodiments, the cell surface molecule is aberrantly expressed on tumor cells, e.g., includes deleted, added or mutated molecules compared to the corresponding molecule expressed on normal cells. In some embodiments, the tumor antigen may be expressed exclusively on the tumor cell surface, intact or as a fragment (e.g., MHC / peptide), and not synthesized or expressed on the normal cell surface. In some embodiments, the CAR of the present application may include an antigen binding domain that binds to a peptide presented by MHC, such as an antibody or antibody fragment. Normally, peptides derived from endogenous proteins bind to major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. Virus-specific and / or tumor-specific peptide / MHC complexes can be used as specific classes of cell surface targets for immunotherapy. (See Sastry et al. J Virol. 2011 85 (5) :1935-1942; Sergeeva et al. Blood 2011 117 (16):4262-4272; Verma et al. J Immunol 2010 184 (4) :2156-2165; Willemsen et al. Gene Ther 2001 8 (21):1601-1608; Dao et al. Sci Transl Med 2013 5 (176):176 ra 33; Tassev et al. Cancer Gene Ther 2012 19(2):84-100).

[0097] In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in very small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Unlike typical polyclonal antibody preparations that contain different antibodies against different determinants (tables), each monoclonal antibody was directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized in a hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by a particular method.

[0098] In this application, the term "polyclonal antibody" generally refers to a composition of different antibody molecules. Polyclonal antibodies can bind or react with multiple different specific antigenic determinants on the same or different antigens. The antigen-specific variability of polyclonal antibodies is present in the variable regions of the individual antibodies that make up the polyclonal antibody, such as the complementarity determining region (CDR) 1, CDR 2, and CDR 3 regions. For example, polyclonal antibodies can be prepared by immunizing animals with a target sFGFR or a portion thereof. For example, polyclonal antibodies may be prepared by mixing multiple monoclonal antibodies that are specific for a desired target sFGFR.

[0099] In this application, the term "human antibody" generally refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are known in the art (e.g., van Dijk, MA and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Adult antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing the entire collection or anthology of adult antibodies after immunization in the absence of endogenous immunoglobulin production (e.g., Jakobovits, A. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A. et al., Nature 362 (1993) 255-258; Brueggemann, M. et al., Year Immunol. 7 (1993) 33-40). Adult antibodies (e.g., Hoogenboom, HR and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD et al., J. Mol. Biol. 222 (1991) 581-597) can also be produced in phage display libraries. The term "human antibody" can also include antibodies that are modified in the constant region.

[0100] In the present application, the term "humanized antibody" refers to an antibody that comprises heavy and light chain variable region sequences, usually from a non-human species (e.g., mouse), but where at least a portion of the VH and / or VL sequences have been altered to resemble human germline variable sequences. For example, the term "humanized antibody" refers to an antibody or variant, derivative, analog or fragment thereof that is capable of immunospecifically binding to an antigen of interest and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. The term "essentially" refers, in the case of a CDR, to the amino acid sequence of the CDR being at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises at least one, and usually two, variable regions (Fab, Fab', F(ab.)2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions have human immunoglobulin consensus sequences. In some embodiments, the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin.

[0101] In this application, the term "single domain antibody" generally refers to a class of antibodies that lack the antibody light chain and contain the variable region of the heavy chain. They are also called nanobodies because of their small molecular weight. Single domain antibodies were first discovered in camelids, and similar antibodies have since been discovered in chondrichthyans such as nurse sharks, star sharks, and rays. For example, antibodies that lack the CH1 region, which is both the light chain and the heavy chain constant region of conventional antibodies, are called heavy chain antibodies (HcAb), and HcAb is ubiquitous in various camelids. For example, a heavy chain antibody called IgNAR, a new antigen receptor, is composed of two isoheavy chains consisting of five constant regions and one variable region. The variable region of a heavy chain antibody is composed only of the variable region of the antibody heavy chain, and it specifically binds to antigens like a conventional Fab, so it functions like a conventional antibody.

[0102] As used herein, the term "tumor" generally refers to a growth or parenchymal lesion formed by abnormal cell proliferation. As used herein, a tumor may be a solid tumor or a non-solid tumor. In some embodiments, a tangible mass that is palpable by X-ray, CT, ultrasound, or palpation is referred to as a solid tumor, and a tumor that is not palpable or is palpable by X-ray, CT, and ultrasound is referred to as a non-solid tumor.

[0103] In this application, the term "CD", also called cluster of differentiation, is often used to identify cell surface molecules used as immunogenic markers. CD molecules have many uses and are usually used as important receptors or ligands for cells. Some CDs are involved in cell signaling cascades and change cell behavior, while some CD proteins are not involved in cell signaling but have other functions such as cell adhesion. As of April 21, 2016, the total number of human CD molecules is 371, including CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, which are the source of transmembrane domains in this application, and CD137, CD28, CD134 (OX-40) and CD278 (ICOS), which are the source of costimulatory domains.

[0104] In this application, the term "pharmaceutical acceptable carrier" generally refers to a pharmaceutical acceptable substance, composition, or medium for carrying, storing, transporting, or administering a cell preparation. For example, liquid, semi-solid, or solid fillers, diluents, isotonic agents, solvents, or cyst materials. Pharmaceutically acceptable carriers may include pharmaceutical acceptable salts, where the term "pharmaceutical acceptable salts" includes salts of active compounds prepared with relatively non-toxic acids or bases, such as sodium chloride, depending on the nature of the cells described in this application. Pharmaceutically acceptable carriers may also include organic acids (e.g., lactic acid), biologically active substances (e.g., polypeptides, antibodies, etc.), and antibiotics (penicillin, streptomycin, etc.). Pharmaceutically acceptable carriers may also include hydrogels, such as hydrogels including polyacrylamide. Pharmaceutically acceptable carriers may also include storage solutions, freezing storage solutions, injection solutions, etc., for use with cells. In general, the pharma- ceutically acceptable carrier can maintain the activity of the cells carried therein and does not impede the therapeutic effect. The pharma-ceutically acceptable carrier can promote the preservation, transportation, and proliferation and migration of cells, and is suitable for clinical application.

[0105] As used herein, the term "composition" generally refers to a composition suitable for application to a patient, a human patient. For example, a composition described herein may include immune effector cells described herein, and optionally a pharma- ceutically acceptable carrier. In some embodiments, an acceptable composition is non-toxic to a recipient at the dosages and concentrations used. Compositions of the present application include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0106] As used herein, the term "allogeneic therapy" generally refers to a therapeutic method in which organs, tissues, cells, etc. that do not originate from the subject or patient are administered to the subject or patient to achieve a therapeutic goal.

[0107] In this application, the term "comprising" generally refers to the inclusion of features explicitly specified without the exclusion of other elements.

[0108] As used herein, the term "about" generally refers to a variation of 0.5% to 10% above the specified value, for example, a variation of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

[0109] On the other hand, the present application provides modified immune effector cells, in which the expression and / or activity of the TRAC gene and the HLA-A gene are down-regulated, the expression and / or activity of the B2M gene is not down-regulated, and the expression and / or activity of the CIITA gene is not down-regulated, compared with the expression and / or activity of corresponding genes in the unmodified corresponding cells.

[0110] Meanwhile, the present application also provides a method for producing the modified immune effector cells of the present application, which comprises the steps of: down-regulating the expression and / or activity of TRAC gene and HLA-A gene in the immune effector cells, compared with the expression and / or activity of the corresponding genes in the unmodified corresponding cells, the expression and / or activity of B2M gene is not down-regulated, and the expression and / or activity of CIITA gene is not down-regulated.

[0111] In the present application, this modification downregulates the expression and / or activity of two genes: the TRAC gene and the HLA-A gene.

[0112] Immune effector cells

[0113] In the present application, the immune effector cells may include plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and / or bone marrow derived phagocytes.

[0114] In the present application, the plasma cells refer to effector B cells / antibody-secreting cells, and may include primitive plasma cells, immature plasma cells, Russell bodies, Dutcher bodies, and flame cells.

[0115] In this application, the B cells refer to all B cell types other than plasma cells, such as pre-B cells, immature B cells, mature B cells, activated B cells, etc.

[0116] In the present application, the T cells may include helper T cells (Th) that can assist humoral immunity and cellular immunity; suppressor T cells (Ts) that suppress cellular and humoral immunity; effector T cells (Te), which release lymphokines; cytotoxic T cells (Tc) that can kill target cells; delayed type hypersensitivity T cells (Td) that play a role in type IV allergy; expansion of T cells (Ta) that act on Th and Ts to expand immune effects; virgin or natural T cells, the ability of virgin or natural T cells to differentiate upon contact with an antigen; or memory T cells; memory T cells (Tm) that remember specific antigen stimulation.

[0117] For example, the cytotoxic T cells may have the cell surface marker CD8+.

[0118] For example, a helper T cell may have the cell surface marker CD4+.

[0119] In the present application, the unmodified counterpart cells may include wild-type cells and / or artificially modified cells. The wild-type cells may include naturally occurring or derived cells, such as plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells and / or bone marrow derived phagocytes isolated from a human body, or progenitor or multipotent cells isolated from a human body and induced to differentiate to obtain immune effector cells; the artificially modified cells may be plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells and / or bone marrow derived phagocytes isolated from a human body or induced to differentiate from progenitor / multipotent cells isolated from a human body and then artificially modified. For example, the human body may be a healthy human body. For example, the healthy human body may include a body free of tumors or immune system related diseases or disorders.

[0120] In the present application, the artificial modification may be an artificial modification that does not use the TRAC gene, HLA-A gene, B 2 M gene and CIITA gene as a recognition site / target. For example, the artificial modification does not affect the expression and / or activity of the TRAC gene, HLA-A gene, B 2 M gene and CIITA gene. Not affecting the expression and / or activity of the TRAC gene, HLA-A gene, B 2 M gene and CIITA gene means that the expression and / or activity of the TRAC gene, HLA-A gene, B 2 M gene and CIITA gene is not changed compared to the corresponding cells isolated from the human body or obtained by inducing differentiation from progenitor cells or multipotential cells isolated from the human body and not artificially modified, but the changes in the expression and / or activity are not completely consistent. For example, it is possible to carry out an examination using conventional technical means in the field of expertise to determine whether the expression and / or activity of the TRAC gene, HLA-A gene, B 2 M gene and CIITA gene in both of the two genes is completely consistent. The mRNA quantitative analysis of M gene and CIITA gene was not significantly different (P>0.05), for example, by using the conventional technical means in the relevant field, the corresponding polypeptide / protein quantitative analysis of the TRAC gene, HLA-A gene, B 2 M gene and CIITA gene in both of them was not significantly different (P>0.05). For example, the artificially modified cells may include CAR-T cells. For example, the CAR-T cells may include CAR-T cells whose CAR molecule comprises a binding domain that targets the following molecules: CD19, PSCA, CD123, CD20, CEA, FAP, CD133, EGFR, EGFRVIII, BCMA, PSMA, Her2, CA125, EphA2, C-met, L1CAM, CAR-T, CS1, ROR1, EC, NY-ESO-1, MUC1, LewisY, GPC3, GD2, DLL3, CD99, 5T4, CD22, CD30, CD33, CD138, and / or CD171. CD19, CD133, CD123, CD22, CD30, CD171, CA125, C-met, L1CAM, EC, DLL3, CD99, CS1, 5T4, CD138, CS-1 (also called CD2 subclass 1, CRACC, SLAMF7, CD319, 19A24), C-type lectin-like molecule-1 (CD133 or CD123), CD33, epidermal growth factor receptor variant III (CD171), ganglioside G2 (GD2),Ganglioside GD3, TNF receptor family B cell maturation antigen (CA125), Tn antigen (e.g., C-met, L1CAMα-CD138), prostate-specific membrane antigen (CRACC); receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (SLAMF7), CD38, CD319, carcinoembryonic antigen (also called CD2 subclass 1, CRACC, SLAMF7, CD319, 19A24), epithelial cell adhesion molecule (CD133 or CD123), B7H3 (CD171), KI T(GD2), interleukin-13 receptor subunit alpha-2 (CA125), mesothelin, interleukin-11 receptor alpha (e.g., C-met, L1CAMα-CD138), prostate stem cell antigen (CRACC), protease serine 21, vascular endothelial growth factor receptor 2 (ROR1), CD44v6(Y) antigen, CD24, platelet-derived growth factor receptor beta (FLT3), stage-specific embryonic antigen-4 (CD123), CD20, folate receptor alpha, receptor tyrosine kinase CD171 (CD133), cell surface-associated mucin 1 (CD123) (MUC1), epidermal growth factor receptor (CD171), neural cell adhesion molecule (CA125), CD138, prostatic acid phosphatase (c-met or L1CAM), mutant elongation factor 2 (CD138), liver protein B1002, fibroblast activation protein alpha (CRACC), insulin-like growth factor 1 receptor (SLAMF7), carbonic acid IX (CD133β), cell surface-associated mucin kinase (e.g., proteasome, megalin factor) subprotein, glycoprotein 9, prostatic acid phosphatase (CA125), mutant 9, breakout cluster (CD171), and Oncogene fusion protein composed of C-met murine leukemia viral oncogene homolog 1 (GD2) (CA125), tyrosinase, liver protein type A receptor 2 (e.g., C-met, L1CAMα-CD138), fucosyl-GM1 sialyl-CRACC adhesion molecule (CRACC), transglutaminase 5 (ROR1), macromolecular-melanoma-associated antigen (FLT3), o-acetyl-GD2 ganglioside (SLAMF7), folate receptor β, tumor endothelial marker 1 (also known as CD2 subclass 1, CRACC, SLAMF7, CD319, 19A24),Tumor endothelial marker 7 (CD133 or CD123), tight junction protein 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5 member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta specific 1 (PLAC1), hexose moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), urothelial differentiation specific glycoprotein (CLDN6) 2 (UPK2), and hepatitis A type inflammatory cell receptor 1 (TSHR), adrenergic receptor beta 3 (GPRC5D), pan-binding protein (CXORF61) 3 (CD179a), G protein-coupled receptor 20 (ALK), lymphocyte antigen 6 complex locus K9 (LY6K), olfactory receptor 51E2 (PLAC1), TCR gamma variable open reading frame protein (globoH glycoceramide), Wilm tumor protein (WT1); cancer / testis antigen 1 (UPK2), cancer / testis antigen 2 (CLDN6), melanoma-associated antigen 1 (TSHR), ETS translocation variant gene 6 on chromosome 12p (GPR C5D), sperm protein 17 (CXORF61), X-antigen family 1A (ALK), angiopoietin-binding cell surface receptor 2 (PLAC1), melanoma cancer testis antigen-1 (GloboH), melanoma cancer testis antigen-2 (NY-BR-1), Fos-related antigen 1, p53, p53 mutant, prostate-specific protein (CLDN6), prostate cancer tumor antigen-1 (GloboH), T cell-recognized melanoma antigen 1 (TSHR) rat sarcoma mutant, human telomerase reverse transcriptase (GPRC5D), sarcoma translocation breakpoints, melanoma apoptosis inhibitor protein ( CXORF61), ERG (ALK), N-acetylglucosaminyltransferase V (PLAC1), paired frame protein CD179a (GloboH), androgen receptor, cyclin B1, ALK avian myeloblastosis viral oncogene neuroblastoma-derived homolog (NY-BR-1), Ras homolog family member C (CLDN6), tyrosinase-related protein 2 (UPK2), cytochrome GloboH (CLDN6), T-cell-discriminating squamous cell carcinoma antigen 3 (GPRC5D), paired frame protein TSHR (CXORF61),Precursor protein binding protein sp32 (CD179a), lymphocyte-specific protein tyrosine kinase (ALK), A-kinase anchoring protein 4 (LY6K), synovial sarcoma X breakpoint 2 (PLAC1), receptor for advanced glycation end products (globoHglycoceramide), pea pod protein (WT1), human papillomavirus E6 (GloboH), human papillomavirus E7 (TSHR), endopeptidase E7, carboxylase, intestinal junction protein sp (CLDN6), synovial sarcoma X breakpoint protein, synovial sarcoma protein CLDN6, synovial sarcoma X breakpoint 2, synovial sarcoma protein HAVCR1 mutation , CD72, leukocyte-associated immunoglobulin-like receptor 1 (GPRC5D), Fc fragment of IgA receptor (CXORF61), leukocyte immunoglobulin-like receptor subfamily A member 2 (ALK), ALK molecule-like family member f (PLAC1), C-type lectin domain family 12 member A (GloboH), bone marrow stromal cell antigen 2 (NY-BR-1), mucin-like hormone receptor containing EGF-like module 2 (CLDN6), lymphocyte antigen 75 (UPK2), phosphatidyl alcohol proteoglycan-3 (CLDN6), Fc receptor-like 5 (TSHR) and / or immunoglobulin lambda-like polypeptide 1 (CXORF61).

[0121] TRAC gene, HLA-A gene, B2M gene, and CIITA gene

[0122] In the present application, the expression and / or activity of the TRAC gene and the HLA-A gene was down-regulated compared to the corresponding wild-type cells, the expression and / or activity of the B2M gene was not down-regulated, and the expression and / or activity of the CIITA gene was not down-regulated.

[0123] In the present application, the expression and / or activity of both genes was downregulated compared to corresponding wild-type cells consisting of the TRAC and HLA-A genes.

[0124] In the present application, downregulated expression level and / or activity of said gene includes downregulating the expression and / or activity of a nucleic acid molecule encoding said gene and / or downregulating the expression and / or activity of a protein product encoded by said gene.

[0125] For example, the protein product may include a polypeptide.

[0126] In the present application, the expression and / or activity of the TRAC gene and HLA-A gene are downregulated, or the expression and / or activity of the TRAC gene and HLA-A gene in the downregulated immune effector cells is compared to the corresponding unmodified cells, and the modification may include changing the nucleotide sequence structure of the TRAC gene and HLA-A gene. The nucleotide sequence may include a coding region or a non-coding region, such as a cis-regulatory element sequence, an exon sequence, and the cis-regulatory element sequence may include a promoter. The change may include partial or complete sequence deletion, insertion of an exogenous fragment, base site mutation, etc., and the inserted exogenous fragment replaces or destroys the sequence structure of the TRAC gene and the HLA-A gene, making them unable to be translated normally. The base site mutation may include a frameshift mutation, a missense mutation, a nonsense mutation, etc. The change may include a chemical group modification of the nucleotide sequence, such as methylation, etc. The change in the nucleotide sequence structure may be detected by gene sequencing, such as Sanger sequencing, sulfite sequencing, etc.

[0127] In the present application, the expression and / or activity of the TRAC gene and HLA-A gene are downregulated, or the expression and / or activity of the TRAC gene and HLA-A gene in the downregulated immune effector cells is downregulated, compared to the corresponding cells that are not modified, and the modification also includes reducing the mRNA content of the TRAC gene and HLA-A gene. The mRNA content is obtained by experimental and biostatistical methods known to those skilled in the art, such as molecular probe in situ hybridization, real-time fluorescent quantitative PCR (qPCR, RT-PCR), and the real-time fluorescent quantitative PCR may include SYBR Green method, TaqMan method, double hybridization probe method, and molecular beacon method. The detection result of the mRNA content allows for inevitable experimental or statistical errors, which are known in the art. The error may be within the range of ±10%, such as ±8%, ±6%, ±5%, ±4%, ±3%, etc. The mNNA content is reduced by at least 30%, e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, e.g., no mNNA of the TRAC gene and HLA-A gene is detectable in the modified immune effector cells.

[0128] In the present application, the expression and / or activity of the TRAC gene and HLA-A gene in the downregulated immune effector cells is also included in that the modification reduces the polypeptide content of the TRAC gene and HLA-A gene expression, compared to the unmodified corresponding cells. The polypeptide is a polypeptide having the same structure and function as the polypeptide produced by the expression of the TRAC gene and HLA-A gene in the unmodified corresponding cells, and the polypeptide having a changed function and structure produced by the change in the nucleotide sequence of the TRAC gene and HLA-A gene is not included in the comparison range. The content of the polypeptide is obtained by experimental and biostatistical methods known to those skilled in the art, such as flow cytometry analysis, enzyme-linked immunosorbent assay (ELISA), cell immunofluorescence staining, and immunoblotting (Western blotting, WB). The detection result of the polypeptide content allows for inevitable experimental or statistical errors, which are known in the art. The error may be within the range of ±10%, such as ±8%, ±6%, ±5%, ±4%, ±3%, etc. The reduction in polypeptide content is at least 30%, e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, e.g., no polypeptides of TRAC gene and HLA-A gene expression are detectable in the modified immune effector cells.

[0129] In this application, the expression and / or activity of the TRAC gene and HLA-A gene is downregulated, or the expression and / or activity of the TRAC gene and HLA-A gene in the downregulated immune effector cells may refer to knocking out or knocking down the expression of the TRAC gene and HLA-A gene, or performing other manipulations that disrupt the function of the TRAC protein and HLA-A protein.

[0130] In the present application, the expression and / or activity of the B2M gene is not downregulated and the expression and / or activity of the CIITA gene is not downregulated, or the expression and / or activity of the B2M gene is not downregulated and the expression and / or activity of the CIITA gene is not downregulated may include that the mRNA content of the B2M gene and the CIITA gene is not decreased compared to the corresponding unmodified cells. The mRNA content is obtained by experimental and biostatistical methods known to those skilled in the art, such as molecular probe in situ hybridization, real-time fluorescent quantitative PCR (qPCR, RT-PCR), and the real-time fluorescent quantitative PCR may include SYBR Green method, TaqMan method, double hybridization probe method, and molecular beacon method. The detection result of the mRNA content allows for inevitable experimental or statistical errors, which are known in the art. The error may be within the range of ±10%, such as ±8%, ±6%, ±5%, ±4%, ±3%, etc. The lack of a decrease in the mRNA content may include an increase or no change in the content, and the no change may include that the corresponding mRNA quantitative analysis of the B2M gene and CIITA gene in the two compared does not have a significant difference (P>0.05).

[0131] In the present application, the expression and / or activity of the B2M gene is not downregulated and the expression and / or activity of the CIITA gene is not downregulated, or the expression and / or activity of the B2M gene is not downregulated and the expression and / or activity of the CIITA gene is not downregulated, may include that the polypeptide content of the B2M gene and CIITA gene expression is not decreased compared to the unmodified corresponding cells. The polypeptide is identical in structure and function to the polypeptide produced by the expression of the B2M gene and the CIITA gene in the unmodified corresponding cells. The content of the polypeptide is obtained by experimental and biostatistical methods known to those skilled in the art, such as flow cytometry analysis, enzyme-linked immunosorbent assay (ELISA), cell immunofluorescence staining, and immunoblotting (Western blotting, WB). The detection result of the polypeptide content allows for inevitable experimental or statistical errors, which are known in the art. The error may be within the range of ±10%, such as ±8%, ±6%, ±5%, ±4%, ±3%, etc. The content of the polypeptide not decreasing includes the content increasing or unchanged, and the unchanged includes that the quantitative analysis of the corresponding polypeptides of the B2M gene and the CIITA gene in the comparison of the two does not have a significant difference (P>0.05).

[0132] In the present application, the fact that the expression and / or activity of the B2M gene is not down-regulated and the expression and / or activity of the CIITA gene is not down-regulated, or the expression and / or activity of the B2M gene is not down-regulated and the expression and / or activity of the CIITA gene is not down-regulated, includes artificial intervention in the CIITA gene and the B2M gene, or the mRNA and / or polypeptide encoding the CIITA gene and the B2M gene, to the corresponding unmodified cells, and for example, the artificial intervention may include introducing, into the corresponding unmodified cells, a nucleotide molecule or other compound that can target the CIITA gene and the B2M gene or the mRNA molecule encoded by them and change their structure or content.

[0133] In the present application, the expression and / or activity of the B2M gene is not downregulated and the expression and / or activity of the CIITA gene is not downregulated, or the expression and / or activity of the B2M gene is not downregulated and the expression and / or activity of the CIITA gene is not downregulated, refers to the absence of change in the nucleotide sequence structure of the B2M gene and the CIITA gene compared to the corresponding unmodified cell, and includes the absence of change in the nucleotide sequence structure, which can be detected by gene sequencing, such as Sanger sequencing, bisulfite sequencing, etc. The absence of change in the nucleotide sequence structure includes the absence of artificial change, and may also include changes that occur naturally and do not affect its function.

[0134] In the present application, the expression and / or activity of the B2M gene is not down-regulated and the expression and / or activity of the CIITA gene is not down-regulated, or the expression and / or activity of the B2M gene is not down-regulated and the expression and / or activity of the CIITA gene is not down-regulated, may mean that no modification is targeted to the B2M gene or the protein it encodes, or the CIITA gene or the protein it encodes.

[0135] In the present application, the expression level and / or activity of up to two alleles in the HLA-A gene are downregulated. For example, the two alleles can be a pair of alleles of the HLA-A gene in the immune effector cells. For example, the expression level and / or activity of one allele of the HLA-A gene is downregulated.

[0136] As used herein, the TRAC gene may include the gene designated HGNC:12029 and all allelic forms thereof. For example, the TRAC gene and all allelic forms thereof may include TRAC genotypes that can be present in immune effector cells. For example, the TRAC gene may include the nucleotide sequence designated SEQ ID NO.55.

[0137] For example, the TRAC gene may comprise a nucleotide sequence of human origin having 80% or more homology to the nucleotide sequence set forth in SEQ ID NO. 55, such as 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0138] In the present application, the HLA-A gene may include the gene designated by HGNC:4931 and all allelic forms thereof. For example, the HLA-A gene may include all HLA-A allelic types listed by the IMGT / HLA database version 3.38.0 (https: / / www.ebi.ac.uk / ipd / imgt / hla / ) named by the WHO HLA Factor Naming Committee, and the HLA-A allelic types and sequence information thereof disclosed by the IMGT / HLA database version 3.38.0 are incorporated herein by reference.

[0139] For example, the HLA-A alleles may include any one or more of A*02, A*24, A*01, A*03, A*32, A*11, A*26, A*68, A*23, A*29, A*31, A*33, A*25, A*43, A*74, A*30, A*69.

[0140] For example, the HLA-A alleles may include any one or more of A*02:01, A*03:01, A*01:01, A*24:02, A*68:01, A*11:01, A*31:01:02, A*29:02, A*32:01, A*26, A*23:01, A*30:02, A*25:01, A*33:03.

[0141] For example, the HLA-A alleles may include any one or more of A*02:01:01, A*01:01:01, A*03:01:01, A*24:02:01, A*11:01:01, A*32:01:01, A*29:02:01, A*31:01:02, A*23:01:01, A*26:01:01, A*68:01:02, A*30:01:01, A*68:02:01, A*25:01:01, A*68:01:01, A*02:05:01.

[0142] For example, the HLA-A alleles may include any one or more of A*02, A*30, A*03, A*01, A*24, A*32, A*68, A*11, A*26, A*23, A*31, A*25.

[0143] For example, the HLA-A alleles may include any one or more of A*02:01, A*03:01, A*24:02, A*01:01, A*11:01, A*26:01, A*25:01, A*68:01, A*32:01, A*31:01.

[0144] For example, the HLA-A alleles may include any one or more of A*24, A*33, A*02, A*11, A*26, A*31, A*01, A*24:02, A*02:01, A*33:03, A*11:01, A*26:01, A*02:06, A*31:01:02, A*26:03, A*26:02, A*02:07, A*01:01, A*02:10, A*03:01.

[0145] For example, HLA-A alleles may include A*02, A*24, A*33, A*11, A*26, A*31, A*30, A*03, A*01, A*32, A*29, A*68, A*23, A*25, A*34, A*36, A*43, A*66, A*74.

[0146] For example, the HLA-A alleles can include any one or more of A*24:02, A*33:03, A*02:01, A*11:01, A*02:01, A*31:01, A*26:01, A*02:07, A*30:01, A*26:02, A*01:01, A*03:01, A*30:04, A*26:03.

[0147] For example, the HLA-A alleles may include any one or more of A*02:01, A*11:01, A*24:02, A*30:01, A*26:01, A*23:01, A*02:07, A*02:06, A*03:01, A*01:01, A*31:01:02, A*33:03, A*32:01, A*68:01, A*02:03, A*02:05.

[0148] For example, the HLA-A alleles may include any one or more of A*03:01, A*02:01, A*23:01, A*01:01, A*30:02, A*30:01, A*33:03, A*29:02, A*74:01, A*36:01, A*24:02, A*02:02, A*68:01, A*68:02, A*34:02, A*66:02, A*31:01:02, A*32:01, A*02:05, A*66:01, A*26:01.

[0149] For example, the HLA-A alleles may include any one or more of A*02, A*11, A*24, A*30, A*33, A*03, A*01, A*26.

[0150] For example, the HLA-A alleles may include any one or more of A*11:01, A*24:02, A*02:01, A*02:07, A*33:03, A*02:06, and A*30:01.

[0151] For example, the HLA-A alleles may include any one or more of HLA-A*02:01:01:01, HLA-A*11:01:01:01, HLA-A*24:02:01, HLA-A*30:01:01:01, HLA-A*33:01:01:01, HLA-A*03:01:01:01, HLA-A*01:01:01:01, HLA-A*26:01:01:01.

[0152] For example, the HLA-A gene may comprise a nucleic acid sequence set forth in any of SEQ ID NOs. 56-63.

[0153] For example, the HLA-A gene may comprise a nucleotide sequence of human origin having 80% or more homology to a nucleic acid sequence set forth in any of SEQ ID NOs. 56-63, e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0154] qualification

[0155] In the present application, said modification may include gene knockout and / or gene silencing.

[0156] For example, the modification may include deletion of all or part of a gene, gene mutation, and / or gene silencing.

[0157] For example, the knockout may include a deletion of all or part of a gene, a genetic mutation, and the like.

[0158] For example, the genes may include the HLA-A gene and / or the TRAC gene.

[0159] For example, the modification may include knocking out any one of two TRAC alleles and knocking out any one of two HLA-A alleles in the immune cell.

[0160] For example, the modification may include knocking out two TRAC alleles and knocking out either one of two HLA-A alleles in the immune cell.

[0161] For example, the modification may include knocking out either one of two TRAC alleles and knocking out two HLA-A alleles.

[0162] For example, the modification may include two TRAC alleles being knocked out and two HLA-A alleles being knocked out.

[0163] For example, the gene subsegment deletion may comprise a deletion of ≧1 exon sequence.

[0164] For example, the genetic mutation may include a change in the composition or sequence of base pairs, which may result in, for example, a missense mutation, a frameshift mutation, and / or a nonsense mutation. The missense mutation usually changes a codon that codes for one amino acid to a codon that codes for another amino acid, changing the corresponding amino acid that constitutes a protein, by changing a base pair. The shift mutation usually is an insertion or deletion of one or more bases that are not an integer multiple of three in the DNA strand, resulting in a change in the coding codon. The nonsense mutation usually is a base pair change that changes a codon that codes for an amino acid to a terminator, at which site protein synthesis is prematurely terminated.

[0165] For example, the change in the base pair composition or sequence order may include a change in a single nucleotide or base (also called a point mutation) and / or a change in a polynucleotide or base. For example, the change in a single nucleotide or base may include a substitution of one base or nucleotide with another base or nucleotide, an insertion or deletion of a base. For example, the change in a polynucleotide or base may include a loss of a segment of a base sequence, an insertion of a segment of a base sequence, and / or a rearrangement of a segment of a base sequence. The partial base sequence is a part of any one of the exons and / or introns in a gene. The rearrangement may include a duplication, an inversion, a translocation, etc. of the single base sequence.

[0166] For example, the gene silencing may include transcriptional gene silencing (TGS) and / or post-transcriptional gene silencing (PTGS). The transcriptional silencing may include methylation of DNA molecules, thereby silencing DNA, for example, methylation of promoter sequences. The post-transcriptional gene silencing may include gene expression changes due to specific intervention on target RNA after transcription of the gene. The mRNA level is reduced, for example, by RNA interference (RNAi).

[0167] In the present application, the modification may include a technique or method using homologous recombination to replace an exogenous nucleotide sequence with an endogenous normal gene, thereby inactivating the endogenous normal gene. The exogenous nucleotide sequence is known. For example, the exogenous nucleotide sequence may be a partial fragment of the endogenous normal gene. For example, the exogenous nucleotide sequence may include, in order from 5' to 3', a homologous arm 1, a nucleotide sequence to be inserted, and a homologous arm 2. The nucleotide sequence to be inserted may include a reporter gene, a non-coding sequence, or a mutated sequence of the endogenous normal gene.

[0168] In the present application, the modification may include administering to the immune effector cells one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, and the CRISPR / Cas9 system.

[0169] In the present application, the antisense RNA may be a single-stranded RNA complementary to the transcription product mRNA (messenger RNA).

[0170] For example, at least 60% of the nucleic acid sequence of the antisense RNA is complementary to the mRNA, such as at least 80%, such as at least 90%, such as at least 70%, and such as 100% complementary.

[0171] For example, after binding to the target mRNA, the antisense RNA causes the double-stranded RNA molecule to become more sensitive to RNAse III, thus causing it to be degraded.

[0172] For example, the antisense RNA binds to a non-coding region upstream of an mRNA and inhibits translation of the target mRNA.

[0173] For example, the antisense RNA is artificially prepared.

[0174] For example, the antisense RNA is unable to form a short hairpin structure.

[0175] In the present application, the antisense RNA may comprise a nucleotide sequence set forth in any of SEQ ID NOs:93-96.

[0176] In the present application, the siRNA may be a class of double-stranded non-coding RNA molecules that are about 18-28 base pairs in length.

[0177] For example, the siRNA may cause degradation of mRNA by complementary binding to the mRNA.

[0178] For example, the siRNA may be 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 base pairs in length.

[0179] For example, the siRNA is prepared manually.

[0180] For example, the siRNA can be obtained by treating intracellular long double-stranded RNA or shRNA with Dicer enzyme.

[0181] In the present application, said shRNA refers to a class of RNA that can form short hairpin structures.

[0182] For example, the shRNA may comprise two short inverted repeats and a stem loop sequence located between the two short inverted repeats.

[0183] For example, at least 18 consecutive nucleic acid sequences in the shRNA can complementarily bind to the target mRNA, such as at least 19, for example, at least 20, for example, at least 21, for example, at least 22, for example, at least 23, for example, at least 24, for example, at least 25, for example, at least 26, etc.

[0184] For example, the shRNA does not include the sgRNA of the present application.

[0185] In the present application, the modification may include administering a CRISPR / Cas system to the immune effector cells.

[0186] CRISPR / Cas system

[0187] In the present application, the CRISPR / Cas system may include a guide RNA (gRNA) and a Cas enzyme. The gRNA may include a crRNA and a tracrRNA. When the crRNA and the tracrRNA are located on two different nucleotide molecules, the gRNA is called a dgRNA (dual-molecule gRNA), and when the crRNA and the tracrRNA are located on the same nucleotide molecule, the gRNA is called a sgRNA (unimolecule gRNA).

[0188] In the present application, the CRISPR / Cas system may include a gRNA nucleic acid sequence and a Cas protein, for example, an RNP complex of the gRNA nucleic acid sequence and the Cas protein.

[0189] In the present application, the CRISPR / Cas system may further comprise a nucleic acid sequence encoding the gRNA and a nucleic acid sequence encoding a Cas protein, and the nucleic acid sequence encoding the gRNA and the nucleic acid sequence encoding the Cas protein may be located in a plasmid, a virus (adenovirus, lentivirus, retrovirus, etc.) or other vectors conventionally used in the field to which they belong. For example, the nucleic acid sequence encoding the gRNA and the nucleic acid sequence encoding the Cas protein may be present in the same vector or in different vectors. For example, the nucleic acid sequence encoding the tracrRNA and the sequence encoding the crRNA may be present in the same vector or in different vectors. The promoters for driving the expression of each coded sequence may be the same or different.

[0190] In the present application, the CRISPR / Cas system may include multiple guide RNAs. Each guide RNA may include a different target sequence, such that the CRISPR / Cas system cleaves one or more target sequences. For example, one or more guide RNAs may have the same or different properties, such as activity and stability in the CRISPR / Cas complex. When multiple guide RNAs are used, each guide RNA may be encoded in the same vector or different vectors. The promoters for driving the expression of the presented multiple guide RNAs may be the same or different. For example, the guide RNA includes a guide RNA targeting the HLA-A gene and a guide RNA targeting the TRAC gene.

[0191] In the present application, the modification further comprises administering to the cell a Cas enzyme, which may comprise a Cas protein or a nucleic acid sequence encoding a Cas protein.

[0192] For example, the Cas protein may comprise at least one structural domain that interacts with a guide RNA (gRNA), e.g., the Cas protein may be guided to a target sequence by a guide RNA, e.g., the guide RNA interacts with the Cas protein as well as the target sequence to guide binding of the Cas protein to the target sequence, e.g., the guide RNA provides specificity for target cleavage, and the Cas protein may be generic, pairing with different guide RNAs to cleave different target sequences; e.g., the Cas protein may cleave single-stranded or double-stranded DNA; e.g., the Cas protein may cleave RNA; e.g., the Cas protein may cause RNA / DNA nicking; and / or comprise at least one DNA-binding structural domain and at least one nuclease. Structural domains; for example, the nuclease structural domain may be heterologous to the DNA binding structural domain; for example, the nuclease activity may be altered by modifying the Cas protein; for example, the Cas protein may be used to bind and control DNA expression or activity; for example, the Cas protein may be a Cas nuclease.

[0193] As used herein, the CRISPR / Cas system may comprise class 1 or 2 system components, including ribonucleoprotein complexes (e.g., Makarova et al., Nat Rev Microbiol, 13 (11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). Of these, class 2 CRISPR / Cas systems have a single protein effector. The type II, V and VI Cas proteins can be single protein, RNA-guided nucleases, referred to herein as "class 2 Cas nucleases." Class 2 Cas nucleases include Cas9, Cpf 1, C2c1, C2c2 and C2c3 proteins. Cas9 or Cpf1 protein (Zetsche et al., Cell, 163:1-13 (2015)) can include a RuvC-like nuclease domain or an HNH-like nuclease domain, and the Cpf1 sequence in Zetsche is incorporated herein in its entirety.

[0194] For example, the Cas protein is a protein of a type II CRISPR / Cas system (Cas9 protein of the CRISPR / Cas9 system) or a type V CRISPR / Cas system (e.g. Cpf 1 protein). For example, the Cas protein can originate from a class 2 CRISPR / Cas system, such as the Cas9 protein or the Cpf 1 protein. Class 2 Cas nuclease family proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA as further described herein.

[0195] For example, two class CRISPR / Cas system components can arise from type IIA, type IIB, type IIC, type V, or type VI systems, including Cas9 and its orthologues. The Cas9 protein or its orthologue may be derived from the following exemplary species: Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria in-nocua, Lactobacillus gasseri, Pasteurella multocida, Saccharomyces cerevisiae, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Streptococcus pyogenes, S. kanselii, Listeria in-nocua) (Streptococcus thermophilus), scarlet spirochete (Streptococcus sp.), Nocardia spp. (Staphylococcus aureus), S.Burnetii (Listeria innocua), green Streptococcus (Lactobacillus gasseri), genus Us bacteria (Francisella novicida), Streptococcus bacteria (Wolinella succinogenes), Pseudomycetus bacteria (Neisseria meningitidis), Dedella bacteria (Campylobacter jejuni) (Wolinella succinogenes), Lactobacillus bacteria (Streptococcus pyogenes), Lactobacillus bacteria (Streptococcus thermophilus), Campylobacter bacteria, Campylobacter bacteria, Brucella pylobacter bacteria (Streptococcus sp.), marine bacteria, marine bacteria, Bacterium bacteria, Bacterium Lactobacillus gasseri bacteria, Bacterium genus, Microcystis aeruginosa (Wolinella succinogenes), Paracocytogenes species (Neisseria meningitidis), Arabidopsis (Campylobacter jejuni), Clostridium botulinum of the Clostridium genus (Streptococcus pyogenes), Clostridium difficile (Streptococcus thermophilus), Haeus species (Streptococcus sp.), halophilic nitrifying cocci (Staphylococcus aureus), Daphylococcus species (Listeria in-nocua), Lactobacillus gasseri, Candida species (Francisella novicida), Wolinella succinogenes, Neisseria meningitidis, Corynebacterium difficile species (Campylobacter jejuni), Sphingobacterium species (Streptococcus pyogenes), soil microsphingobacteria (Streptococcus thermophilus), Streptococcus species (Streptococcus sp.), African thermococcal bacteria (Staphylococcus aureus), Listeria in-nocua, Campylobacter pylori (Lactobacillus gasseri) or Francisella novicida.

[0196] For example, the Cas9 protein may be derived from Streptococcus pyogenes; for example, the Cas9 protein may be derived from Thermophilus; for example, the Cas9 protein may be derived from Staphylococcus aureus; for example, the Cpf1 protein may be derived from Pasteurella tultocida (Francisella tularensis), Acidaminococcus, Eubacterium eligens, Leptospira dabilis (Leptospira inadai), Bacteroides (Prevotella dis-iens), or Actinoporphyrinae (Porphyromonas macacae). For example, the Cpf1 protein may be derived from the genus Acidaminococcus or the family Lachnospiraceae.

[0197] For example, the Cas9 protein may comprise an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to Streptococcus pyogenes Cas9.

[0198] For example, the Cas9 protein may comprise the amino acid sequence shown in SEQ ID NO:65.

[0199] For example, exemplary nucleic acid sequences encoding Cas9 proteins are described in the following documents: Cong et al., SCIENCE 2013, 399 (6121):819-823; Wang et al., CELL 2013, 153 (4):910-918; Mali et al., SCIENCE 2013, 399 (6121):823-826; Jinek et al., SCIENCE 2012, 337 (6096):816-821.

[0200] For example, the nucleotide sequence encoding the Cas9 protein is set forth in SEQ ID NO:64.

[0201] For example, the Cas9 protein may be modified, for example, by amino acid substitution, to form a fusion protein with another polypeptide fragment, which may include a PEST sequence, ubiquitin, polyubiquitin, or a nuclear localization signal (NLS).

[0202] In the present application, the crRNA may include a target sequence of a gRNA that can target any target sequence by the target sequence of the crRNA. For example, the degree of complementarity between the target sequence and the target sequence on the target nucleic acid molecule may be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. For example, the target sequence and the target sequence on the target nucleic acid molecule may be 100% complementary. For example, the target sequence and the target sequence on the target nucleic acid molecule may include at least one mismatch. For example, the target sequence and the target sequence on the target nucleic acid molecule may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches. For example, the target sequence and the target sequence on the target nucleic acid molecule may include 1 to 6 mismatches. For example, the target sequence and the target sequence on the target nucleic acid molecule may include 5 or 6 mismatches. For example, the target sequence and the target sequence on the target nucleic acid molecule do not include any mismatches.

[0203] In the present application, the length of the target sequence may depend on the CRISPR / Cas system and components used. For example, different Cas proteins from different bacterial species have different optimal target sequence lengths. Furthermore, the target sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 nucleotides in length. For example, the target sequence may comprise 18-24 nucleotides in length. For example, the target sequence may comprise 19-21 nucleotides in length. For example, the target sequence may comprise 20 nucleotides in length.

[0204] In the present application, the crRNA may further comprise a crRNA flagpole sequence, which may comprise any sequence crRNA that can be sufficiently complementary to the tracrRNA to promote the formation of a CRISPR / Cas complex. For example, the flag sequence may comprise all or a portion of a sequence of a naturally occurring crRNA that complements the tracrRNA in the same CRISPR / Cas system (also referred to as "tagging" or "handle"). For example, the flag sequence may comprise all or a portion of a repeat sequence from a naturally occurring CRISPR / Cas system. For example, the sequence may comprise a shortened or modified tag or handle sequence. The portion of the tracrRNA that is complementary to the crRNA pole sequence may be referred to as the tracrRNA pole sequence. For example, the degree of complementarity between the tracrRNA and the flag portion that hybridizes to the tracrRNA along the shorter of the two sequences may be about 40%, 50%, 60%, 70%, 80% or more. For example, the flag portion of the hybridization between tracrRNA and tracr RNA was not 100% complementary along the shorter of the two sequences. The length of the crRNA varies depending on the CRISPR / Cas system or tracrRNA being used. For example, the crRNA flag sequence may comprise 10-50 nucleotides in length or more than 50 nucleotides in length. For example, the crRNA flag sequence may comprise 15-40 nucleotides in length. For example, the crRNA flag sequence may comprise 20-30 nucleotides in length. For example, the crRNA flag sequence may comprise 22 nucleotides in length. When using double guide RNA, there is no upper limit to the length of the crRNA flag sequence.

[0205] In the present application, the tracrRNA may comprise all or part of the wild-type tracrRNA sequence from a naturally occurring CRISPR / Cas system. For example, the tracrRNA may comprise a truncated or modified variant of the wild-type tracrRNA. The length of the tracrRNA varies depending on the CRISPR / Cas system used. For example, the tracr RNA may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides in length. For example, the tracr RNA is at least 20 nucleotides in length. For example, the tracr RNA is at least 40 nucleotides in length. For example, the tracr RNA may comprise a secondary structure such as one or more hairpin or stem ring structures, or one or more bulge structures.

[0206] In the present application, the guide RNA may comprise two RNA molecules, referred to herein as "dual guide RNA" or "dgRNA." For example, the dgRNA may comprise a first RNA molecule comprising a crRNA and a second RNA molecule comprising a tracrRNA. The first and second RNA molecules can form an RNA duplex by base pairing between flagpole sequences on the crRNA and tracrRNA.

[0207] For example, a first RNA molecule from 5' to 3' may include a target sequence complementary to the target sequence, a crRNA flag sequence, and a second RNA molecule from 5' to 3' may include a tracrRNA flag sequence complementary to the crRNA flag sequence, a nuclease binding sequence, which may be, for example, a Cas nuclease such as Cas9.

[0208] In the present application, the guide RNA may comprise a single RNA molecule, referred to as a "single-molecule gRNA" or "sgRNA." For example, the sgRNA may comprise a tracrRNA, a crRNA covalently linked to the tracrRNA. For example, the crRNA and the tracrRNA may be covalently connected via a linker nucleic acid sequence. For example, the single-molecule gRNA may comprise a stem-loop structure formed by base pairing between flag sequences on the crRNA and the tracrRNA. For example, the sgRNA is a "Cas9sgRNA" capable of mediating DNA cleavage via the Cas9 protein. For example, the sgRNA is a "Cpf1sgRNA" capable of mediating DNA cleavage via the Cpf 1 protein.

[0209] For example, the 5' to 3' single molecule gRNA or sgRNA may include a crRNA, a loop, and a tracrRNA. The crRNA 5' to 3' may include a target sequence complementary to the target sequence, a crRNA flagpole sequence. The 5' to 3' tracrRNA may be a Cas nuclease, such as Cas9, and may include, for example, a tracrRNA flag sequence complementary to the crRNA flag sequence, a nuclease binding sequence.

[0210] For example, from 5' to 3', a single molecule gRNA or sgRNA may include a target sequence complementary to the target sequence, a crRNA ribosome sequence, a loop, a tracrRNA ribosome sequence complementary to the crRNA ribosome sequence, and a nuclease binding sequence.

[0211] The gRNA may also include modified nucleosides or nucleotides, as used herein, such as by modifying (e.g., replacing) one or two non-linked phosphate oxygens and / or one or more linking phosphate oxygens of a backbone phosphodiester bond; by substituting a moiety of the ribose, such as by substituting the 2' hydroxyl group of the ribose; by substituting, e.g., a phosphate moiety with a dephosphorylated joint; by modifying or substituting, e.g., a naturally occurring nucleobase; by substituting or modifying, e.g., a phosphoribose backbone; by modifying, e.g., the 3' or 5' end of an oligonucleotide, e.g., by removing, modifying or replacing a terminal phosphate group or a joint moiety, by adding a cap or joint (e.g., a 3' or 5' cap modification may include a sugar and / or a backbone modification); by modifying or substituting a sugar.

[0212] For example, the introduction of the modified nucleosides or nucleotides increases stability against nucleases. For example, the introduction of the modified nucleosides or nucleotides reduces the innate immune response. The innate immune response includes cellular responses to exogenous nucleic acids (including single-stranded nucleic acids) and can include the expression and release of cytokines (especially interferons) and induction of cell death.

[0213] For example, the modification may include administering to the immune effector cells an sgRNA that targets an exon portion of the HLA-A gene.

[0214] For example, the sgRNA targeting an exon portion of the HLA-A gene may comprise a nucleotide sequence shown in any of SEQ ID NOs. 16-54, 91-92.

[0215] For example, the sgRNA targeting an exon portion of the HLA-A gene may comprise a nucleotide sequence that is at least 70% homologous to a nucleotide sequence set forth in any of SEQ ID NOs. 16-54, 91-92, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%.

[0216] For example, the modification may include administering to the immune effector cells an sgRNA that targets an exon portion of the TRAC gene.

[0217] For example, an sgRNA targeting an exon portion of the TRAC gene may comprise a nucleotide sequence set forth in any of SEQ ID NOs. 1-15.

[0218] For example, the sgRNA targeting an exon portion of the TRAC gene may comprise a nucleotide sequence that is at least 70% homologous to a nucleotide sequence set forth in any of SEQ ID NOs. 1-15, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%.

[0219] Chimeric antigen receptors (CARs)

[0220] In the present application, the immune effector cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory structure and a primary signaling domain.

[0221] For example, the antigen binding domain specifically binds to a tumor antigen, for example, the tumor antigen is selected from the group consisting of CD19, CD123, CD22, CD30, CD171, CA125, C-met, L1CAM, EC, DLL3, CD99, 5T4, CD138, CS-1 (also known as CD2 subclass 1, CRACC, SLAMF7, CD319, 19A24), C-type lectin-like molecule-1 (CLL-1 or CD123), CD33, epidermal growth factor receptor variant III (CD171), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member. B-cell maturation antigen (CA125), Tn antigen (e.g., C-met, L1CAMα-CD138), prostate-specific membrane antigen (CRACC); receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (SLAMF7), CD38, CD319, carcinoembryonic antigen (CD2 subclass 1, also known as CRACC, SLAMF7, CD319, 19A24), epithelial cell adhesion molecule (CLL-1 or CD123), B7H3 (CD171), KIT (GD2) , interleukin-13 receptor subunit alpha-2 (CA125), mesothelin, interleukin-11 receptor alpha (e.g., C-met, L1CAMα-CD138), prostate stem cell antigen (CRACC), protease serine 21, vascular endothelial growth factor receptor 2 (ROR1), CD44v6(Y) antigen, CD24, platelet-derived growth factor receptor beta (FLT3), stage-specific embryonic antigen-4 (CD123), CD20, folate receptor alpha, receptor tyrosine kinase CD171 (CLL-1), cell surface-associated mucin 1 (CD123) ( MUC1), epidermal growth factor receptor (CD171), neural cell adhesion molecule (CA125), CD138, prostatic acid phosphatase (C-met or L1CAM), mutant elongation factor 2 (CD138), liver complement protein B2, fibroblast activation protein alpha (CRACC), insulin-like growth factor 1 receptor (SLAMF7), carbonic acid IX (CLL-1β), cell surface-associated mucin kinase (e.g., proteasome, megalin factor) subprotein 100, glycoprotein 9, prostatic acid phosphatase (CA125), mutant 9,Oncogene fusion protein consisting of breakout cluster (CD171) and C-met murine leukemia viral oncogene homolog 1 (GD2) (CA125), tyrosinase, liver protein type A receptor 2 (e.g., C-met, L1CAMα-CD138), fucosyl-GM1 sialyl-CRACC adhesion molecule (CRACC), transglutaminase 5 (ROR1), macromolecular-melanoma-associated antigen (FLT3), o-acetyl-GD2 ganglioside (SLAMF7), folate receptor β, tumor endothelial marker 1 (CD2 subclass 1, CRACC, SLAM F7, also known as CD319, 19A24), tumor endothelial marker 7-related (CLL-1 or CD123), tight junction protein 6 (CD171), thyroid-stimulating hormone receptor (GD2), G protein-coupled receptor class C group 5 member D (CA125), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), globoH glycoceramide hexose moiety (GloboH), mammary differentiation antigen (NY-BR-1), urothelial Differentiation-specific glycoprotein (uroplakin) 2 (UPK2), Hepatitis A virus cellular receptor 1 (HAVCR1), Adrenergic receptor beta 3 (ADRB3), Ubiquitin (CXORF61) 3 (CD179a), G protein-coupled receptor 20 (ALK), Lymphocyte antigen 6 complex locus K9 (LY6K), Olfactory receptor 51E2 (PLAC1), TCR gamma open reading frame protein (globoHglycoceramide), Wilm's tumor protein (WT1) Cancer / testis antigen 1 (UPK2), Cancer / testis antigen 2 (HAVCR1), Melanoma-associated antigen ADRB3, ETS translocation mutant gene 6 on chromosome 12p (CXORF61), sperm protein 17 (CXORF61), X-antigen family member 1A (ALK), angiopoietin-binding cell surface receptor 2 (PLAC1), melanoma cancer testis antigen-1 (GloboH), melanoma cancer testis antigen-2 (NY-BR-1), Fos-related antigen 1, p53, p53 mutant, prostate-specific protein (uroplakin), prostate cancer tumor antigen-1 (GloboH), T cell-recognized melanoma antigen 1 (NY-BR-1) rat sarcoma (ADRB3) mutant,Human telomerase reverse transcriptase (CXORF61), sarcoma translocation breakpoints, melanoma apoptosis inhibitor protein, ERG (ALK), N-acetylglucosaminyltransferase V (PLAC1), paired frame protein CD179a (GloboH), androgen receptor, cyclin B1, ALK avian myeloblastosis viral oncogene neuroblastoma-derived homolog (NY-BR-1), Ras homolog family member C (uroplakin), tyrosinase-related protein protein 2 (UPK2), cytochrome GloboH (HAVCR1), squamous cell carcinoma antigen 3 for T cell recognition (uroplakin), paired frame protein HAVCR1 (HAVCR1), precursor protein binding protein sp32 (CXORF61), lymphocyte-specific protein tyrosine kinase (ALK), A-kinase anchor protein 4 (LY6K), synovial sarcoma X breakpoint 2 (PLAC1), receptor for advanced glycation end products (globoHglycoceramide) , pea pod protein (WT1), human papillomavirus E6 (GloboH), human papillomavirus E7 (NY-BR-1), intestinal glycoprotein-binding protein spuroplakin (CD179a), lymphocyte-specific protein tyrosine kinase, ETV6-AML heat shock protein (ras) 72, leukocyte-associated immunoglobulin-like receptor 1 (CXORF61), Fc fragment of IgA receptor (CXORF61), leukocyte immunoglobulin-like receptor subfamily A melanoma These include ALK, ALK molecule-like family member f (PLAC1), C-type lectin domain family 12 member A (GloboH), bone marrow stromal cell antigen 2 (NY-BR-1), mucin-like hormone receptor 2 containing an EGF-like module (uroplakin), lymphocyte antigen 75 (UPK2), phosphatidylinositol proteoglycan-3 (HAVCR1), Fc receptor-like 5 (ADRB3) and / or immunoglobulin lambda-like polypeptide 1 (HAVCR1).

[0222] In the present application, the antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof that specifically binds to the tumor antigen. For example, the specific binding GPC3 antibody or an antigen-binding fragment thereof described in the present application includes, but is not limited to, a recombinant antibody, a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a single-chain antibody, a diantibody, a triabody, a tetraabody, an Fv fragment, an scFv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a camelid single domain antibody.

[0223] For example, the antibody may be a humanized antibody, which is an antibody or variant, derivative, analog or fragment thereof that immunospecifically binds to an associated antigen (e.g., CD 19, BCMA, GPC 3) and comprises a frame (FR) region essentially of the amino acid sequence of a human antibody and a complementarity determining region (CDR) essentially of the amino acid sequence of a non-human antibody, where "essentially" in the case of a CDR means that the amino acid sequence of the CDR is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the non-human antibody CDR.

[0224] For example, the antigen-binding fragment may include a Fab, a Fab', a F(ab)2, an Fv fragment, a F(ab.)2, a scFv, a di-scFv, and / or a dAb.

[0225] For example, the single chain antibody is an scFv.

[0226] For example, the antigen-binding domain targets a solid tumor. For example, the solid tumor is selected from the group consisting of liver cancer, gastric cancer, lung cancer, breast cancer, colon cancer, rectal cancer, renal cell carcinoma, hepatoma, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, vulvar cancer, childhood solid tumor, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, glioma, pituitary sarcoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and metastases thereof.

[0227] For example, the antigen-binding domain targets a non-solid tumor. For example, the non-solid tumor is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, polyhilar cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasma cell lymphoma, plasmacytoid cell neoplasm, and Waldenstrom's macroglobulinemia.

[0228] In the present application, the transmembrane domain may comprise a transmembrane domain selected from the following proteins: CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0229] In the present application, the costimulatory domain can include a costimulatory domain selected from the following proteins: CD137, CD28, 4-1BB, OX-40, and ICOS.

[0230] In the present application, the intracellular signaling domain may comprise a signaling domain derived from CD3ζ.

[0231] In the present application, the hinge region connects the antigen binding domain and the transmembrane domain, and the hinge region comprises a hinge region selected from the following proteins: IgG1, IgG4, IgD, CD8.

[0232] Composition, Application

[0233] The present application also provides compositions, which include the modified immune effector cells described herein and a pharma- ceutically acceptable carrier.

[0234] For example, the composition can include a population of cells, wherein the population of cells includes a modified immune effector cell as described herein.

[0235] For example, the ratio of the number of modified immune effector cells to the total number of cells in the cell population is at least 0.001%, at least 0.01%, at least 0.1%, at least 1%, at least 5%, at least 10%, 55%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least, at least 50%, at least, at least 80%, at least 58%. 83%, at least 60%, at least 63%, at least 65%, at least 68%, at least 70%, at least 45%, at least 73%, at least 90%, at least, at least 75%, at least 53%, at least 78%, at least 93%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least, at least 89%, at least 99%, at least, at least, at least 91%, at least 92%, at least 94%, at least 95%, at least 96%, at least, at least 97%, at least, at least, at least 100%.

[0236] For example, the population of cells may include the modified immune effector cells and corresponding unmodified immune effector cells.

[0237] For example, modified immune effector cells may include cells in which either of two TRAC alleles have been knocked out and either of two HLA-A alleles have been knocked out, cells in which two TRAC alleles have been knocked out and either of two HLA-A alleles have been knocked out, cells in which either of two TRAC alleles have been knocked out and two HLA-A alleles have been knocked out, cells in which two TRAC alleles have been knocked out and two HLA-A alleles have been knocked out, cells in which two TRAC alleles have been knocked out and two HLA-A alleles have been knocked out.

[0238] For example, the cell population may be a cell population obtained after the immune effector cell population is treated with a genetic engineering procedure, and the genetic engineering procedure may include administering the antisense RNA, the siRNA, the shRNA and / or the CRISPR / Cas9 system of the present application to the immune effector cell population. For example, the CRISPR / Cas9 system may include an sgRNA targeting an exon portion of an HLA-A gene, an sgRNA targeting an exon portion of a TRAC gene, and a Cas9 protein.

[0239] For example, the cell population can include a cell population obtained after the immune effector cells are edited by a CRISPR / Cas9 system, wherein the editing efficiency of the editing is at least 30%, at least 35% 68%, at least 40%, at least 45% 73%, at least 50%, at least 53%, at least 55%, at least 58%, at least 60%, at least, at least 65%, at least 88%, at least 70%, at least 90%, at least 75%, at least 78%, at least 80%, at least 85%, at least 63%, at least 86%, at least 96%, at least, at least 87%, at least 98%, at least 89%, at least, at least 91%, at least 92%, at least 93%, at least, at least 83%, at least 94%, at least 95%, at least, at least 97%, at least 99%.

[0240] For example, editing efficiency can be obtained by Sanger sequencing, TA cloning, and flow cytometry.

[0241] For example, the cell population includes a cell population of immune effector cells obtained after application of the antisense RNA, siRNA, or shRNA of the present application, wherein the cell population has a reduction in mRNA expression of at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 53%, at least at least 58%, at least 60%, at least 63%, at least 65%, at least 87%, at least at least 68%, at least 88%, at least at least 70%, at least 73%, at least 75%, at least 78%, at least 55%, at least 80%, at least 83%, at least at least 85%, at least 86%, at least at least 89%, or at least 90% compared to a corresponding cell population of immune effector cells prior to application.

[0242] For example, the cell population can include a cell population of immune effector cells obtained after application of the antisense RNA, siRNA, or shRNA of the present application, wherein the cell population has a reduction in protein expression of at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 53%, at least 58%, at least 60%, at least 63%, at least 65%, at least 68%, at least 70%, at least 73%, at least 75%, at least 78%, at least 55%, at least 80%, at least 83%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% compared to a corresponding cell population of immune effector cells prior to application.

[0243] For example, such acceptable compositions are non-toxic to recipients at the dosages and concentrations employed.Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

[0244] For example, the pharma- ceutically acceptable carrier may be composed of any solvent, dispersion medium, isotonicity agent, and absorption retardant that is compatible with the immune effector cells, generally safe, non-toxic, and biologically or otherwise undesirable. For example, the pharma-ceutically acceptable carrier may include a 2°C-8°C storage solution, a frozen storage solution, an injection solution, and the like. For example, the carrier may include the following components: adenosine, sodium chloride, albumin, interleukin-15, angiotensin-II, short peptide and polypeptide compounds in serum-free culture medium from human umbilical cord mesenchymal stem cells, and the like. For example, the carrier may further include Normosol R (Abbott), Plasma-Lyte A (Baxter) injection, 5% dextrose water, or Ringer's lactate solution. For example, the carrier may also include glycerin or DMSO.

[0245] For example, the compositions include parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration or injection directly into tissue. For example, the compositions can be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition can be administered in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the pharmaceutical composition can be administered continuously. Continuous (or continuous) administration can be achieved by a small pump system worn by the patient to meter the therapeutic agent into the patient's body, as described in WO2015 / 036583.

[0246] The present application also provides an application of the modified immune effector cells of the present application in the preparation of CAR-T cells.

[0247] The present application also provides the application of the modified immune effector cells of the present application in the preparation of a drug, the drug comprising Homeopathic Therapy Used for.

[0248] The present application also provides an application of the modified immune effector cells of the present application in preparing a drug, the drug being used in tumor treatment.

[0249] For example, the tumor includes a solid tumor and a non-solid tumor. The types of the solid tumor and the non-solid tumor are as described above.

[0250] Without intending to be limited by any theory, the following embodiments are not intended to limit the scope of the invention of the present application, but are intended to illustrate the modified immune effector cells, production methods, and uses of the present application. The embodiments do not include detailed descriptions of conventional methods, such as how to construct vectors and plasmids, how to insert genes encoding proteins into such vectors and plasmids, or how to introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

[0251] Working Example

[0252] Example 1 Guide RNA Design

[0253] The corresponding gene sequences (SEQ ID No.55-63) can be downloaded from the website https: / / www.ncbi.nlm.nih.gov / . This gene sequence can be opened using SnapGene software to design sgRNAs in different exons of the target gene. The sgRNA non-restrictive design principle of the CRISPR / Cas9 system adopted in this embodiment is 5'-NNN(20)-NGG-3', where NGG is called the protospacer adjacent motif (PAM), where N represents A, T, C or G. Since many sgRNAs are devised for the same exon and sgRNAs consisting of 20 nucleotide sequences may appear in duplicate in the genome, the site http: / / crispr.cos.uni-heidelberg.de was used to devise and evaluate sgRNAs, the exon sequence was pasted on this site, and a site called sgRNA was devised and predicted evaluation was performed. The higher the evaluation score, the higher the editing efficiency and the lower the risk of off-targeting may be, and the sgRNAs with the highest scores were selected and tested. The sgRNAs targeting the TRAC gene were synthesized by GenScript as follows: SEQ ID No. 1-15, the sgRNAs targeting the HLA-A02 gene were synthesized as follows: SEQ ID No. 16-37, the sgRNAs targeting the HLA-A11 gene were synthesized as follows: SEQ ID No. 38-46, and the sgRNAs targeting the HLA-A24 gene were synthesized as follows: SEQ ID No. 47-54.

[0254] Example 2: CD3 + 1. Preparation of T Cells

[0255] (1) Isolation of PBMCs from peripheral blood

[0256] Peripheral blood was collected from healthy donors and diluted 1:1 with PBS buffer. A cell separation solution (Ficoll) of 1 / 3 of the diluted blood volume was placed in a new 50 ml centrifuge tube, and the blood cell dilution solution was then added very slowly along the tube wall, and centrifuged at 800 g for 20 minutes at room temperature (centrifuge setting speed increase 1, speed decrease 0). After centrifugation, the liquid in the centrifuge tube was separated into PBS and serum layer, white blood cell layer, lymphocyte separation solution, and red blood cell layer from the top. The PBS and serum layer were removed, the white blood cell layer was transferred to a new 50 ml centrifuge tube, PBS was added to 40 ml of washed cells, and centrifuged at 450 g for 10 minutes. After centrifugation, the supernatant was discarded to obtain peripheral blood mononuclear cells. Cell counts were performed after the cells were resuspended.

[0257] (2) Resuscitation of frozen healthy human PBMCs

[0258] Frozen healthy PBMC cells were resuscitated in a 37°C incubator, and after complete thawing, they were aspirated into a 15ml centrifuge tube containing 10ml of X-VIVO15 medium (purchased from LONZA) containing 10% FBS, centrifuged at 400g for 8 minutes, the supernatant was removed, and 2ml of X-VIVO15 medium (containing 10% FBS and DNase I at a final concentration of 100μg / ml) was added. The supernatant was removed, 2ml of X-VIVO15 medium (containing 10% FBS and DNase I at a final concentration of 100μg / ml) was added, and incubated at room temperature for 15 minutes with constant shaking. The solution at the end of incubation was filtered through a 40μm filter, 10ml of PBS buffer was aspirated to resuspend the cells at the bottom, and then added to the filter, examined, centrifuged at 400g for 8 minutes, the supernatant after centrifugation was discarded, the cells were resuspended, and the cell count was counted.

[0259] (3) CD3+ T cell fraction

[0260] T cells were extracted from peripheral blood mononuclear cells (PBMCs) using EasySep™ Human T Cell Sorting Kit (purchased from StemCellTechnologies, part number: 17951). The PBMC density was adjusted to 5 × 107 cells / ml, and the range of PBS buffer added was 0.25-2 ml. First, add the cocktail and mix uniformly, then add the isolation cocktail at 50 μl / ml and mix uniformly, then leave at room temperature for 5 minutes; scroll RapidSpheres in a vortex vibrator for 30 s, then add 40 μl / ml to the cells and mix uniformly; replenish the buffer to a multiple of 2.5 ml and blow gently up and down 2-3 times; place 2.5 ml per tube into a cryopreservation tube, place the cryopreservation tube on a magnetic frame, and leave at room temperature for 3 minutes. Gently open the cryopreservation tube cover, carefully hold both sides and hold the magnetic frame, invert it and hold it for 2-3 s, and transfer the cell solution into a new centrifuge tube at once; resuspend the cells in 10-20 ml of buffer (photoreceptor cell volume), then centrifuge at 300 g for 10 minutes, discard the supernatant, and transfer the CD3 + T cells were obtained.

[0261] (4) T cell activation

[0262] The activating agent was arranged in a volume ratio of culture medium:Transact=99:1, the culture medium was X-VIVO15 culture medium (containing 5% FBS, 200 U / ml IL2, 10 ng / ml IL7, and 5 ng / ml IL15), and Transact was purchased from MiltenyiBiotec. T cells were thoroughly suspended in 1 ml of activation reagent (containing 10 μl Transact) for every 1×106 cells, and then incubated at 37°C in a 5% CO2 culture box for 3 days.

[0263] Example 3. Preparation of single gene knockout T cells

[0264] The RNP complex was electrically transferred to the activated T cells prepared in Example 2 using an electrotransfer kit (purchased from LONZA, product number: V4XXP-3024). The medium (X-VIVO15 medium + 10% FBS + IL2 (200 U / ml) + IL7 (10 ng / ml) + IL15 (5 ng / ml)) was preheated in the orifice 30 minutes before. The electrotransfer buffer was arranged as Nucleofector Solution:Supplement = 82:18. Preparation of RNP complex: The sgRNA sequence of TRAC is sg9 (shown in SEQ ID No.1), and the sgRNA sequence of HLA-A is HLA-A02Sg2 (shown in SEQ ID No.17) or HLA-A02Sg5 (shown in SEQ ID No.18) or HLA-A11sg21 (shown in SEQ ID No.91). As shown) or HLA-A11Rsg2 (shown in SEQ ID No.92), 20 μg of sgRNA was added to a PCR tube (RNAase-free), followed by 10 μg of Cas9 protein (purchased from thermo, part number: A36499), mixed gently, and incubated at room temperature for 12 minutes. The activated T cells cultured in Example 2 were counted, centrifuged at 300 g for 8 minutes, the supernatant was discarded, PBS heavy suspension cells were added, 1E7 cells were aspirated and centrifuged again at 300 g for 8 minutes, the supernatant was discarded, and the cells were heavy suspension in 100 μl of electrophoresis buffer. The incubated RNP complex was added to the above cell suspension, mixed gently, and the mixture was carefully transferred to an electroporation cuvette. The electroporation cuvette was placed on a Lonza-4D and spun by selecting the EO-115 program. Add pre-warmed medium to the electroporation cuvette and transfer the cells using a suitable pipette into the pre-warmed medium in the well plate. Incubate at 37°C and 5% CO. 2 The mice were placed in an incubator.

[0265] Example 4. Comparison of methods for measuring knockout efficiency

[0266] (1) Sanger sequencing

[0267] The number of cells is 3–5 × 10 4 Harvest 100 cells, centrifuge at 2000r / min for 5 min, remove as much clean supernatant as possible, add 20μl DE lysis solution to each tube, add cells to PCR tube after lysis, centrifuge instantaneously, and run on PCR machine. The machine conditions are 65℃ 30min, 4℃ 30s, 95℃ 2min, 16℃ infinite. PCR was performed with primer pair TRAC-For / TRAC-Rev or HLA-A For / HLA-A Rev as template for the fragment, and the PCR primer sequence was as follows: SEQ ID NO.66-81, and the PCR product was sent to Kin Wei-ji for Sanger sequencing. After obtaining the results of sanger sequencing, use the EditR editor on the website: https: / / moriaritylab.shinyapps.io / editr_v10 / to predict where editing occurs and the editing efficiency.

[0268] (2) TA clone sequence measurement

[0269] After purifying the PCR product using AxyPrepTM PCR Product Cleaning Kit (AXYGEN), the purified PCR product was ligated to the T vector with a sticky end using a kit (DNAA-Tailing Kit, TaKaRa) using DNA Ligation Kit Ver2.1 (purchased from TaKaRa). The ligated product was transformed into recipient cells (DH5alpha) using (pMDTM19-TVector Cloning Kit from TaKaRa), coated on an LB plate containing ampicillin resistance, and cultured at 37°C for about 12 hours. Single colonies were picked and sequenced at GENEWIZ, Inc. Knockout efficiency = number of mutant clones / total number of clones.

[0270] (3) Flow cytometry

[0271] 10E5 to 10E8 cells were centrifuged at 2000 rpm for 5 minutes, the supernatant was removed, and then the cells were suspended in 100 μl of PBS buffer in each tube. Furthermore, 5 μl of anti-human ABTCR-APC (purchased from eBioscience) antibody, HLA-A02 Monoclonal Antibody (BB7.2), APC, eBioscince (trademark) (Purchased from Invitrogen) 5μl of antibody was added, mixed uniformly, and incubated at room temperature for 10 minutes. After centrifugation at 2000rpm for 5 minutes, the cells were washed twice with PBS buffer, resuspended, and assayed with BD FACSAria flow cytometer to determine the positive expression rates of TCR and HLA-A02 on the cell surface. Knockout efficiency = (AB) / A × 100%; A is the positive expression rate of the control group; B is the positive expression rate of the knockout group.

[0272] The three detection results of TRAC single gene knockout are shown in Figures 1 to 3, and the calculation results of knockout efficiency are shown in Table 1. The three detection methods are basically the same, and subsequent experiments only adopt Sanger sequencing to detect the editing efficiency. [Table 1]

[0273] The results of Sanger sequencing for HLA-A02 gene editing are shown in Figures 4-5, and the editing efficiency was 90% in both cases. The results of Sanger sequencing for HLA-A11 gene editing are shown in Figures 6-7.

[0274] Example 5. Preparation of TRAC gene and HLA-A gene double knockout T cells

[0275] The RNP complex was electrically transferred to the activated T cells prepared in Example 2 using an electrotransfer kit (LONZA, product number: V4XXP-3024). 30 minutes beforehand, the medium (X-VIVO15 medium + 10% FBS + IL2 (200 U / ml) + IL7 (10 ng / ml) + IL15 (5 ng / ml)) was preheated in the orifice. The current buffer was arranged in a ratio of Nucleofector Solution:Supplement = 82:18. Preparation of RNP complex: 20 μg TRACsgRNA (TRACSg9), 20 μg HLA-AsgRNA (HLA-A02Sg2 or HLA-A02Sg5 or HLA-A11sg21 or HLA-A*24:02:01, HLA-A*30:01:01, HLA-A*33:01:01:01, HLA-A*03:01:01:01, HLA-A*01:01:01:01 or HLA-A*26:01:01sgRNA) were placed in a PCR tube (without RNA), 10 μg of each Cas9 protein (purchased from thermo, product number: A36499) was added, mixed gently, and incubated at room temperature for 12 minutes. The activated T cells cultured in Example 2 were counted, centrifuged at 300 g for 8 minutes, the supernatant was discarded, PBS heavy suspension cells were added, 1E7 cells were aspirated and centrifuged again at 300 g for 8 minutes, the supernatant was discarded, and the cells were heavy suspension in 100 μl of electrophoresis buffer. The incubated TRAC and HLA-A RNP complex was added to the above cell suspension, and after shaking gently, the mixture was carefully transferred to an electroporation cuvette. The electroporation cuvette was placed on a Lonza-4D and rotated by selecting the EO-115 program. Pre-warmed medium was added to the electroporation cuvette, and the cells were transferred to the pre-warmed medium in the well plate using a suitable pipette, and incubated at 37 °C, 5% CO 2 The mice were placed in an incubator.

[0276] The double knockout efficiency was measured by sequencing, and TRAC-negative, HLA-A-negative T cells with a double knockout efficiency of 80% or more could be obtained. The results are shown in Figures 8-9. Here, Figure 8A shows the results of knocking out HLA-A02 using HLA-A02 Sg5, the upper row shows the results of the control group (i.e., no knockout using HLA-A02 Sg5); the lower row shows the results of simultaneous knockout of HLA-A02 and TRAC; here, Figure 8B shows the results of knocking out TRAC using TRAC Sg9, the upper row shows the results of the control group (i.e., no knockout using TRAC Sg9); the lower row shows the results of simultaneous knockout of HLA-A02 and TRAC. Figures 9A-9B show knockout HLA-A02 and knockout of TRAC protein levels, where NEG is the negative control, WT is no knockout treatment, and TRAC+HLA-A double knockout is the result of simultaneous knockout of HLA-A02 and TRAC.

[0277] Example 6. Differentiation of the expression of TRAC, HLA-A, B2M and CIITA genes in double knockout T cells and the corresponding genes in corresponding cells

[0278] (1) The activated T cells prepared in Example 2 are divided into two groups, one of which is a control group, and the other is prepared as a double knockout T cells of TRAC gene and HLA-A gene according to the method of Example 5, and Sanger sequencing is performed according to step (1) of Example 4. The double knockout T cells of TRAC and HLA-A are obtained from the sequencing results. The prepared double knockout T cells are incubated with the corresponding TRAC and HLA-A antibodies, and the double knockout cell line can be obtained by flow cytometry selection or magnetic bead selection.

[0279] (2) Changes in mRNA expression levels in double knockout T cells were examined in comparison with the control group. RNA was extracted using an RNA extraction kit (purchased from QIAGEN, product number: 74004) and reverse transcribed using a reverse transcription kit (purchased from Applied Biosystems, product number: 4368814) to obtain cDNA. The cDNA was used as a template for quantitative PCR.

[0280] (3) The changes in protein expression levels of double knockout T cells were examined compared to the control group. Proteins were extracted using a total protein extraction reagent (purchased from ThermoScientific, part number: 87787), and protein expression was measured using Western Blot or flow cytometry with TRAC antibody (purchased from eBioscience, part number: 17-9986-42), HLA-A antibody (purchased from Merck, part number: 17-9876-41), B2M antibody (purchased from Invitrogen, part number: A15770), and CIITA antibody (purchased from OriGene, part number: CF812200).

[0281] Sange sequencing showed that the nucleotide sequences of TRAC and / or HLA-A genes in double knockout T cells were altered compared to the control group. Quantitative PCR showed that the mRNA expression levels of TRAC and / or HLA-A genes were downregulated in double knockout T cells, but not that of B2M and / or CIITA genes. FACS and Western Blot results showed downregulation of protein expression levels in double knockout T cells, but not downregulation of B2M and / or CIITA protein expression levels.

[0282] The results are shown in Figures 10-11. Here, Figure 10 shows the mRNA level of gene expression, and Figures 10A-10D show the mRNA levels of TRAC, HLA-A, B2M, and CIITA, respectively; here, WT refers to the situation where nothing is knocked out, and double knockout refers to the results of T cells with double knockout of TRAC gene and HLA-A gene. Figure 11 shows the protein level measurement of gene expression, of which Figures 11A-11B show the protein expression levels of B2M and CIITA, respectively; here, NEG refers to the negative control group, WT refers to the situation where nothing is knocked out, and TRAC+HLA-A double knockout refers to the results of T cells with double knockout of TRAC gene and HLA-A gene.

[0283] Example 7. Preparation of TRAC gene, HLA-A / B2M gene, and CIITA gene knockout T cells and verification of the expression changes of the corresponding three genes

[0284] (1) According to the method of step (1) of Example 6, a control group, triple knockout cells of TRAC gene, HLA-A gene and CIITA gene, and triple knockout cells of TRAC gene, B2M gene and CIITA gene are prepared.

[0285] (2) Examine and measure the changes in protein expression levels by FACS and Western Blot methods according to the method in Example 6, step (3).

[0286] Compared to control cells, the protein expression levels of TRAC, HLA-A and CIITA genes were down-regulated in T cells with the triple knockout of TRAC, HLA-A and CIITA genes.Compared to control cells, the protein expression levels of TRAC, HLA-A and CIITA genes were down-regulated in T cells with the triple knockout of TRAC, B2M and CIITA genes.

[0287] (3) The knockout efficiencies of the double knockout cells of Example 6 and the two triple knockout cells of this Example were measured by flow cytometry using TRAC (purchased from eBioscience, product code: 17-9986-42), HLA-A (purchased from Merck, product code: 17-9876-41) and B2M (purchased from Invitrogen, product code: A15770) antibodies, which showed that the simultaneous multiple knockout efficiency at the single cell level was significantly higher for double knockout than for triple knockout.

[0288] The results are shown in Figures 12A-12D. Here, Figures 12A-12C show the knockout of TRAC, HLA-A, and B2M protein levels. Here, WT refers to the situation where nothing is knocked out, and TRAC+HLA-A double knockout refers to the results of T cells with TRAC gene and HLA-A gene double knockout. TRAC+HLA-A+CIITA triple knockout shows the results of T cells with TRAC, HLA-A, and CIITA triple knockout; here, TRAC+B2M+CIITA triple knockout refers to the results of T cells with B2M, CIITA, and TRAC triple knockout. TRAC+HLA-A knockdown refers to the results of T cells with TRAC gene and HLA-A gene knockdown prepared in Example 9. Figure 12D shows the knockout of CIITA protein level.

[0289] The results in Figure 12 show that the protein levels of TRAC, HLA-A, CIITA, and B2M are downregulated compared with the WT control group, while the knockout efficiency of TRAC+HLA-A double knockout is higher than that of TRAC+HLA-A+CIITA triple knockout and TRAC+B2M+CIITA triple knockout.

[0290] Example 8 Design of antisense RNA sequences

[0291] Obtain the transcript RNA sequences (such as SEQ ID NO. 82-90) of the corresponding genes (TRAC gene and HLA-A gene) from the database https: / / www.ncbi.nlm.nih.gov / or www.ensembl.org / , and design siRNAs based on the following principles:

[0292] If possible, sequences 50-100 base pairs downstream of the start codon and 100 bases upstream of the stop codon should be avoided;Select sequences less than 30 nucleotides in length;Avoid more than four consecutive identical bases;Avoid intronic regions;Avoid duplicated sequences;Avoid single nucleotide polymorphism (SNP) sites;Sequence GC content between 30% -60%,Preferentially select sequence patterns AA (N 19), NA (N 21) or NAR (N 17) YNN, where A is adenylic acid; T is thymidylic acid; R is adenylic acid or guanylic acid (purines); Y is thymidylic acid or cytidic acid (pyrimidines); N is adenylic acid, thymidylic acid, guanylic acid, or cytidic acid;Perform homology comparison analysis on the selected sequence to avoid antisense RNA having significant homology with other genes or sequences, which would result in off-target effects. Homology analysis was performed using the NCBI Blast tool: Nucleotide-nucleotide BLAST (blastn), UCSC Blat tool or Ensembl Blast.

[0293] Design of antisense RNA sequences including HLA-A-homo-551 (containing the nucleotide sequence shown in SEQ ID NO.93); HLA-A-homo-NEG (containing the nucleotide sequence shown in SEQ ID NO.94); TRAC-homo-375 (containing the nucleotide sequence shown in SEQ ID NO.95); TRAC-homo-NEG (containing the nucleotide sequence shown in SEQ ID NO.96).

[0294] Example 9 Preparation of TRAC gene and HLA-A gene knockdown T cells

[0295] Double knockdown was performed using the antisense RNA designed in Example 8. The company prepared lentivirus (Zima) with TRAC gene and HLA-A gene antisense RNA sequences. + T cells (day D0) were prepared and activated with CD3 / CD28 antibody beads, and the lentivirus carrying antisense RNA sequences of TRAC gene and HLA-A gene (SEQ ID NO.95 and SEQ ID NO.93) was transfected into the activated T cells (day D1). The lentivirus vector was washed out on day D2, and the cells were continuously cultured until day D5. The T cells cultured until day D5 were collected, and the gene knockdown efficiency was detected by quantitative PCR, Western Blot, etc. The obtained T cells were labeled with corresponding TRAC and HLA-A antibodies, and the knockdown T cells of TRAC gene and HLA-A gene could be obtained by streaming sorting or magnetic bead sorting. The results showed that the mRNA and protein expression levels of TRAC and HLA-A were downregulated in both TRAC and HLA-A knockdown groups. Figures 13A-13B show the TRAC and HLA-A mRNA levels. Here, WT refers to a state where nothing is knocked out, and TRAC+HLA-A double knockout refers to the result of T cells with both the TRAC gene and the HLA-A gene knocked out. Here, the knockout level, which is the protein level of TRAC and HLA-A, can be seen from the results in Figure 12.

[0296] Example 10. Discrimination of Different T Cell Activities

[0297] No knockout, double knockout, triple knockout, and double knockout T cells of Examples 2, 5, 7, and 9 were prepared, and the cell numbers of each group were compared for multiple T cell activities. 6Cells were seeded in a 24-well plate, and PHA (0.3 μg / ml) (ionomycin+) or 5 ng / ml PMA and 50 ng / ml ionomycin were added to the cells in each well. After 5 hours of incubation, the activation status of the cells was detected by flow cytometry using antibodies against CD69 (early activation) (purchased from BD Biosciences, product number: FN50) and CD137 (late bias) (purchased from BD Biosciences, product number: 4B4-1). The results showed that the activity of double knockout and double knockdown T cells was superior to that of triple knockout T cells.

[0298] The expression status of CD69 and CD137 protein levels can be seen in Figures 14A-14B, respectively. Here, WT refers to the situation where nothing is knocked out, and TRAC+HLA-A double knockout refers to the results of T cells with TRAC gene and HLA-A gene double knockout. TRAC+HLA-A+CIITA triple knockout refers to the results of T cells with TRAC, HLA-A, and CIITA triple knockout; here, TRAC+B2M+CIITA triple knockout refers to the results of T cells with B2M, CIITA, and TRAC triple knockout. TRAC+HLA-A knockout refers to the results of T cells with TRAC gene and HLA-A gene knockout, as prepared in Example 9.

[0299] Example 11 Differentiation of reactivity of different T cells to heterologous NK cells

[0300] The non-knockout, double knockout, triple knockout and double knockdown T cells in Examples 2, 5, 7 and 9 were labeled with CFSE (Invitrogen, C34554) and counted at 1*10 6 The cells were then co-cultured with NK cells (NK92MI) at a 1:1 ratio. After 24 hours, the cells from each co-culture group were collected and the proportion of CFSE-positive cells in the mixed cells was examined and measured.

[0301] The results show that the killing toxicity of NK cells against double knockout and double knockdown T cells is lower than that of triple knockout T cells. The results are shown in Figure 15. Here, NK+T refers to the case where NK cells are co-cultured with T cells that have not been knocked out; NK+TRAC+HLA-A knockdown refers to the case where NK cells are co-cultured with the T cells that have been knocked out of the TRAC gene and the HLA-A gene, as prepared in Example 9; NK+TRAC+HLA-A double knockout refers to the case where NK cells are co-cultured with T cells that have been double knocked out of the TRAC gene and the HLA-A gene; NK+TRAC+HLA-A+CIITA triple knockout refers to the case where NK cells are co-cultured with T cells that have been triple knocked out of TRAC, HLA-A, and CIITA; NK+TRAC+B2M+CIITA triple knockout refers to the case where NK cells are co-cultured with T cells that have been triple knocked out of B2M, CIITA, and TRAC.

[0302] Example 12. Differentiation of Different T Cell Allogeneic Immune Rejection Responses

[0303] Peripheral blood preparation from donor 1. Examples 2, 5, 7 and 9 show non-knockout, double knockout, triple knockout and double knockdown T cells. CD3 + T cells were prepared. Each group of cells was prepared from the peripheral blood of donor 1, and CD3 T cells were prepared from the peripheral blood of donor 2 according to Example 2. + The T cells were mixed in equal amounts, and the expression levels of IFN-γ in the cell mixture were measured 24 hours later. The results showed that the expression levels of IFN-γ in the double knockout T cells were lower than those in the triple knockout T cells.

[0304] The results are shown in Figure 16, which shows the results of TRAC+HLA-A double knockout TRAC gene and HLA-A gene double knockout T cells when WT is not treated with any knockout treatment; TRAC+HLA-A+CIITA triple knockout refers to the results of TRAC, HLA-A, CIITA triple knockout T cells; here, TRAC+B2M+CIITA triple knockout refers to the results of B2M, CIITA and TRAC triple knockout T cells. TRAC+HLA-A knockdown refers to the results of TRAC gene and HLA-A gene knockdown T cells prepared in Example 9.

[0305] Embodiment 13. Preparation of two knockout CAR-T cells of TRAC gene and HLA-A gene, three knockout CAR-T cells of TRAC gene, HLA-A gene and CIITA gene, and knockout CAR-T cells of TRAC gene, B2M gene and CIITA gene

[0306] (1) CD3 as in Example 2 + T cells were obtained (day D0) and activated with CD3 / CD28 antibody magnetic beads. After activation, lentivirus vectors (lentiviruses containing CD19-CAR, CD20-CAR, or BCMA-CAR, etc.) were transfected on day D1, the lentivirus vectors were washed off on day D2, and CAR-positive T cells were selected on day D3 and cultured until day D5.

[0307] (2) D Using the CAR-T cells collected after 5 days as initial cells, two-gene knockout cells of the TRAC gene and the HLA-A gene, three-gene knockout cells of the TRAC gene, the HLA-A gene, and the CIITA gene, and three-gene knockout cells of the TRAC gene, the B2M gene, and the CIITA gene were prepared in Example 5 and Example 7, respectively.

[0308] (3) The double knockout and triple knockout CAR-T cells were detected by flow cytometry technology, among which the yield of double knockout CAR-T cells was higher than that of triple knockout CAR-T cells.

[0309] The results are shown in Figure 17A-17D. Here, Figure 17A-17C are knockouts at the TRAC, HLA-A, and B2M protein levels. Figure 17D shows knockouts at the CIITA protein level. Here, WT refers to the situation where nothing is knocked out, and TRAC+HLA-A double knockout refers to the results of CAR-T cells with TRAC gene and HLA-A gene double knockout. TRAC+HLA-A+CIITA triple knockout refers to the results of CAR-T cells with TRAC, HLA-A, and CIITA triple knockout; here, TRAC+B2M+CIITA triple knockout refers to the results of HLA-A cells with B2M, CIITA, and TRAC triple knockout.

[0310] Here, the transfection efficiency of CD19CAR is shown in Figures 18A-18B, where CAR30%+ represents the transfection efficiency of CD19 CAR.

[0311] The amplification fold of different cells is shown in Figure 19. Among them, the amplification fold of CAR-T cells with both TRAC and HLA-A gene knockout was the highest.

[0312] Example 14. Antitumor effect of CAR-T cells with double knockout of TRAC gene and HLA-A gene

[0313] In Example 14, prepare the CAR-T cells (targeting CD19, CD20, or BCMA) with double knockout of TRAC gene and HLA-A gene, inoculate the target cells expressing luciferase gene (target gene positive leukemia or lymphoma cell lines such as Raji, Jurkat, MM 1 S, etc.) into the orifice, and then add the double knockout CAR-T cells, triple knockout CAR-T cells, or T cells without gene knockout at different target ratios (1:2.5, 1:1, 5:1, 10:1), and after 24 hours of co-culture, transfer the cells into the orifice, add luciferase substrate, and measure the fluorescence value with an enzyme labeling machine. Killing efficiency = 1-target cell T cell co-culture fluorescence value / target cell fluorescence value of single culture.

[0314] As a result, CAR-T cells with both the TRAC gene and the HLA-A gene knocked out had a remarkable killing effect on tumor cells.

[0315] Figure 20 shows the killing effect of Raji-Luciferase on CD19 target cells, among which the killing effect of CAR-T cells with double knockout of TRAC and HLA-A genes was the most significant. The results correspond to the AD annotations from left to right under each E / T ratio.

[0316] Example 15. Antitumor effect of CAR-T cells with double knockout of TRAC gene and HLA-A gene

[0317] Tumor cells were injected intravenously into NSG mice, and after tumors were successfully established, CAR-T cells with double knockout of the TRAC gene and HLA-A gene, triple knockout CAR-T cells, or T cells without gene knockout were injected into the mice, and the tumor volume in the mice was monitored.

[0318] Mice that received the double knockout CAR-T cells showed a significant slowing of tumor growth.

[0319] The results are shown in Figures 21-22. Here, Figure 21 shows the administration mode to mice, iv means intravenous injection, and CAR-T cells mean double knockout CAR-T cells expressing CD19CAR and triple knockout CAR-T cells. Figure 20 shows the volume of tumor in vivo after CAR-T cells are administered to mice. Among them, Figure 20 shows the volume of tumor in vivo in mice using saline, unmodified T cells, TRAC gene and HLA-A knockout CD19CAR-T cells, TRAC, HLA-A and CIITA knockout CD19CAR-T cells, B2M, CIITA and TRAC knockout CD19CAR-T cells, respectively, from left to right. As a result, the mice reinjected with TRAC gene and HLA-A gene double knockout CAR-T cells showed an obvious slowdown in tumor growth.

[0320] The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the embodiments described herein will be apparent to those skilled in the art and remain within the scope of the appended claims and their equivalents.

Claims

1. 1. A modified immune effector cell, comprising: the expression and / or activity of the TRAC gene and the HLA-A gene in the cells is downregulated compared to the expression and / or activity of the TRAC gene and the HLA-A gene in unmodified immune effector cells; the expression and / or activity of the B2M gene in said cells is not downregulated compared to the expression and / or activity of the B2M gene in unmodified immune effector cells; and the expression and / or activity of the CIITA gene in said cell is not downregulated compared to the expression and / or activity of the CIITA gene in an unmodified immune effector cell; The modification is carried out by introducing an sgRNA targeting an exon portion of the TRAC gene and an sgRNA targeting an exon portion of the HLA-A gene into an unmodified immune effector cell; The sgRNA targeting the exon portion of the TRAC gene comprises the nucleotide sequence shown in SEQ ID No. 1; The sgRNA targeting the exon portion of the HLA-A gene comprises a nucleotide sequence shown in any one of SEQ ID Nos. 17-18 and 91-92; The modified immune effector cells.

2. The modified immune effector cell of claim 1 , wherein the immune effector cell is a T cell.

3. The modified immune effector cell of claim 1 or 2, wherein the modification further comprises treating the unmodified immune effector cell with a Cas enzyme.

4. The modified immune effector cell of claim 3 , wherein the Cas enzyme comprises a Cas9 protein.

5. the immune effector cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR); The modified immune effector cell of any one of claims 1 to 4, wherein the CAR comprises an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain and a primary signaling domain.

6. The modified immune effector cell of claim 5 , wherein the antigen binding domain specifically binds to a tumor antigen.

7. The modified immune effector cell of claim 6, wherein the tumor antigen is selected from the group consisting of CD19, CD20 and BCMA.

8. A method for producing the modified immune effector cells according to any one of claims 1 to 7 in vitro, comprising the steps of: introducing into an unmodified immune effector cell an sgRNA targeting an exon portion of the TRAC gene and an sgRNA targeting an exon portion of the HLA-A gene; wherein the sgRNA targeting the exon portion of the TRAC gene comprises the nucleotide sequence shown in SEQ ID No. 1; and The sgRNA targeting the exon portion of the HLA-A gene comprises a nucleotide sequence shown in any one of SEQ ID Nos. 17-18 and 91-92; The method.

9. The method of claim 8, wherein the modification further comprises treating the unmodified immune effector cells with a Cas enzyme.

10. 10. The method of claim 9, wherein the Cas enzyme comprises a Cas9 protein.

11. A composition comprising the modified immune effector cells of any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

12. The composition of claim 11, comprising a cell population comprising a modified immune effector cell of any one of claims 1 to 7.

13. An allogeneic therapeutic composition comprising the modified immune effector cells of any one of claims 1 to 7.

14. A composition for treating tumors comprising the modified immune effector cells according to any one of claims 1 to 7.

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