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 the same. [Background technology]

[0002] Antitumor immunotherapy has shown broad potential for inducing robust and durable responses in a variety of malignancies and can be used to treat many different types of cancer. Current antitumor immunotherapy approaches primarily include immune cell-targeted monoclonal antibody (mAb) therapy and adoptive cell therapy (ACT). ACT involves in vitro stimulation and expansion of autologous or allogeneic lymphocytes, followed by infusion into the body to achieve antitumor effects. This therapy is known to be relatively effective only in patients with MHC-matched lymphocytes. CAR-T is a novel, effective, MHC-independent adoptive cell therapy. CARs, also known as chimeric antigen receptors, are artificial receptors that mimic TCR function and specifically recognize antigens on tumor cell surfaces to target tumor cells. However, the timely and successful production and infusion of autologous CAR T cells remains a major obstacle to effective CAR T cell therapy. For example, if a patient has previously been treated with chemotherapy, the chemotherapy drugs can cause the patient's T cells to have difficulty expanding or function poorly in vitro, resulting in insufficient quantities of CAR T cell products or relatively poor quality. Furthermore, while autologous CAR-T is currently primarily used in patients with ALL or CLL, its application in patients with solid tumors faces significant challenges, including tumor antigen heterogeneity. To increase CAR flexibility and broaden the range of antigen recognition, generic CAR-T has emerged. Generic CAR T cells are primarily designed to generate tumor antigen-specific T cells from allogeneic healthy donors. While this method improves the proliferation efficiency and survival rate of CAR T cells, the endogenous TCR of allogeneic T cells can recognize alloantigens as receptors, leading to graft-versus-host disease (GVHD). Furthermore, the expression of HLA on the surface of allogeneic T cells can also trigger rapid rejection (HVGR) by the host immune system. Therefore, the issue of immune rejection following allogeneic cell therapy requires further investigation. DISCLOSURE OF THE INVENTION [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 the corresponding genes in the unmodified corresponding 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 corresponding wild-type cells.

[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 allele of the HLA-A gene is 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 comprises 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 administering a Cas protein to the cell.

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

[0022] In some embodiments, the immune effector cells comprise a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises 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. 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 α (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 β (CEAβ), stage-specific embryonic antigen-4 (e.g., proteasome, megalin factor), CD20, folate receptor α, 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), carbonic acid enzyme IX (EPCAM), cell surface-associated mucin 1 (CD276), oxidized glycoprotein 9, prostatic acid phosphatase, mutant IL-100 glycoprotein 9, oncogene fusion protein (IL-13Ra2 or CD213A2) consisting of breakout cluster (CD276) and IL-13Ra2 murine leukemia viral oncogene homolog 1 (CD117), tyrosinase,Liver protein type A receptor 2 (IL-11Ra), fucosyl-GM1; sialyl-PSCA adhesion molecule (PSCA), transglutaminase 5 (TAG72), polymeric melanoma-associated antigen (CEAβ), o-acetyl-GD2 ganglioside (TAG72), folate receptor β, tumor endothelial marker 1 (CEA), tumor endothelial marker 7-related (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 cell receptor 1 (IL-13Ra2), adrenergic receptor β3 (CD213A2), ubiquitin (PSCA) 3 (TAG72), G protein-coupled receptor 20 (sLe), lymphocyte antigen 6 complex locus K9 (TGS5), Olfactory receptor 51 (TAG72), TCRγ 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-related antigen 1, p53, p53 mutant, prostate-specific protein (CEA), prostate cancer tumor antigen-1 (MUC1), T cell-recognized melanoma antigen 1 (IL-13Ra2) rat sarcoma (CD117) mutant, human telomerase reverse transcriptase (IL-13Ra2 or CD213A2), sarcoma translocation breakpoint, 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-identifying 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 junction 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 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, and 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: CD3ζ, FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, 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 for producing the modified immune effector cells described herein, comprising the steps of: down-regulating the expression and / or activity of the TRAC gene and the HLA-A gene in the immune effector cells relative to the expression and / or activity of the corresponding genes in the unmodified corresponding cells, wherein 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.

[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 corresponding wild-type cells.

[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 administering 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 pharmaceutically acceptable carrier.

[0050] In some embodiments, the composition comprises a population of cells comprising 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 an application of the modified immune effector cells of the present application in the preparation of a drug, which is used in the treatment of allogeneic tumors.

[0053] The present application also provides methods of allogeneic therapy comprising administering to a patient or subject the modified immune effector cells described herein.

[0054] The present application also provides the modified immune effector cells described herein for allogeneic therapy.

[0055] The present application also provides an application of the modified immune effector cells of the present application in preparing a drug, and the drug is used for 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 to treat 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 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.

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

[0061] The following detailed description shows and describes only exemplary embodiments of the present application. Those skilled in the art will recognize that the present application will enable them to make modifications to the particular embodiments disclosed without departing from the spirit and scope of the present invention. Accordingly, the accompanying drawings and description of the present application are to be taken as illustrative only and not restrictive. [Brief explanation of the drawings]

[0062] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention 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 of the TRAC gene after Sg9 RNA editing. [Figure 2]This shows the results of TA cloning in which the TRAC gene was edited with Sg9RNA. [Figure 3] 1 shows the results of flow cytometry of the TRAC gene edited with Sg9 RNA. [Figure 4] 1 shows the results of Sanger sequencing after Sg2 RNA editing of the HLA-A02 gene. [Figure 5] 1 shows the results of Sanger sequencing after Sg5 RNA 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] Protein levels of B2M and CIITA in modified immune effector cells are shown. [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] 1 shows the protein levels of CD69 and CD137 in modified immune effector cells. [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] Figure 1 shows the efficiency of infection of modified immune effector cells with CAR. [Figure 19] The fold expansion of modified immune effector cells is shown. [Figure 20] 1 shows the killing effect of modified immune effector cells on CD19-positive target cells. [Figure 21] 1 shows a schedule for administering modified immune effector cells. [Figure 22] 1 shows the killing effect of modified immune effector cells on tumors in mice. DETAILED DESCRIPTION OF THE INVENTION

[0064] In the following, embodiments of the present invention will be described using certain specific embodiments, but those familiar with the art will be able to easily understand other advantages and effects of the present invention from the disclosure herein.

[0065] The present application will be further described below: In the present invention, unless otherwise specified, scientific and technical terms used in the present application have the meanings 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 widely used and routine procedures in the respective fields. In addition, definitions and explanations of relevant terms are provided below to better understand the present invention.

[0066] As used herein, the term "immune effector cell" generally refers to an immune cell that participates in an immune response and 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] As used herein, the term "modify" generally refers to a change that alters the state or structure of a cell and / or a change that alters the state or structure of a cell. The modification typically refers to a change in the state or structure of a cell compared to a corresponding cell that does not have the modification. The modification may include, for example, a change in the expression level or function of an endogenous gene by genetic engineering techniques that downregulate, upregulate, or eliminate the expression of the cell's endogenous gene. The genetic engineering techniques may include gene editing using homologous recombination, the CRISPR / CAS9 system, or the like. The modification 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 the corresponding protein achieved by changing the expression level or function of the endogenous gene, for example, a change in protein expression, structure, or function achieved by regulating protein translation or post-translational modification. The modification may also include the introduction of an exogenous gene, the expression of an exogenous protein, or the like.

[0068] As used herein, the term "TRAC" generally refers to the T cell receptor alpha constant region (T cell receptor alpha constant region). T cell receptors (TCRs) are specific receptors typically present on the surface of T cells that recognize antigens bound to major histocompatibility complex (MHC) molecules. TCRs are typically composed of two distinct protein chains (heterodimers). In humans, the majority of T cells have TCRs consisting of one alpha chain and one beta chain (encoding TRA and TRB, respectively), and are referred to as αβ T cells. A minority of T cells have TCRs consisting of one gamma chain and one delta chain (encoding TRG and TRD, respectively), and are referred to as γδ T cells. Typically, αβ T cells account for approximately 95% of the total T cell population, and γδ T cells account for approximately 5% of the total T cell population; however, this ratio varies during individual development and pathological conditions (e.g., leukemia), and also varies between species. Each chain that makes up the TCR contains a variable region and a constant region. In humans, the α chain-encoding gene (TRA, e.g., HGNC:12027) is located on chromosome 14 and consists of multiple gene segments, including a variable region (V), a joining region (J), and a constant region (C). The TRAC gene generally refers to the gene sequence encoding the T cell receptor α chain constant region (C) (e.g., HGNC:12029), which is located on chromosome 14 (14q11.2; 14:22, 547, 505-22, 552, 131). Typically, one of the variable region (V) genes encoding the antigen recognition region of the N fragment rearranges with one of the joining regions (J) to produce a functional V region exon. This exon is transcribed and spliced ​​to join with the constant region (C), thereby forming the T cell receptor α chain coding sequence.

[0069] As used herein, the term "HLA-A" generally refers to the human leukocyte antigen polypeptide chain encoded by the HLA-A gene located on human chromosome 6p21.3 (e.g., the information shown in HGNC:4931). HLA-A is one of three major polypeptide types that constitute human cell surface class I MHC molecules; the others are 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 B2M gene constitutes 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 and α2 domains bind to peptide fragments and 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 as of December 2013, 2432 HLA-A alleles encoding 1740 activating proteins and 117 inactivating proteins have been identified. In this application, HLA-A alleles may include sequence information of different HLA-A alleles named by the WHO HLA Factor Naming Committee, which are 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" generally refers to β2-microglobulin, which is part of a class MHC I molecule. β2-microglobulin (also called the β chain) can form class MHC I molecules with an HLA-encoded α chain. B2M is normally expressed in all nucleated cells. In humans, β2-microglobulin is encoded by the B2M gene located at 15q21.1 (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). This transactivator is a protein with an acidic transcriptional activation domain, four LRRs (leucine-rich repeats), and a GTP-binding domain. This protein can be located in the cell nucleus and is referred to as the "master regulator" of class II major histocompatibility complex (MHCII) gene transcription as a positive regulator of these gene expression. This protein can also bind to GTP and is transported into the cell nucleus using this GTP binding; once in the cell nucleus, it normally acts as a coactivator via its acetyltransferase (AT) activity. In humans, this protein is encoded by a gene located at 16p13.13 (e.g., information provided 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 exists 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] As used herein, the terms "nucleic acid," "polynucleotide," or "nucleic acid molecule" generally refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise specified, the term may include nucleic acids containing analogs of naturally occurring nucleotides, which have similar binding properties to those of the reference nucleic acid (e.g., for which sequence information is provided) and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise specified, the sequence of a nucleic acid may include conservatively modified variants thereof, such as truncated codon substitutions, alleles, orthologs, SNPs, complementary sequences, and the sequence specified.

[0075] As used herein, the term "expression" generally refers to the transcription and / or translation of a particular nucleotide sequence.

[0076] As used herein, the term "gene mutation" generally refers to a change in the base pair composition or sequence order that occurs structurally in a gene, such as a point mutation resulting from a single base change, or a deletion, duplication, or insertion of multiple bases.

[0077] In this application, the term "gene silencing" generally refers to the inhibition of expression of a specific gene through a regulatory mechanism. It mainly includes two types: transcriptional gene silencing (TGS), which is mediated by factors such as DNA methylation, heterochromatinization, and position effects, and post-transcriptional gene silencing (PTGS), which affects gene expression by specifically interfering with target RNA at the post-transcriptional level. Typically, when a gene is silenced, the expression of the corresponding gene is downregulated / reduced. When a gene is knocked out, it usually appears to be absent; for example, when all alleles of a particular gene are knocked out, the expression of that gene appears to be abolished. Gene silencing is generally considered a gene knockdown mechanism, and a commonly used method for gene silencing is RNAi.

[0078] As used herein, the term "endogenous" refers to any substance 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 the biological, cellular, histological, or system.

[0080] As used herein, the term "antisense RNA" generally refers to single-stranded RNA complementary to the transcript mRNA (messenger RNA). Antisense RNA binds to mRNA and inhibits gene expression. For example, after binding to a target mRNA, antisense RNA increases the susceptibility of this double-stranded RNA molecule to RNAse III, causing its degradation. For example, antisense RNA can bind to the upstream non-coding region of an mRNA and directly inhibit the translation of the target mRNA.

[0081] In this application, the term "siRNA" generally refers to the abbreviation for small interfering RNA or short interfering RNA. siRNA is a type of double-stranded non-coding RNA molecule, approximately 18-28 base pairs in length, that binds to mRNA via complementary binding, causing mRNA degradation and 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 is degraded and the anti-meaning strand can bind to the complementary target sequence, thereby achieving gene silencing.

[0082] In this application, the term "shRNA" is generally an abbreviation for short hairpin RNA, i.e., "short hairpin RNA." shRNAs typically contain two short inverted repeats, separated in between by a stem loop sequence that forms a hairpin structure. They may also contain five to six T bases as transcription terminators for RNA polymerase III. In some embodiments, shRNAs enter cells via a viral vector or plasmid, where they are transcribed by polymerase II or polymerase III. The transcripts are exported from the cell nucleus (usually via Exportin 5) and then transported to RISC via Dicer processing. The sense strand is degraded, and the antisense strand binds to the complementary target sequence, thereby achieving gene silencing.

[0083] As used herein, the term "CRISPR / Cas system" generally refers to a group of molecules including an RNA-guided nuclease or other effector molecule and a gRNA molecule that can direct the RNA-guided nuclease or other effector molecule to a target sequence, e.g., to cause degradation of the target sequence. In some embodiments, a CRISPR system includes a gRNA and a Cas protein, e.g., a Cas9 protein. A system including 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," and "gRNA" 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, a 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, a 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] As used herein, the term "Cas protein" generally refers to an enzyme responsible for DNA cleavage in a CRISPR / Cas system, and may include enzymes from type I, II, and III CRISPR / Cas systems, such as "Cas3," "Cas9," and "Cas10."

[0086] As used herein, 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 variants thereof.

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

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

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

[0090] As used herein, the term "specific binding" refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that allows the determination of the presence of the target 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 other embodiments, specific binding can include, but does not require, exclusive binding.

[0091] As used herein, the term "transmembrane domain" generally refers to a polypeptide or protein encoded by at least one exon at the DNA level, which comprises an extracellular region, a transmembrane region, and an intracellular region. A transmembrane domain generally comprises three distinct structural regions: an N-terminal extracellular region, a conserved transmembrane stretch in the middle, and a C-terminal cytoplasmic region. A transmembrane domain may also include an intracellular or cytoplasmic domain.

[0092] As used herein, the term "hinge region" generally refers to the region located between the binding domain and the transmembrane domain in the CAR structure. Hinge regions are typically derived from the IgG family, such as IgG1 and IgG4, and some are derived from IgD and CD8, but typically 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" generally refers to the activation source of second signal transduction by lymphocytes, which usually occurs through the interaction of a costimulatory molecule with its receptor on the surface of immune cells involved in adaptive immunity (between T cells and B cells or between antigen-presenting cells and T cells). For example, the full activation of T cells depends on dual signal transduction and the action of cytokines. The first signal for T cell activation comes from the specific binding of its receptor TCR with an antigen, i.e., T cell antigen recognition; the second signal for 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] As used herein, the term "costimulatory domain" generally refers to the intracellular portion of a receptor for 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 and transmits a costimulatory signal after the extracellular binding domain in the CAR structure binds to the corresponding antigen.

[0095] As used herein, the term "primary signaling domain" generally refers to an intracellular amino acid sequence capable of generating a signal that promotes the immune effector function of a cell containing a CAR, e.g., a CAR-T cell. For example, examples of immune effector function in a CAR-T cell may include accessory activities, including cytolytic activity and cytokine secretion. 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 entire chain, provided it is still capable of transmitting the effector function signal. The term primary signaling domain refers to any short portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0096] As used herein, the term "tumor antigen" refers to a molecule (e.g., a protein, sugar, lipid) that is typically expressed intact or as a fragment on the surface of tumor cells and can be used to target drugs to tumor cells. In some embodiments, a tumor antigen may be a marker expressed on both normal and cancer cells, such as a lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed in tumor cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more than that of normal cells. In some embodiments, the cell surface molecule is aberrantly expressed on tumor cells, e.g., contains a deleted, added, or mutated molecule compared to the corresponding molecule expressed on normal cells. In some embodiments, a tumor antigen may be expressed exclusively intact or as a fragment (e.g., MHC / peptide) on the surface of tumor cells and is not synthesized or expressed on the surface of normal cells. In some embodiments, a CAR of the present application may comprise an antigen-binding domain that binds to an MHC-presented peptide, such as an antibody or antibody fragment. Typically, 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 class 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] As used herein, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Unlike typical polyclonal antibody preparations, which contain different antibodies directed against different determinants, each monoclonal antibody targets 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 uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method.

[0098] As used herein, the term "polyclonal antibody" generally refers to a composition of different antibody molecules. Polyclonal antibodies can bind to 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 can be prepared by mixing multiple monoclonal antibodies specific to a desired target sFGFR.

[0099] As used herein, 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] As used herein, the term "humanized antibody" generally refers to an antibody comprising heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which 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 a relevant antigen and comprises framework (FR) regions having substantially the amino acid sequence of a human antibody and complementarity-determining regions (CDRs) having substantially the amino acid sequence of a non-human antibody. The term "essentially," in the case of a CDR, indicates that the amino acid sequence of the CDR is 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 substantially 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 also comprises 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 but contain the heavy chain variable region. Due to their small molecular weight, they are also called nanobodies. Single-domain antibodies were first discovered in camelids, and similar antibodies have since been discovered in cartilaginous fishes such as nurse sharks, star sharks, and rays. For example, antibodies lacking the CH1 region of both the light chain and the heavy chain constant region of conventional antibodies are called heavy-chain antibodies (HcAbs), and HcAbs are ubiquitous in various camelids. For example, heavy-chain antibodies, known as Ig new antigen receptors (IgNARs), consist of two isoheavy chains, each consisting of five constant regions and one variable region. The variable regions of heavy-chain antibodies are composed only of the variable regions of the antibody heavy chains. They specifically bind to antigens like conventional Fabs, and thus function similarly to conventional antibodies.

[0102] As used herein, the term "tumor" generally refers to a growth or solid 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, while 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 known as cluster of differentiation, is often used to identify cell surface molecules used as immunogenic markers. CD molecules have many uses and are typically used as important cellular receptors or ligands. While some CDs participate in cell signaling cascades and alter cell behavior, 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 sources of transmembrane domains in this application, and CD137, CD28, CD134 (OX-40), and CD278 (ICOS), which are the sources of costimulatory domains.

[0104] As used herein, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable substance, composition, or medium for transporting, storing, transferring, or administering a cell preparation. Examples include liquid, semisolid, or solid fillers, diluents, isotonic agents, solvents, or cyst materials. Pharmaceutically acceptable carriers may also include pharmaceutically acceptable salts, where the term "pharmaceutically 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 herein. 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 polyacrylamide-containing hydrogels. Pharmaceutically acceptable carriers may also include storage solutions, freezing solutions, injection solutions, etc., for cells. Generally, the pharmaceutically acceptable carrier can maintain the activity of the cells carried therein, and does not interfere with the therapeutic effect. The pharmaceutically acceptable carrier may also promote the preservation, transportation, 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, typically a human patient. For example, a composition described herein may include immune effector cells described herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, an acceptable composition is non-toxic to the 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 the features expressly 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, compared with the expression and / or activity of the corresponding genes in the unmodified corresponding cells, 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.

[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 the TRAC gene and the HLA-A gene in the immune effector cells compared to the expression and / or activity of the corresponding genes in the unmodified corresponding cells, while not down-regulating the expression and / or activity of the B2M gene and not down-regulating the expression and / or activity of the CIITA gene.

[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 this 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 this application, the T cells may include helper T cells (Th) that can support humoral and cellular immunity; suppressor T cells (Ts) that suppress cellular and humoral immunity; effector T cells (Te) that 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, which have the ability to differentiate upon contact with antigens; and memory T cells (Tm), which remember specific antigen stimulation.

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

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

[0119] In the present application, the unmodified counterpart cells can include wild-type cells and / or artificially modified cells. The wild-type cells can include naturally occurring or naturally 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 that have been induced to differentiate to obtain immune effector cells; the artificially modified cells can 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 obtained by inducing differentiation from progenitor / multipotent cells isolated from a human body and then further artificially modified. For example, the human body can be a healthy human body. For example, the healthy human body can include a human body that is free from tumors or immune system-related diseases or conditions.

[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, or CIITA gene as a recognition site / target. For example, the artificial modification does not affect the expression / activity of the TRAC gene, HLA-A gene, B 2 M gene, or CIITA gene. Not affecting the expression / activity of the TRAC gene, HLA-A gene, B 2 M gene, or CIITA gene means that the expression / activity of the TRAC gene, HLA-A gene, B 2 M gene, or CIITA gene is unchanged compared to corresponding cells isolated from the human body or obtained by inducing differentiation from progenitor cells or multipotential cells isolated from the human body that have not been artificially modified. The changes in the expression / activity do not necessarily coincide. For example, it is possible to determine whether the TRAC gene, HLA-A gene, B 2 M gene, or CIITA gene is completely different from the corresponding cells isolated from the human body or obtained by inducing differentiation from progenitor cells or multipotential cells isolated from the human body. The mRNA quantitative analysis of the M gene and CIITA gene showed no significant difference (P>0.05). For example, by using conventional technical means in the relevant field, the corresponding polypeptide / protein quantitative analysis of the TRAC gene, HLA-A gene, B2M gene and CIITA gene in the two genes showed no significant difference (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 one of 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 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 family B-cell maturation antigen (CA125), Tn antigens (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 known as CD2 subclass 1, CRACC, SLAMF7, CD319, and 19A24), epithelial cell adhesion molecule (CD133 or CD123), B7H3 (CD171), KI T(GD2), interleukin-13 receptor subunit α-2 (CA125), mesothelin, interleukin-11 receptor α (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 β (FLT3), stage-specific embryonic antigen-4 (CD123), CD20, folate receptor α, 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), high molecular weight 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, and 19A24),Tumor endothelial marker 7 (CD133 or CD123), tight junction protein 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor 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 liver type A antigen 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), Wilms 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 (TSHR) mutant, human telomerase reverse transcriptase (GPRC5D), sarcoma translocation breakpoint, melanoma apoptosis inhibitor protein ( CXORF61), ERG (ALK), N-acetylglucosaminyltransferase V (PLAC1), paired frame protein CD179a (GloboH), androgen receptor, cyclin B1, ALK avian myelocytosis 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 anchor protein 4 (LY6K), synovial sarcoma X breakpoint 2 (PLAC1), receptor for advanced glycation end products (globoH glycoceramide), 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 2 containing EGF-like module (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 HLA-A gene was downregulated compared to corresponding wild-type cells, the expression and / or activity of the B2M gene was not downregulated, and the expression and / or activity of the CIITA gene was not downregulated.

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

[0124] As used herein, down-regulated gene expression level and / or activity includes down-regulating the expression and / or activity of a nucleic acid molecule encoding the gene and / or down-regulating the expression and / or activity of a protein product encoded by the gene.

[0125] For example, the protein product may comprise 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 lower than that in the unmodified corresponding cells. The modification may involve altering the nucleotide sequence structure of the TRAC gene and HLA-A gene. The nucleotide sequence may include coding or non-coding regions, such as cis-regulatory element sequences and exon sequences, and the cis-regulatory element sequence may include a promoter. The alteration may include partial or complete sequence deletion, insertion of an exogenous fragment, or base mutation, where the inserted exogenous fragment replaces or disrupts the sequence structure of the TRAC gene and HLA-A gene, preventing normal translation. The base mutation may include frameshift mutation, missense mutation, nonsense mutation, etc. The alteration may include chemical group modification of the nucleotide sequence, such as methylation. The alteration of the nucleotide sequence structure can be detected by gene sequencing, such as Sanger sequencing or sulfite sequencing.

[0127] In the present 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 is downregulated compared to the corresponding unmodified cells. The mRNA content is measured by experimental and biostatistical methods known to those skilled in the art, such as molecular probe in situ hybridization and real-time fluorescent quantitative PCR (qPCR, RT-PCR). The real-time fluorescent quantitative PCR may include SYBR Green, TaqMan, double hybridization probe, and molecular beacon methods. The results of detecting the mRNA content are subject to inevitable experimental or statistical errors, which are known in the art. The error may be within a range of ±10%, such as ±8%, ±6%, ±5%, ±4%, or ±3%. The mRNA 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 mRNAs of the TRAC gene and HLA-A gene are 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 is 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 unmodified corresponding cells. The modification also includes reducing the polypeptide content of the TRAC gene and HLA-A gene expression. The polypeptide is identical in structure and function to the polypeptide produced by the expression of the TRAC gene and HLA-A gene in the unmodified corresponding cells, and does not include polypeptides with altered function or structure produced by altering the nucleotide sequences of the TRAC gene and HLA-A gene. The polypeptide content can be determined 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 Western blotting (WB). The results of detecting the polypeptide content are subject to inevitable experimental or statistical errors, which are well known in the art. The margin of error may be within ±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 expressed by the TRAC gene and HLA-A gene 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 being 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 this application, "not down-regulating the expression and / or activity of the B2M gene and not down-regulating the expression and / or activity of the CIITA gene," or "not down-regulating the expression and / or activity of the B2M gene and not down-regulating the expression and / or activity of the CIITA gene," may mean that the mRNA content of the B2M gene and the CIITA gene is not reduced compared to that of the unmodified corresponding 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 and real-time fluorescent quantitative PCR (qPCR, RT-PCR). The real-time fluorescent quantitative PCR may include the SYBR Green method, TaqMan method, double hybridization probe method, and molecular beacon method. The detection results of the mRNA content are subject to inevitable experimental or statistical errors, which are known in the art. The error may be within a range of ±10%, such as ±8%, ±6%, ±5%, ±4%, ±3%, etc. The absence of a decrease in the mRNA content may include an increase or no change in the content, and the no change may include the fact that the corresponding mRNA quantitative analysis of the B2M gene and CIITA gene in the two compared does not show a significant difference (P>0.05).

[0131] In the present application, "not down-regulated in the expression and / or activity of the B2M gene and not down-regulated in the expression and / or activity of the CIITA gene," or "not down-regulated in the expression and / or activity of the B2M gene and not down-regulated in the expression and / or activity of the CIITA gene," may mean that the polypeptide content of the B2M gene and CIITA gene expression is not reduced compared to that of the unmodified corresponding cells. The polypeptide is identical in structure and function to the polypeptide produced by the expression of the B2M gene and CIITA gene in the unmodified corresponding cells. The polypeptide content can be 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 Western blotting (WB). The detection results of the polypeptide content are subject to inevitable experimental or statistical errors, which are well known in the art. The error may be within the range of ±10%, such as ±8%, ±6%, ±5%, ±4%, ±3%, etc. The absence of a decrease in the content of the polypeptide includes an increase or no change in the content, and the no change includes no significant difference (P>0.05) between the quantitative analysis of the corresponding polypeptides of the B2M gene and CIITA gene compared between the two.

[0132] In the present application, the fact that 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 fact that 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, includes artificial intervention in the CIITA gene and B2M gene, or the mRNA and / or polypeptides encoding the CIITA gene and B2M gene, in the corresponding unmodified cells; 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 B2M gene or the mRNA molecules encoded by them and change their structure or content.

[0133] In the present application, "not down-regulating the expression and / or activity of the B2M gene and not down-regulating the expression and / or activity of the CIITA gene," or "not down-regulating the expression and / or activity of the B2M gene and not down-regulating the expression and / or activity of the CIITA gene," means that there is no change in the nucleotide sequence structures of the B2M gene and the CIITA gene compared to the corresponding unmodified cells, and includes no change in the nucleotide sequence structures, which can be detected by gene sequencing, such as Sanger sequencing, sulfite sequencing, etc. The no change in the nucleotide sequence structures includes no artificial change, and may also include changes that occur naturally and do not affect their function.

[0134] 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, can mean that no targeted modifications are made 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 of 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 that has 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 of its allele types. For example, the HLA-A gene may include all of the HLA-A allele types listed in 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 allele types and their sequence information disclosed in 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 may 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 the 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 that has 80% or more homology to the 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 involve deletion of all or part of a gene, genetic mutation, or the like.

[0158] For example, the gene 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 can result in, for example, a missense mutation, a frameshift mutation, and / or a nonsense mutation. A missense mutation typically involves a base pair change that changes a codon encoding one amino acid to a codon encoding another amino acid, thereby changing the corresponding amino acid that makes up the protein. A shift mutation typically involves the insertion or deletion of one or more bases in a DNA strand that is not an integer multiple of three, resulting in a change in the coding codon. A nonsense mutation typically involves a base pair change that changes a codon encoding an amino acid into a terminator, causing premature termination of protein synthesis at that site.

[0165] For example, the change in 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, or 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 part of any one exon and / or intron in a gene. The rearrangement may include a duplication, inversion, transposition, etc. of the single base sequence.

[0166] For example, this gene silencing may include transcriptional gene silencing (TGS) and / or post-transcriptional gene silencing (PTGS). Transcriptional silencing may involve methylation of DNA molecules, thereby silencing the DNA, such as methylation of promoter sequences. Post-transcriptional gene silencing may involve altering gene expression by specific intervention on target RNA after gene transcription. mRNA levels may be 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 inserted nucleotide sequence 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 a 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%, 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 susceptible 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 to 28 base pairs in length.

[0177] For example, the siRNA can 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, or 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 repeat sequences and a stem loop sequence located between the two short inverted repeat sequences.

[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 this 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 gRNA), and when the crRNA and the tracrRNA are located on the same nucleotide molecule, the gRNA is called a sgRNA (unimolecular 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 include a nucleic acid sequence encoding the gRNA and a nucleic acid sequence encoding a Cas protein. 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 driving the expression of each coding 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 so 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 or stability in the CRISPR / Cas complex. When multiple guide RNAs are used, each guide RNA may be encoded on the same vector or on different vectors. The promoters driving the expression of the multiple guide RNAs may be the same or different. For example, the guide RNAs may include a guide RNA targeting the HLA-A gene and a guide RNA targeting the TRAC gene.

[0191] In this 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- or double-stranded DNA; e.g., the Cas protein may cleave RNA; e.g., the Cas protein may cause RNA / DNA nicking; 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 regulate 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 Class 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. 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, and others. Cas9 or Cpf1 protein (Zetsche et al., Cell, 163:1-13 (2015)) can contain 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 (e.g., the Cas9 protein of the CRISPR / Cas9 system) or a type V CRISPR / Cas system (e.g., the Cpf1 protein). For example, the Cas protein can originate from a class 2 CRISPR / Cas system, such as the Cas9 protein or the Cpf1 protein. Class 2 Cas nuclease family proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave desired nucleic acid targets by designing an appropriate guide RNA, as further described herein.

[0195] For example, two classes of CRISPR / Cas system components can arise from Type IIA, Type IIB, Type IIC, Type V, or Type VI systems, including Cas9 and its orthologs. The Cas9 protein or its ortholog may be derived from the following exemplary species: Streptococcus pyogenes, thermophilic Streptococcus, Streptococcus sp., Staphylococcus aureus, harmless Listeria monocytogenes, Lactobacillus gasseri, Pasteurella multocida, Saccharomyces cerevisiae, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Streptococcus pyogenes, S. kanselii, Lactobacillus gasseri, Pasteurella multocida, Saccharomyces cerevisiae, S. succinogenes, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Streptococcus pyogenes, S. kanselii, Lactobacillus pyogenes, S. kanselii, Lactobacillus gasseri, Pasteurella multocida, S. pyogenes ... in-nocua) (Streptococcus thermophilus), scarlet spirochete (Streptococcus sp.), Nocardia spp. (Staphylococcus aureus), S.burnetii (Listeria in-nocua), green Streptococc (Lactobacillus gasseri) 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 genus (Streptococcus pyogenes), Clostridium difficile (Streptococcus thermophilus), Haeus genus species (Streptococcus sp.), halophilic nitrifying cocci (Staphylococcus aureus), Daphylococcus species (Listeria in-nocua), foamy cyanobacteria (Lactobacillus gasseri), Candida species (Francisella novicida), Wolinella succinogenes, discoid cyanobacteria (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 diphtheria bacteria (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, Actinomyces (Eubacterium eligens), Leptospira dabilis (Leptospira inadai), Bacteroides (Prevotella disiens), or Actinoporphyrins (Porphyromonas macacae). For example, the Cpf1 protein may be derived from Acidaminococcus or 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 set forth 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 an amino acid substitution, to form a fusion protein with another polypeptide fragment, which may contain a PEST sequence, ubiquitin, polyubiquitin, or a nuclear localization signal (NLS).

[0202] In the present application, the crRNA may include a gRNA target sequence, which allows the crRNA to target any target sequence. 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 contain at least one mismatch. For example, the target sequence and the target sequence on the target nucleic acid molecule may contain 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 contain 1 to 6 mismatches. For example, the target sequence and the target sequence on the target nucleic acid molecule may contain 5 or 6 mismatches. For example, the target sequence and the target sequence on the target nucleic acid molecule may contain no 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] As used herein, the crRNA may further comprise a crRNA flagpole sequence, which may include any sequence of the crRNA that is sufficiently complementary to the tracrRNA to promote CRISPR / Cas complex formation. For example, the flagpole sequence may include all or a portion of a sequence of a naturally occurring crRNA that complements the tracrRNA within the same CRISPR / Cas system (also referred to as a "tagging" or "handle" sequence). For example, the flag sequence may include all or a portion of a repeat sequence from a naturally occurring CRISPR / Cas system. For example, the sequence may include 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 hybrid between tracrRNA and tracrRNA 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 used. For example, the crRNA flag sequence may be 10 to 50 nucleotides in length or more than 50 nucleotides in length. For example, the crRNA flag sequence may be 15 to 40 nucleotides in length. For example, the crRNA flag sequence may be 20 to 30 nucleotides in length. For example, the crRNA flag sequence may be 22 nucleotides in length. When using double-guide RNA, there is no upper limit to the length of the crRNA flag sequence.

[0205] As used herein, tracrRNA can comprise all or part of a wild-type tracrRNA sequence from a naturally occurring CRISPR / Cas system. For example, tracrRNA can comprise a truncated or modified variant of wild-type tracrRNA. The length of the tracrRNA varies depending on the CRISPR / Cas system being used. For example, tracrRNA can 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, tracrRNA can be at least 20 nucleotides in length. For example, tracrRNA can be at least 40 nucleotides in length. For example, tracrRNA can comprise secondary structures such as one or more hairpin or stem loop structures, or one or more bulges.

[0206] In the present application, the guide RNA can comprise two RNA molecules, referred to herein as "dual guide RNA" or "dgRNA." For example, the dgRNA can 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 the flagpole sequences on the crRNA and tracrRNA.

[0207] For example, the 5' to 3' portion of a first RNA molecule may include a target sequence complementary to the target sequence, a crRNA flag sequence, and the 5' to 3' portion of a second RNA molecule 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 this 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 or a crRNA covalently linked to the tracrRNA. For example, the crRNA and 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 the flag sequences on the crRNA and tracrRNA. For example, the sgRNA may be a "Cas9sgRNA" that can mediate DNA cleavage via the Cas9 protein. For example, the sgRNA may be a "Cpf1sgRNA" that can mediate DNA cleavage via the Cpf1 protein.

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

[0211] The gRNA may also include modified nucleosides or nucleotides, such as those modified (e.g., replaced) at one or two non-linked phosphate oxygens and / or at one or more linking phosphate oxygens of a backbone phosphodiester bond; those ribose moieties, such as those replacing the 2' hydroxyl group of the ribose; those replacing the phosphate moiety with a dephosphorylated joint; those modifying or substituting a naturally occurring nucleobase; those substituting or modifying the phosphoribose backbone; those modifying the 3' or 5' end of the oligonucleotide, such as by removing, modifying, or substituting a terminal phosphate group or a splice moiety, or those adding a cap or joint (e.g., a 3' or 5' cap modification may include a sugar and / or backbone modification). For example, those 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 the 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 the exon portion of the HLA-A gene may comprise the 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 the 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%, or 99% homologous.

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

[0219] Chimeric antigen receptor (CAR)

[0220] In this application, the immune effector cells comprise 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, and 19A24), C-type lectin-like molecule-1 (CLL-1 or CD123), CD33, epidermal growth factor receptor variant III (CD171), ganglioside G2 (GD2), ganglioside GD3, and TNF receptor family members. 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 known as CD2 subclass 1, CRACC, SLAMF7, CD319, or 19A24), epithelial cell adhesion molecule (CLL-1 or CD123), B7H3 (CD171), and KIT (GD2). , interleukin-13 receptor subunit α-2 (CA125), mesothelin, interleukin-11 receptor α (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 β (FLT3), stage-specific embryonic antigen-4 (CD123), CD20, folate receptor α, 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 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 (CA125) consisting of breakout cluster (CD171) and C-met murine leukemia viral oncogene homolog 1 (GD2), tyrosinase, liver protein type A receptor 2 (e.g., C-met, L1CAMα-CD138), fucosyl-GM1 sialyl-CRACC adhesion molecule (CRACC), transglutaminase 5 (ROR1), polymeric 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), adrenoceptor 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 on chromosome 12p 6 (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 myelocytosis viral oncogene neuroblastoma-derived homolog (NY-BR-1), Ras homolog family member C (uroplakin), tyrosinase-related protein protein 2 (UPK2), cytochrome GloboH (HAVCR1), T-cell-recognized squamous cell carcinoma antigen 3 (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 membrane 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 antigen-binding fragment thereof that specifically binds to the tumor antigen. For example, the specific-binding GPC3 antibody or 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, an Fab fragment, an Fab' fragment, an F(ab')2 fragment, and a camelid single-domain antibody.

[0223] For example, the antibody may be a humanized antibody, which immunospecifically binds to an associated antigen (e.g., CD19, BCMA, GPC-3) and comprises a framework (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, or a variant, derivative, analog, or fragment thereof, 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, antigen-binding fragments may include Fab, Fab', F(ab)2, Fv fragments, F(ab.)2, scFv, di-scFv, and / or dAbs.

[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, liver cancer, 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 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 pharmaceutically acceptable carrier.

[0234] For example, the composition comprises a population of cells, wherein the population of cells comprises a modified immune effector cell 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 cell population may include the modified immune effector cells and the corresponding unmodified immune effector cells.

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

[0238] For example, the cell population may be a cell population obtained after the immune effector cell population is treated by genetic engineering, and the genetic engineering treatment 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 the HLA-A gene, an sgRNA targeting an exon portion of the 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 using 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 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 87%, at least 98%, at least 89%, at least 91%, at least 92%, at least 93%, at least 83%, at least 94%, at least 95%, at least 97%, or at least 99%.

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

[0241] For example, the cell population includes a cell population of immune effector cells obtained after using the antisense RNA, siRNA, or shRNA of the present application, wherein the mRNA expression of the cell population is reduced by 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 87%, at least 68%, at least 88%, 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 89%, or at least 90% compared to a corresponding immune effector cell population before use.

[0242] For example, the cell population includes a cell population obtained after the antisense RNA, siRNA, or shRNA of the present application is applied to the immune effector cell population, and 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 immune effector cell population before 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 pharmaceutically acceptable carrier may be composed of any solvent, dispersion medium, isotonic agent, and absorption delaying agent that is compatible with the immune effector cells, generally safe, non-toxic, and biologically or otherwise undesirable. For example, the pharmaceutically acceptable carrier may include a 2°C-8°C storage solution, a frozen storage solution, an injection solution, etc. For example, the carrier may contain the following ingredients: 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, etc. For example, the carrier may further contain Normosol® (Abbott), Plasma-Lyte A (Baxter) injection, 5% dextrose in water, or Ringer's lactate solution. For example, the carrier may also contain glycerin or DMSO.

[0245] For example, the compositions may be administered parenterally, transdermally, intracavitary, intraarterially, intrathecally, and / or intranasally, or by direct injection into tissue. For example, the compositions may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical compositions may be administered in different ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the pharmaceutical compositions may be administered continuously. Continuous (or continuous) administration may be achieved by a small pump system worn by the patient to meter the therapeutic agent into the patient's body, as described in WO 2015 / 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 an application of the modified immune effector cells of the present application in the preparation of a drug, which is used in the treatment of allogeneic tumors.

[0248] The present application also provides an application of the modified immune effector cells of the present application in preparing a drug, and the drug is used for 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 intended to illustrate the modified immune effector cells, production methods, and uses of the present invention, without limiting the scope of the present invention. The embodiments do not include detailed descriptions of conventional methods, such as how to construct vectors and plasmids, how to insert protein-encoding genes 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 detail in Sambrook, J., Fritsch, E. F., and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0251] Example

[0252] Example 1 Guide RNA design

[0253] The corresponding gene sequences (SEQ ID Nos. 55-63) can be downloaded from the website https: / / www.ncbi.nlm.nih.gov / . Opening this gene sequence using SnapGene software allows for the design of sgRNAs for different exons of the target gene. The non-restrictive sgRNA design principle for the CRISPR / Cas9 system employed 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. Because multiple sgRNAs can be designed for the same exon and sgRNAs consisting of 20 nucleotide sequences may appear redundantly in the genome, sgRNAs were designed and evaluated using the website http: / / crispr.cos.uni-heidelberg.de. The exon sequence was then inserted into the website, and a predictive evaluation of sgRNAs was performed. A higher evaluation score indicated higher editing efficiency and lower off-target risk. From these, sgRNAs with higher scores were selected for testing. The sgRNAs targeting the TRAC gene were synthesized by GenScript as follows: SEQ ID No. 1-15, sgRNAs targeting the HLA-A02 gene were synthesized as follows: SEQ ID No. 16-37, sgRNAs targeting the HLA-A11 gene were synthesized as follows: SEQ ID No. 38-46, and sgRNAs targeting the HLA-A24 gene were synthesized as follows: SEQ ID No. 47-54.

[0254] Example 2: CD3 + 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. One-third of the diluted blood volume was placed in a new 50 ml centrifuge tube with cell separation solution (Ficoll). The diluted blood was then poured very slowly along the tube wall, and centrifuged at 800 g for 20 minutes at room temperature (centrifuge speed setting: 1 for acceleration, 0 for deceleration). After centrifugation, the liquid in the tube was separated into PBS and serum layer, white blood cell layer, lymphocyte separation solution, and red blood cell layer. The PBS and serum layer was removed, and the white blood cell layer was transferred to a new 50 ml centrifuge tube. PBS was added to 40 ml of washed cells, and the tube was 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 PBMCs from healthy donors

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

[0259] (3) CD3+ T cell fraction

[0260] T cells were extracted from peripheral blood mononuclear cells (PBMCs) using the EasySep™ Human T Cell Sorting Kit (StemCell Technologies, product number 17951). The PBMC density was adjusted to 5 × 10 cells / ml, and 0.25–2 ml of PBS buffer was added. The cocktail was first added and mixed thoroughly, followed by 50 μl / ml of isolation cocktail. After mixing thoroughly, the mixture was left at room temperature for 5 minutes. RapidSpheres were then swirled in a vortex centrifuge for 30 seconds, followed by 40 μl / ml of the isolation cocktail. The buffer was then added to the cells to a volume of 2.5 ml and gently blown up and down 2–3 times. 2.5 ml of each tube was placed in a cryopreservation tube, which was then placed in a magnetic frame and left at room temperature for 3 minutes. Gently open the cryopreservation tube lid, carefully hold both sides of the magnetic frame, invert it, and hold for 2-3 s. Transfer the cell solution all at once into a new centrifuge tube. 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 activator was placed in a culture medium:Transact volume ratio of 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). Transact was purchased from Miltenyi Biotec. T cells were thoroughly suspended in 1 ml of activation reagent (containing 10 μl of Transact) for every 1 × 106 cells and then incubated at 37°C in a 5% CO2 incubator for 3 days.

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

[0264] The RNP complex was electrotransferred into 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 prior to transfection. The electrotransfer buffer was arranged in a Nucleofector Solution:Supplement ratio of 82:18. Preparation of RNP complex: The sgRNA sequence for TRAC was sg9 (shown in SEQ ID No. 1), and the sgRNA sequences for HLA-A were HLA-A02Sg2 (shown in SEQ ID No. 17), HLA-A02Sg5 (shown in SEQ ID No. 18), or HLA-A11sg21 (shown in SEQ ID No. 91). 20 μg of sgRNA (as indicated) or HLA-A11Rsg2 (as indicated in SEQ ID No. 92) was added to a PCR tube (RNAase-free), followed by 10 μg of Cas9 protein (purchased from Thermo Scientific, 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 discarded, and PBS-suspended cells added. 1E7 cells were aspirated and centrifuged again at 300 g for 8 minutes. The supernatant was discarded, and the cells were resuspended in 100 μl of electrophoresis buffer. The incubated RNP complex was added to the cell suspension and gently mixed. The mixture was then carefully transferred to an electroporation cuvette. The electroporation cuvette was placed on a Lonza-4D and spun using the EO-115 program. Pre-warmed medium was added to the electroporation cuvette, and the cells were transferred using a suitable pipette to the pre-warmed medium in the well plate, which was then placed in a 37°C, 5% CO2 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 Cells were harvested and centrifuged at 2000 rpm for 5 minutes. As much of the supernatant as possible was removed, 20 μl of DE lysis buffer was added to each tube. After lysis, the cells were added to PCR tubes, centrifuged briefly, and then run on a PCR machine. The conditions were 65°C for 30 minutes, 4°C for 30 seconds, 95°C for 2 minutes, and 16°C for infinity. PCR was performed using the primer pair TRAC-For / TRAC-Rev or HLA-A For / HLA-A Rev as the template. The PCR primer sequences were SEQ ID NOs. 66-81. The PCR products were sent to Kin Wei-ji for Sanger sequencing. After obtaining the Sanger sequencing results, the EditR editor at the website: https: / / moriaritylab.shinyapps.io / editr_v10 / was used to predict the location of editing and the editing efficiency.

[0268] (2) TA clone sequence measurement

[0269] After purifying the PCR product using the AxyPrep™ PCR Product Cleanup Kit (AXYGEN), the purified PCR product was ligated to a T vector using a sticky end kit (DNAA-Tailing Kit, TaKaRa) with the DNA Ligation Kit Ver. 2.1 (purchased from TaKaRa). The ligated product was transformed into recipient cells (DH5alpha) using the pMDTM19-TVector Cloning Kit from TaKaRa. The ligated product was coated onto an ampicillin-resistant LB plate and cultured at 37°C for approximately 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, and the supernatant was removed. Then, the cells were suspended in 100 μl of PBS buffer in each tube. Five μl of anti-human ABTCR-APC (purchased from eBioscience) antibody and five μl of HLA-A02 Monoclonal Antibody (BB7.2), APC, eBioscince™ were added. Five microliters of antibody (purchased from Invitrogen) was added, mixed uniformly, and incubated at room temperature for 10 minutes. After centrifugation at 2000 rpm for 5 minutes, the cells were washed twice with PBS buffer, resuspended, and assayed using a BD FACSAria flow cytometer to determine the positive rates of TCR and HLA-A02 expression on the cell surface. Knockout efficiency = (A−B) / A × 100%; A is the positive rate of expression in the control group; B is the positive rate of expression in the knockout group.

[0272] The three detection results for TRAC single gene knockout are shown in Figures 1 to 3, and the calculation results for knockout efficiency are shown in Table 1. The three detection methods are basically the same, and subsequent experiments only use Sanger sequencing to detect 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 into the activated T cells prepared in Example 2 using an electrotransfer kit (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 prior to transfer. The current buffer was arranged in a Nucleofector Solution:Supplement ratio of 82:18. Preparation of RNP complex: 20 μg TRAC sgRNA (TRACSg9) and 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*26:01:01 sgRNA) were placed in a PCR tube (without RNA), 10 μg of each Cas9 protein (purchased from Thermo Scientific, 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, and the supernatant discarded. PBS-suspended cells were added, and 1E7 cells were aspirated and centrifuged again at 300 g for 8 minutes. The supernatant was discarded, and the cells were resuspended in 100 μl of electrophoresis buffer. The incubated TRAC and HLA-A RNP complex was added to the cell suspension, gently mixed, and then carefully transferred to an electroporation cuvette. The electroporation cuvette was placed on a Lonza-4D and the EO-115 program was selected for rotation. Pre-warmed medium was added to the electroporation cuvette, and the cells were transferred to the pre-warmed medium in a well plate using a compatible pipette and placed in an incubator at 37°C and 5% CO2.

[0276] Double knockout efficiency was measured by sequencing, and TRAC-negative, HLA-A-negative T cells with a double knockout efficiency of over 80% were obtained. The results are shown in Figures 8-9. Figure 8A shows the results of knockout of HLA-A02 using HLA-A02 Sg5. The top row shows the control results (i.e., no knockout using HLA-A02 Sg5); the bottom row shows the results of simultaneous knockout of HLA-A02 and TRAC. Figure 8B shows the results of knockout of TRAC using TRAC Sg9. The top row shows the control results (i.e., no knockout using TRAC Sg9); the bottom row shows the results of simultaneous knockout of HLA-A02 and TRAC. Figures 9A-9B show knockout HLA-A02 and TRAC protein levels. 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 their corresponding genes in matched cells

[0278] (1) The activated T cells prepared in Example 2 were divided into two groups: one group was used as a control group, and the other group was used to prepare double knockout T cells for the TRAC gene and HLA-A gene using the method of Example 5, followed by Sanger sequencing according to step (1) of Example 4. Based on the sequencing results, TRAC and HLA-A double knockout cells were identified. The prepared double knockout T cells were then incubated with the corresponding TRAC and HLA-A antibodies, and the double knockout cell lines were obtained by flow cytometry or magnetic bead selection.

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

[0280] (3) The changes in protein expression levels in double knockout T cells were examined compared with 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 revealed altered nucleotide sequences of TRAC and / or HLA-A genes in double knockout T cells compared with controls. Quantitative PCR showed that mRNA expression levels of TRAC and / or HLA-A genes were downregulated in double knockout T cells, but not B2M and / or CIITA genes. FACS and Western blot results showed downregulation of protein expression levels in double knockout T cells, but not B2M and / or CIITA protein expression levels.

[0282] The results are shown in Figures 10-11. Figure 10 shows the mRNA levels 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 no knockout was performed, and double knockout refers to the results of T cells with a double knockout of the TRAC gene and the HLA-A gene. Figure 11 shows the protein level measurements 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 no knockout was performed, and TRAC+HLA-A double knockout refers to the results of T cells with a double knockout of the TRAC gene and the 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 the TRAC gene, the HLA-A gene and the CIITA gene, and triple knockout cells of the TRAC gene, the B2M gene and the CIITA gene are prepared.

[0285] (2) Following the method of Example 6, step (3), the changes in protein expression level are examined and measured by FACS and Western Blot methods.

[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 number: 17-9986-42), HLA-A (purchased from Merck, product number: 17-9876-41), and B2M (purchased from Invitrogen, product number: A15770) antibodies, and showed that the simultaneous multiple knockout efficiency at the single cell level was significantly higher for double knockouts than for triple knockouts.

[0288] The results are shown in Figures 12A-12D. Figures 12A-12C show the knockout results for TRAC, HLA-A, and B2M protein levels. Here, WT refers to the absence of any knockouts, and TRAC+HLA-A double knockout refers to the results for T cells with double knockouts of the TRAC and HLA-A genes. TRAC+HLA-A+CIITA triple knockout refers to the results for T cells with triple knockouts of TRAC, HLA-A, and CIITA; here, TRAC+B2M+CIITA triple knockout refers to the results for T cells with triple knockouts of B2M, CIITA, and TRAC. TRAC+HLA-A knockdown refers to the results for T cells with knockdowns of the TRAC and HLA-A genes, as prepared in Example 9. Figure 12D shows the knockout results for CIITA protein levels.

[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. Meanwhile, 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] The transcript RNA sequences (such as SEQ ID NOs. 82-90) of the corresponding genes (TRAC gene and HLA-A gene) are obtained from the database https: / / www.ncbi.nlm.nih.gov / or www.ensembl.org / , and siRNAs are designed based on the following principles:

[0292] If possible, avoid sequences 50-100 base pairs downstream of the start codon and 100 bases upstream of the stop codon; select sequences no longer than 30 nucleotides; avoid sequences with four or more consecutive identical bases; avoid intronic regions; avoid duplicated sequences; avoid single nucleotide polymorphisms (SNPs); and select sequences with a GC content between 30% and 60%. Preferentially select sequences with the following sequence patterns: AA (N19), NA (N21), or NAR (N17) YNN, where A is adenylic acid; T is thymidylic acid; R is adenylic acid or guanylic acid (purines); Y is thymidylic acid or cytidylic acid (pyrimidines); and N is adenylic acid, thymidylic acid, guanylic acid, or cytidylic acid. Perform a homology comparison analysis of the selected sequence to avoid significant homology of the antisense RNA to other genes or sequences, which could 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); and 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 lentivirus (Zima) containing the TRAC gene and HLA-A gene antisense RNA sequences was prepared in-house. + T cells (day D0) were prepared and activated with CD3 / CD28 antibody beads. Lentivirus carrying antisense RNA sequences for the 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 lentiviral vector was washed out on day D2 and cultured until day D5. T cells cultured until day D5 were harvested and gene knockdown efficiency was detected by quantitative PCR and Western blotting. The resulting T cells were labeled with the corresponding TRAC and HLA-A antibodies, and TRAC and HLA-A gene knockdown T cells were isolated by streaming sorting or magnetic bead sorting. Results showed that the mRNA and protein expression levels of TRAC and HLA-A were downregulated in both the TRAC and HLA-A knockdown groups. Figures 13A-13B show the TRAC and HLA-A mRNA levels. Here, WT refers to a state in which nothing has been knocked out, and TRAC+HLA-A double knockout refers to the results of T cells in which the TRAC gene and the HLA-A gene have been double knocked out. Here, the knockout levels, which are the protein levels of TRAC and HLA-A, can be seen in Figure 12.

[0296] Example 10: Distinguishing between different T cell activities

[0297] No knockout, double knockout, triple knockout, and double knockout T cells were prepared in Examples 2, 5, 7, and 9, and the cell numbers in each group were compared for multiple T cell activities. 6Cells were seeded into 24-well plates, and either PHA (0.3 μg / ml) (ionomycin+) or 5 ng / ml PMA and 50 ng / ml ionomycin was added to 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 activation) (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] See Figures 14A-14B for the protein expression of CD69 and CD137. Here, WT refers to the absence of any knockouts, and TRAC + HLA-A double knockout refers to the results of T cells with a double knockout of the TRAC gene and the HLA-A gene. TRAC + HLA-A + CIITA triple knockout refers to the results of T cells with a triple knockout of TRAC, HLA-A, and CIITA; here, TRAC + B2M + CIITA triple knockout refers to the results of T cells with a triple knockout of B2M, CIITA, and TRAC. TRAC + HLA-A knockout refers to the results of T cells with a knockout of the TRAC gene and the HLA-A gene, as prepared in Example 9.

[0299] Example 11: Differentiation of reactivity of different T cells to different 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 x 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 percentage of CFSE-positive cells in the mixed cells was examined and measured.

[0301] The results showed that double-knockout and double-knockdown NK cells had lower killing toxicity against T cells than triple-knockout T cells. The results are shown in Figure 15. Here, NK+T refers to NK cells co-cultured with untreated T cells; NK+TRAC+HLA-A knockdown refers to NK cells co-cultured with T cells in which the TRAC gene and HLA-A gene were knocked down, as prepared in Example 9; NK+TRAC+HLA-A double knockout refers to NK cells co-cultured with T cells in which the TRAC gene and HLA-A gene were double-knocked out; NK+TRAC+HLA-A+CIITA triple knockout refers to NK cells co-cultured with T cells in which the TRAC gene, HLA-A gene, and CIITA gene were triple-knocked out; and NK+TRAC+B2M+CIITA triple knockout refers to NK cells co-cultured with T cells in which the B2M gene, CIITA gene, and TRAC gene were triple-knocked out.

[0302] Example 12: Distinguishing between 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. + Equal amounts of T cells were mixed, and 24 hours later, the IFN-γ expression levels in the cell mixture were measured. The results showed that the IFN-γ expression levels in the double-knockout T cell group were lower than those in the triple-knockout T cell group.

[0304] The results are shown in Figure 16. These are the results of TRAC+HLA-A double knockout TRAC gene and HLA-A gene double knockout T cells when no WT was knocked out; TRAC+HLA-A+CIITA triple knockout refers to the results of TRAC, HLA-A, and CIITA triple knockout T cells; 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 TRAC gene and HLA-A gene double knockout CAR-T cells, TRAC gene, HLA-A gene and CIITA gene triple knockout CAR-T cells, and TRAC gene, B2M gene and CIITA gene knockout CAR-T cells

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

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

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

[0309] The results are shown in Figures 17A-17D. Figures 17A-17C show 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 no knockouts were performed, and TRAC+HLA-A double knockout refers to the results of CAR-T cells with double knockouts of the TRAC gene and the HLA-A gene. TRAC+HLA-A+CIITA triple knockout refers to the results of CAR-T cells with triple knockouts of TRAC, HLA-A, and CIITA; and TRAC+B2M+CIITA triple knockout refers to the results of HLA-A cells with triple knockouts of B2M, CIITA, and TRAC.

[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, CAR-T cells with double knockout of the TRAC gene and HLA-A gene (targeting CD19, CD20, or BCMA) were prepared. Luciferase-expressing target cells (target gene-positive leukemia or lymphoma cell lines such as Raji, Jurkat, and MM1S) were inoculated into the orifice. Then, double knockout CAR-T cells, triple knockout CAR-T cells, or non-knockout T cells were added at different target ratios (1:2.5, 1:1, 5:1, and 10:1). After 24 hours of co-culture, the cells were transferred to the orifice, luciferase substrate was added, and fluorescence was measured using an enzyme labeling instrument. Killing efficiency = 1 - fluorescence value of target cells and T cells co-cultured / fluorescence value of target cells cultured alone.

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

[0315] Figure 20 shows the killing effect of Raji-luciferase on CD19 target cells, with the most notable effect being seen in CAR-T cells with a double knockout of the TRAC gene and HLA-A gene. The results correspond to the AD annotations below each E / T ratio, from left to right.

[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 successful tumor establishment, CAR-T cells with double knockout of the TRAC gene and HLA-A gene, triple knockout of 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. Figure 21 shows the administration method, with iv representing intravenous injection and CAR-T cells representing double-knockout and triple-knockout CD19CAR-expressing CAR-T cells. Figure 20 shows the tumor volume in mice after CAR-T cell administration. Figure 20 shows the tumor volume in mice treated with saline, unmodified T cells, TRAC and HLA-A knockout CD19CAR-T cells, TRAC, HLA-A, and CIITA knockout CD19CAR-T cells, and B2M, CIITA, and TRAC knockout CD19CAR-T cells, respectively, from left to right. The results showed that mice reinjected with TRAC and HLA-A double-knockout CAR-T cells showed a significant slowing of 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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