Immune cells enriched with dual shRNA and compositions containing same

Abstract

To provide a vector for producing immune cells comprising a genetically engineered antigen receptor that specifically binds to a target antigen and a genetic disruption agent that reduces or is capable of reducing the expression in the immune cell of a gene that weakens the function of the immune cell.SOLUTION: A vector comprises: a) a base sequence encoding two types of short hairpin RNA (shRNA) which inhibit the expression of at least one gene that weakens the function of immune cells, one of the two types of shRNA targeting the expression of T cell immunoreceptor with Ig and ITIM domains (TIGIT); and b) a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Related Applications This application is a Korean patent application number 10-2018-000 filed on January 12, 2018. No. 4,238, the disclosure of which is incorporated herein by reference in its entirety. Be absorbed.

[0002] Reference to an electronically submitted sequence listing This application was created on January 5, 2019 and is 145 bytes in size, 88,751 bytes. A text file titled 70-001-228_SEQ_LISTING.txt The sequence listing submitted with this application is incorporated by reference as a part of this application.

[0003] Technical Field The present disclosure relates generally to the field of cancer immunotherapy. For example, the present invention generally relates to the use of immunomodulators of cancer target antigens. Genetically engineered antigen receptors that specifically bind to the immune cells and genetic immunization that weakens the function of immune cells and a gene disrupting agent that reduces or is capable of reducing expression in the cell. Regarding cells. [Background technology]

[0004] Isolating T cells or NK cells (natural killer cells) from the body of a patient or donor, These cells were cultured in vitro and then infused back into the patient. Anti-cancer therapy is currently attracting a lot of attention as a new method of cancer treatment. A process in which new genetic information is injected using a method such as cloning, and then cultured in a test tube. It has been reported that immune cells that have been subjected to steroid therapy have a greater anti-cancer effect than cells that have not. Here, the genetic information injected into T cells usually gives them high affinity for the target antigen. Chimeric antigen receptors (hereafter referred to as CARs) or monoclonal T cells engineered to have These modified immune cells are restricted to unique antigen specificity. Without being affected, they recognize and attack cancer cells expressing target antigens, inducing cell death. The method of genetically engineering T cells using CARs was first described in 1989 by Eshhar et al. al. and was called the "T-body".

[0005] Provided herein are methods that address the problems of conventional simultaneous immune cell therapy as noted above. The problem is that the high cost of immune cell compositions and methods places a significant economic burden on patients. It acts on T cells other than CAR-T, reducing the risk of autoimmune symptoms and cytokine release syndrome. In short, for example, disclosed herein are high yield and low production Furthermore, it is possible to suppress the function of immune cells with higher probability and effectiveness. By inhibiting the molecules responsible for cell proliferation, the disclosure herein addresses the need for technologies that provide effective cell therapy. The technical problem that the present disclosure aims to solve is not limited to the above technical problem. Other technical problems not mentioned will be apparent to those skilled in the art from the following. Summary of the Invention

[0006] Provided herein are vectors, immune cells, pharmaceutical compositions comprising the immune cells, and and compositions comprising immune cells. Also provided herein are methods for producing immune cells. and methods of treatment and use of the immune cells.

[0007] In one aspect, provided herein are vectors. The study provides two types of short hairpin RNAs that inhibit the expression of genes that weaken immune cell function. A (shRNA) encoding a chimeric antigen receptor (CAR) or T cell receptor Receptor (TCR), e.g., monoclonal T cell receptor (mTCR) In some embodiments, the vector comprises a CAR or TCR, e.g., mT Targets for CR are selected from among the elevated antigens that show increased expression in cancer, or from the cancer, e.g. For example, mutated antigens found in cancer cells, cancer tissues, and / or the tumor microenvironment The human tumor antigen is selected from the following forms:

[0008] In some embodiments, the expression of two shRNAs is controlled by two different promoters. Each of these is controlled by a separate controller.

[0009] In some embodiments, the two promoters are promoters for RNA polymerase III. In some embodiments, the two promoters are a U6 promoter, e.g., a For example, U6 promoters from different species. In some embodiments, the promoters are oriented in the same direction as each other on the vector. The two promoters may be oriented in different directions on the vector. In this configuration, the promoters are oriented in a head-to-head manner. In this embodiment, the promoter is oriented in an end-to-end direction.

[0010] In some embodiments, the gene that attenuates immune cell function is an immune checkpoint inhibitor. It can be a receptor or a ligand.

[0011] In some embodiments, the immune checkpoint receptor or ligand is PD1, PD-L1, CTLA4, TIM3, CEACAM(CEACAM-1, CEACAM- 3 or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR 1, CD160, CD96, MerTK and 2B4.

[0012] In some embodiments, the gene that attenuates immune cell function is FAS, CD45, PP 2A, SHIP1, SHIP2, DGK alpha, DGK zeta, Cbl-b, CD14 7, LRR1, TGFBR1, IL10R alpha, KLGR1, DNMT3A and A2 aR.

[0013] In some embodiments, the targeting of one or more genes that attenuate immune cell function. In some embodiments, two shRNAs are utilized. They target single genes that weaken the function of immune cells, or In some embodiments, the two shRNAs target different genes involved in PD-1. In some embodiments that include two shRNAs, one shRNA targets One shRNA targets PD-1, and the second shRNA targets TIM-3. In some embodiments, including one shRNA targets PD-1 and a second shRNA targets PD-1. NA targets TIGIT.

[0014] shRNAs have a hairpin structure containing sense and antisense shRNA sequences. In some embodiments, the shRNA encoding the shRNA described herein forms a The base sequence to be used includes a sequence selected from the group consisting of SEQ ID NOs: 2 to 219. In one embodiment, the base sequence encoding the shRNA described herein is SEQ ID NO: 2 to SEQ ID NO: 219, wherein the sequence comprises a sense shRNA sequence. In certain embodiments, salts encoding shRNAs described herein are The base sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 2 to 219, wherein the sequence is Encodes the antisense shRNA sequence.

[0015] In some embodiments, the vector comprises either the nucleic acid sequence of SEQ ID NO: 220 or 221. In some embodiments, the vector comprises a plasmid vector or a virus vector. viral vectors, e.g., lentiviral vectors, e.g., retroviral vectors, adenoviral vectors, The vector is a viral vector or an adeno-associated viral vector.

[0016] In another aspect, provided herein is a CAR or TCR, e.g., an mTCR. These are immune cells containing vectors expressing the shRNA. The expression of the target genes of the two types of shRNA is The expression of the target gene is reduced to 40% or less compared to the control group that does not express shRNA. In some embodiments, the immune cells are selected from among T cells and NK cells of human origin.

[0017] In another aspect, provided herein is a method for immunotherapy of a human patient comprising the immune cells described above. In some embodiments, the immune cells are originally derived from a patient. In some embodiments, the patient is treated with a CAR or TCR expressed in a cell, e.g. In tumors or tumors where increased or altered levels of cancer antigens targeted by mTCR are detected or have cancer.

[0018] In another aspect, provided herein are engineered antibodies that specifically bind to a target antigen. It reduces the expression of genes in immune cells that weaken immune cell function and inhibit the expression of antigen receptors in immune cells. or a gene disrupting agent capable of reducing the expression of the gene.

[0019] In another aspect, provided herein are engineered antibodies that specifically bind to a target antigen. It reduces the expression of genes in immune cells that weaken immune cell function and inhibit the expression of antigen receptors in immune cells. or a gene disrupting agent capable of reducing the expression of the gene.

[0020] In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). ) or T cell receptor (TCR).

[0021] In some embodiments, the engineered antigen receptor is a CAR. In embodiments, a CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen recognition domain of the CAR comprises: It specifically binds to the target antigen.

[0022] In some embodiments, the intracellular signaling domain of the CAR is CD3 zeta (CD In some embodiments, the intracellular domain of the CAR comprises the intracellular domain of the CAR 3ζ) chain. In some embodiments, the delivery domain further comprises a costimulatory molecule. S, OX40, CD137(4-1BB), CD27, and CD28 In some embodiments, the costimulatory molecule is CD137 (4-1BB). In some embodiments, the costimulatory molecule is CD28.

[0023] In some embodiments, the engineered antigen receptor is a TCR. In an embodiment, the TCR is a monoclonal TCR (mTCR).

[0024] In some embodiments, the target antigen is a target antigen of a cancer cell, cancer tissue, and / or tumor microenvironment. expressed in or on the surface of

[0025] In some embodiments, the target antigen is 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (alpha-fetoprotein), AlbZIP (androgen-induced bZIP) ), HPG1 (human prostate-specific gene-1), α5β1-integrin, α5β6-integrin Integrin, α-methylacyl-coenzyme A racemase, ART-4 (ADP-ribosyltransferase), B7H4 (v-set domain-containing T-cell activation inhibitor 1), BAGE-1 (B melanoma antigen-1), BCL-2 (B-cell lymphoma / CLL-2) ), BING-4 (WD repeat domain 46), CA15-3 / CA27-29 (mucin 1 ), CA19-9 (cancer antigen 19-9), CA72-4 (cancer antigen 72-4), CA12 5 (cancer antigen 125), calreticulin, CAMEL (antigen recognized by CTL on melanoma) , CASP-8 (caspase 8), cathepsin B, cathepsin L, CD19 (cluster of differentiation antigen) 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD 55, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride chloride Nel Accessory 2), CML28 (chronic myeloid leukemia tumor antigen 28), coactosin-like Protein, collagen XXIII, COX-2 (cyclooxygenase-2), CT- 9 / BRD6 (cancer / testis antigen 9), Cten (c-terminal tensin-like protein), Cyclin B1, cyclin D1, cyp-B, CYPB1 (cytochrome p450 family) -1 subfamily b member 1), DAM-10 / MAGE-B1 (melanoma-associated antigen receptor 1), GenB1), DAM-6 / MAGE-B2, EGFR / Her1 (epidermal growth factor receptor), EMMPRIN (basigin), EpCam, EphA2 (EPH receptor A2), Eph A3, ErbB3 (Erb-B2 receptor tyrosine kinase 3), EZH2 (zeste2 Enhancer of Polycomb Repressive Complex 2 Subunit), FGF-5 (fibroblast proliferation factor 5), FN (fibronectin), Fra-1 (Fos-related antigen-1), G250 / CAIX (carbonic anhydrase 9), GAGE-1 (G antigen-1), GAGE-2, GAGE- 3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GD EP (differentially expressed gene in prostate), GnT-V (gluconate kinase), g p100 (melanocyte lineage-specific antigen GP100), GPC3 (glypican 3), HA GE (helical antigen), HAST-2 (sulfotransferase family 1A member) 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K-MEL, HNE (medullasin), homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPVE7 , HST-2 (Sirtuin-2), hTERT, iCE (Caspase-1), IGF-1R (insulin-like growth factor-1 receptor), IL-13Ra2 (interleukin-13 receptor Subunit α2), IL-2R (interleukin-2 receptor), IL-5 (interleukin -leukin-5), immature laminin receptor, kallikrein 2, kallikrein 4, Ki67 , KIAA0205 (lysophosphatidylglycerol acyltransferase 1), KK-LC-1 (Kitakyushu lung cancer antigen-1), KM-HN-1, LAGE-1 (L antigen family) Libermember-1), Livin, MAGE-A1, MAGE-A10, MAGE-A12, MAGEA2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, M AGE-B1, MAGE-B10, MAGE-B16, MAGEB17, MAGE-B2 , MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1 , MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4 , MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2( Melanoma antigen family L2), mammaglobin A, MART-1 / Melan-A (T -melanoma antigen recognized by cell-1), MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor) factor), mesothelin, MG50 / PXDN (peroxidasin), MMP11 (matrix Metalloproteinase 11), MN / CA IX-antigen (carbonic anhydrase 9), MRP- 3 (multidrug resistance-associated protein-3), MUC1 (mucin 1), MUC2, NA88-A ( VENT-like homeobox 2 pseudogene 1), N-acetylglucosaminyltransferase Neo-poly(A) polymerase V, Neo-PAP (Neo-poly(A) polymerase), NGEP (expressed in the prostate) newly expressed gene), NMP22 (nuclear matrix protein 22), NPM / ALK (nucleophosmin), NSE (neuron-specific enolase), NY-ESO-1, N Y-ESO-B, OA1 (osteoarthritis QTL1), OFA-iLRP (carcinoembryonic antigen receptor p53 (OFA)-iLRP) Immature laminin receptor protein), OGT (O-GlcNAc transferase), OS-9 (endoplasmic reticulum lectin), osteocalcin, osteopontin, p15 (CDK inhibitor) Inhibitor 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (Pro Lasminogen activator inhibitor-1), PAI-2, PAP (prostatic acid phosphatase ), PART-1 (prostate androgen-regulated transcript 1), PATE (expressed in the prostate and testis) PDEF (prostate-derived Ets factor), Pim-1-kinase (provirus-derived Ets factor), rus integration site 1), Pin1 (peptidyl-prolyl cis-trans isomer NIMA-interacting 1), POTE (expressed in the prostate, ovary, testis, and placenta), PRAME (preferentially expressed antigen in melanoma), prostein, proteinase- 3. PSA (Prostate Specific Antigen), PSCA (Prostate Stem Cell Antigen), PSGR (Prostate Specific atypical G-protein-coupled receptors), PSM, PSMA (prostate-specific membrane antigen), RAGE -1 (renal tumor carcinoma antigen), RHAMM / CD168, RU1 (renal ubiquitous protein 1), R U2, SAGE (sarcoma antigen), SART-1 (squamous epithelial cell antigen recognized by T-cells-1) Sperm carcinoma antigen), SART-2, SART-3, Sp17 (sperm protein 17), SS X-1 (SSX Family Member 1), SSX-2 / HOM-MEL-40, SSX- 4. STAMP-1 (STEAP2 metalloreductase), STEAP, survivin, Survivin-213, TA-90 (tumor-associated antigen-90), TAG-72 (tumor-associated glycoprotein Protein-72), TARP (TCRγ alternative reading frame protein), TGF b (transforming growth factor beta), TGFbR11 (transforming growth factor beta receptor 11), TGM -4 (transglutaminase 4), TRAG-3 (taxol resistance-associated gene 3), T RG (T-cell receptor gamma locus), TRP-1 (transient receptor potential-1), T RP-2 / 6b, TRP-2 / INT2, Trp-p8, tyrosinase, UPA (U-proline) lasminogen activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FL K-1, and WT1 (Wilms' tumor 1). In some embodiments, the target antigen is CD19 or CD22. is CD19.

[0026] In some embodiments, the target antigen is expressed in cancer cells, cancer tissues, and / or tumors. It is a cancer antigen that increases within or on the surface of the tumor microenvironment.

[0027] In some embodiments, the target antigen is α-actinin-4 / m, ARTC1 / m, b cr / abl, beta-catenin / m, BRCA1 / m, BRCA2 / m, CASP-5 / m, CASP-8 / m, CDC27 / m, CDK4 / m, CDKN2A / m, CML6 6, COA-1 / m, DEK-CAN, EFTUD2 / m, ELF2 / m, ETV6-A ML1, FN1 / m, GPNMB / m, HLA-A*0201-R170I, HLA-A 11 / m, HLA-A2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LDLR-FUT, MART2 / m, ME1 / m, MUM-1 / m, MUM-2 / m , MUM-3 / m, myosin class 1 / m, neo-PAP / m, NFYC / m, NR as / m, OGT / m, OS-9 / m, p53 / m, Pml / RARa, PRDX5 / m , PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT- SSX-2, TEL-AML1, TGFbRII, and TPI / m In this case, the target antigen is expressed in or on cancer cells, cancer tissues, and / or the tumor microenvironment. It is a mutated form of a cancer antigen expressed on the surface of the tumor.

[0028] In some embodiments, the expression of a gene that attenuates immune cell function includes one or more of the following: cause: (i) inhibition of immune cell proliferation; (ii) induction of cell death in immune cells; (iii) inhibiting the ability of immune cells to recognize and / or be activated by target antigens; (iv) inducing differentiation of immune cells into cells that do not induce an immune response against the target antigen; (v) a decreased response of immune cells to molecules that promote the immune response of immune cells; or (vi) Increased immune cell response to molecules that suppress immune cell immune responses.

[0029] In some embodiments, the gene that attenuates immune cell function is PD1, PD-L1, C TLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEA CAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160 , CD96, MerTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGK alpha, DGK zeta, Cbl-b, Cbl-c, CD148, LRR1, TG Selected from the group consisting of FBR1, IL10RA, KLGR1, DNMT3A, and A2aR will be done.

[0030] In some embodiments, the gene that attenuates the function of an immune cell suppresses the immune response of the immune cell. Enhances immune cell response to inhibitory molecules.

[0031] In some embodiments, the response of an immune cell to a molecule that inhibits the immune response of the immune cell. Genes that increase immune checkpoint activity encode receptors or ligands for immune checkpoints.

[0032] In some embodiments, the immune checkpoint receptor or ligand is PD1, PD-L1, CTLA4, TIM3, CEACAM(CEACAM-1, CEACAM- 3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAI R1, CD160, CD96, MerTK and 2B4.

[0033] In some embodiments, the gene disrupting agent increases the expression of immune cells relative to immune cells in the absence of the gene disrupting agent. In total, we have at least 30, 40, or 50,000 genes in immune cells that weaken their function. Reduce by 50, 60, 70, 80, 90, or 95%.

[0034] In some embodiments, the gene disrupting agent is directed against a molecule that suppresses the immune response of an immune cell. It reduces the expression of genes that enhance immune cell responses.

[0035] In some embodiments, the gene disrupting agent is an immune checkpoint receptor or ligand. It reduces the expression of genes encoding these proteins.

[0036] In some embodiments, the gene disrupting agent is selected from the group consisting of PD1, PD-L1, CTLA4, TIM 3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), L AG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, Me The expression of a gene selected from the group consisting of rTK and 2B4 is reduced.

[0037] In some embodiments, the gene disrupting agent is activated by RNA interference (RNAi) in immune cells. In some embodiments, the expression of more than one gene is reduced. The disruptor reduces the expression of genes in immune cells by RNAi, which impairs immune cell function. Lower it.

[0038] In some embodiments, the gene disrupting agent targets a single gene that attenuates immune cell function. targeting different genes that target or attenuate immune cell function, while A gene disruptor targets a first gene, a second gene disruptor targets a second gene, and or any combination thereof.

[0039] In some embodiments, RNAi is mediated by short hairpin RNA (shRNA). In some embodiments, RNAi is mediated by more than one shRNA. In an embodiment, the RNAi is mediated by two shRNAs.

[0040] In some embodiments, the two shRNAs target PD-1. In this configuration, the first shRNA targets PD-1 and the second shRNA targets TIM-3. In some embodiments, the first shRNA targets PD-1 and the second shRNA targets PD-2. The hRNA targets CTLA-4. In some embodiments, the first shRNA is P In some embodiments, one shRNA targets D-1 and the second shRNA targets LAG-3. The first shRNA targets PD-1 and the second shRNA targets TIGIT. do.

[0041] In some embodiments, the immune cells comprise a nucleotide sequence encoding the shRNA. In some embodiments, the immune cells contain more than one shRNA-encoding nucleotide sequence. In some embodiments, the immune cells contain nucleic acids encoding two shRNAs. Unless otherwise specified, as used herein, "nucleotide sequence" and "nucleotide sequence" include The terms "nucleotide sequence" and "nucleotide sequence" are interchangeable.

[0042] In some embodiments, the nucleotide sequence encoding the shRNA is SEQ ID NO:2- 219 and 238-267.

[0043] In some embodiments, the nucleotide sequence encoding the shRNA is on a vector. do.

[0044] In some embodiments, the expression of different shRNAs is driven by different promoters. In some embodiments, the expression of two different shRNAs is regulated by two different In some embodiments, the two promoters are regulated by different promoters. In some embodiments, the promoter is an RNA polymerase III promoter. In some embodiments, the two promoters are U6 promoters. In some embodiments, the two promoters are from different species. For example, in certain embodiments, the promoters are oriented head-to-head. In another embodiment, the promoter is oriented in an end-to-end direction. do.

[0045] In some embodiments, the engineered antigen receptor and the gene disrupting agent each comprise a vector. In some embodiments, engineered antigen receptors and The gene disruptor is expressed from the same vector.

[0046] In some embodiments, the vector is a plasmid vector or a viral vector. In some embodiments, the viral vector is a lentiviral vector or an adenoviral vector. In some embodiments, the lentiviral vector is a retroviral vector. It is an illus vector.

[0047] In some embodiments, the immune cells consist of T cells and natural killer (NK) cells. In some embodiments, the immune cell is a T cell. In embodiments, the T cells are CD4+ T cells or CD8+ T cells.

[0048] In some embodiments, the immune cells encode two shRNAs and one CAR. In some embodiments, the two shRNAs are contained on the same vector. Two different RNA polymerase III promoters oriented in different directions For example, in certain embodiments, the promoter is In another embodiment, the promoter is oriented in an end-to-end direction. In some embodiments, the CAR targets CD19 and the first shRNA is One shRNA targets PD-1, and the second shRNA targets TIGIT.

[0049] In another aspect, provided herein are methods for treating immune cells. (1) a gene encoding a genetically engineered antigen receptor that specifically binds to a target antigen; (2) Reduce or decrease the expression in immune cells of genes that weaken immune cell function. and a gene disrupting agent capable of disrupting the expression of the gene, which can be introduced simultaneously or sequentially in any order; This results in the expression of genetically engineered antigen receptors, which weaken the function of immune cells. and generating immune cells in which expression of the gene is reduced.

[0050] In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). ) or T cell receptor (TCR).

[0051] In some embodiments, the engineered antigen receptor is a CAR. In embodiments, a CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen recognition domain of the CAR comprises: It specifically binds to the target antigen.

[0052] In some embodiments, the intracellular signaling domain of the CAR is CD3 zeta (CD In some embodiments, the intracellular domain of the CAR comprises the intracellular domain of the CAR 3ζ) chain. In some embodiments, the delivery domain further comprises a costimulatory molecule. S, OX40, CD137(4-1BB), CD27, and CD28 In some embodiments, the costimulatory molecule is CD137 (4-1BB). In some embodiments, the costimulatory molecule is CD28.

[0053] In some embodiments, the engineered antigen receptor is a TCR. In an embodiment, the TCR is a monoclonal TCR (mTCR).

[0054] In some embodiments, the target antigen is a target antigen of a cancer cell, cancer tissue, and / or tumor microenvironment. expressed in or on the surface of

[0055] In some embodiments, the target antigen is 5T4 (trophoblast glycoprotein), 707-AP, 9 D7, AFP (alpha-fetoprotein), AlbZIP (androgen-induced bZIP) , HPG1 (human prostate-specific gene-1), α5β1-integrin, α5β6-integrin Tegrin, α-methylacyl-coenzyme A racemase, ART-4 (ADP-ribosyltransferase), B7H4 (v-set domain-containing T-cell activation inhibitor 1), B AGE-1 (B-melanoma antigen-1), BCL-2 (B-cell CLL / lymphoma-2) , BING-4 (WD repeat domain 46), CA15-3 / CA27-29 (mucin 1) , CA19-9 (cancer antigen 19-9), CA72-4 (cancer antigen 72-4), CA125 (cancer antigen 125), calreticulin, CAMEL (antigen recognized by CTL on melanoma), CASP-8 (caspase 8), cathepsin B, cathepsin L, CD19 (cluster of differentiation 1 9), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD5 5, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel accessory 2), CML28 (chronic myeloid leukemia tumor antigen 28), coactosin-like protein Protein, Collagen XXIII, COX-2 (cyclooxygenase-2), CT-9 / BRD6 (cancer / testis antigen 9), Cten (c-terminal tensin-like protein), cyclin Cyclin B1, cyclin D1, cyp-B, CYPB1 (cytochrome p450 family) 1 subfamily b member 1), DAM-10 / MAGE-B1 (melanoma-associated antigen B1), DAM-6 / MAGE-B2, EGFR / Her1 (epidermal growth factor receptor), E MMPRIN (basigin), EpCam, EphA2 (EPH receptor A2), EphA 3. ErbB3 (Erb-B2 receptor tyrosine kinase 3), EZH2 (zeste2 receptor) Enhancer of ribosomal repressive complex 2 subunit), FGF-5 (fibroblast growth factor 5), FN (fibronectin), Fra-1 (Fos-related antigen-1), G250 / C AIX (carbonic anhydrase 9), GAGE-1 (G antigen-1), GAGE-2, GAGE-3 , GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDE P (differentially expressed gene in the prostate), GnT-V (gluconate kinase), gp 100 (melanocyte lineage-specific antigen GP100), GPC3 (glypican 3), HAG E (helical antigen), HAST-2 (sulfotransferase family 1A member 1) ), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2 ), HERV-K-MEL, HNE (medullasin), homeobox NKX3.1, H OM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPVE7, HST-2 (Sirtuin-2), hTERT, iCE (Caspase 1), IGF-1R ( insulin-like growth factor-1 receptor), IL-13Ra2 (interleukin-13 receptor) subunit α2), IL-2R (interleukin-2 receptor), IL-5 (interleukin leukin-5), immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205 (lysophosphatidylglycerol acyltransferase 1), K K-LC-1 (Kitakyushu lung cancer antigen-1), KM-HN-1, LAGE-1 (L antigen family) Member-1), Livin, MAGE-A1, MAGE-A10, MAGE-A12, M AGEA2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MA GE-B1, MAGE-B10, MAGE-B16, MAGEB17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2 Melanoma antigen family L2), mammaglobin A, MART-1 / Melan-A (T- melanoma antigen recognized by mitochondrial cells-1), MART-2, matrix protein 2 2. MC1R (melanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor) mesothelin, MG50 / PXDN (peroxidasin), MMP11 (matrix Metalloproteinase 11), MN / CA IX-antigen (carbonic anhydrase 9), MRP-3 (multidrug resistance-associated protein-3), MUC1 (mucin 1), MUC2, NA88-A(V ENT-like homeobox 2 pseudogene 1), N-acetylglucosaminyltransferase Ze-V, Neo-PAP (Neo-poly(A) polymerase), NGEP (expressed in the prostate New genes that are being investigated), NMP22 (nuclear matrix protein 22), NPM / ALK ( Nucleophosmin), NSE (neuron-specific enolase), NY-ESO-1, NY -ESO-B, OA1 (osteoarthritis QTL1), OFA-iLRP (carcinoembryonic antigen receptor 1 (OFA-iLRP) Mature laminin receptor protein), OGT (O-GlcNAc transferase), O S-9 (endoplasmic reticulum lectin), osteocalcin, osteopontin, p15 (CDK inhibitor) Agent 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (P Prostatic acid phosphatase (PAP) , PART-1 (prostate androgen-regulated transcript 1), PATE (expressed in the prostate and testis) 1), PDEF (prostate-derived Ets factor), Pim-1-kinase (proviral integration site 1), Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1), POTE (expressed in the prostate, ovary, testis, and placenta), P RAME (antigen preferentially expressed in melanoma), prostein, proteinase-3 , PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate-specific G-protein-coupled receptors), PSM, PSMA (prostate-specific membrane antigen), RAGE- 1 (renal tumor carcinoma antigen), RHAMM / CD168, RU1 (renal ubiquitous protein 1), RU 2. SAGE (sarcoma antigen), SART-1 (squamous cell antigen recognized by T-cell-1) Sperm carcinoma antigen), SART-2, SART-3, Sp17 (sperm protein 17), SSX -1 (SSX Family Member 1), SSX-2 / HOM-MEL-40, SSX-4 , STAMP-1 (STEAP2 metalloreductase), STEAP, survivin, sa Vivin-213, TA-90 (tumor-associated antigen-90), TAG-72 (tumor-associated glycoprotein Protein-72), TARP (TCRγ alternative reading frame protein), TGFb (transforming growth factor beta), TGFbR11 (transforming growth factor beta receptor 11), TGM- 4 (transglutaminase 4), TRAG-3 (taxol resistance-associated gene 3), TR G (T-cell receptor gamma locus), TRP-1 (transient receptor potential-1), TR P-2 / 6b, TRP-2 / INT2, Trp-p8, tyrosinase, UPA (U-pla Sminogen activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK -1, and WT1 (Wilms' tumor 1). In some embodiments, the target antigen is CD19 or CD22. It's CD19.

[0056] In some embodiments, the target antigen is expressed in cancer cells, cancer tissues, and / or tumors. It is a cancer antigen that increases within or on the surface of the tumor microenvironment.

[0057] In some embodiments, the target antigen is α-actinin-4 / m, ARTC1 / m, b cr / abl, beta-catenin / m, BRCA1 / m, BRCA2 / m, CASP-5 / m, CASP-8 / m, CDC27 / m, CDK4 / m, CDKN2A / m, CML6 6, COA-1 / m, DEK-CAN, EFTUD2 / m, ELF2 / m, ETV6-A ML1, FN1 / m, GPNMB / m, HLA-A*0201-R170I, HLA-A 11 / m, HLA-A2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LDLR-FUT, MART2 / m, ME1 / m, MUM-1 / m, MUM-2 / m , MUM-3 / m, myosin class 1 / m, neo-PAP / m, NFYC / m, NR as / m, OGT / m, OS-9 / m, p53 / m, Pml / RARa, PRDX5 / m , PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT- SSX-2, TEL-AML1, TGFbRII, and TPI / m In this case, the target antigen is expressed in or on cancer cells, cancer tissues, and / or the tumor microenvironment. It is a mutated form of a cancer antigen expressed on the surface of the tumor.

[0058] In some embodiments, the expression of a gene that attenuates immune cell function includes one or more of the following: cause: (i) inhibition of immune cell proliferation; (ii) induction of cell death in immune cells; (iii) inhibiting the ability of immune cells to recognize and / or be activated by target antigens; (iv) inducing differentiation of immune cells into cells that do not induce an immune response against the target antigen; (v) a decreased response of immune cells to molecules that promote the immune response of immune cells; or (vi) Increased immune cell response to molecules that suppress immune cell immune responses.

[0059] In some embodiments, the gene that attenuates immune cell function is PD1, PD-L1, C TLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEA CAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160 , CD96, MerTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGK alpha, DGK zeta, Cbl-b, Cbl-c, CD148, LRR1, TG Selected from the group consisting of FBR1, IL10RA, KLGR1, DNMT3A, and A2aR will be done.

[0060] In some embodiments, the gene that attenuates the function of an immune cell suppresses the immune response of the immune cell. Enhances immune cell response to inhibitory molecules.

[0061] In some embodiments, the response of an immune cell to a molecule that inhibits the immune response of the immune cell. Genes that increase immune checkpoint activity encode receptors or ligands for immune checkpoints.

[0062] In some embodiments, the immune checkpoint receptor or ligand is PD1, PD-L1, CTLA4, TIM3, CEACAM(CEACAM-1, CEACAM- 3 or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR 1, CD160, CD96, MerTK and 2B4.

[0063] In some embodiments, the gene disrupting agent increases the expression of immune cells relative to immune cells in the absence of the gene disrupting agent. In total, we have at least 30, 40, or 50,000 genes in immune cells that weaken their function. Reduce by 50, 60, 70, 80, 90, or 95%.

[0064] In some embodiments, the gene disrupting agent is directed against a molecule that suppresses the immune response of an immune cell. It reduces the expression of genes that enhance immune cell responses.

[0065] In some embodiments, the gene disrupting agent is an immune checkpoint receptor or ligand. It reduces the expression of genes encoding these proteins.

[0066] In some embodiments, the gene disrupting agent is selected from the group consisting of PD1, PD-L1, CTLA4, TIM 3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), L AG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, Me The expression of a gene selected from the group consisting of rTK and 2B4 is reduced.

[0067] In some embodiments, the gene disrupting agent is activated by RNA interference (RNAi) in immune cells. In some embodiments, the expression of more than one gene is reduced. The disruptor reduces the expression of genes in immune cells by RNAi, which impairs immune cell function. Lower it.

[0068] In some embodiments, the gene disrupting agent targets a single gene that attenuates immune cell function. targeting different genes that target or attenuate immune cell function, while A gene disruptor targets a first gene, a second gene disruptor targets a second gene, and or any combination thereof.

[0069] In some embodiments, RNAi is mediated by short hairpin RNA (shRNA). In some embodiments, RNAi is mediated by more than one shRNA. In an embodiment, the RNAi is mediated by two shRNAs.

[0070] In some embodiments, the two shRNAs target PD-1. In this configuration, the first shRNA targets PD-1 and the second shRNA targets TIM-3. In some embodiments, the first shRNA targets PD-1 and the second shRNA targets PD-2. The hRNA targets CTLA-4. In some embodiments, the first shRNA is P In some embodiments, one shRNA targets D-1 and the second shRNA targets LAG-3. The first shRNA targets PD-1 and the second shRNA targets TIGIT. do.

[0071] In some embodiments, the immune cells comprise a nucleotide sequence encoding the shRNA. In some embodiments, the immune cells contain more than one shRNA-encoding nucleotide sequence. In some embodiments, the immune cells contain nucleic acids encoding two shRNAs. Contains an octide sequence.

[0072] In some embodiments, the nucleotide sequence encoding the shRNA is SEQ ID NO:2- 219 and 238-267.

[0073] In some embodiments, the nucleotide sequence encoding the shRNA is on a vector. do.

[0074] In some embodiments, the expression of different shRNAs is driven by different promoters. In some embodiments, the expression of two different shRNAs is regulated by two different In some embodiments, the two promoters are regulated by different promoters. In some embodiments, the promoter is an RNA polymerase III promoter. In some embodiments, the two promoters are U6 promoters. In some embodiments, the two promoters are from different species. For example, in certain embodiments, the promoters are oriented head-to-head. In another embodiment, the promoter is oriented in an end-to-end direction. do.

[0075] In some embodiments, the engineered antigen receptor and the gene disrupting agent each comprise a vector. In some embodiments, engineered antigen receptors and The gene disruptor is expressed from the same vector.

[0076] In some embodiments, the vector is a plasmid vector or a viral vector. In some embodiments, the viral vector is a lentiviral vector or an adenoviral vector. In some embodiments, the lentiviral vector is a retroviral vector. It is an illus vector.

[0077] In some embodiments, the immune cells consist of T cells and natural killer (NK) cells. In some embodiments, the immune cell is a T cell. In embodiments, the T cells are CD4+ T cells or CD8+ T cells.

[0078] In some embodiments, the immune cells encode two shRNAs and one CAR. In some embodiments, the two shRNAs are contained on the same vector. Two different RNA polymerase III promoters oriented in different directions For example, in certain embodiments, the promoter is In another embodiment, the promoter is oriented in an end-to-end direction. In some embodiments, the CAR targets CD19 and the first shRNA is One shRNA targets PD-1, and the second shRNA targets TIGIT.

[0079] In another aspect, provided herein is a composition comprising the engineered immune cells. In one aspect, provided herein is a pharmaceutical comprising immune cells and a pharmaceutically acceptable carrier. It is a composition.

[0080] In another aspect, provided herein is a method for administering immune cells or In some embodiments, the method comprises administering a genetically engineered The engineered antigen receptor specifically binds to an antigen associated with a disease or condition. In some embodiments, the disease or condition is cancer or a tumor.

[0081] In another aspect, provided herein are immunosuppressants for use in treating a disease or condition. In another aspect, provided herein are immune cells or compositions for treating a disease or condition. The use of immune cells or compositions in the manufacture of a medicament for treating In an embodiment, the engineered antigen receptor specifically binds to an antigen associated with a disease or condition. In some embodiments, the disease or condition is cancer or a tumor. Further non-limiting embodiments are presented below. 1. A vector comprising: Two short hairpin Rs inhibit the expression of at least one gene that weakens immune cell function a base sequence encoding NA (shRNA); Encoding chimeric antigen receptors (CARs) or monoclonal T cell receptors (mTCRs) and a base sequence as set forth above. 2. The expression of the two types of shRNAs is controlled by two different promoters. 2. The vector of embodiment 1, wherein each of the vectors is regulated. 3. The embodiment in which the two promoters are promoters for RNA polymerase III A vector according to embodiment 2. 4. Embodiment 2, wherein the two promoters are U6 promoters from different species. The vector described in 5. An embodiment in which the two promoters are oriented in different directions on the vector A vector according to embodiment 2. 6. The gene that weakens the function of immune cells is an immune checkpoint receptor or ligand 2. The vector of embodiment 1, wherein the vector is a vector. 7. The immune checkpoint receptor or ligand is PD1, PD-L1, CT LA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEAC AM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 7. The vector of embodiment 6, wherein the vector is selected from the group consisting of CD96, MerTK, and 2B4. . 8. Genes that weaken immune cell function include FAS, CD45, PP2A, SHIP1, and S. HIP2, DGK alpha, DGK zeta, Cbl-b, CD147, LRR1, TGF BR1, IL10R alpha, KLGR1, DNMT3A and A2aR 2. The vector of embodiment 1, selected from the group consisting of: 9. The two types of shRNA target a single gene that weakens the function of immune cells, or or an embodiment in which the two shRNAs target different genes that attenuate immune cell function. The vector according to embodiment 1. 10. The vector of embodiment 1, wherein the two shRNAs target PD-1. 11. Of the two types of shRNA, (i) one shRNA targets PD-1; , the second shRNA targets TIM-3, or (ii) one shRNA is PD 1. The vector of embodiment 1, wherein the first shRNA targets TIGIT and the second shRNA targets TIGIT. Tar. 12. The base sequence encoding one of the two types of shRNA is A sequence selected from the group consisting of SEQ ID NOs: 2 to 219, encoding a second shRNA. In an embodiment, the base sequence includes a different sequence selected from the group consisting of SEQ ID NOs: 2 to 219. The vector according to embodiment 1. 13. The target of the CAR or mTCR is a cancer cell, cancer tissue, and / or tumor. It is a human tumor antigen that shows increased expression in the microenvironment, or in cancer cells, cancer tissues, and and / or the vector of embodiment 1, which is a mutated form of an antigen found in the tumor microenvironment. . 14. The method of claim 1, wherein the vector comprises the nucleic acid sequence of SEQ ID NO: 220 or 221. Vector of. 15. The vector is a plasmid vector, a lentivirus vector, an adenovirus vector, or vector, adeno-associated virus vector, or retroviral vector 1. The vector described in 1. 16. An immune cell comprising the vector of embodiment 1, wherein the expression of one or more genes is The immune cells, wherein the expression is reduced to 40% or less compared to that in the absence of shRNA. 17. The immune cells are human-derived T cells or natural killer (NK) cells. 17. An immune cell according to embodiment 16. 18. A pharmaceutical composition comprising an immune cell according to any one of embodiments 1 to 17. 19. A method for treating a patient in need of immunotherapy, wherein the immune cells are originally obtained from the patient. 19. The pharmaceutical composition of embodiment 18 for the treatment of 20. The patient is administered the target and mTCR of the CAR or mTCR expressed on the immune cell. and / or subjects having a tumor or cancer in which elevated or altered levels of said target are detected. 20. The pharmaceutical composition of Form 19. 21. An immune cell having a genetically engineered antigen receptor that specifically binds to a target antigen. or reducing the expression in said immune cells of genes that attenuate the function of said immune cells. and a gene disrupting agent capable of lowering the immune cell. 22. The engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor. 22. The immune cell of embodiment 21, which is a receptor (TCR). 23. The immune cell of embodiment 22, wherein the engineered antigen receptor is a CAR. . 24. The CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. 24. The immune cell of embodiment 23, comprising a transduction domain. 25. A method for specifically binding the extracellular antigen recognition domain of the CAR to the target antigen. 25. The immune cell of embodiment 24. 26. The intracellular signaling domain of the CAR is a CD3 zeta (CD3ζ) chain. 25. The immune cell of embodiment 24, comprising an intracellular domain. 27. An embodiment in which the intracellular signaling domain of the CAR further comprises a costimulatory molecule 27. The immune cell according to aspect 26. 28. The costimulatory molecule is ICOS, OX40, CD137 (4-1BB), CD27, and CD28. 29. The immune cell of embodiment 28, wherein the costimulatory molecule is CD137 (4-1BB). Cell. 30. The immune cell of embodiment 28, wherein the costimulatory molecule is CD28. 31. The immune cell of embodiment 22, wherein the engineered antigen receptor is a TCR. . 32. The immunogen of embodiment 31, wherein the TCR is a monoclonal TCR (mTCR). Epidemic cells. 33. The target antigen is detected in or on cancer cells, cancer tissues and / or tumor microenvironments. 33. The immune cell of embodiment 31 or 32, wherein the antigen is expressed on the surface of the immune cell. 34. The target antigen is 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AF P (α-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α-Methylacyl-coenzyme A racemase, ART-4 (ADP-ribosyltransferase ze-4), B7H4 (v-set domain-containing T-cell activation inhibitor 1), BAGE-1 (B melanoma antigen-1), BCL-2 (B-cell CLL / lymphoma-2), BING- 4 (WD repeat domain 46), CA15-3 / CA27-29 (mucin 1), CA19- 9 (cancer antigen 19-9), CA72-4 (cancer antigen 72-4), CA125 (cancer antigen 1 25), calreticulin, CAMEL (a CTL-recognized antigen on melanoma), CASP-8 (caspase 8), cathepsin B, cathepsin L, CD19 (cluster of differentiation 19), CD2 0, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56 , CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel accessory -2), CML28 (chronic myeloid leukemia tumor antigen 28), coactosin-like protein, cyclooxygenase-2 (COX-2), CT-9 / BRD6 ( Cancer / testis antigen 9), Cten (c-terminal tensin-like protein), cyclin B1, Icrin D1, cyp-B, CYPB1 (cytochrome p450 family 1 subfamily) Lee b member 1), DAM-10 / MAGE-B1 (melanoma-associated antigen B1), DA M-6 / MAGE-B2, EGFR / Her1 (epidermal growth factor receptor), EMMPRIN (basigin), EpCam, EphA2 (EPH receptor A2), EphA3, ErbB 3 (Erb-B2 receptor tyrosine kinase 3), EZH2 (zeste2 polycomb repression Enhancer of sex complex 2 subunit), FGF-5 (fibroblast growth factor 5), FN (fibronectin), Fra-1 (Fos-related antigen-1), G250 / CAIX (carbonate Dehydratase 9), GAGE-1 (G antigen-1), GAGE-2, GAGE-3, GAGE- 4. GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP (prostate differentially expressed genes), GnT-V (gluconate kinase), gp100 (metabolites), Glypican 3 (GPC3), helical antigen 100 (GP100), helical antigen 200 (HAGE), helical antigen 300 (HAGE), helical antigen 100 (HAGE), helical antigen 2 ... Hara), HAST-2 (sulfotransferase family 1A member 1), hepsin , Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV -K-MEL, HNE (Medullasin), Homeobox NKX 3.1, HOM-TE S-14 / SCP-1, HOM-TES-85, HPV-E6, HPVE7, HST-2 (Sirtuin-2), hTERT, iCE (caspase 1), IGF-1R (insulin -like growth factor-1 receptor), IL-13Ra2 (interleukin-13 receptor subunit α2), IL-2R (interleukin-2 receptor), IL-5 (interleukin- 5), immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0 205 (lysophosphatidylglycerol acyltransferase 1), KK-LC- 1 (Kitakyushu lung cancer antigen-1), KM-HN-1, LAGE-1 (L antigen family member) -1), Livin, MAGE-A1, MAGE-A10, MAGE-A12, MAGEA2 , MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1 , MAGE-B10, MAGE-B16, MAGEB17, MAGE-B2, MAGE- B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE- C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE- E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2 (melanoma antigen protease family L2), mammaglobin A, MART-1 / Melan-A (T-cell-1 melanoma antigen recognized by MC1), MART-2, matrix protein 22, MC1 R (melanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor), meso Thelin, MG50 / PXDN (peroxidasin), MMP11 (matrix metalloproteinase anhydrase 11), MN / CA IX-antigen (carbonic anhydrase 9), MRP-3 (multidrug resistance related protein-3), MUC1 (mucin 1), MUC2, NA88-A (VENT-like host Meobox 2 pseudogene 1), N-acetylglucosaminyltransferase-V, N eo-PAP (Neo-poly(A) polymerase), NGEP (a new enzyme expressed in the prostate) NMP22 (nuclear matrix protein 22), NPM / ALK (nucleophore Sumin), NSE (neuron-specific enolase), NY-ESO-1, NY-ESO- B, OA1 (osteoarthritis QTL1), OFA-iLRP (carcinoembryonic antigen immature laminin receptor protein) receptor protein), OGT (O-GlcNAc transferase), OS-9 (small Plasma lectin), osteocalcin, osteopontin, p15 (CDK inhibitor 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor) Prostate activator inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PART -1 (prostate androgen-regulated transcript 1), PATE (prostate and testis expressed 1) , PDEF (prostate-derived Ets factor), Pim-1-kinase (proviral integration site 1), Pin1 (peptidyl-prolyl cis-trans isomerase NIMA- Interaction 1), POTE (expressed in the prostate, ovary, testis, and placenta), PRAME ( antigens preferentially expressed in melanoma), prostein, proteinase-3, PSA ( Prostate-specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate-specific G-tan Protein-coupled receptor), PSM, PSMA (prostate-specific membrane antigen), RAGE-1 (renal tumor Carcinoma antigen), RHAMM / CD168, RU1 (renal ubiquitous protein 1), RU2, SAG E (sarcoma antigen), SART-1 (squamous cell carcinoma antigen recognized by T-cell-1) ), SART-2, SART-3, Sp17 (sperm protein 17), SSX-1 (SS X family member 1), SSX-2 / HOM-MEL-40, SSX-4, STAM P-1 (STEAP2 metalloreductase), STEAP, survivin, survivin- 213, TA-90 (tumor-associated antigen-90), TAG-72 (tumor-associated glycoprotein-7 2), TARP (TCRγ alternative reading frame protein), TGFb (transforming growth factor β), TGFbR11 (transforming growth factor β receptor 11), TGM-4 (transforming growth factor β receptor 11) Glutaminase 4), TRAG-3 (Taxol resistance-associated gene 3), TRG (T-cell Transient receptor potential-1 (TRP-1), TRP-2 / 6 b, TRP-2 / INT2, Trp-p8, tyrosinase, UPA (U-plasminogen activator) activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK-1, and 34. The immune cell of embodiment 33, selected from the group consisting of WT1 (Wilms' tumor 1). 35. The immune cell of embodiment 35, wherein the target antigen is CD19 or CD22. 36. The immune cell of embodiment 36, wherein the target antigen is CD19. 37. The target antigen is a cancer antigen, and the expression of the cancer antigen is detected in cancer cells, cancer tissues, and the like. Embodiment 3, wherein the antigen is a cancer antigen that is increased in or on the tissue and / or tumor microenvironment. Any of 4 to 36 immune cells. 38. The target antigen is α-actinin-4 / m, ARTC1 / m, bcr / abl, beta-catenin / m, BRCA1 / m, BRCA2 / m, CASP-5 / m, CASP -8 / m, CDC27 / m, CDK4 / m, CDKN2A / m, CML66, COA-1 / m, DEK-CAN, EFTUD2 / m, ELF2 / m, ETV6-AML1, FN1 / m, GPNMB / m, HLA-A*0201-R170I, HLA-A11 / m, HL A-A2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LDLR- FUT, MART2 / m, ME1 / m, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class 1 / m, neo-PAP / m, NFYC / m, N-Ras / m, OG T / m, OS-9 / m, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, T EL-AML1, TGFbRII, and TPI / m, wherein The target antigen is a cancer antigen, and the cancer antigen is a cancer cell, a cancer tissue, and / or a tumor microorganism. The immunization of embodiment 33, which is a mutated form of the antigen that is expressed in the environment or on a surface. Epidemic cells. 39. The expression of the gene that attenuates the function of the immune cell is selected from the following: (i) inhibiting the proliferation of said immune cells; (ii) inducing cell death of said immune cells; (iii) inhibiting the ability of said immune cells to recognize and / or be activated by said target antigen. , (iv) inducing differentiation of the immune cells into cells that do not induce an immune response against the target antigen. , (v) a decrease in the responsiveness of said immune cells to molecules that stimulate the immune response of said immune cells; and teeth (vi) increasing the response of the immune cells to molecules that suppress the immune response of the immune cells; The immune cell according to any one of embodiments 21 to 38, which causes the above. 40. The gene that weakens the function of the immune cells is PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5 ), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96 , MerTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGK Al Fa, DGK zeta, Cbl-b, Cbl-c, CD148, LRR1, TGFBR1, IL10RA, KLGR1, DNMT3A, and A2aR. 39. The immune cell of claim 39. 41. The gene that weakens the function of the immune cell suppresses the immune response of the immune cell. 40. The immune cell of embodiment 39, wherein the immune cell enhances a response to a molecule. 42. Before enhancing the response of the immune cell to a molecule that suppresses the immune response of the immune cell. 42. The method of claim 41, wherein the gene encodes a receptor or ligand of an immune checkpoint. Immunologic cells. 43. The immune checkpoint receptor or ligand is PD1, PD-L1, C TLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEA CAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160 , CD96, MerTK, and 2B4. cell. 44. The gene disrupting agent induces a proliferation of the immune cell, compared to the immune cell in the absence of the gene disrupting agent. The expression of a gene in the immune cell that attenuates the function of the immune cell is increased by at least 30, 40 , 50, 60, 70, 80, 90, or 95% reduction in the Any immune cell. 45. The gene disrupting agent is a gene disrupting agent for the immune cell that inhibits the immune response of the immune cell. 45. The immune cell of embodiment 44, wherein the expression of a gene that enhances the response of the cell is reduced. 46. ​​The gene disrupting agent encodes a receptor or ligand of an immune checkpoint. 46. ​​The immune cell of embodiment 45, wherein the expression of a gene is reduced. 47. The gene disrupting agent is selected from the group consisting of PD1, PD-L1, CTLA4, TIM3, and CEACA. M (CEACAM-1, CEACAM-3, or CEACAM-5), LAG3, VIS TA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B 47. The immune cell of embodiment 46, wherein the immune cell reduces expression of a gene selected from the group consisting of: 48. The gene disrupting agent weakens the function of the immune cells by RNA interference (RNAi). 48. The immune cell according to any one of embodiments 45 to 47, wherein the expression of the gene containing the gene is reduced. More than 49.1 gene disrupting agents induce the expression of the immune cells in the immune cells by RNAi. 49. The immune cell of embodiment 48, wherein the expression of said gene is reduced, which impairs the function of said immune cell. 50. The gene disrupting agent targets a single gene to attenuate the function of the immune cell. Alternatively, different gene disrupting agents may target different genes that impair the function of the immune cells, e.g. For example, a first gene disrupting agent targets a first gene, and a second gene disrupting agent targets a second gene. 50. The immune cell of embodiment 49, which targets 51. Embodiments 48 to 5, wherein the RNAi is mediated by short hairpin RNA (shRNA). 10. An immune cell according to any one of claims 1 to 9. 52. The immune cell of embodiment 51, wherein the RNAi is mediated by more than one shRNA. Cell. 53. The immune cell of embodiment 52, wherein the RNAi is mediated by two shRNAs. 54. Any of embodiments 52-53, wherein two shRNAs target PD-1. immune cells. 55. The first shRNA targets PD-1 and the second shRNA targets TIM-3. The immune cell according to any one of embodiments 52 to 53. 56. One shRNA targets PD-1 and the second shRNA targets CTLA-4. The immune cells according to any one of embodiments 52 to 53, which are targeted. 57. The first shRNA targets PD-1 and the second shRNA targets LAG-3. The immune cell according to any one of embodiments 52 to 53. 58. The first shRNA targets PD-1 and the second shRNA targets TIGIT. The immune cell according to any one of embodiments 52 to 53. 59. Embodiments 51 to 59, wherein the immune cells comprise a nucleotide sequence encoding an shRNA. 58. An immune cell according to any one of 58. 60. The method according to claim 1, wherein the immune cells comprise a nucleotide sequence encoding more than one shRNA. 60. The immune cell of claim 59. 61. The embodiment in which the immune cells comprise nucleotide sequences encoding two shRNAs 59. An immune cell according to 59. 62. The nucleotide sequence encoding the shRNA(s) is selected from SEQ ID NOs: 2 to 62. 219 and 238-267. 62. The immune cell according to any one of 9 to 61. 63. The nucleotide sequence encoding the shRNA(s) is / are present on a vector. The immune cell according to any one of embodiments 59 to 62. 64. Embodiment 63, in which the expression of different shRNAs is regulated by different promoters. The immune cell according to any one of the preceding claims. 65. Expression of two different shRNAs is regulated by two different promoters An immune cell described in embodiment 64. 66. The two different promoters are promoters for RNA polymerase III. 66. The immune cell of embodiment 65. 67. The method of embodiment 66, wherein the two different promoters are U6 promoters. immune cells. 68. The immune cell of embodiment 67, wherein the U6 promoter is derived from a different species. 69. The method of any one of embodiments 65 to 68, wherein the two promoters are oriented in different directions. Any of the immune cells. 70. The engineered antigen receptor and the gene disrupting agent(s) are delivered from a vector. 70. The immune cell according to any one of embodiments 21 to 69, wherein each of the following is expressed: 71. The engineered antigen receptor and the gene disrupting agent(s) are / are contained in the same vector. 71. The immune cell of embodiment 70, wherein the immune cell is expressed from 72. Embodiment 7, wherein the vector is a plasmid vector or a viral vector. 72. The immune cell according to any one of 0 to 71. 73. The viral vector is a lentiviral vector, an adenoviral vector, or 73. The immune cell of embodiment 72, wherein the vector is an adeno-associated viral vector. 74. The method of embodiment 73, wherein the lentiviral vector is a retroviral vector. Immunologic cells. 75. The immune cells are selected from the group consisting of T cells and natural killer (NK) cells. The immune cell according to any one of embodiments 21 to 74. 76. The immune cell of embodiment 75, wherein the immune cell is a T cell. 77. The method of embodiment 76, wherein the T cells are CD4+ T cells or CD8+ T cells. immune cells. 78. The immune cells are transfected with two shRNAs and a nucleic acid encoding a CAR or mTCR. 78. The immune cell of any of embodiments 76 or 77, comprising a nucleotide sequence on the same vector. . 79. The two shRNAs are two different RNA polymerases oriented in different directions. 79. The immune cells of embodiment 78, each regulated by a promoter of amylase III. Cell. 80. The CAR targets CD19 and the first shRNA targets PD-1. 80. The immune cell of embodiment 79, wherein the second shRNA targets TIGIT. 81. A method for producing immune cells, comprising: (1) a gene encoding a genetically engineered antigen receptor that specifically binds to a target antigen; (2) a gene disrupting agent or a gene whose expression attenuates the function of said immune cell; or a gene disrupting agent capable of reducing or reducing expression in sequentially in any order into said immune cells, This results in the expression of a genetically engineered antigen receptor, which weakens the function of the immune cells. The method further comprises producing the immune cell in which expression of a gene is reduced. 82. The engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor. 82. The method of embodiment 81, wherein the receptor (TCR) is a TCR receptor. 83. The method of embodiment 82, wherein the engineered antigen receptor is a CAR. 84. The CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. 84. The method of embodiment 83, comprising a transduction domain. 85. A method for specifically binding the extracellular antigen recognition domain of the CAR to the target antigen. 85. The method of embodiment 84. 86. The intracellular signaling domain of the CAR is a CD3 zeta (CD3ζ) chain. 85. The method of embodiment 84, comprising an intracellular domain. 87. The embodiment wherein the intracellular signaling domain of the CAR further comprises a costimulatory molecule. 86. The method according to claim 86. 88. The costimulatory molecule is ICOS, OX40, CD137 (4-1BB), CD27, 88. The method of embodiment 87, wherein the antibody is selected from the group consisting of CD28 and CD28. 89. The method of embodiment 88, wherein the costimulatory molecule is CD137 (4-1BB). 90. The method of embodiment 88, wherein the costimulatory molecule is CD28. 91. The method of embodiment 82, wherein the engineered antigen receptor is a TCR. 92. The method of embodiment 91, wherein the TCR is a monoclonal TCR (mTCR). Law. 93. The target antigen is present in or on cancer cells, cancer tissues and / or the tumor microenvironment. 93. The method of any of embodiments 81-92, wherein the antibody is expressed on the surface. 94. The target antigen is 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AF P (α-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α-Methylacyl-coenzyme A racemase, ART-4 (ADP-ribosyltransferase ze-4), B7H4 (v-set domain-containing T-cell activation inhibitor 1), BAGE-1 (B melanoma antigen-1), BCL-2 (B-cell CLL / lymphoma-2), BING -4 (WD repeat domain 46), CA15-3 / CA27-29 (mucin 1), CA19 -9 (cancer antigen 19-9), CA72-4 (cancer antigen 72-4), CA125 (cancer antigen 125), calreticulin, CAMEL (CTL-recognized antigen on melanoma), CASP- 8 (caspase 8), cathepsin B, cathepsin L, CD19 (cluster of differentiation 19), CD 20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD5 6, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel accession number 2) Lee 2), CML28 (chronic myeloid leukemia tumor antigen 28), coactosin-like protein, Collagen XXIII, COX-2 (cyclooxygenase-2), CT-9 / BRD6 (cancer / testis antigen 9), Cten (c-terminal tensin-like protein), cyclin B1, Cyclin D1, cyp-B, CYPB1 (cytochrome p450 family 1 subfamily) Milli b member 1), DAM-10 / MAGE-B1 (melanoma-associated antigen B1), D AM-6 / MAGE-B2, EGFR / Her1 (epidermal growth factor receptor), EMMPRI N (basidin), EpCam, EphA2 (EPH receptor A2), EphA3, Erb B3 (Erb-B2 receptor tyrosine kinase 3), EZH2 (zeste2 polycomb repressor) Enhancer of regulatory complex 2 subunit), FGF-5 (fibroblast growth factor 5), F N (fibronectin), Fra-1 (Fos-related antigen-1), G250 / CAIX (charcoal Acid anhydrase 9), GAGE-1 (G antigen-1), GAGE-2, GAGE-3, GAGE -4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP (prostate GnT-V (gluconate kinase), gp100 (metabolite), and Raninocyte lineage-specific antigen GP100), GPC3 (glypican 3), HAGE (helix antigen), HAST-2 (sulfotransferase family 1A member 1), Hepsi Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HER VK-MEL, HNE (medullasin), Homeobox NKX 3.1, HOM-T ES-14 / SCP-1, HOM-TES-85, HPV-E6, HPVE7, HST- 2 (Sirtuin-2), hTERT, iCE (Caspase 1), IGF-1R (Insulin Interleukin-like growth factor-1 receptor), IL-13Ra2 (interleukin-13 receptor subunit IL-2R (interleukin-2 receptor), IL-5 (interleukin -5), immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA 0205 (lysophosphatidylglycerol acyltransferase 1), KK-LC -1 (Kitakyushu lung cancer antigen-1), KM-HN-1, LAGE-1 (L antigen family member -1), Livin, MAGE-A1, MAGE-A10, MAGE-A12, MAGEA 2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B 1, MAGE-B10, MAGE-B16, MAGEB17, MAGE-B2, MAGE -B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE -C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE -E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2 (melanoma antigen family L2), mammaglobin A, MART-1 / Melan-A (T-cell-1 melanoma antigen recognized by MC-1), MART-2, matrix protein 22, MC 1R (melanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor), Sothelin, MG50 / PXDN (peroxidasin), MMP11 (matrix metalloproteinase) Proteinase 11), MN / CA IX-antigen (carbonic anhydrase 9), MRP-3 (multidrug resistance) Sex-associated protein-3), MUC1 (mucin 1), MUC2, NA88-A (VENT-like Homeobox 2 pseudogene 1), N-acetylglucosaminyltransferase-V, Neo-PAP (Neo-poly(A) polymerase), NGEP (Neo-poly(A) polymerase expressed in the prostate) novel gene), NMP22 (nuclear matrix protein 22), NPM / ALK (nucleoprotein kinase C / KLK) phosmin), NSE (neuron-specific enolase), NY-ESO-1, NY-ESO -B, OA1 (osteoarthritis QTL1), OFA-iLRP (carcinoembryonic antigen immature lamina propria) receptor protein), OGT (O-GlcNAc transferase), OS-9 ( Endoplasmic reticulum lectin), osteocalcin, osteopontin, p15 (CDK inhibitor 2B) , p53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator I (PI-1) Prostatic acid phosphatase inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PAR T-1 (prostate androgen-regulated transcript 1), PATE (prostate and testis expressed 1 ), PDEF (prostate-derived Ets factor), Pim-1-kinase (proviral integration factor) site 1), Pin1 (peptidyl-prolyl cis-trans isomerase NIMA -Interaction 1), POTE (expressed in the prostate, ovary, testis, and placenta), PRAME (antigen preferentially expressed in melanoma), prostein, proteinase-3, PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate-specific G-tag Protein-coupled receptor), PSM, PSMA (prostate-specific membrane antigen), RAGE-1 (nephroma) tumor carcinoma antigen), RHAMM / CD168, RU1 (renal ubiquitous protein 1), RU2, SA GE (sarcoma antigen), SART-1 (squamous cell carcinoma antigen recognized by T-cell-1) SART-2, SART-3, Sp17 (sperm protein 17), SSX-1 (S SX family members 1), SSX-2 / HOM-MEL-40, SSX-4, STA MP-1 (STEAP2 metalloreductase), STEAP, survivin, survivin -213, TA-90 (tumor-associated antigen-90), TAG-72 (tumor-associated glycoprotein 72), TARP (TCRγ alternative reading frame protein), TGFb (transforming Transforming growth factor β), TGFbR11 (transforming growth factor β receptor 11), TGM-4 (transforming growth factor β receptor 11) Glutaminase 4), TRAG-3 (Taxol resistance-associated gene 3), TRG (T- TRP-1 (transient receptor potential-1), TRP-2 / 6b, TRP-2 / INT2, Trp-p8, tyrosinase, UPA (U-plasminogen activator) gene activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK-1, and 94. The method of embodiment 93, wherein the IL-11 nucleotide sequence is selected from the group consisting of WT1 (Wilms' tumor 1) and WT1 (Wilms' tumor 1). 95. The method of embodiment 95, wherein the target antigen is CD19 or CD22. 96. The method of embodiment 96, wherein the target antigen is CD19. 97. The target antigen is a cancer antigen, and the expression of the cancer antigen is detected in cancer cells and cancer tissues. and / or antigens increased in or on the surface of the tumor microenvironment. 7. A method according to any one of 6. 98. The target antigen is α-actinin-4 / m, ARTC1 / m, bcr / abl, beta-catenin / m, BRCA1 / m, BRCA2 / m, CASP-5 / m, CASP -8 / m, CDC27 / m, CDK4 / m, CDKN2A / m, CML66, COA-1 / m, DEK-CAN, EFTUD2 / m, ELF2 / m, ETV6-AML1, FN1 / m, GPNMB / m, HLA-A*0201-R170I, HLA-A11 / m, HL A-A2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LDLR- FUT, MART2 / m, ME1 / m, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class 1 / m, neo-PAP / m, NFYC / m, N-Ras / m, OG T / m, OS-9 / m, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, T EL-AML1, TGFbRII, and TPI / m, wherein The target antigen is a cancer antigen, and the cancer antigen is a cancer cell, a cancer tissue, and / or a tumor microorganism. The method of embodiment 93, which is a mutated form of the antigen that is expressed in the environment or on a surface. Law. 99. The expression of the gene that weakens the function of the immune cell is one of the following: (i) inhibiting the proliferation of said immune cells; (ii) inducing cell death of said immune cells; (iii) inhibiting the ability of said immune cells to recognize and / or be activated by said target antigen. , (iv) inducing differentiation of the immune cells into cells that do not induce an immune response against the target antigen. , (v) a decrease in the responsiveness of said immune cells to molecules that stimulate the immune response of said immune cells; and teeth (vi) increasing the response of the immune cells to molecules that suppress the immune response of the immune cells; The method according to any one of embodiments 81 to 98, wherein the method causes the above. 100. The gene that weakens the function of the immune cells is PD1, PD-L1, or CTLA4 , TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM- 5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD9 6, MerTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGK a Lufa, DGK zeta, Cbl-b, Cbl-c, CD148, LRR1, TGFBR1 , IL10RA, KLGR1, DNMT3A, and A2aR. 99. The method of claim 99. 101. The gene that weakens the function of the immune cell suppresses the immune response of the immune cell. 100. The method of embodiment 99, wherein the immune cell is stimulated to react with a molecule that stimulates the immune cell to react with the molecule. 102. Enhancement of the response of the immune cells to molecules that suppress the immune response of the immune cells. embodiment 101, wherein the gene encodes a receptor or ligand for an immune checkpoint. The method described below. 103. The immune checkpoint receptor or ligand is PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CE ACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD16 0, CD96, MerTK and 2B4 method. 104. The gene disrupting agent is a gene disrupting agent that inhibits the immune cell in the absence of the gene disrupting agent(s). at least one gene in the immune cell that weakens the function of the immune cell, compared to 30, 40, 50, 60, 70, 80, 90, or 95% reduction in embodiment 81-1 03. A method according to any one of the preceding paragraphs. 105. The gene disrupting agent is a gene disrupting agent for the immune cell that inhibits the immune response of the immune cell. 105. The method of embodiment 104, wherein the expression of a gene that enhances immune cell response is reduced. 106. The gene disrupting agent encodes a receptor or ligand of an immune checkpoint. 106. The method of embodiment 105, wherein the expression of a gene is reduced. 107. The gene disrupting agent is selected from the group consisting of PD1, PD-L1, CTLA4, TIM3, and CEAC. AM (CEACAM-1, CEACAM-3, or CEACAM-5), LAG3, VI STA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2 107. The method of embodiment 106, wherein the expression of a gene selected from the group consisting of: B4 is reduced. 108. The gene disrupting agent inhibits the function of the immune cells by RNA interference (RNAi). 108. The method of any of embodiments 105-107, wherein the expression of said gene that is attenuated is reduced. More than 109.1 gene disrupting agents induce the immune response in the immune cells by RNAi. 109. The method of embodiment 108, wherein the expression of said gene is reduced, which impairs the function of the cell. 110. The gene disrupting agent targets a single gene to attenuate the function of the immune cell. Alternatively, the first gene disrupting agent may target a different gene that attenuates the function of the immune cell, a first gene disrupting agent targets a second gene, and a second gene disrupting agent targets a third gene, or 110. The method of embodiment 109, wherein any combination of 111. Embodiment 108, wherein the RNAi is mediated by short hairpin RNA (shRNA). A method according to any one of claims 1 to 110. 112. The method of embodiment 111, wherein the RNAi is mediated by more than one shRNA. Law. 113. The method of embodiment 112, wherein said RNAi is mediated by two shRNAs. 114. The method of embodiment 112 or 113, wherein two shRNAs target PD-1. How to do it. 115. The first shRNA targets PD-1 and the second shRNA targets TIM-3. 114. The method of embodiment 112 or 113, wherein 116. The first shRNA targets PD-1 and the second shRNA targets CTLA-4. The method of embodiment 112 or 113, wherein the targeting 117. The first shRNA targets PD-1 and the second shRNA targets LAG-3. 114. The method of embodiment 112 or 113, wherein 118. The first shRNA targets PD-1 and the second shRNA targets TIGIT. 114. The method of embodiment 112 or 113, wherein 119. Embodiment 11, wherein the immune cells comprise a nucleotide sequence encoding an shRNA. 118. A method according to any one of 1 to 118. 120. The immune cells are a method for detecting a subject's immune system comprising administering to the subject a method for detecting a subject's immune system the ... 120. The method of embodiment 119. 121. An embodiment in which the immune cells contain nucleotide sequences encoding two shRNAs. 119. The method according to claim 119. 122. The nucleotide sequence encoding the shRNA(s) is SEQ ID NO: 2 Embodiments 119-12 comprise a sequence selected from the group consisting of 219 and 238-267 1. The method according to any one of claims 1 to 9. 123. A method for producing a shRNA, wherein the nucleotide sequence encoding the shRNA is present on a vector. The method according to any one of embodiments 119 to 122. 124. Experiments in which the expression of different shRNAs is regulated by different promoters 124. The method of any one of embodiments 123. 125. Two different shRNAs were expressed by two different promoters. The method of embodiment 124, wherein the modulation is 126. The two different promoters are RNA polymerase III promoters. 126. The method of embodiment 125. 127. The method of embodiment 126, wherein the two promoters are U6 promoters. Law. 128. The method of embodiment 127, wherein the U6 promoter is derived from a different species. 129. Embodiments 125-126, in which the two promoters are oriented in different directions from each other. 28. A method according to any one of claims 1 to 28. 130. The engineered antigen receptor and the gene disrupting agent(s) are vectors. 130. The method of any of embodiments 81-129, wherein each of the following is expressed by: 131. The engineered antigen receptor and the gene disrupting agent(s) are in the same vector. The method of embodiment 130, wherein the gene is expressed from a target gene. 132. An embodiment in which the vector is a plasmid vector or a viral vector. 130. A method according to any one of 130 to 131. 133. The viral vector is a lentiviral vector, an adenoviral vector, or or an adeno-associated virus vector. 134. Embodiment 133, wherein the lentiviral vector is a retroviral vector. The method described below. 135. The immune cells are selected from the group consisting of T cells and natural killer (NK) cells. The method according to any one of embodiments 81 to 134. 136. The method of embodiment 135, wherein the immune cells are T cells. 137. The method of embodiment 136, wherein the T cells are CD4+ T cells or CD8+ T cells. How to post. 138. The immune cells contain a nucleotide sequence encoding two shRNAs and a CAR. 138. The method of any of embodiments 136 or 137, comprising on the same vector. 139. The two shRNAs are two different RNA fragments oriented in different directions. 139. The method of embodiment 138, wherein each of the two is regulated by a promoter for ribosomal enzyme III. Law. 140. The CAR targets CD19 and the first shRNA targets PD-1. 140. The method of embodiment 139, wherein said second shRNA targets TIGIT. 141. A composition comprising immune cells according to any of embodiments 21 to 80. 142. A method for producing a human immunodeficiency virus (HIV)-infected human HIV-1 virus comprising administering to a subject an immunodeficiency virus (HIV)-infected human HIV-1 virus, an immune cell, or a human HIV-1 virus, according to any one of embodiments 21 to 80, and a pharmaceutically acceptable carrier. 10. A pharmaceutical composition comprising: 143. A subject having a disease or condition requiring immunotherapy is administered any of the methods of embodiments 21 to 80. administering the immune cells of any of the above or the composition of any of the above embodiments 141 or 142. A treatment method comprising: 144. The engineered antigen receptor binds to an antigen associated with the disease or condition. 144. The method of embodiment 143, wherein the specific binding 145. Embodiment 143 or 14, wherein the disease or condition is cancer, e.g., a tumor. 4. The method described in 4. 146. A compound according to any one of embodiments 21 to 80 for use in treating a disease or condition. or the composition according to embodiments 141-142. 147. Any of embodiments 21 to 80 in the manufacture of a medicament for treating a disease or condition. Use of any of the immune cells or compositions according to embodiments 121-122. 148. The engineered antigen receptor binds to an antigen associated with the disease or condition. The immune cell or composition of embodiment 146 or embodiment 147 specifically binds Use as described in. 149. The method of embodiment 147 or embodiment 148, wherein the disease or condition is cancer, for example, a tumor. The use, composition, or immune cell according to embodiment 148. [Brief explanation of the drawings]

[0082] [Figure 1A] Generation of cell-specific PD-1-blocked CAR-T cells. Schematic of a two-in-one CAR vector. [Figure 1B] Generation of cell-intrinsic PD-1-blocked CAR-T cells. Expression of LNGFR and CAR was analyzed 4 days after transduction. [Figure 1C] Generation of cell-intrinsic PD-1-blocked CAR-T cells. Expression of LNGFR and CAR was analyzed 4 days after transduction. [Figure 1D] Generation of cell-specific PD-1-blocked CAR-T cells. CAR-T cells were sorted using LNGFR magnetic beads and seeded at 2 x 105 cells / ml. The number of accumulated CAR-T cells was assessed by trypan blue staining. [Figure 1E] Generation of cell-intrinsic PD-1-blocked CAR-T cells. LNGFR+ CAR-T cells were mixed with γ-irradiated NALM-6 without exogenous cytokines. [Figure 1F] One of the three PD-1-targeting shRNA candidates, shPD-1#1, did not affect the differentiation status (CCR7 / CD45RA) or CD4 / CD8 composition of CAR-T cells expressing the shRNA.

[0083] [Figure 2] FIG. 1 shows the effect of Pol III promoter type on cell-intrinsic PD-1 blockade.

[0084] [Figure 3A] In vitro cytotoxicity and proliferation under CD19 and PD-L1 stimulation with PD-1 blockade. LNGFR+CAR-T cells were mixed with live NALM-6 or NALM-6-PDL1 cells at E:T ratios of 1:1, 0.3:1, and 0.1:1. [Figure 3B] In vitro cytotoxicity and proliferation under CD19 and PD-L1 stimulation with PD-1 blockade. LNGFR+CAR-T cells were mixed with γ-irradiated NALM-6-PDL1, NALM-6-PDL1-CD80, or K562-CD19-PDL1 at a 1:1 E:T ratio without exogenous cytokines. [Figure 3C]Measurement of the expression level of CD80, a representative costimulatory ligand, on target cells showed that K562-CD19 cells express CD80, but Nalm-6 cells do not.

[0085] [Figure 4] FIG. 1 shows the in vivo antitumor function of CAR-T cells by cell-intrinsic PD-1 blockade.

[0086] [Figure 5A] FIG. 1 shows a decrease in in vivo cytokine production upon disruption of cell-intrinsic PD-1 in CAR-T cells. [Figure 5B] FIG. 1 shows that cell-intrinsic PD-1 disruption delays in vivo expansion of CAR-T cells.

[0087] [Figure 6A] Function of CD28 / CD3ζ or 4-1BB / CD3ζ CAR-T cells in cell-intrinsic PD-1 ablation. Schematic diagram of G28z, GBBz, P28z, and PBBz vectors. [Figure 6B] Figure 1 shows the function of CD28 / CD3ζ or 4-1BB / CD3ζ CAR-T cells in cell-intrinsic PD-1 ablation. Figure 2 shows flow cytometry analysis demonstrating LNGFR expression in transduced T cells 4 days after transduction.

[0088] [Figure 7A] PD-1 expression levels upon costimulation with CD28 or 4-1BB. LNGFR+CAR-T cells were incubated with γ-irradiated NALM-6 or K562-CD19 without exogenous cytokines. After 3 days of incubation, PD-1 expression on LNGFR+CAR-T cells was analyzed. [Figure 7B] PD-1 expression levels upon costimulation with CD28 or 4-1BB. Quantitative real-time PCR performed using PD-1 primers.

[0089] [Figure 8A]Establishment of NFAT or NF-κB reporter systems. Schematic diagram of the NFAT-RE3×-eGFP and NF-κB-RE5×-eGFR reporter vectors. [Figure 8B] Establishment of a reporter system for NFAT or NF-κB. Figure 1 shows the fold change in reporter activity calculated using eGFP gMFI in LNGFR+CART cells.

[0090] [Figure 9A] CD28 costimulation, but not 4-1BB costimulation, activated NFAT signaling. Reporter-transduced T cells were restimulated and transduced with G28z or GBBz. [Figure 9B] CD28 but not 4-1BB costimulation activated NFAT signaling. mRNA levels of NFAT target genes in CAR T cells were assessed by qPCR.

[0091] [Figure 10] FIG. 1 shows NF-κB signaling activated by co-stimulation with both CD28 and 4-1BB.

[0092] [Figure 11A] The TGF-β signaling intensity of G28z CART is slightly higher than that of BBz CART. After 4 or 24 hours of incubation with NALM-6 at a 1:1 E:T cell ratio, phosphorylated SMAD2 / 3 in CAR-T cells was analyzed by intracellular flow cytometry. [Figure 11B] The TGF-β signaling strength of G28z CART is slightly higher than that of BBz CART. Flow cytometry analysis showing PD-1 expression in G28z and GBBz CAR T cells with 10 ng / ml recombinant human TGF-β1. [Figure 11C] The TGF-β signaling strength of G28z CART is slightly higher than that of BBz CART. The mRNA levels of TGF-β1, TGFBR1, and TGFBR2 in CAR T cells were assessed by qPCR.

[0093] [Figure 12A] Figure 1 shows the retained cytotoxicity and proliferation potential of PBBz CAR T cells in vitro under repeated stimulation with CD19 and PD-L1. Cytotoxicity. (Top) After magnetic selection of LNGFR, day 12 primary LNGFR+ CAR T cells were mixed with NALM-6-PDL1 at E:T ratios of 1:1, 0.3:1, and 0.1:1. (Bottom) LNGFR+ CAR T cells were stimulated with γ-irradiated K562-CD19-PDL1 at an E:T ratio of 1:1. [Figure 12B] Figure 1 shows the retained in vitro cytotoxicity and proliferative capacity of PBBz CAR T cells under repeated stimulation with CD19 and PD-L1. LNGFR+CAR T cells were repeatedly stimulated with γ-irradiated NALM-6-PDL1-CD80 or K562-CD19-PDL1 at a 1:1 E:T ratio without exogenous cytokines.

[0094] [Figure 13A] Figure 1 shows that CAR-derived regulatory T cells are less sensitive to TGF-β-mediated dysfunction in PBBz CAR T cells and are under-produced in vitro. Figure 2 shows TGF-beta-mediated suppression of CAR-T proliferation. [Figure 13B] Figure 1 shows the in vitro generation of CAR-derived regulatory T cells with reduced sensitivity to TGF-beta-mediated dysfunction in PBBz CAR T cells. [Figure 13C] Figure 1 shows the in vitro generation of CAR-derived regulatory T cells, which are less sensitive to TGF-β-mediated dysfunction in PBBz CAR T cells. Figure 2 shows the effect of PDL1 / PD-1 on Treg induction.

[0095] [Figure 14] Figure 1 shows sustained inhibition of leukemia progression by PBBz CAR T cells.

[0096] [Figure 15] This is a diagram of the configuration of two vectors encoding two types of shRNA, one that inhibits PD-1 expression and the other that inhibits TIM-3 expression, and a CD19 CAR expression cassette.

[0097] [Figure 16] FIG. 1 shows the process of CAR-T cell preparation. ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6 cells and ΔLNGFR-CART19 / shTIM-3-mU6←→hU6-shPD-1 cells were prepared and isolated as described herein.

[0098] [Figure 17] This figure shows flow cytometry data from CAR-T cells containing the vectors shown in Figure 15 (ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6 cells and ΔLNGFR-CART19 / shTIM-3-mU6←→hU6-shPD-1 cells).

[0099] [Figure 18A] FIG. 10 shows flow cytometry data for ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6 cells and ΔLNGFR-CART19 / shTIM-3-mU6←→hU6-shPD-1 cells generated using the method of Example 8. [Figure 18B] FIG. 1 shows the expression of PD-1 and TIM-3 in CAR-T cells.

[0100] [Figure 19] This is flow cytometry data showing that CAR-T cells were repeatedly stimulated with target cells, and then the degree of cell differentiation was confirmed using antibodies against CD45RA and CCR7.

[0101] [Figure 20A] 1 shows an evaluation of CAR-T cells generated using the method of Example 8. FIG. 2 shows transduction efficiency. [Figure 20B] 1 shows an evaluation of CAR-T cells prepared using the method of Example 8. FIG. 1 shows proliferation ability. [Figure 20C] 1 shows an evaluation of CAR-T cells generated using the method of Example 8. FIG. 1 shows the survival rate.

[0102] [Figure 21A] Selection of shRNAs targeting CTLA-4, LAG-3, TIGIT, and TIM-3 is shown. T cells stimulated with CD3 / CD28 for two days were electroporated with 21-nt siRNAs targeting CTLA-4, LAG-3, TIGIT, and TIM-3. Two days after transfection, the siRNA-mediated knockdown efficiency was confirmed. Shading: Based on the sequences of the selected siRNAs, a dual-function vector expressing CAR and shRNA was constructed. Shading indicates the initially selected siRNAs. [Figure 21B] Selection of shRNAs targeting CTLA-4, LAG-3, TIGIT, and TIM-3 is shown. T cells were transduced with dual-function vectors containing shRNA for CTLA-4, LAG-3, TIGIT, or TIM-3 and selected with LNGFR magnetic beads. After seeding at 2x105 cells / ml, the number of LNGFR+CAR T cells was measured every 3 days. [Figure 21C] Figure 1 shows a selection of shRNAs targeting CTLA-4, LAG-3, TIGIT, and TIM-3. Figure 2 shows Tim3 expression (%).

[0103] [Figure 22A] Figure 1 shows the generation of dual immune checkpoint-disrupted CAR T cells. Schematic diagram of a dual vector with two functions in one. [Figure 22B] Figure 1 shows generation of dual immune checkpoint-disrupted CAR T cells. Four days after dual transduction, the % LNGFR+ T cells were analyzed. [Figure 22C]Figure 1 shows the generation of dual immune checkpoint-disrupted CAR T cells. Dual KD (knockdown) CAR-T cells were sorted and seeded at 2x105 / ml. The number of accumulated CAR T cells was assessed by trypan blue staining. [Figure 22D] Generation of dual immune checkpoint-disrupted CAR T cells. LNGFR+CAR T cells were analyzed for LAG-4, PD-1, TIGIT, or TIM-3 expression on day 3 after co-culture with γ-irradiated NALM-6 or K562-CD19. CTLA-4 expression was analyzed by intracellular flow cytometry. [Figure 22E] Generation of dual immune checkpoint-disrupted CAR T cells. LNGFR+CAR-T cells were analyzed for LAG-4, PD-1, TIGIT, or TIM-3 expression on day 3 after co-culture with γ-irradiated NALM-6 or K562-CD19. CTLA-4 expression was analyzed by intracellular flow cytometry.

[0104] [Figure 23] FIG. 1 shows the in vivo treatment of CD19+ blood cancers with dual KD CAR-T cells targeting two immune checkpoints.

[0105] [Figure 24] FIG. 1 shows the treatment of cancer in solid tumor models with dual-KD CAR-T cells targeting PD-1 and TIGIT. DETAILED DESCRIPTION OF THE INVENTION

[0106] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding will be obtained from the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized. For a complete understanding of the disclosure set forth herein, reference may be made to: To facilitate clarity, a number of terms are defined below.

[0107] Briefly, in one aspect, disclosed herein are immune checkpoint receptors. Two types of antibodies that inhibit the expression of one or more genes that impair the function of immune cells, including their receptors and ligands. Short hairpin RNA (shRNA) coding sequence and chimeric antigen receptor (CAR) or T cell receptors (TCRs), such as monoclonal T cell receptors (mTCRs). a vector containing a base sequence encoding an antigen receptor; a gene that specifically binds to a target antigen; Genetically engineered antigen receptors and a gene or genes that weaken immune cell function one or more genes that decrease or can decrease expression in immune cells of Immune cells comprising a destructive agent; a method for producing immune cells; e.g., for immunotherapy of human patients a composition or pharmaceutical composition comprising the immune cells; and administering the immune cells to a subject having a disease or condition. The immune cells, composition, or pharmaceutical composition may be administered to one or more These include genetic disruptors, such as those activated by cancer cells and disrupting immune cell function. Two shRs that reduce the expression of genes for two immune checkpoint molecules that can be attenuated These genes encode NAs, so the potential for these effects may be due to the use of separate inhibitors against these genes. Eliminate serious systemic adverse reactions such as certain cytokine release syndromes and autoimmune conditions This will also alleviate the burden of increased treatment costs arising from expensive concurrent treatments. This provides a more effective cell therapy than expression of a single shRNA while reducing the risk of developing a new gene.

[0108] 1.General techniques The techniques and procedures described or referenced herein are well known to those skilled in the art. well understood and / or commonly employed using conventional methods, See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual(4th ed.2012);Current P rotocols in Molecular Biology(Ausubel et al. al.eds.,2003);Therapeutic Monoclonal An tibodies:From Bench to Clinic(An ed.2009 );Monoclonal Antibodies:Methods and Prot ocols(Albitar ed.2010); and Antibody Engine ering,Vols.1 and 2 (Kontermann and Dubel eds.,2nd ed.2010).Molecular Biology of A widely used method described in the Cell (6th Ed., 2014) This includes the law.

[0109] 2.Definition Unless otherwise specified, all technical and scientific terms used herein are understood by those skilled in the art. For purposes of interpreting this specification, the term The following explanations apply and wherever appropriate, terms used in the singular also include the plural: All patents, applications, published applications and other publications are incorporated by reference. The disclosure of any of the terms mentioned is incorporated herein by reference. Therefore, in the event of a conflict with any document incorporated herein, the use set forth below shall prevail. The description in the text shall take precedence.

[0110] The terms used in this disclosure are used only to describe particular embodiments and are not intended to limit the scope of the present invention. The singular expressions "a," "an," and "the" are not intended to limit the scope of the invention unless clearly indicated by the context. The present invention is not limited to the specific methods, protocols, and methods described herein. It should be understood that the present specification is not limited to the above and may vary depending on the reagents and the like. The terminology used in this document is for the purpose of describing particular embodiments only and They are not intended to limit the scope of the invention, which is defined solely by the claims.

[0111] As used herein, the articles "a," "an," and "the" refer to: To refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an element" refers to one element or one It means the element that transcends.

[0112] The use of alternatives (e.g., "or") means one, both, or any of the alternatives. It should be understood to mean any combination of:

[0113] The term "and / or" means either one or both of the alternatives. It should be understood as follows.

[0114] As used herein, the terms "about" or "approximately" refer to a reference amount, level, or ,Value, number, frequency, percentage, dimension, size, amount, weight, or length, 15% , fluctuate by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% Indicates the amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the term "about" or "approximately" refers to a reference amount, level, value, or number. ,±15%,± for frequency, percentage, dimension, size, amount, weight, or length. 10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ± 1% of the amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length It refers to the range of size.

[0115] Throughout this specification, the terms "one embodiment" and "one embodiment" are used interchangeably. "an embodiment," "a particular embodiment," "a particular embodiment," "related embodiment," "a particular embodiment," in embodiment), "additional embodiments," or "further embodiments" ", or combinations thereof, etc., are incorporated herein by reference in their entirety. that a certain feature, structure, or characteristic is included in at least one embodiment of the present invention. Therefore, the above phrases appearing in various places throughout this specification necessarily Furthermore, the specific features, structures, or The features may be combined in any suitable manner in one or more embodiments.

[0116] A "construct" is a polynucleoside that is delivered to a target cell either in vitro or in vivo. As used herein, "vector" refers to a macromolecule or complex of molecules that contains a vector. , any nucleic acid construct capable of directing the delivery or transfer of foreign genetic material into a target cell " refers to a gene that can be replicated and / or expressed therein." The term "vector" as used herein includes a delivery construct. can be a linear or circular molecule. Vectors can be integrating or non-integrating. The main types of vectors are plasmids, episomal vectors, and vector-specific vectors. Examples of vectors include, but are not limited to, virus vectors, cosmids, and artificial chromosomes. Viral vectors include adenovirus vectors, adeno-associated virus vectors, and retrovirus vectors. Examples include virus vectors, lentivirus vectors, and Sendai virus vectors. However, the present invention is not limited to these.

[0117] The "two-in-one vectors" as described herein are capable of transducing immune cells One or more short hairpins that inhibit the expression of a gene or genes, impairing their function The base sequence encoding the RNA (shRNA) and the chimeric antigen receptor (CAR) or T cell a salt encoding a T cell receptor (TCR), e.g., a monoclonal T cell receptor (mTCR) A "two-in-one" vector as described herein is a vector containing a base sequence. A "dual vector" contains two types of short hairpins that block the expression of genes that weaken the function of immune cells. The base sequence encoding shRNA and the chimeric antigen receptor (CAR) and T any one of the monoclonal T cell receptors (TCR), e.g., monoclonal T cell receptors (mTCR) and a vector containing a base sequence encoding one of the two described herein. A dual vector with two functions is a vector with two functions in one.

[0118] "RNAi" (post-transcriptional gene silencing (PTGS), quelling, or co-suppression) (also known as ribosomal transcription) is a process in which an RNA molecule typically causes the destruction of a specific mRNA molecule. It is a process of post-transcriptional gene silencing that inhibits gene expression in a sequence-specific manner. The active component of RNAi is a short duplex called small interfering RNA (siRNA). It is a double stranded RNA (dsRNA), which is usually 15-30 nucleotides (e.g., 19- 25, 19-24, or 19-21 nucleotides) and 2 nucleotides 3' over These short RNA species contain hangovers and match the nucleic acid sequence of the target gene. It may also be generated naturally in vivo by Dicer-mediated cleavage of large dsRNA. , which are functional in mammalian cells. DNA expression plasmids were used to express the siRs of the present disclosure. Stably expressing NA duplexes or dsRNA in cells for long-term inhibition of target gene expression In one embodiment, the sense and antisense strands of the siRNA duplex are A short hairpin RNA (shRNA) is a RNA that induces the expression of a stem-loop structure. The hairpin is connected by a short spacer sequence. The hairpin is recognized and cleaved by Dicer. Thus, mature siRNA molecules are generated.

[0119] The term "shRNA" refers to a gene that contains several self-complementary sequences bound together in a tightly coupled chain with its stem. The RNA molecule that creates the pin structure is approximately 80 bp in length. Once expressed in cells, shRNA is processed through a series of steps to It becomes a small interfering RNA (siRNA) that acts as a silencing guide. In other words, when shRNA is expressed, it is processed by the Drosha complex in the cell and then transduced into proteins. The resulting shRNA is then transported out of the nucleus, where it is further processed by Dicer. This becomes siRNA, which then becomes single-stranded and is then inserted into the RISC (RNA-induced silencing complex). The antisense strand of the siRNA is then loaded by the RISC complex. It acts as a guide for attaching to the mRNA of the target gene, and R that is attached in this way When the ISC complex cleaves mRNA, gene silencing occurs. shRNAs enable sustained and specific gene silencing of specific genes. and is included in a vector for the purpose of inhibiting a target gene.

[0120] The term "promoter" refers to the upstream region of a gene that is involved in initiating transcription of the gene. The two types of shRNA mentioned above also regulate the expression of the promoter. The expression of hRNAs is regulated by two different promoters. If there is cloning with the same base sequence using repeated inserts, If this happens, proper cloning will not occur due to the binding between these identical base sequences, resulting in recombination or It is highly likely that a deletion or deletion will occur. Depending on which promoter binds and initiates transcription, the promoter is a promoter for RNA polymerase I. -, RNA polymerase II promoter or RNA polymerase III promoter The two promoters mentioned above can be RNA polymerase II promoters. It may be characterized as being a promoter of pol III (hereinafter referred to as pol III promoter). Pol III promoters can be used without a 5' cap or without adding a poly(A) tail to the 3' end of the RNA transcribed under the control of the promoter. It can be engineered to transcribe precisely from the 5' to the 3' end. The types of promoters include the U6 promoter, H1 promoter, and 7SK promoter. Examples include, but are not limited to:

[0121] As used herein, the term "G28z" refers to a vector containing an shGFP expression cassette and a CD2 This refers to a construct containing the CD3ζ domain and the 8 costimulatory domain (Figure 6A). In the term "G28z," "G" represents shGFP; "28" represents CD28. "z" represents CD3ζ. Following the same pattern, "P28" as used herein The term "z" refers to the combination of the shPD-1 expression cassette, the CD28 costimulatory domain, and the CD3ζ domain (Figure 6A), "P" represents shPD-1; "28" represents " "z" represents CD3ζ. As used herein, the term "GBBz" refers to The term refers to a shGFP expression cassette, a 4-1BB costimulatory domain, a CD3ζ domain, and (Figure 6A), "G" stands for shGFP; "BB" stands for "4-1BB" "z" represents CD3ζ. As used herein, the term "PBBz" refers to s It contains an hPD-1 expression cassette, a 4-1BB costimulatory domain, and a CD3ζ domain. The constructs refer to the constructs containing the PD-1 gene (Figure 6A), "P" stands for shPD-1; "BB" stands for "4-1BB." "z" represents CD3ζ.

[0122] Generally, when present on immune cells, CARs target the immune cells to target cells (usually cancer cells). While providing specificity to the target cell, polypeptides that cause signal transduction within the cell At a minimum, a CAR consists of an extracellular domain that recognizes the target antigen described below. It comprises an antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, and The signaling domains are derived from stimulatory or costimulatory molecules described below. The set comprising polypeptides may be linked or may be dimerized upon stimulation. The stimulatory molecule may be in a form linked via a TCR enzyme as described above. "CD19 CAR" is a CAR that targets the CD19 cancer antigen. be.

[0123] As used herein, the term "T cell receptor (TCR)" refers to an alpha (α) It refers to a protein receptor on T cells that is composed of a heterodimer of a β-chain and a β-chain. In some cells, the TCR consists of a gamma chain and a delta chain (γ / δ). TCRs are used to differentiate into various types of T cells, such as helper T cells, cytotoxic T cells, memory T cells, and inhibitory T cells. Any cell containing a TCR, including T cells, natural killer T cells, and gamma delta T cells. It may be modified with cells.

[0124] As used herein, the term "monoclonal T cell receptor (mTCR)" refers specifically to It refers to a T cell receptor (TCR) that is genetically engineered to specifically target a certain antigen. It can also be called an antigen-specific TCR. T cells with mTCR are used in adoptive T cell therapy. It has been reported to be used in immunotherapy for viral infections such as HIV and cancer. Retroviral transfer of chimeric single-chain antibody constructs (scFv) has been shown to enhance the expression of antibodies with defined antigen specificity. have been used as a strategy to generate T cells with chimeric sc Fv constructs linked to the intracellular signaling domains of FcR-gamma or CD3 zeta and induce T cell effector function. The CD3 zeta domain binds, for example, to CD28 , combined with the signaling domain of a costimulatory molecule such as 4-1BB or OX40. Monoclonal T cell receptors (mTCRs) and their applications in cancer therapy Stauss et al., 2007, Molecular Therapy, 1 5(10):1744-50,Zhang and Morgan,2012,Adva nced Drug Delivery Reviews,64(8):756-762 , and Liddy et al., 2012, Nature Medicine, 18( 6):980-7, the contents of each of which are incorporated herein by reference in their entirety. will be incorporated into

[0125] As used herein, the term "ΔLNGFR" refers to a cell in which the above insertion has been made. The cytoplasmic domain-less LNGFR (low affinity nerve growth factor receptor) used for purification of Point.

[0126] "Immune cells" include killer T cells, helper T cells, gamma delta T cells, B cells, and lymphocytes such as neural killer cells, mast cells, eosinophils, and basophils, Phagocytes may be characterized herein as including, but not limited to, macrophages. T cells include CD4+ T cells and CD8+ T cells. Contains cells.

[0127] As used herein, the terms "T lymphocyte" and "T cell" are interchangeable. They are used to complete maturation in the thymus and to identify specific foreign antigens in the body and to stimulate other immune cells A major type of leukocyte that has various roles in the immune system, including activating and inactivating lymphocytes. A T cell refers to any T cell, e.g., a cultured T cell, e.g., a primary T cell, or T cells derived from cultured T cell lines, e.g., Jurkat, SupT1, etc., or mammalian The T cells can be CD3+ cells. The cells can be any type of T cell, and can be CD4+ / CD8+ double positive T cells. CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumors Infiltrating lymphocytes (TIL), memory T cells, naive T cells, suppressor T cells, gamma-delta at any stage of development, including, but not limited to, T cells (γδ T cells), etc. Additional types of helper T cells include Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include central memory T cells. memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells) T cells include cells such as T cell receptors (TCRs) or chimeric antigen receptors (CACRs). Refers to genetically engineered T cells, such as T cells modified to express a CAR (Carbohydrate-Associated Immunoglobulin) T cells can also be differentiated from stem or progenitor cells.

[0128] "CD4+ T cells" express CD4 on their surface and are involved in cell-mediated immune responses. They are characterized by their secretory profile after stimulation and are sensitive to IFN- This includes the secretion of cytokines such as γ, TNF-α, IL2, IL4, and IL10. "CD4" is a 55-kD glycoprotein originally defined as a differentiation antigen on T lymphocytes. It is a protein, but is also found on other cells, including monocytes / macrophages. Hara is a member of the immunoglobulin supergene family and is involved in the MHC (major histocompatibility complex) It is involved as a relevant recognition element in class II-restricted immune responses. , they define a subset of helpers / inducers.

[0129] "CD8+ T cells" express CD8 on their surface and are restricted by MHC class I. The CD8 molecule is a subset of T cells that function as cytotoxic T cells. It is a differentiation antigen found on T lymphocytes, cytotoxic T lymphocytes, and suppressor T lymphocytes. The D8 antigen is a member of the immunoglobulin supergene family and is involved in major histocompatibility complexes. Relevant recognition elements in sex complex class I restricted interactions.

[0130] As used herein, the term "NK cells" or "natural killer cells" is defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As used herein, "adaptive NK cells" refers to a subset of peripheral blood lymphocytes that are capable of activating immune cells. and "memory NK cells" are interchangeable and represent phenotypically CD3- and CD56 + and express at least one of NKG2C and CD57, and optionally CD16, NK cells lacking expression of one or more of the following: PLZF, SYK, FceRγ, and EAT-2 In some embodiments, an isolated subset of CD56+ NK cells The group consists of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, and N Kp40, NKp46, activating and inhibitory KIR, NKG2A and / or DNAM-1 CD56+ may be weakly or clearly expressed.

[0131] As used herein, the term "immune checkpoint" refers to a molecule present in the immune system. These molecules can turn on or off the immune response. It is a fail-safe mechanism that regulates the excessive activation of immune cells that can lead to autoimmune reactions. The immune checkpoint molecules of are stimulatory immune checkpoint molecules that enhance the immune response, They can be broadly classified into inhibitory immune checkpoint molecules that inhibit immune responses and inhibitory immune checkpoint molecules that inhibit immune responses. For example, immune checkpoint receptors and ligands include PD1 (programmed cell death protein). Protein 1), PD-L1 (programmed death ligand 1), CTLA4 (cytotoxic T lymphocyte antigen 4) T-cell associated protein 4), TIM-3 (T cell immunoglobulin and mucin domain containing-3) ), CEACAM (three subtypes: CEACAM-1, CEACAM-3 or CEA Carcinoembryonic antigen-related cell adhesion molecule (CAM-5), LAG3 (lymphocyte activation gene 3) ), VISTA (V domain Ig suppressor of T cell activation), BTLA (B and T lymphocyte activation) TIGIT (T cell immunoreceptor with Ig and ITIM domains) , LAIR1 (leukocyte-associated immunoglobulin-like receptor 1), CD160 (cluster of differentiation 160 ), CD96 (cluster of differentiation 96), MerTK (proto-oncogene tyrosine protein kinase MER), and 2B4 (NK cell activation-inducing ligand). , for example, may be selected between PD1 and TIM3.

[0132] The term "culture" or "cell culture" refers to the maintenance, growth, and maintenance of cells in an in vitro environment. "Cell culture medium," "culture medium" (in either case, the singular "medium" is used) "), "nutritional supplement" and "media supplement" refer to a nutritional composition in which a cell culture is cultivated. The terms "cultivating" or "maintaining" refer to the process of culturing a cell outside of a tissue or body, e.g., by killing the cell. Sterile plastic (or coated plastic) cell culture dishes or plates The ability to sustain, proliferate (grow) and / or differentiate cells in a "Cultivating" or "maintaining" refers to the act of culturing a culture medium to grow and / or maintain cells. may be used as a source of nutrients, hormones and / or other factors that help sustain good.

[0133] The "pharmaceutical compositions" described herein for immunotherapy in human patients include those that contain immune cells. In addition to the cells, other pharmaceutically acceptable salts, carriers, excipients, etc., which may further improve the immune response may also be used. It is obvious that excipients, vehicles, and other additives may be added to the pharmaceutical composition. , the detailed description of which should be omitted.

[0134] The term "subject" refers to a human, non-human primate, canine, or other mammal that will be the recipient of a particular treatment. refers to any animal (e.g., mammal), including but not limited to, cats, rodents, etc. Generally, the terms "subject" and "patient" are used interchangeably herein to refer to a human subject. are used interchangeably.

[0135] The terms "treating" or "treat" refer to the condition, symptoms, or both of the disease being treated. suppressing, eliminating, or reducing the severity and / or frequency of symptoms of As used herein, "treat," "treatment," and The term "treating" refers to the progression of a disease or condition resulting from the administration of one or more therapeutic modalities. , refers to a reduction or improvement in severity and / or duration.

[0136] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and are not intended to limit the scope of the invention. and a T cell, such as those described herein, effective to achieve a biological result of Such results may be achieved by any method known in the art. The effects of the present invention may include, but are not limited to, inhibition of cancer as determined by any suitable means. I can't.

[0137] "Administering" or "administration" refers to administering a substance to a subject when it is present outside the body, e.g., mucosal delivery, Intradermal delivery, intravenous delivery, intramuscular delivery and / or other methods described herein or in the art. The substance may be injected into the patient by any other physical delivery method known in the art. Refers to the act of shooting or otherwise physically delivering.

[0138] 3. Dual-function vectors targeting one or more immune checkpoints Tumor cells express various immune checkpoints, e.g., checkpoint ligands. Therefore, even if one immune checkpoint is inhibited, other immune checkpoints may not be inhibited. It may be difficult to expect sustained effects of CAR-T through point activation. It primarily uses a combination of monoclonal antibodies to inhibit multiple immune checkpoints. Its antitumor effects have been continuously reported (J Clin. Invest., 2019). 015.,Chauvin,JM;PNAS,2010,Curran MA;Bloo d,2018,Wierz,M;Cancer cell.2014,Johnston However, it is known that therapeutic antibodies can induce excessive immune responses throughout the body. Furthermore, CAR-T cell therapy has been shown to reduce the risk of life-threatening cytokine release syndrome (CRS). S) and neurotoxicity (Nat Rev Clin Oncol, 2017 ,Neelapu SS) This means that the combination of CAR-T and antibody therapy may have side effects. Furthermore, conventional simultaneous immune cell therapy has The cost and the effects of these on T cells other than CAR-T, leading to autoimmune symptoms and cytokine release This places a significant economic burden on patients due to the risk of developing glaucoma. The present invention has been devised to address the above problems.

[0139] In one embodiment, provided herein is a two-in-one vector, The vector contains one or more short hairpin molecules that inhibit the expression of genes that attenuate immune cell function. The base sequence encoding shRNA and the chimeric antigen receptor (CAR), T cell receptor Receptor (TCR), e.g., monoclonal T cell receptor (mTCR) Columns and

[0140] Vectors include DNA, RNA, plasmids, lentiviral vectors, and adenovirus. The vector may be selected from among vectors and retroviral vectors, e.g., lentiviruses. Viral and retroviral vectors are used to insert genes into the genomic DNA of cells and transmit the genes. In some embodiments, for example, two-in-one functions are achieved by Using a lentiviral vector, for example, a dual vector with two functions in one, The gene on the vector can be inserted into the genome of the cell.

[0141] In some embodiments, provided are antibodies that inhibit the expression of genes that attenuate immune cell function. The base sequences encoding two types of short hairpin RNA (shRNA) that inhibit the chimeric antigen receptor receptors (CARs) and T cell receptors (TCRs), e.g., monoclonal T cell receptors (mCRs) and a base sequence encoding one of the TCRs.

[0142] In some embodiments, the expression of two shRNAs is controlled by two different promoters. In some embodiments, two The promoter is a promoter for RNA polymerase III. In this example, the two promoters are U6 promoters from different species. In an embodiment, the two promoters are oriented in different directions from each other on the vector. For example, in certain embodiments, the promoters are oriented in a head-to-head manner. In embodiments, the promoter is oriented in an end-to-end direction. , genes that weaken immune cell function are receptors or ligands of immune checkpoints .

[0143] In some embodiments, the immune checkpoint receptor or ligand is PD1, PD-L1, CTLA4, TIM3, CEACAM(CEACAM-1, CEACAM- 3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAI R1, CD160, CD96, MerTK and 2B4. In some embodiments, the gene that attenuates immune cell function is FAS, CD45, PP2A, S HIP1, SHIP2, DGK alpha, DGK zeta, Cbl-b, CD147, LR R1, TGFBR1, IL10Ralpha, KLGR1, DNMT3A and A2aR is selected from the group consisting of:

[0144] In some embodiments, the two shRNAs are directed against a single gene that attenuates immune cell function. target different parts of the immune system, or they target different genes that weaken immune cell function. In some embodiments, the two shRNAs target different parts of PD-1. In some embodiments, the two shRNAs are PD-1 and TIM-3, respectively. In some embodiments, the base sequences encoding the two shRNAs are: It comprises different sequences selected from the group consisting of SEQ ID NOs: 2-219.

[0145] In some embodiments, the target of the CAR or TCR, e.g., mTCR, is a Among the cancer antigens that are increased in the tumors, or mutated forms of cancer antigens found in the tumors The human tumor antigen is selected from the following:

[0146] In some embodiments, the vector comprises either the nucleic acid sequence of SEQ ID NO: 220 or 221. In some embodiments, the vector may be DNA, RNA, a plasmid, or Among lentiviral vectors, adenoviral vectors, and retroviral vectors be selected.

[0147] 3.1 RNA interference and short hairpin RNA RNAi (post-transcriptional gene silencing (PTGS), quelling, or co-suppression) (also known as mitochondrial DNA fragments) occur when an RNA molecule typically causes the destruction of a specific mRNA molecule. It is the process of post-transcriptional gene silencing that inhibits gene expression in a sequence-specific manner. The active component of RNAi is a short double-stranded RNA called small interfering RNA (siRNA). NA (dsRNA), which is usually 15-30 nucleotides (e.g., 19-25 , 19-24 or 19-21 nucleotides) and a dinucleotide 3' overhang These short RNA species contain the same sequence as the target gene and match the nucleic acid sequence of the target gene. It may be naturally generated in vivo by Dicer-mediated cleavage of dsRNA, These are functional in mammalian cells. DNA expression plasmids can be used to express the vectors described herein. The siRNA duplex or dsRNA containing the nucleotides is stably expressed in cells to inhibit target gene expression. Long-term inhibition can be achieved. In one embodiment, the sense and antisense strands of the siRNA duplex The sense strand usually induces the expression of a stem-loop structure called short hairpin RNA (shRNA). The hairpins are connected by a short spacer sequence that provides the hairpin recognition and It is cleaved to generate the mature siRNA molecule.

[0148] Short hairpin RNA (shRNA), as used herein, refers to any RNA having some self-complementarity. The sequence of the RNA molecule creates a tight hairpin structure with its stem. The shRNA molecules are typically about 40-120 nucleotides in length, e.g., about 70-90 nucleotides. In an exemplary embodiment, the shRNA may be 80 nucleotides in length. shRNAs can be of any length. They are designed to activate microinteracting proteins that are endogenous triggers of the RNAi pathway. miRNAs (Lu et al., 2005, Advances in nces in Genetics,54:117-142,Fewell et al. .,2006,Drug Discovery Today,11:975-982). Once an shRNA is expressed in a cell, it is processed through a series of steps to silence the gene. The resulting small interfering RNA (siRNA) acts as a guide for the transcription of the target gene. Once expressed, the hRNA is processed by the Drosha complex within the cell to form pre-shRNAs. A, which is then transported out of the nucleus, where it is further processed by Dicer to form siRNP A, then single-stranded, and then negatively reacted with RISC (RNA-induced silencing complex). Here, the antisense strand of the siRNA is transported by the RISC complex to the mRNA of the target gene. This acts as a guide for the attachment of NA to the RISC complex, and the RISC complex attached in this way is Cleavage of RNA results in gene silencing. This allows for sustained and specific gene silencing of a specific gene, making it possible to It is included in the vector for the purpose of inhibition.

[0149] Naturally expressed small RNAs called microRNAs (miRNAs) are the building blocks of mRNA. RISC-containing m The iRNA is composed of nucleotides 2–7 of the 5′ region of the miRNA, called the seed region. miRNs target mRNAs that display sequence complementarity and other base pairs in their 3' region. A-mediated downregulation of gene expression is mediated by target mRNA cleavage, translational inhibition of target mRNA, and or mRNA decay. The sequences targeted by miRNAs are usually , located in the 3'-UTR of target mRNAs. A single miRNA originates from various genes. Over 100 transcripts may be targeted, and one mRNA may be targeted by many different miRNAs. It may also be a target.

[0150] siRNA duplexes, or dsRNA, that target specific mRNAs are engineered in vitro. Elb may be synthesized and introduced into cells to activate the RNAi process. Ashir et al. developed a 21-nucleotide siRNA duplex (called a small interfering RNA) However, it has been shown to induce potent and specific gene knockdown in mammalian cells without inducing an immune response. It has been demonstrated that this can be achieved (Elbashir, SM et al., Nat Since this first report, siRNA-mediated Post-transcriptional gene silencing is a powerful tool for genetic analysis in mammalian cells. It is quickly emerging as a promising new treatment option.

[0151] For example, polyglutamines that cause polyglutamine expansion diseases such as Huntington's disease RNAi molecules designed to target nucleic acid sequences encoding repeat proteins have been US Patent Nos. 9,169,483 and 9,181,544, and International Patent Publication WO2 015179525, the contents of each of which are incorporated by reference in their entirety. Nos. 9,169,483 and 9,181,544, which are incorporated herein by reference. and International Patent Publication No. WO2015179525, respectively, describe the first strand of RNA ( a first strand of RNA (e.g., at least 15 contiguous nucleotides) and a second strand of RNA (e.g., at least 1 and providing an isolated RNA duplex comprising a nucleotide sequence complementary to two adjacent nucleotides of the RNA duplex. The first strand of RNA and the second strand of RNA are about 15-30 base pairs in length. operably linked by a fragment (about 4 to 50 nucleotides) and inserted into an expression cassette. Non-limiting examples of loop portions include those described in the literature, the contents of which are incorporated herein by reference. SEQ ID NO: 1 of U.S. Patent No. 9,168,483, which is incorporated herein in its entirety by reference. 9 to 14. A complete RNA strand that may be used to form an RNA duplex may be Non-limiting examples of any suitable sequence or portion of a sequence include those described in U.S. Pat. No. 9,169,483. SEQ ID NOs: 1 to 8 and SEQ ID NOs: 1 to 11, 33 to 59 of U.S. Patent No. 9,181,544; 208-210, 213-215, and 218-221, and within each of these The contents of which are incorporated herein by reference in their entirety. Non-limiting examples of RNAi molecules include Sequence numbers 1 to 8 in US Patent No. 9,169,483, and sequences in US Patent No. 9,181,544. Column numbers 1 to 11, 33 to 59, 208 to 210, 213 to 215, and 218 to 221, and and SEQ ID NOs: 1, 6, 7 and 35-38 of International Patent Publication No. WO2015179525. and US Pat. No. 6,229,999, the contents of each of which are incorporated herein by reference in their entirety.

[0152] siRNA molecules synthesized in vitro are introduced into cells to activate RNAi. The exogenous siRNA duplex may be expressed in the same manner as endogenous dsRNA when it is introduced into a cell. Once assembled, they form a complex that is complementary to one of the two strands of the siRNA duplex (all of which are complementary to one of the two strands of the siRNA duplex). RNA-guided subunits, which are assembly complexes that interact with RNA sequences (i.e., antisense strands), During this process, the siRNA can form a ribosomal isoform (RISC). The sense strand (or passenger strand) is lost from the complex, while the anti- The sense strand (or guide strand) corresponds to its complementary RNA. The ISC complex targets mRNAs that display perfect sequence complementarity. -mediated gene silencing occurs by cleaving, releasing, and degrading the target do.

[0153] Consists of a sense strand that is homologous to the target mRNA and an antisense strand that is complementary to the target mRNA The siRNA duplex may be a single-stranded (ss)-siRNA (e.g., an antisense strand RNA or in terms of the efficiency of target RNA destruction compared to the use of antisense oligonucleotides offer a much greater advantage than the corresponding duplex, which often provides more effective gene silencing. To achieve the desired efficacy, even higher concentrations of ss-siRNA are required.

[0154] Guidelines for designing siRNAs exist in the art. The primer generally consists of a 19-nucleotide sequence that targets the region of the gene to be silenced. a double-stranded region, a symmetric 2-3 nucleotide 3' overhang, a 5'-phosphate group and a 3 It is recommended to generate a '-hydroxyl group. The preference of the siRNA sequence may be controlled. Other rules include: (i) A / U at the 5' end of the antisense strand; (ii) A / U at the 5' end of the sense strand; (iii) at least five A's in the 5'-end one-third of the antisense strand; / U residues; (iv) absence of GC stretches greater than 9 nucleotides in length, In accordance with such considerations, the mammalian genome may be expressed in combination with the specific sequence of the target gene. Highly effective siRNA molecules essential for silencing target gene expression in animals are readily available. It may be designed.

[0155] As provided herein, dual-function vectors can be used to inhibit immune cell function. one or more types of short hairpin RNA (shRNA) that inhibit the expression of one or more genes that attenuate ) and a chimeric antigen receptor (CAR) or T cell receptor (TCR) For example, a nucleotide sequence encoding any one of the monoclonal T cell receptors (mTCR) Columns and

[0156] In some embodiments, the nucleic acid sequence inhibits the expression of a gene that attenuates immune cell function. In some embodiments, the base sequence encodes a single shRNA that is expressed in an immune cell. The gene encodes two types of shRNA that inhibit the expression of two genes that weaken their functions. In another embodiment, the vector is referred to as a "two-in-one dual vector." The sequence inhibits the expression of more than two genes that weaken immune cell function. Encodes hRNA.

[0157] In some embodiments, two or more types of shRNAs are used to identify immune cell functions. Attenuating a single gene, e.g., by targeting different parts of a single gene For example, two or more types of shRNAs may be used to target various PD-1 receptors, e.g., PD-1. In other embodiments, two or more shRNAs can target different regions. , which target different genes that weaken immune cell function, e.g., PD-1 and T It may be characterized by targeting IM-3.

[0158] In an exemplary embodiment, the base sequences encoding two or more types of shRNAs are are various sequences selected from the group consisting of SEQ ID NOs: 2 to 219 and 238 to 267 For example, they may be selected from the group consisting of SEQ ID NOs: 2 to 117 and 238 to 267. For example, they may comprise different sequences selected from SEQ ID NOs: 2 to 12, 7 and may include various sequences selected from the group consisting of 0 to 75 and 266 to 267. It may be characterized as being good.

[0159] In some embodiments, the expression of two shRNAs is controlled by two different promoters. Each gene is regulated by a different gene, minimizing the chance of recombination or deletion during cloning. The method may be characterized by keeping the number of times of the noise to a minimum.

[0160] In some embodiments, the RNA polymerase binds to the promoter and initiates transcription. Depending on which promoter initiates the transcription, the promoter can be an RNA polymerase I promoter, an RNA polymerase II promoter, or an RNA polymerase III promoter. The promoter may be a promoter for RNA polymerase II or a promoter for RNA polymerase III. The above two promoters are RNA polymerase III promoters (hereafter referred to as The promoter may be a Pol III promoter. The motor can translocate without a 5'-end cap or under promoter control. from the 5' end to the 3' end without adding a poly(A) tail to the 3' end of the transcribed RNA Pol III promoters can be engineered to transcribe precisely at the 6 promoter, H1 promoter, 7SK promoter, etc., but are not limited to these. The two promoters contained in the vector may be different, and the above three types and the same type of promoter may be selected from Pol III promoters including If two promoters are selected, they may be from different species. For example, is a U6 promoter, such as the U6 promoter from human and mouse, It may be a U6 promoter from a different species. The shRNA is then transported to the Drosha complex, which is present in the nucleus, where it is then transported to the nucleus. It is believed that this can promote the process of pre-shRNA processing. .

[0161] In some embodiments, two or more promoters are oriented in different directions relative to one another on the vector. For example, in certain embodiments, the promoter may be In another embodiment, the promoter is oriented in a two-headed (→←) direction. It is directed towards the end (←→). In a dual vector with two functions, the vector The fact that they are directed in different directions on the chromosome means that their expression is regulated by two promoters. When each shRNA is transcribed, the RNA polymerase moves in a single direction. In an exemplary embodiment, two promoters are oriented in different directions in a nucleic acid molecule. In another exemplary embodiment, the motor can be in the →← direction (FIG. 15A). The promoters can be in a ←→ orientation (Figure 15B). For example, two promoters can be assumed to be in the →← direction of the vector.

[0162] In some embodiments, the expression of the target gene(s) of one or more shRNAs is compared with that of a control group. The expression of the target gene is reduced to about 90% or less of that of the control group, for example, the expression of the target gene is reduced to about 80% of that of the control group. Below, 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20 % or less, and about 10% or less.

[0163] Usually, shRNAs contain a sequence highly homologous to a part of the mRNA sequence of the target gene (hereafter referred to as the "seq"). The sequences were selected based on the base sequence of the shRNA, sequences that can generate sharp hairpins, and sequences with high homology. It is designed to have a complementary sequence (hereafter referred to as the antisense shRNA base sequence). The non-covalent bond between the self-complementary portions forms a stem structure, allowing the shRNA to be expressed in cells and When processed, the antisense shRNA sequence mediates the gene silencing process. For example, shR acts as a guide for mRNA of a target gene in the target gene. The nucleotide sequence of the cassette used for expression of NA is NNNNNNNNNNNNNNNNNN NNN (21 bases) - loop sequence -NNNNNNNNNNNNNNNNNNNNNN (19 bases) In one embodiment, the 21-base stretch can include a sense shRNA salt structure. The 19 base stretch encodes a base sequence, and the 19 base stretch is complementary or substantially complementary to the 21 base stretch. In another embodiment, a 19-base segment encodes an antisense shRNA sequence. The 21-nt section encodes the sense shRNA sequence, and the 21-nt section is complementary to the 19-nt section. is substantially complementary to and encodes an antisense shRNA sequence. Upon expression, the resulting RNA forms a stem and loop structure. Such sense or antisense sequences of the target gene (of human origin) may be included in the cassette. The shRNA base sequence is selected from the group consisting of SEQ ID NOs: 1 to 219. In an embodiment, the base sequence of the cassette used to express the shRNA herein is SEQ ID NO:2 It can be selected from the group consisting of 20 to 224.

[0164] In some embodiments, the complete shRNA sequence is expressed as a mouse or human U6 promoter. can be placed at the 3' end of the promoter to terminate transcription by the U6 promoter. The required TTTTT can be placed at the 3' end of any shRNA sequence.

[0165] In some embodiments, the nucleic acid sequence of each shRNA is in addition to the sequences described herein. In addition, at least 50%, particularly at least 70%, and more particularly is at least 80%, even more specifically at least 90%, and most specifically at least The nucleic acid sequence may contain a nucleic acid sequence that exhibits at least 95% sequence homology. In the case of small interfering RNAs (shRNAs) and shRNAs that are processed intracellularly to become siRNAs, Mutations, especially those at the 5' end, are tolerated and result in successful knockdown of the target gene. and have a structure similar to miRNA, which plays a role in gene silencing. Mutations in siRNA and shRNA induce more effective target gene knockdown Furthermore, it is obvious to those skilled in the art that the use of vectors The reason is that variations within the vector, i.e., the clones used to introduce specific sequences into the vector, additions, modifications or deletions of base sequences that may occur during the scanning process, or The construction of components to improve the ease of use of vectors to express the intended gene. It is a change or introduction.

[0166] There are various mechanisms of action for genes that weaken immune cell function. inhibiting proliferation or causing cell death, and activating immune cell responses This reduces the reaction with molecules that are necessary for immune cells to recognize their target. Instead of inhibiting the expression of essential genes and triggering an immune response against specific targets, These cells induce differentiation into different types of immune cells with different functions. Typical examples include molecules related to immune checkpoints, as described below. Not limited to these.

[0167] In some embodiments, the gene that attenuates immune cell function is one that inhibits immune checkpoint activity. The immune checkpoint may be characterized as a receptor or ligand for a specific immune checkpoint. These are molecules present in the immune system that can turn the immune response on or off. Viewed as a fail-safe mechanism that regulates excessive activation of immune cells that can lead to cell death and autoimmune reactions These immune checkpoint molecules act as stimulatory immune checkpoints that enhance immune responses. These are broadly divided into inhibitory immune checkpoint molecules and inhibitory immune checkpoint molecules that inhibit immune responses. Many cancer cells express inhibitory immune checkpoint signals, especially immune By activating the receptors and ligands of inhibitory immune checkpoints on immune cells, It has been reported that these cells can evade the immune system, and therefore are suitable for immune cell therapy targeting specific cancers. The method may be effective by countering this evasive action of cancer, which is called suppression. By inhibiting the activation of regulatory immune checkpoint receptors and their ligands This may be achieved by increasing or decreasing their expression. The receptors and ligands of the checkpoints are PD1 (programmed cell death protein 1), PD- L1 (programmed death ligand 1), CTLA4 (cytotoxic T lymphocyte-associated protein 4) ), TIM-3 (T-cell immunoglobulin and mucin domain-containing-3), CEACAM ( Cancers containing the three subtypes CEACAM-1, CEACAM-3, or CEACAM-5 fetal antigen-related cell adhesion molecule), LAG3 (lymphocyte activation gene 3), VISTA (T V domain Ig suppressor of cell activation), BTLA (B and T lymphocyte attenuating factor), TIGIT (T cell immunoreceptor with Ig and ITIM domains), LAIR1 (leukemia leukocyte-associated immunoglobulin-like receptor 1), CD160 (cluster of differentiation 160), CD96 ( antigen group 96), MerTK (proto-oncogene tyrosine protein kinase MER), and 2B 4 (NK cell activation-inducing ligand), for example, PD1, A choice may be made between TIM3 and TIGIT.

[0168] In another embodiment, the gene that attenuates immune cell function is one that is expressed by a gene that attenuates immune cell function upon repeated stimulation by a TCR. Thus, negative regulatory factors for activated T lymphocytes, such as FAS (CD95, AICD (activating apoptosis antigen-1) acts as apoptosis inhibitor. The cell death inhibitor may be characterized by encoding a receptor capable of promoting mitochondrial cell death (mitochondrial cell death). In some embodiments, the gene that attenuates immune cell function is one that inhibits TCR signal activation. For example, the factor may encode a factor that inhibits CD45, PP2 A, SHP1, SHP2, DGK alpha, DGK zeta, Cbl-b, Cbl-c and In some embodiments, the inhibitors of the present invention can be selected from the group consisting of CD148, CD150, CD160, CD200, CD300, CD400, CD500, CD600, CD800, CD90, CD101, CD112, CD113, CD114, CD120, CD135, CD148, CD151, CD152, CD153, CD154, CD155 The gene that inhibits 4-1BB signaling is thought to be involved in CAR and / or TCR, e.g. For example, the gene may be characterized by encoding a protein that suppresses the efficacy of mTCR. The BB binds to a costimulatory molecule described herein, such as LRR1 (Leucine-Rich In some embodiments, the gene that attenuates immune cell function is a repeat protein 1). , whose ligands are cytokines and receptors that inhibit T cells, such as TGFBR1 ( Transforming growth factor beta receptor 1) and IL10R alpha (IL-10R subunit In some embodiments, the immune cells may be characterized by encoding a nucleotide sequence encoding a nucleotide sequence (nucleotide sequence alpha). The gene whose function is attenuated inhibits the proliferation and cytotoxicity of T cells and NK cells. Characterized by encoding a receptor, such as KLGR1 (killer cell lectin-like receptor G1) In some embodiments, the gene that attenuates immune cell function may be knocked out. A novel DNA methyltransferase that has been reported to suppress T cell exhaustion when knocked out Regulatory factors related to transcription, such as TNMT3a (DNA methyltransferase 3a) In some embodiments, the function of the immune cell is enhanced by encoding a gene encoding a target gene. When activated, genes that attenuate this inhibit the cytotoxic and cytokine-producing capacity of T cells. Adenosine receptors, such as A2aR (Adenosine receptors), are present in excess in the tumor microenvironment. It may be characterized by encoding adenosine receptor subtype A2a).

[0169] In some embodiments, the gene that attenuates immune cell function is selected from the group consisting of FAS, CD4 5, PP2A, SHP1, SHP2, DGK alpha, DGK zeta, Cbl-b, Cb lc, CD148, LRR1, TGFBR1, IL10RA, KLGR1, DNMT3 A and A2aR.

[0170] 3.2 Chimeric Antigen Receptors (CARs) and T Cell Receptors, e.g., Monoclonal T Cells Receptor (mTCR) As provided herein, dual-function vectors can be used to inhibit immune cell function. one or more types of short hairpin RNA (shRNA) that inhibit the expression of one or more genes that attenuate ) and a chimeric antigen receptor (CAR) or T cell receptor (TCR) For example, a nucleotide sequence encoding any one of the monoclonal T cell receptors (mTCR) Columns and

[0171] CARs generally, when present on immune cells, direct the immune cells to target cells (usually cancer cells). While having specificity for the target, it is also a polypeptide that causes signal transduction within the cell. At a minimum, a CAR is a set of extracellular antigens that recognize the target antigens listed below. It contains a recognition domain, a transmembrane domain, and an intracellular signaling domain, and The null transduction domain is derived from a stimulatory or costimulatory molecule.

[0172] The CAR structure commonly used in clinical applications today is a single-chain antibody that confers specificity to an antigen. The fragment variable domain (hereinafter referred to as scFv) and the spacer that controls the distance between the scFv and the cell membrane. The third domain, the transmembrane domain, and the intracellular signaling domain (hereafter referred to as ISD) The ISD then contains one or more costimulatory domains that contribute to the in vivo proliferation and longevity of T cells. (CD28, CD137, or OX40) and TCR signaling that contributes to T cell activation The CA prepared in this manner contains a transduction domain (CD3 zeta, CD3ζ). T cells engineered to express R can target cancer cells expressing the target antigen with high specificity. and activates the IL-1 receptor by recognizing it, effectively inducing the death of such cancer cells, while They can grow exponentially in the body and survive for a long time. For example, B cell-specific CAR-T cells (CART-19) engineered to target the antigen CD19 When administered to patients with B-cell leukemia, the cells multiplied 1,000 to 10,000 times, As a result, CART-19 has been shown to be effective against conventional chemotherapy and other cancers. This study was conducted on patients with terminal acute lymphoblastic leukemia (B-ALL) for whom other treatments were ineffective. Clinical trials showed a 90% complete response rate, and investigator-initiated early clinical trials This led to a rare case of licensing to a global pharmaceutical company in 2017. It became the first CAR-T cell therapy approved by the US FDA, but it was later approved by the second AR-T was also approved.

[0173] Immune cells, such as T cells, have CTLA-4 (cytotoxic T lymphocyte-associated protein 4) on their surface. immune checkpoints such as protein 4 (PD-1) or PD-1 (programmed cell death protein 1) These receptors primarily prevent excessive T cell activation and cell death, as well as However, cancer cells, especially Solid tumors have been reported to use this to evade immune surveillance by T cells. For example, When cancer cells express PD-L1 (programmed death ligand 1) on their surface, the receptor T cells expressing PD-1 recognize cancer cells and are activated, but the activation from PD-1 These immune checkpoint receptors are quickly exhausted by inhibitory signals. To prevent these signals from inhibiting T cell activity, the immune checkpoint receptors are targeted. Monoclonal antibodies against CTLA4 or PD-1, etc., that inhibit signal transduction by By using these immune checkpoint receptor inhibitors, Therapies that improve overall immune function of T cells through immune checkpoint blockade are It has also shown efficacy against a variety of solid cancers.

[0174] CAR-T cells are also a treatment that ultimately depends on the cytotoxicity of activated T cells. The presence of an immunosuppressive environment around CAR-T cells is a major obstacle to therapeutic efficacy. In fact, unlike the therapeutic effects shown in B-cell leukemia, targeting solid tumors CAR-T cells engineered in this way have rarely shown promising therapeutic effects. This is because, unlike blood cancers, solid tumors create an immunosuppressive tumor microenvironment that can inhibit CAR- It is thought that this is because it suppresses the activity and proliferation of T cells. Even among those with acute lymphoblastic leukemia, nearly 90% responded to treatment with CART-19. Unlike patients with hematologic malignancies (ALL), treatment efficacy is similar to that of patients with lymphoma (20-50% response rate) or The response rate was relatively low in patients with chronic lymphoblastic leukemia (CLL, around 20%). It has been reported.

[0175] Furthermore, PD-L1 and other immunosuppressive ligands are involved in the regulation of tumor microenvironment formed by lymphoma. It has been reported that T cells in cancerous tissues are functionally exhausted due to the expression of T cells in the microenvironment. Furthermore, T cells obtained from CLL patients express PD-1, CD160, and CD244 are already substantially exhausted by high expression of immune checkpoint receptors such as It has been reported that:

[0176] To restore this decreased activity of CAR-T cells, CAR-T cells and anti-CTLA- Preclinical results suggest that the simultaneous use of anti-PD-1 inhibitors improves anti-cancer efficacy. Although clinical trials using these combinations have been reported, clinical trials using these combinations are currently underway. The challenge with such simultaneous antibody and CAR-T cell therapy is the ability of antibodies to spread throughout the body. This affects not only CAR-T cells but also all other T cells present in the body, e.g. For example, cytokine release syndrome and severe, systemic adverse reactions such as autoimmune conditions. Another challenge that has been pointed out is the cost of cell therapy and expensive antibodies. The increased cost of treatment resulting from the combination of therapies.

[0177] Therefore, to enable immune checkpoint suppression in CAR-T cells, Recent attempts have been made to regulate gene expression within the cells. 69282 is a group consisting of TCR α chain, TCR β chain, β-2 microglobulin, and FAS. and further comprising a nucleic acid capable of down-regulating endogenous gene expression selected from the group consisting of: Encoding a modified T cell receptor (TCR) that contains affinity for a surface antigen on a target cell. Electroporation of nucleic acids encoding a chimeric antigen receptor (CAR) The publication discloses compositions and methods for generating T cells containing the nucleic acid. Knock out endogenous gene expression using genetic scissors like SPR / Cas9 However, the method for preparing CAR-T cells disclosed in the patent publication is quite complicated. However, they state that there are problems such as low production yield and high production costs.

[0178] On the other hand, International Patent Publication WO2015 / 090230 discloses a compound that further suppresses the function of T cells. A single short hairpin RNA (shRNA) that inhibits the gene expression was used in cells expressing CAR. The cost of cell therapy is high, so if it fails, the patient may be However, a single shRNA can be used to target such molecules. In some embodiments, the activity of the poly In some embodiments, a set comprising a polypeptide may be linked. The set is a form in which they are linked via a switch that dimerizes upon stimulation. In some embodiments, the CAR comprises an extracellular antigen recognition domain and a transmembrane domain. The protein may be a fusion protein comprising an amino acid sequence and an intracellular signaling domain. In embodiments, the CAR fusion protein may further comprise a leader sequence at the N-terminus. The driver sequence is cleaved during the process of CAR expression and becoming anchored to the cell membrane. This may be done.

[0179] The TCRs described herein, e.g., mTCRs, can be selected from α, β, γ, and δ chains. In some embodiments, the chains may comprise a target antigen, as described below. , CD3, and zeta chains, as well as costimulatory molecules, which are Selected from COS, OX40, CD137 (4-1BB), CD27, or CD28 This may also be done.

[0180] In some embodiments, retroviral delivery of chimeric single chain antibody constructs (scFv) is used. This allows the generation of TCRs with defined antigen specificity, e.g., mTCRs. In a further embodiment, the chimeric scFv construct binds to the intracellular domain of FcR-gamma or CD3ζ. Linked to signaling domains, they can induce T cell effector functions. In CD3ζ, the domain can be combined with the signaling domain of a costimulatory molecule. In this case, antibody binding can trigger the function of effector T cells, and co-stimulatory In a further embodiment, the costimulatory molecule is CD28, 4-1 You can choose from BB and OX40.

[0181] In some embodiments, the dual-function vector comprises a CD3 ζ domain. The chains of the TCR, e.g., mTCR, can be non-covalently bound to the CD3 and zeta (ζ) chains. The antigen is recognized through the antigen recognition site of the chain. The CD3 and zeta chains are expressed in the cytoplasm of immune cells where such TCR complexes are expressed. signal to induce functional activation.

[0182] In some embodiments, the intracellular signaling domain further comprises a co-stimulatory molecule derived intracellular signaling domain. In some embodiments, the promoter may comprise one or more functional signaling domains. The molecule may be the zeta chain of the TCR described above.

[0183] CAR and TCR, e.g., mTCR, are cell surface receptors. In some embodiments, The target of the CAR or TCR, e.g., mTCR, is a target whose expression is specifically increased in cancer. In some embodiments, the CAR or The targets of TCRs, e.g., mTCRs, are cancer antigens that exist in mutated forms in cancer. The present invention may be characterized by the above.

[0184] In some embodiments, the target of a CAR or TCR, e.g., an mTCR, is its expression The target may be a human tumor antigen that is elevated in the cancer to be treated. Target antigens are 5T4 (trophoblast glycoprotein), 707-AP, 9D7, and AFP (alpha-fetoprotein). protein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate-specific Allergic gene-1), α5β1-integrin, α5β6-integrin, α-methylaspartate leu-Coenzyme A racemase, ART-4 (ADP-ribosyltransferase-4), B7 H4 (v-set domain-containing T-cell activation inhibitor 1), BAGE-1 (B melanoma antigen-1), BCL-2 (B-cell CLL / lymphoma-2), BING-4 (WD repeat domain 46), CA15-3 / CA27-29 (mucin 1), CA19-9 (cancer antigen 19-9), CA72-4 (cancer antigen 72-4), CA125 (cancer antigen 125), Cal Reticulin, CAMEL (a CTL-recognized antigen on melanoma), CASP-8 (caspase 8), cathepsin B, cathepsin L, CD19 (cluster of differentiation 19), CD20, CD22 , CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel accessory 2), CM L28 (chronic myeloid leukemia tumor antigen 28), coactosin-like protein, collagen XX III, COX-2 (cyclooxygenase-2), CT-9 / BRD6 (cancer / testicular anti- Hara9), Cten (c-terminal tensin-like protein), cyclin B1, cyclin D1 , cyp-B, CYPB1 (cytochrome p450 family 1 subfamily b member -1), DAM-10 / MAGE-B1 (melanoma-associated antigen B1), DAM-6 / MA GE-B2, EGFR / Her1 (epidermal growth factor receptor), EMMPRIN (basigin ), EpCam, EphA2 (EPH receptor A2), EphA3, ErbB3 (Erb- B2 receptor tyrosine kinase 3), EZH2 (zeste2 polycomb repressive complex 2) enhancer of subunits), FGF-5 (fibroblast growth factor 5), FN (fibrone Cutin), Fra-1 (Fos-related antigen-1), G250 / CAIX (carbonic anhydrase 9) , GAGE-1 (G antigen-1), GAGE-2, GAGE-3, GAGE-4, GAGE GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP (differentially expressed in the prostate GnT-V (gluconate kinase), gp100 (melanocyte lineage expressed gene) Specific antigen GP100), GPC3 (glypican 3), HAGE (helical antigen), HAS T-2 (sulfotransferase family 1A member 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K-MEL , HNE (medullasin), homeobox NKX3.1, HOM-TES-14 / SC P-1, HOM-TES-85, HPV-E6, HPVE7, HST-2 (Siltuin- 2), hTERT, iCE (caspase 1), IGF-1R (insulin-like growth factor-1 receptor), IL-13Ra2 (interleukin-13 receptor subunit α2), IL -2R (interleukin-2 receptor), IL-5 (interleukin-5), immature leukin Minin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205 (lysophore Sphatidylglycerol acyltransferase 1), KK-LC-1 (Kitakyushu Lung Cancer antigen-1), KM-HN-1, LAGE-1 (L antigen family member-1), livin , MAGE-A1, MAGE-A10, MAGE-A12, MAGEA2, MAGE-A 3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MAGE-B 10, MAGE-B16, MAGEB17, MAGE-B2, MAGE-B3, MAGE -B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE -C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE -E2, MAGE-F1, MAGE-H1, MAGEL2 (melanoma antigen family L 2), mammaglobin A, MART-1 / Melan-A (recognized by T-cell-1) melanoma antigen), MART-2, matrix protein 22, MC1R (melanocorticoid 1 receptor), M-CSF (macrophage colony-stimulating factor), mesothelin, MG5 0 / PXDN (peroxidasin), MMP11 (matrix metalloproteinase 11) ), MN / CA IX-antigen (carbonic anhydrase 9), MRP-3 (multidrug resistance-associated protein -3), MUC1 (mucin 1), MUC2, NA88-A (VENT-like homeobox 2 Pseudogene 1), N-acetylglucosaminyltransferase-V, Neo-PAP( Neo-poly(A) polymerase), NGEP (novel gene expressed in prostate), N MP22 (nuclear matrix protein 22), NPM / ALK (nucleophosmin), NS E (neuron-specific enolase), NY-ESO-1, NY-ESO-B, OA1 (variant Osteoarthritis QTL1), OFA-iLRP (Carcinoembryonic Antigen-Immature Laminin Receptor Protein) Protein), OGT (O-GlcNAc transferase), OS-9 (endoplasmic reticulum lectin) , osteocalcin, osteopontin, p15 (CDK inhibitor 2B), p53, PAG E-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor) PAI-1), PAI-2, PAP (prostatic acid phosphatase), PART-1 (prostatic acid phosphatase) androgen-regulated transcript 1), PATE (prostate and testis expressed 1), PDEF (prostate Ets factor from prostate gland), Pim-1-kinase (proviral integration site 1), Pi n1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1), POTE (expressed in the prostate, ovaries, testes, and placenta), PRAME (predominant in melanoma) prostate-specific antigen (PSA), prostein, proteinase-3, and PSA (prostate-specific antigen) Gen), PSCA (prostate stem cell antigen), PSGR (prostate-specific G-protein coupled receptor body), PSM, PSMA (prostate-specific membrane antigen), RAGE-1 (renal tumor carcinoma antigen), R HAMM / CD168, RU1 (renal ubiquitous protein 1), RU2, SAGE (sarcoma antigen) , SART-1 (squamous cell carcinoma antigen recognized by T-cells-1), SART- 2. SART-3, Sp17 (sperm protein 17), SSX-1 (SSX family member) Member 1), SSX-2 / HOM-MEL-40, SSX-4, STAMP-1 (STE AP2 metalloreductase), STEAP, survivin, survivin-213, TA- 90 (tumor-associated antigen-90), TAG-72 (tumor-associated glycoprotein-72), TARP (TCRγ alternative reading frame protein), TGFb (transforming growth factor beta), TGFbR11 (transforming growth factor beta receptor 11), TGM-4 (transglutaminase TRAG-3 (Taxol resistance-associated gene 3), TRG (T-cell receptor gamma gene TRP-1 (transient receptor potential-1), TRP-2 / 6b, TRP-2 / INT2, Trp-p8, tyrosinase, UPA (U-plasminogen activator) , VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK-1, and WT1 (Willow tumours1) or found in the cancer to be treated, and Catenin-4 / m, ARTC1 / m, bcr / abl, beta-Catenin / m, B RCA1 / m, BRCA2 / m, CASP-5 / m, CASP-8 / m, CDC27 / m , CDK4 / m, CDKN2A / m, CML66, COA-1 / m, DEK-CAN, E FTUD2 / m, ELF2 / m, ETV6-AML1, FN1 / m, GPNMB / m, H LA-A*0201-R170I, HLA-A11 / m, HLA-A2 / m, HSP70 -2M, KIAA0205 / m, K-Ras / m, LDLR-FUT, MART2 / m, ME1 / m, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class 1 / m , neo-PAP / m, NFYC / m, N-Ras / m, OGT / m, OS-9 / m, p 53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, TEL-AML1, TGFb It may also be a mutated form of a human tumor antigen selected from among RII and TPI / m. For example, the target antigen may be selected from between CD19 or CD22.

[0185] 3.3 Two-in-one vector components As provided herein, dual-function vectors can be used to inhibit immune cell function. one or more types of short hairpin RNA (shRNA) that inhibit the expression of one or more genes that attenuate ) and a chimeric antigen receptor (CAR) or T cell receptor (TCR) , for example, a nucleotide sequence encoding a monoclonal T cell receptor (mTCR).

[0186] In some embodiments, the two-in-one vector is used for insertion into the host cell genome. In some embodiments, the nucleic acid sequence may include a sequence encoding a factor that facilitates the insertion of the nucleic acid sequence. In some embodiments, the sequences are located at one or both ends of the vector gene. The sequence is an LTR (long terminal repeat).

[0187] In some embodiments, the two-in-one vector is a vector as described above. Contains a domain encoding a protein used for purification of cells into which the insert is carried In some embodiments, the domain is a ΔLNGFR domain, wherein: ΔLNGFR is a cytoplasmic nucleotide sequence used to purify cells carrying the insertion described above. It is a domainless LNGFR (low affinity nerve growth factor receptor).

[0188] In some embodiments, the dual-function vector is characterized by persistent expression. A promoter that drives the expression of both ΔLNGFR and CAR, such as the promoter shown in FIG. 22A The EF1α promoter induces the expression of both ΔLNGFR and CD19 CAR as shown in In a further embodiment, the ΔLNGFR and CD19 CAR are first transcribed in a form in which they exist on a single mRNA. In such embodiments where the above cistron is present, an IRES (internal ribosome entry site) ) may be inserted between them to cause expression of both cistrons. However, it has been reported that excessively long IRES reduces the expression efficiency of downstream cistrons. In some embodiments, components other than an IRES, such as P2A (2a peptide), are used. This drawback can be overcome by using a ribosome-specific C2A fragment. It passes through without forming a peptide bond at the end, allowing the downstream gene to be expressed later.

[0189] Some exemplary single-function dual-function vectors are shown in Figures 1, 6, 14, and 22A. The vector is a two-in-one vector as shown in SEQ ID NO: 220 or 221. An exemplary vector containing the base sequences of SEQ ID NOs: 220 and 221 is shown. The processor can have the structure shown in Figures 1A and 1B. Plasmids are listed in Table 1. Table 1. Exemplary plasmids. [Table 1]

[0190] In some embodiments, the nucleic acid sequences contained in the vectors described above, and each The nucleic acid sequences of shRNAs may be any of the sequences described herein, as well as any combination of these sequences. at least 50%, specifically at least 70%, more specifically at least 80%, More particularly, at least 90%, and most particularly, at least 95% sequence homology This may include nucleic acid sequences that exhibit specificity. For shRNAs that are processed specifically to become siRNAs, some mutations, especially at the 5' end, are present. The mutation is tolerated, resulting in normal knockdown of the target gene and gene silencing. siRNA and shR are designed to have structures similar to miRNAs that play a role in signaling. It has been reported that NA mutations induce more effective target gene knockdown. Furthermore, in using vectors, it is obvious to those skilled in the art that Variation, i.e., in the cloning process to introduce specific sequences into a vector additions, modifications or deletions of base sequences that may occur or that may prevent the intended gene from being expressed. The modification or introduction of components to improve the usability of the vector to a certain extent.

[0191] Production and evaluation of CAR-T cells targeting immune checkpoints 4.1 and above As provided herein, dual-function vectors can be used to inhibit immune cell function. One or more types of short hairpin RNA (shRNA) are encoded to inhibit the expression of genes that attenuate the and chimeric antigen receptors (CARs) and T cell receptors (TCRs), e.g., and a base sequence encoding any one of the monoclonal T cell receptors (mTCR). The vector is used to create immune cells that inhibit the expression of genes that weaken the function of immune cells. In some embodiments, the vector may be a DNA, RNA, a plasmid, Consists of lentiviral vectors, adenoviral vectors, and retroviral vectors is selected from the group.

[0192] In one aspect, the immune cells described herein comprise the vector and are capable of expressing a CAR or expressing a TCR, e.g., mTCR, and one or more target genes of shRNA In some embodiments, the expression of one or more shRNs is reduced. The expression of the target gene in A is reduced to about 90% or less of that in the control group, e.g., the expression of the target gene The expression of the IL-11 gene is about 80% or less, 70% or less, about 60% or less, about 50% or less, or about 10% or less of that of the control group. The reduction is 40% or less, about 30% or less, about 20% or less, and about 10% or less. In embodiments, the immune cells are selected from among T cells and NK cells of human origin.

[0193] In another aspect, provided herein are methods. In some embodiments, the methods The target antigens are (1) antigen receptors encoding engineered antigen receptors that specifically bind to target antigens. (2) Reduce the expression in immune cells of genes that weaken immune cell function. or gene disrupting agents capable of reducing the level of HIV-1, HIV-1, or HIV-2. cells, thereby expressing the engineered antigen receptor and enhancing immune cell function. methods for producing immune cells, including producing immune cells with reduced expression of genes that weaken immune function It is the law.

[0194] In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). ) or T cell receptor (TCR). In some embodiments, engineered antibodies The original receptor is a CAR. In some embodiments, the CAR comprises an extracellular antigen recognition domain. a transmembrane domain and an intracellular signaling domain.

[0195] In some embodiments, the extracellular antigen recognition domain of the CAR specifically binds to a target antigen. Combine.

[0196] In some embodiments, the intracellular signaling domain of the CAR is CD3 zeta (CD In some embodiments, the intracellular domain of the CAR comprises the intracellular domain of the CAR 3ζ) chain. The delivery domain further comprises a costimulatory molecule.

[0197] In some embodiments, the costimulatory molecule is ICOS, OX40, CD137 (4-1BB ), CD27, and CD28. In some embodiments, the co-stimulatory In some embodiments, the costimulatory molecule is CD137 (4-1BB). It is 28.

[0198] In some embodiments, the target antigen is a cancer cell, cancer tissue, and / or tumor microcirculation. In some embodiments, the target antigen is expressed on the surface of cells in the cancer cell line. Cancer antigens that are increased in cells, cancer tissues, and / or the tumor microenvironment, or that are mutated This is the form.

[0199] In some embodiments, the expression is in cancer cells, cancer tissues, and / or the tumor microenvironment. The cancer antigens that increase with this are 5T4 (trophoblast glycoprotein), 707-AP, 9D7, and AFP. (α-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1( Human prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α -methylacyl-coenzyme A racemase, ART-4 (ADP-ribosyltransferase -4), B7H4 (v-set domain-containing T-cell activation inhibitor 1), BAGE-1 ( B-melanoma antigen-1), BCL-2 (B-cell CLL / lymphoma-2), BING- 4 (WD repeat domain 46), CA15-3 / CA27-29 (mucin 1), CA19- 9 (cancer antigen 19-9), CA72-4 (cancer antigen 72-4), CA125 (cancer antigen 1 25), calreticulin, CAMEL (a CTL-recognized antigen on melanoma), CASP-8 (caspase 8), cathepsin B, cathepsin L, CD19 (cluster of differentiation 19), CD2 0, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56 , CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel accessory -2), CML28 (chronic myeloid leukemia tumor antigen 28), coactosin-like protein, cyclooxygenase-2 (COX-2), CT-9 / BRD6 ( Cancer / testis antigen 9), Cten (c-terminal tensin-like protein), cyclin B1, Icrin D1, cyp-B, CYPB1 (cytochrome p450 family 1 subfamily) Lee b member 1), DAM-10 / MAGE-B1 (melanoma-associated antigen B1), DA M-6 / MAGE-B2, EGFR / Her1 (epidermal growth factor receptor), EMMPRIN (basigin), EpCam, EphA2 (EPH receptor A2), EphA3, ErbB 3 (Erb-B2 receptor tyrosine kinase 3), EZH2 (zeste2 polycomb repression Enhancer of sex complex 2 subunit), FGF-5 (fibroblast growth factor 5), FN (fibronectin), Fra-1 (Fos-related antigen-1), G250 / CAIX (carbonate Dehydratase 9), GAGE-1 (G antigen-1), GAGE-2, GAGE-3, GAGE- 4. GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP (prostate differentially expressed genes), GnT-V (gluconate kinase), gp100 (metabolites), Glypican 3 (GPC3), helical antigen 100 (GP100), helical antigen 200 (HAGE), helical antigen 300 (HAGE), helical antigen 100 (HAGE), helical antigen 2 ... Hara), HAST-2 (sulfotransferase family 1A member 1), hepsin , Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV -K-MEL, HNE (medullasin), homeobox NKX3.1, HOM-TES -14 / SCP-1, HOM-TES-85, HPV-E6, HPVE7, HST-2( Sirtuin-2), hTERT, iCE (caspase 1), IGF-1R (insulin-like Growth factor-1 receptor), IL-13Ra2 (interleukin-13 receptor subunit α2), IL-2R (interleukin-2 receptor), IL-5 (interleukin-5 ), immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA02 05 (lysophosphatidylglycerol acyltransferase 1), KK-LC-1 (Kitakyushu Lung Cancer Antigen-1), KM-HN-1, LAGE-1 (L antigen family member 1), Livin, MAGE-A1, MAGE-A10, MAGE-A12, MAGEA2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MAGE-B10, MAGE-B16, MAGEB17, MAGE-B2, MAGE-B 3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C 2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E 1. MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2 (melanoma antigens) Family L2), mammaglobin A, MART-1 / Melan-A (T-cell-1 melanoma antigen recognized by IgG, MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor), mesothelial cells Phosphorus, MG50 / PXDN (peroxidasin), MMP11 (matrix metalloproteinase) anhydrase 11), MN / CA IX-antigen (carbonic anhydrase 9), MRP-3 (multidrug resistance related Mucin 1 (MUC1), MUC2, NA88-A (VENT-like homeostasis factor-3), obox2 pseudogene 1), N-acetylglucosaminyltransferase-V, Ne o-PAP (Neo-poly(A) polymerase), NGEP (new prostate-expressed gene), NMP22 (nuclear matrix protein 22), NPM / ALK (nucleophos Min), NSE (neuron-specific enolase), NY-ESO-1, NY-ESO-B , OA1 (osteoarthritis QTL1), OFA-iLRP (carcinoembryonic antigen immature laminin receptor protein), OGT (O-GlcNAc transferase), OS-9 (vesicle body lectin), osteocalcin, osteopontin, p15 (CDK inhibitor 2B), p 53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor) activator inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PART- 1 (prostate androgen-regulated transcript 1), PATE (prostate and testis expressed 1), PDEF (prostate-derived Ets factor), Pim-1 kinase (proviral integration site) Position 1), Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-phase interaction 1), POTE (expressed in the prostate, ovary, testis, and placenta), PRAME ( antigens preferentially expressed in leukocytes), prostein, proteinase-3, PSA (previously prostate-specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate-specific G-protein Protein-coupled receptor), PSM, PSMA (prostate-specific membrane antigen), RAGE-1 (renal tumor cancer) tumor antigen), RHAMM / CD168, RU1 (renal ubiquitous protein 1), RU2, SAGE (sarcoma antigen), SART-1 (squamous cell carcinoma antigen recognized by T-cells-1) , SART-2, SART-3, Sp17 (sperm protein 17), SSX-1 (SSX Family member 1), SSX-2 / HOM-MEL-40, SSX-4, STAMP -1 (STEAP2 metalloreductase), STEAP, survivin, survivin-2 13, TA-90 (tumor-associated antigen-90), TAG-72 (tumor-associated glycoprotein-72) ), TARP (TCRγ alternative reading frame protein), TGFb (transforming factor Transforming growth factor β), TGFbR11 (transforming growth factor β receptor 11), TGM-4 (transforming growth factor β receptor 11) glutaminase 4), TRAG-3 (Taxol resistance-associated gene 3), TRG (T-cell receptor γ locus), TRP-1 (transient receptor potential-1), TRP-2 / 6b , TRP-2 / INT2, Trp-p8, tyrosinase, UPA (U-plasminogen) activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK-1, and W T1 (Wilms' tumor 1).

[0200] In some embodiments, the target antigen is CD19 or CD22. In this embodiment, the target antigen is CD19.

[0201] In some embodiments, the mutated form of the tumor antigen is α-actinin-4 / m, AR TC1 / m, bcr / abl, beta-catenin / m, BRCA1 / m, BRCA2 / m , CASP-5 / m, CASP-8 / m, CDC27 / m, CDK4 / m, CDKN2A / m, CML66, COA-1 / m, DEK-CAN, EFTUD2 / m, ELF2 / m , ETV6-AML1, FN1 / m, GPNMB / m, HLA-A*0201-R170 I, HLA-A11 / m, HLA-A2 / m, HSP70-2M, KIAA0205 / m , K-Ras / m, LDLR-FUT, MART2 / m, ME1 / m, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class 1 / m, neo-PAP / m, NFY C / m, N-Ras / m, OGT / m, OS-9 / m, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX -1, SYT-SSX-2, TEL-AML1, TGFbRII, and TPI / m The compound is selected from the group consisting of:

[0202] In some embodiments, the expression of a gene that attenuates immune cell function is one of the following: (i) inhibiting immune cell proliferation; (ii) inducing immune cell death (iii) the target antigens required for immune cells to recognize and / or be activated; (iv) inhibiting the function of a molecule; instead of eliciting an immune response against the target antigen, (v) induce differentiation of immune cells into different types with specific functions; (v) stimulate immune cell responses (vi) reducing the immune cell's response to a molecule that promotes immune response; or It enhances immune cell responses to molecules that stimulate immune cells.

[0203] In some embodiments, the gene that attenuates immune cell function is PD1, PD-L1, C TLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEA CAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160 , CD96, MerTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGK alpha, DGK zeta, Cbl-b, Cbl-c, CD148, LRR1, TG Selected from the group consisting of FBR1, IL10RA, KLGR1, DNMT3A, and A2aR In some embodiments, the gene that attenuates the function of an immune cell is a gene that attenuates the immune response of an immune cell. In some embodiments, the immune cells enhance the response of the immune cells to molecules that inhibit the immune response. Genes that enhance immune cell responses to molecules that suppress immune responses are receptors of immune checkpoints. The nucleotide sequence encodes a target molecule or ligand.

[0204] In some embodiments, the immune checkpoint receptor or ligand is PD1, PD-L1, CTLA4, TIM3, CEACAM(CEACAM-1, CEACAM- 3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAI R1, CD160, CD96, MerTK and 2B4.

[0205] In some embodiments, the gene disrupting agent increases the expression of immune cells relative to immune cells in the absence of the gene disrupting agent. In total, we have at least 30, 40, or 50,000 genes in immune cells that weaken their function. In some embodiments, the gene expression level is reduced by 50, 60, 70, 80, 90, or 95%. Gene disrupting agents are molecules that suppress the immune response of immune cells and genes that enhance the response of immune cells. In some embodiments, the gene disrupting agent reduces expression of an immune checkpoint receptor. In some embodiments, the expression of a gene encoding a receptor or ligand is reduced. Gene disruptors include PD1, PD-L1, CTLA4, TIM3, and CEACAM (CEAC AM-1, CEACAM-3 or CEACAM-5), LAG3, VISTA, BTL A, a group consisting of TIGIT, LAIR1, CD160, CD96, MerTK, and 2B4 The expression of a gene selected from

[0206] In some embodiments, the gene disrupting agent is activated by RNA interference (RNAi) in immune cells. In some embodiments, the expression of more than one gene is reduced. The disruptor reduces the expression of genes in immune cells by RNAi, which impairs immune cell function. In some embodiments, the gene disrupting agent is a single gene that attenuates immune cell function. targeting different parts of the immune system, targeting different genes that weaken immune cell function, and In some embodiments, the RNAi In some embodiments, RNAi is mediated by short hairpin RNA (shRNA). is mediated by more than one shRNA.

[0207] In some embodiments, the RNAi is mediated by two shRNAs. In some embodiments, the two shRNAs target different parts of PD-1. In this study, two shRNAs target PD-1 and TIM-3, respectively. In an embodiment, the two shRNAs target PD-1 and CTLA-4, respectively. In some embodiments, the two shRNAs target PD-1 and LAG-3, respectively. In some embodiments, the two shRNAs target PD-1 and TIGIT, respectively. Let's say.

[0208] In some embodiments, the base sequence encoding the shRNA is SEQ ID NO: 1 to 219 and and 238-267.

[0209] In some embodiments, the expression of different shRNAs is driven by different promoters. In some embodiments, the expression of two different shRNAs is regulated by two different In some embodiments, the two promoters are regulated by different promoters. In some embodiments, the promoter is an RNA polymerase III promoter. In some embodiments, the two promoters are U6 promoters from different species. In this state, the two promoters are oriented in different directions from each other.

[0210] In some embodiments, the engineered antigen receptor and the gene disrupting agent each comprise a vector. In some embodiments, engineered antigen receptors and In some embodiments, the vector is a DNA disruptor. , RNA, plasmids, lentiviral vectors, adenoviral vectors and retroviral vectors In some embodiments, the vector is selected from the group consisting of lentivirus vectors. It is a viral vector.

[0211] "Immune cells" include killer T cells, helper T cells, gamma delta T cells, B cells, and Selected from lymphocytes such as neural killer cells, mast cells, eosinophils, and basophils Phagocytes include macrophages, neutrophils, and dendritic cells. The cells include CD4+ T cells and CD8+ T cells. In some embodiments, B cells Follicular lymphoma is classified as diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma (PCL), or PMBL, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, mediastinal gray zone In some embodiments, the T-cell lymphoma is leukemia, leukemia, or tuberous sclerosis (HL). , anaplastic large cell lymphoma (ALCL), peripheral T-cell lymphoma not otherwise specified (P TCL-NOS), or angioimmunoblastic T-cell lymphoma (AITL). In embodiments, the immune cells are T cells or T lymphocytes and natural killer ( In some embodiments, the immune cell is a T cell. In some embodiments, the T cells are CD4+ T cells or CD8+ T cells.

[0212] In some embodiments, the immune cells are generated from cells originally derived from the patient. In some embodiments, the subject can be a human. In other embodiments, the subject has a tumor or cancer. wherein the CAR or TCR expressed in the cell, e.g., Increased or fluctuating levels of cancer antigens targeted by mTCR are detected. In some embodiments, the cells are cultured using methods described, for example, in U.S. Pat. Nos. 6,352,694, 6,534, ,055, No. 6,905,680, No. 6,692,964, No. 5,858, No. 358, No. 6,887,466, No. 6,905,681, No. 7,144,5 No. 75, No. 7,067,318, No. 7,172,869, No. 7,232,56 No. 6, No. 7,175,843, No. 5,883,223, No. 6,905,874 Nos. 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 2 The method may be generally used to produce the oocytes described in US Pat. No. 5,995,525, and the oocytes may be grown in a manner similar to that described in US Pat. No. 5,995,525. It is also acceptable to do so.

[0213] In some embodiments, CAR-T cells are generated. The generation of AR-T cells includes providing peripheral blood monoclonal cells. In some embodiments, peripheral blood monoclonal cells can be isolated from a whole blood sample. In embodiments, the generation of CAR-T cells described herein involves the use of antibodies to induce peripheral blood immunization. For example, a step of stimulating the monoclonal cells includes stimulating the monoclonal cells with an agent that provides a primary stimulatory signal. is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-C In some embodiments, the antibody is a D28 antibody or antigen-binding fragment thereof. The production of CAR-T cells involves the step of transducing a CAR, during which the CAR is can target any of the targets described herein and other known targets of CARs, For example, the CAR can be a CD19 CAR that targets CD19. In this embodiment, the generated CAR-T cells are isolated.

[0214] Suitable conditions for T cell culture include serum (e.g., fetal bovine or human serum), interferon, and erythrocyte sequestration. leukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CS F, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or those skilled in the art Contains factors necessary for growth and survival, including any other known additives for cell growth. Suitable culture media (e.g., minimal essential medium, RPMI medium 1640, or X-viv) may also be used. Other additives for cell growth include surfactants, Plasmanate, as well as, for example, N-acetylcysteine ​​and 2-mercaptoethanol The media may contain, but are not limited to, amino acids, reducing agents such as phosphate buffer, phosphate buffer, and phosphate buffer. sodium bicarbonate and vitamins are added, and the product is serum-free or contains an appropriate amount of serum (also or plasma) or a defined set of hormones, and / or T cell proliferation and expansion RPMI 1640 supplemented with sufficient amounts of cytokine(s), AI MV, DMEM, MEM, α-MEM, F-12, X-Vivo15, and X-Viv o20, Optimizer, etc. Antibiotics, such as penicillin and Streptomycin is included only in experimental cultures, not in cultures of cells injected into subjects. The target cells are grown under the conditions necessary to support growth, e.g., an appropriate temperature (e.g., The T cells are maintained in a 6% CO atmosphere (e.g., air + 5% CO2) at 37°C. Several stimulation cycles may also be desired, such as for 0 days or more.

[0215] T cells exposed to different stimulation times may exhibit different characteristics. Typical blood products or apheresis peripheral blood mononuclear cells contain cytotoxic T cells or serotonin. The population of helper T cells (TH, CD8) is larger than the population of suppressor T cells (TC, CD8). 4+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors. Before about day 8-9, the T cell population is composed primarily of TH cells, whereas after about day 8-9, the T cell population is composed primarily of TH cells. From day 9 onwards, the population of T cells comprises a progressively larger population of TC cells. Depending on the purpose of treatment, it may be useful to inject a T cell population that is predominantly composed of TH cells into the subject. Similarly, if an antigen-specific subset of TC cells has been isolated, It may be beneficial to expand this subset to a greater extent. In addition to the D4 and CD8 markers, other phenotypic markers are expressed during the cell proliferation process. Although the changes are significant, most of them are reproducible. This allows for the ability to tailor activated T cell preparations for specific purposes.

[0216] Various assays can be used to evaluate CAR-T cells, e.g., proliferation following antigen stimulation. the ability to sustain T cell proliferation in the absence of restimulation, and the ability to induce appropriate in vitro and Anti-cancer activity can be assessed in animal models; assays are described in more detail below. is described in.

[0217] In some embodiments, Western blot analysis of CAR expression in primary T cells is performed. can be used to detect the presence of monomers and dimers. See, e.g., Milone, et al. al.,Molecular Therapy,17(8):1453-1464(2 See 009).

[0218] In some embodiments, in vitro expansion of CAR+ T cells following antigen stimulation is performed using flow cytometry. For example, the number of CD4+ T cells and CD8+ T cells can be measured by immunohistochemistry. The mixture was stimulated with αCD3 / αCD28 and then subjected to the expression of the promoter to be analyzed. This is followed by transduction with a lentiviral vector expressing GFP under control. The promoters include CMV IE genes, EF-1a, ubiquitin C, or phosphoglycerol. GFP fluorescence is detected by CD4+ and / or C D8+ T cell subsets are assessed by flow cytometry on day 6 of culture. For example, Milone, et al., Molecular Therapy, 17(8 ):1453-1464(2009). Alternatively, CD4+ and CD8+ T The cell mixture was transfected on day 0 with αCD3 / αCD28 coated magnetic beads. The cells were stimulated to express CAR together with eGFP using the 2A ribosomal skipping sequence. Transduce the cells with CAR on day 1 using a bicistronic lentiviral vector containing CAR. The culture medium may be, for example, hCD32 and 4-IBB in the presence of anti-CD3 and anti-CD28 antibodies. They were then restimulated with K562 cells expressing L (K562-BBL-3 / 28). Exogenous IL-2 is added to the cultures at 100 IU / ml every other day, followed by washing. T cells are enumerated by flow cytometry using a bead-based count. For example, Milone, et al., Molecular Therapy, 17(8 ):1453-1464 (2009). CAR+ T cells in the absence of restimulation Sustained proliferation of the cells can also be measured.

[0219] Assessment of cell proliferation and cytokine production can be performed using the methods described, for example, in Milone, et al., M Ocular Therapy, 17(8):1453-1464(2009) Briefly, assessment of CAR-mediated proliferation was performed using microtiter platelets. Washed T cells were plated with target cells, e.g., K562-Meso, Ovcar3 , Ovcar8, SW1990, Panc02.03 cells or CD32 and CD137 (KT32-BBL) at a final T cell:target cell ratio of 1:1 These signals support long-term ex vivo CD8+ T cell proliferation, and anti-CD 3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies were used as KT3 It was added to cultures with 2-BBL cells to serve as a positive control for stimulating T cell proliferation. CountBright™ fluorescent beads as described by the manufacturer (Invitrogen, Carlsbad, CA) and flow cytometry. T cells are counted in the culture. CAR+ T cells express CAR linked to eGFP-2A. Using T cells engineered with lentiviral vectors expressing GFP, The expression of CD4+ and CD8+ on T cells is identified by specific monoclonal antibodies ( Cytokine measurements were also performed by BD Biosciences. A human TH1 / TH2 cytokine cytometry bead array kit (B D Biosciences, San Diego, CA) for 24 hours after restimulation. Fluorescence was measured using a FACScalibur flow cytometer. The data are analyzed according to the manufacturer's instructions.

[0220] Cytotoxicity can be measured, for example, by the methods described herein in the Examples or by standard methods. Something like that 51 It can be evaluated by Cr release assay (Milone, et al. ,Molecular Therapy,17(8):1453-1464(2009) Briefly, target cells (e.g., BHK or CHO cells) are injected with 51Cr (NaCr O4, New England Nuclear, Boston, MA) at 37°C for 2 h. Load into a microtiter plate, wash twice with complete RPMI, and mix frequently between plates. Effector T cells are generated at various effector:target cell ratios (E:T). Mix with target cells in the well in whole RPMI. Add an additional well containing medium only (natural release, SR) or a solution of 1% triton-X 100 surfactant (total release, TR) After incubation at 37°C for 4 hours, the supernatant from each well is collected. , released 51 Cr is measured using a gamma particle counter (Packard Instrument Co., Waltham, MA). Each condition was performed in at least triplicate. The percentage of dissolution was calculated using the formula: % Dissolution = (ER-SR) / (TR-SR), where ER is the average released in each experimental condition 51 Flow-based cytotoxicity assay Alternative cytotoxicity assays such as may also be used.

[0221] Those described in the Examples section herein, as well as those known in the art, Other assays, including those described herein, may also be used to evaluate the CAR-T cells generated herein. can be used for.

[0222] In some embodiments, the immune cells generated herein contain two types of shRNA and for diseases in which the antigen targeted by the CAR or TCR, e.g., mTCR, is expressed. In a further embodiment, the immune cells can be used to treat a human patient. In some embodiments, pharmaceutical compositions for immunotherapy of a patient can be provided. Drug compositions can cause serious adverse reactions and impose high costs on patients. It has demonstrated therapeutic efficacy against targeted diseases without the need for immune checkpoint inhibitors. It is possible.

[0223] In some embodiments, the immune cells can be used as immunotherapy agents; Such immune cells are commonly used in, but not limited to, cancer treatment. In embodiments, to make these immune cells recognize cancer, they target cancer antigens. The cells are modified to express a cell surface receptor.

[0224] In another aspect, provided herein is an engineered immune cell as described above. The composition comprises:

[0225] Treatment using CAR-T cells targeting immune checkpoints 5.1 or higher As provided herein, dual-function vectors can be used to inhibit immune cell function. One or more types of short hairpin RNA (shRNA) are encoded to inhibit the expression of genes that attenuate the and chimeric antigen receptors (CARs) and T cell receptors (TCRs), e.g., and a base sequence encoding any one of the monoclonal T cell receptors (mTCR). According to one embodiment, the vectors can be used to generate immune cells, whereby immune cells The cells have reduced expression of immune checkpoint receptors and express target-specific CARs or or expressing a TCR, such as mTCR. A composition can be provided.

[0226] Introducing immune cells as described herein into a subject suitable for adoptive immunotherapy. In some embodiments, the provided The generated CAR-T cells are for allogeneic adoptive cell therapy. Provided are methods for administering the compositions described herein to a subject suitable for adoptive cell therapy, including introducing the compositions into the subject. Therapeutic uses of the compositions described herein, wherein the subject is a patient suffering from an autoimmune disease; a hematological malignancy; solid tumors; or HIV, RSV, EBV, CMV, adenovirus, BK polyoma Having a viral infection.

[0227] Examples of hematological malignancies include acute and chronic leukemias (acute myeloid leukemia (AML), acute lymphoblastic leukemia (AL), and leukemia-associated malignancies (LE). Lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, non-Hodgkin's lymphoma These include non-Hodgkin's disease, multiple myeloma, and myelodysplastic syndromes. Examples of solid cancers include, but are not limited to, brain, prostate, breast, lung, colon, uterus, skin, Liver, bones, pancreas, ovaries, testes, bladder, kidneys, head, neck, stomach, cervix, rectum, larynx, and Examples of various autoimmune disorders include, but are not limited to, cancer of the esophagus. Alopecia, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), juvenile idiopathic rheumatoid arthritis some forms of idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic Thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / Pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, Rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus thyroiditis, some forms of thyroiditis, some forms of uveitis, vitiligo, polyangiitis Infections caused by viral infections include, but are not limited to, granulomatous disease (Wegener's disease). Examples of diseases include HIV (human immunodeficiency virus), HSV (herpes simplex virus), KSHV (Kaposi's sarcoma-associated herpesvirus), RSV (respiratory syncytial virus), EBV (Epstein-Barr virus), CMV (cytomegalovirus), VZV (varicella-zoster virus), adenovirus, lentivirus, BK polyomavirus Examples of diseases include, but are not limited to, diseases associated with rhesus.

[0228] Acute leukemia is characterized by the rapid proliferation of immature blood cells. This overcrowding causes the bone marrow to The body is unable to produce healthy blood cells. Acute forms of leukemia can occur in children and young adults. In fact, it is a more common cause of death among American children than any other type of malignancy. Acute leukemia requires immediate treatment due to the rapid progression and accumulation of malignant cells. The malignant cells then spill into the bloodstream and spread to other organs of the body. Although involvement is rare, the disease can occasionally cause cranial nerve palsies. It is distinguished by the excessive accumulation of mature but still abnormal blood cells. It takes months to years for the cells to develop, and they are produced at a much faster rate than normal cells, As a result, there are many abnormal white blood cells in the blood. Chronic leukemia occurs mainly in older people, but Theoretically, it can occur at any age. Acute leukemia requires immediate treatment, whereas However, chronic forms should be monitored for some time before treatment to ensure maximum effectiveness of treatment. In addition, the disease affects certain types of bone marrow cells that normally progress to form lymphocytes. Lymphocytic or lymphoblastic, which indicates that cancerous changes have occurred, and usually erythroid, Cancerous changes occur in certain types of bone marrow cells that go on to form certain types of white blood cells, platelets. Classified as bone marrow-originated or myeloid (see Lymphoid and Myeloid Cells) indicating can be.

[0229] Acute lymphocytic leukemia (also known as acute lymphoblastic leukemia, or ALL) is a small It is the most common type of leukemia in children. The disease also occurs in adults, especially those over 65. Chronic lymphocytic leukemia (CLL) occurs most commonly in adults over the age of 55. Acute myeloid leukemia (AML) occurs occasionally in young adults but rarely in children. Myeloid leukemia (AML), also known as leukemia, occurs more often in adults than in children. This type of leukemia was previously called "acute nonlymphocytic leukemia." Chronic myeloid leukemia (CML) ) occurs primarily in adults. A small number of children also develop the disease.

[0230] Lymphomas originate in lymphocytes (a type of white blood cell in the immune system of vertebrates). There are many types of lymphoma. According to the National Institutes of Health, Hodgkin's lymphoma accounts for approximately 5% of all cancer cases in the United States, and Hodgkin's lymphoma accounts for approximately 10% of all cancer cases in the United States. It accounts for less than 1% of all cancer cases. The lymphatic system is part of the body's immune system, so H Patients with weakened immune systems due to IV infections or certain medications or drugs are also at higher risk of developing lymphoma. Has an incidence rate.

[0231] In the 19th and 20th centuries, it was discovered by Thomas Hodgkin in 1832, This disease was called Hodgkin's disease. Colloquially, lymphoma is divided into Hodgkin's lymphoma and non-Hodgkin's lymphoma. Lymphoma is broadly classified as lymphoma (all other types of lymphoma). The classification is more detailed. The older classification referred to histiocytic lymphoma, but these are now classified as B, T, and Or they are recognized in a newer classification as being of the NK cell lineage.

[0232] Patients with cancers that express a target molecule of a CAR or TCR, e.g., mTCR When the pharmaceutical composition is administered to the patient, the pharmaceutical composition contains a gene that weakens the function of immune cells related to cancer cells. It does not activate genes and is caused by the activation-inhibitory signal transduction that it induces. These cells can recognize cancer and have immune activity without problems like exhaustion.

[0233] In some embodiments, the pharmaceutical composition comprising immune cells is an immune checkpoint inhibitor. The expression of the chimeric antigen receptor can be more effectively suppressed while at the same time This can maximize the effectiveness of anti-cancer immune cell therapy. In embodiments, the expression of an immune checkpoint receptor is inhibited as described above. The cells are used in pharmaceutical compositions, and other inhibitors of immune checkpoint receptors are also Severe and potentially life-threatening conditions such as cytokine release syndrome or autoimmune conditions that may result from the use of and the increased costs of treatment resulting from the combination of expensive antibody therapy with cell therapy. It is possible to eliminate the burden of the increase.

[0234] In addition to the cells, other pharmaceutically acceptable salts, carriers, excipients that may further improve the immune response. It is obvious that vehicles and other additives may be added to the pharmaceutical composition. A detailed description of the above should be omitted.

[0235] In some embodiments, the pharmaceutical composition comprises two immunosuppressants as described herein. These include dual CAR-T cells that target checkpoints. For example, dual immune checkpoint The objective of this study was to investigate the role of PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), and 1), CTLA4 (cytotoxic T lymphocyte-associated protein 4), TIM-3 (T cell immune response immunoglobulin and mucin domain-containing-3), CEACAM (3 subtypes of CEAC Carcinoembryonic antigen-related cell adhesion molecules, including AM-1, CEACAM-3, or CEACAM-5 ), LAG3 (lymphocyte activation gene 3), VISTA (V domain I of T cell activation) g suppressor), BTLA (B and T lymphocyte attenuating factor), TIGIT (Ig and IT T cell immunoreceptor with IM domain), LAIR1 (leukocyte-associated immunoglobulin-like receptor CD160 (cluster of differentiation 160), CD96 (cluster of differentiation 96), MerTK (proto-oncogene tyrosine protein kinase MER), and 2B4 (NK cell activation-inducing ligand and PD1 and TIM3. That's fine.

[0236] In some embodiments, the type of immune checkpoint targeted determines the anti-tumor efficacy of the pharmaceutical composition. For example, pharmaceutical compositions targeting PD-1 and TIM3 may affect the efficacy of PD-1. and can exhibit a different level of antitumor effect from pharmaceutical compositions targeting TIGIT. In a further embodiment, the difference in anti-tumor effect is due to the same immune checkpoint unpredictable from known knowledge about the antitumor effects of other drugs targeting e.g. Other agents include antibodies that target immune checkpoints. In fact, targeting two specific immune checkpoints has surprisingly potent antitumor effects. In an exemplary embodiment (Example 10), PD-1 and TIG CAR-T cells targeting IT bind to PD-1 and CTLA-4, PD-1 and LAG-3, and and other dual-KD CAR-T cells targeting a combination of PD-1 and TIM-3. It shows surprisingly excellent antitumor effects.

[0237] In one aspect, the compositions described herein can be provided in unit dosage form, with each A dosage unit, e.g., an injection, contains a predetermined amount of a composition, either alone or in appropriate combination with other active agents. The term "unit dosage form" as used herein refers to a dosage form for human and animal use. refers to physically discrete units suitable as unit dosages for an individual, each unit having a desired effect. a predetermined amount of the composition described herein alone calculated to be sufficient to produce or in combination with other active agents, in association with a pharmaceutically acceptable diluent, carrier or vehicle as appropriate. The specifications for the novel unit dosage forms of the cells or compositions described herein are particularly The efficacy and safety of a particular compound will depend on the particular pharmacodynamics associated with the compound in a particular subject.

[0238] In some embodiments, preferred medicaments for the cells or compositions described herein The dosage form may be determined based on the contents of this disclosure and general knowledge of the pharmaceutical arts, as well as the intended route of administration. Regardless of the method of administration, the patient's weight may be determined according to the delivery method and the target dose. The effective dose may be calculated according to the size, surface area, or organ size. Calculations to determine the appropriate dosage for treatment using each dosage form, as well as additional Purification of is practiced daily in the art and is within the scope of work practiced daily in the art. Appropriate dosages may be identified through use of appropriate dose-response data.

[0239] The pharmaceutical compositions described herein may be used alone or in combination with other known compounds useful in the treatment of cancer. It can be used alone or in combination with other agents. Whether delivered or not, the pharmaceutical compositions described herein can be administered via a variety of routes. and can be delivered via a variety of routes to various parts of the mammalian, particularly human, body to achieve specific effects. Those skilled in the art will appreciate that more than one route of administration may be used, but that a particular route is preferred. will recognize that it may provide a more rapid and effective response than alternative routes. For example, intradermal delivery may be used advantageously over inhalation for the treatment of melanoma. Alternatively, systemic delivery may involve application or instillation of a formulation into a body cavity, inhalation or blowing of an aerosol. or by administration including intramuscular, intravenous, intraportal, intrahepatic, intraperitoneal, subcutaneous, or This can be achieved by parenteral administration, including intradermal administration. Routes of administration include intravenous (IV) injection and local (intratumoral, intraperitoneal) administration. In this embodiment, the pharmaceutical composition can be administered via injection into a solid tumor.

[0240] In some embodiments, the genomically engineered immune cells as provided herein are and antibodies or antibody fragments that target antigens associated with a condition, disease, or indication. Additional therapeutic agents may be used in combination therapy with these effector cells. In some embodiments, the antibody is a monoclonal antibody. The antibody is a humanized antibody, a human antibody, a monoclonal antibody, or a chimeric antibody. In another embodiment, the antibody or antibody fragment specifically binds to a viral antigen. or antibody fragments that specifically bind to tumor antigens. A suitable antibody for combination therapy as an additional therapeutic agent for engineered immune cells is anti-CD 20 (rituximab, veltuzumab, ofatumumab, ublituximab, ocaratu anti-HER2 (trastuzumab, pertuzumab), anti-CD52 (anti-HER2) lemtuzumab), anti-EGFR (celtuximab), anti-GD2 (dinutuximab), anti-PD L1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab); and their Humanized or Fc-modified variants or fragments, or functional equivalents thereof, and These include, but are not limited to, biosimilars and biosimilars.

[0241] Desirably, an effective amount or sufficient number of isolated transduced T cells are present in the composition. and long-term specific antitumor responses have been established that would otherwise be observed in the absence of such treatment. reducing the size of the tumor to less than that which would occur in a conventional treatment, eliminating tumor growth or regrowth; Desirably, the amount of transduced T cells reintroduced into the subject is 10% reduction in tumor size compared to otherwise identical conditions in the absence of transduced T cells %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98 %, or 100% reduction.

[0242] Therefore, the amount of transduced T cells administered should take into account the route of administration. and a sufficient number of transduced T cells are introduced to achieve the desired therapeutic response. Furthermore, the amount of each active agent contained in the compositions described herein must be The amount (e.g., amount per cell contacted or amount per specific body weight) varies depending on the response. Generally, the concentration of transduced T cells is preferably At least about 1 x 10 6 pieces~approx. 1×10 9 Transduced T cells, even more desirable The figure is approximately 1 x 10 7 pieces~approx. 5×10 8 providing transduced T cells to the subject to be treated The amount should be sufficient to 8 More than individual cells, or 1×10 7 Fewer than 100 cells can be used. These can be based on cell-based therapies (e.g., Topalian and Rose nberg, 1987; see U.S. Pat. No. 4,690,915), or alternative A continuous infusion strategy can be employed.

[0243] These values ​​may be utilized by the practitioner in optimizing the methods described herein. This provides general guidance on the range of transduced T cells that may be used. The specification may include more or fewer recitations as may be justified in a particular application. This does not in any way preclude the use of amounts of ingredients. For example, actual doses and schedules may vary. , depending on whether the composition is administered in combination with other pharmaceutical compositions, or on the pharmacokinetics, These may vary depending on individual differences in the kinetics and metabolism of substances. , and can easily make necessary adjustments according to the exigencies of a particular situation.

[0244] Any of the compositions described herein may be included in a kit. In embodiments, the CAR-T cells are incubated with reagents suitable for expanding the cells, such as media, aA PCs, growth factors, antibodies (e.g., for selecting or characterizing CAR-T cells), and / or a plasmid encoding a CAR or a transposase. It is provided in the form of a .

[0245] Non-limiting examples include chimeric receptor expression constructs, One or more reagents, cells for transfection of the expression construct, and / or transfection of the expression construct one or more instruments for obtaining allogeneic cells for the purpose of the invention (such instruments may include syringes, pipettes, may be forceps and / or such medically approved devices).

[0246] In some embodiments, expression constructs, constructs, One or more reagents for generating the construct, and / or CAR+ T cells are provided in the kit In some embodiments, a nucleic acid encoding the zinc finger nuclease(s) is / are In some embodiments, the kit includes an expression construct for electroporating cells. This includes reagents or equipment for the analysis.

[0247] The kit may comprise one or more suitably aliquoted compositions described herein or a combination of the compositions described herein. The kit may include reagents for producing the compositions as described in the document. The container means of the kits may contain the components. At least one bar may be filled with at least one portion, which may be suitably divided into equal parts in certain embodiments. The container may include a vial, test tube, flask, bottle, syringe, or other container means. If there is more than one component in the kit, the kit generally includes additional components that may be packaged separately. It may also contain a second, third, or other additional container. Various combinations may be contained in the vials. The kits described herein also include Typically, the chimeric receptor construct and any other reagents are tightly confined for commercial sale. Such a container may also include a means for containing the desired vial. The container may include an injection molded or blow molded plastic container in which the bottle is held. .

[0248] In one aspect, provided herein is a method for treating a rheumatoid arthritis, comprising administering to a subject a therapeutically effective amount of an immune cell as described above. and an acceptable carrier. A pharmaceutical composition for immunotherapy of a human patient comprising the immune cells described above. In some embodiments, the immune cells are originally derived from the patient. The level of a cancer antigen targeted by a CAR or TCR expressed in a target cell, e.g., mTCR, is Have a tumor or cancer in which an increase or fluctuation in blood pressure is detected.

[0249] In another aspect, provided herein are methods. In some embodiments, the methods The immune cells or the immune system cells described above are administered to a subject having a disease or condition. In some embodiments, a method of treatment includes administering the described composition. The engineered antigen receptor specifically binds to an antigen associated with a disease or condition. In some embodiments, the disease or condition is cancer or a tumor.

[0250] In another aspect, provided herein are immune cells and compositions. In one embodiment, provided is a compound as described above for use in treating a disease or condition. Immune cells and compositions.

[0251] In another aspect, provided herein are uses of the immune cells or compositions. In some embodiments, provided are drugs for use in methods of treating a disease or condition. The use of the immune cells or compositions described above in the manufacture of some In embodiments, the engineered antigen receptor is specific for an antigen associated with a disease or condition. In some embodiments, the disease or condition is cancer or a tumor.

[0252] The above description of the invention is intended to be illustrative and not restrictive, and those skilled in the art will readily appreciate the technical capabilities of the invention. The present invention may be readily modified into specific other forms without altering its spirit or essential characteristics. It should be understood that the above-described embodiments are exemplary and should not be construed as limiting the scope of the present invention. It should be understood that the above is not limiting in any way. Each component described as being combined may be, and should be, implemented separately. Components that are described as being combined may also be implemented as an integrated unit.

[0253] The scope of the present invention is defined by the appended claims, and the meaning of the claims and their equivalents is intended to be construed as limiting the scope of the present invention. and all modifications or variations thereof that fall within the scope of the present invention are included within the scope of the present invention. should be interpreted as follows. Sequence Listing [Table 2] TIFF0007763824000003.tif247170TIFF0007763824000004.tif248170TIFF0007763824000005.tif24817 0TIFF0007763824000006.tif247170TIFF0007763824000007.tif247170TIFF0007763824000008.tif18517 0TIFF0007763824000009.tif218170TIFF0007763824000010.tif235170TIFF0007763824000011.tif9817 0TIFF0007763824000012.tif247170TIFF0007763824000013.tif247170TIFF0007763824000014.tif77170 [Example]

[0254] Examples related to the present invention are described below: In most cases, alternative techniques are used The examples are intended to be illustrative and not limiting or restrictive on the scope of the invention. It is not intended to be limiting.

[0255] General method Cell lines and culture. Nalm-6, Nalm-6GL (GFP and firefly luciferase) expressing), K562, K562-CD19, IM-9, Raji, and Daudi Cell lines were incubated in a humidified incubator at 37°C with 5% CO2 in 10% heat-inactivated fetal bovine serum. and RPMI supplemented with 2 mM L-glutamine and 1% penicillin / streptomycin. The Lenti-X™ 293T cell line was purchased from Takara. 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mM non- Essential amino acids, 1 mM sodium pyruvate and 1% penicillin / streptomycin The cells were maintained in DMEM supplemented with CD19. + PD-L1 + To generate cell lines, 562-CD19 cells or NALM-6-GL cells were transfected with a human PD-L1-encoding antibody. The mice were transduced with the Nalm-6-PDL1-CD virus (NM_014143.3). 80 cell lines were transfected with lentivirus (NM_005191.3) encoding human CD80. It was generated by transduction of Nalm-6-PDL1 cells with

[0256] Plasmid construction. Anti-CD19 scFv (FMC63) and CD8α hinge and transmembrane domains. pLV containing the cytoplasmic domains of 4-1BB (CD137) and CD3ζ and the common region The construction of the -CD19-BBz vector has been previously described (PNAS, 2016, M a JSY). To generate pLV-CD19-28z, the cytoplasmic domain of 4-1BB was cloned. The sequence of the human CD28 costimulatory domain was replaced with that of the human CD28 costimulatory domain. For detection and purification, the ΔLNGFR (cytoplasmic domain truncated CD271) sequence was cloned using p Amplified from the MACS-ΔLNGFR (Milteny Biotec) vector, P2A Insert the CAR transgene via the sequence pLV-ΔLNGFR-CD19-2 8z or pLV-ΔLNGFR-CD19-BBz was obtained.

[0257] A dual-function LV vector encoding both CAR and shRNA expression cassettes was developed. To generate vectors, shRNA (linking sequence: TTCAAGAGA, termination sequence: TT TTT) and a sh containing Pol III promoter (mU6, hU6, or hH1) Synthesize an RNA expression cassette and insert a CAR upstream of the central polypurine tract (cPPT). The vectors were subcloned into LV vectors encoding different promoters (mU6 and hU6 ) to generate a dual-function vector expressing two shRNAs. To do this, we cloned the BstZ171-Xba1-Nde1-Bmt1-Spe1 MCS sequence into pLV-hU6-shPD-1_ΔLNGFR-CD19-BB downstream of the 6 promoter The second mU6-shRNA cassette fragment was subcloned into the MCS. I did it.

[0258] To establish the reporter vector, pGL2_NFAT-Luc reporter (add The NFATREx3 sequence derived from the NFATREx3 gene (gene #10959) is amplified by PCR. The sequence of NF-kB-RE5x(5'-GGGAATTTCC-3') and miniP was The EF-1a vector of pLV-eGFP was synthesized (IDT Technologies). The promoter was replaced with these reporter fragments, creating pLV-NFAT-RE 3×-e GFP or pLV-NF-kB-RE 5×-eGFP reporter vector was obtained.

[0259] Selection of siRNA or shRNA sequences. Inhibitory immune checkpoint (CTLA- 4, 21-nt-long siRNA specific for LAG-3, TIGIT, and TIM-3 Candidate sequences were analyzed using BLOCK-iT™ RNAi Designer or S RNA oligomers designed using the fold program. Targeting CTLA-4. The siRNAs used were selected from the group consisting of SEQ ID NOs: 255 to 260. The siRNA targeting TIGIT was selected from the group consisting of SEQ ID NOs: 244 to 254. The siRNA targeting TIM-3 was selected from the group consisting of SEQ ID NOs: 238 to 243. The targeted siRNA was selected from the group consisting of SEQ ID NOs: 261 to 264 (IDT Te To analyze the dynamics of immune checkpoint expression, PBMCs were used. in the presence of human recombinant IL-2 using Dynabeads Human T-Activator or CD3 / CD28 (Thermofisher) or 4 μg / ml anti-CD3 The cells were stimulated with 2 μg / ml of anti-CD28 antibody and 2 μg / ml of anti-CD28 antibody. The cells were analyzed for 12 days (days 3, 6, and 12). To achieve this, 2 days after stimulation, PBMCs were transfected with Neon® Transfection S siRNA oligomers were electroporated using a system (Thermofisher). The knockdown efficiency of siRNA was evaluated by flow cytometry 2 days after transfection. Based on their efficiency, two or three immune checkpoints were measured. Select three siRNA sequences and convert them into shRNA format to create a two-in-one doublet. Lentiviral vectors were generated to verify the efficacy of shRNA-mediated knockdown. To achieve this, lentiviral-transduced T cells were transduced in γ-irradiated K562 cells at a 1:1 ratio. -CD19 cells were stimulated for 3 days and analyzed by flow cytometry to detect immune checkpoint Its expression level was analyzed.

[0260] Flow cytometry. The expression level of anti-CD19 CAR was measured by AF647-conjugated antibody. Mouse F(ab')2 antibody (115-606-072, Jackson ImmunoR Research) or AF647-conjugated streptavidin (405237, Bi Biotin-conjugated rhCD19-Fc (CD9-H5259) combined with ΔLNGFR expression was analyzed by a cytotoxic assay (ACRO Biosystems). or FITC-conjugated anti-CD271 antibody (ME20.4-1.H4; Mitenyi The expression of immune checkpoints in CAR-T cells was analyzed by a randomized controlled trial (NCT02224262). The following antibodies were used: PD-1 (PE, clone J105; Thermofisher), T IM-3 (PE, clone 344823; R&D Systems), LAG-3 (PE , clone 7H2C65; Biolgend), TIGIT (PE, clone MBSA4 3; Thermofisher) by conventional flow cytometry. .

[0261] CTLA-4 expression in CAR-T cells was significantly increased in irradiated NALM-6 cells or K56 After 3 days of incubation with 2-CD19 cells at a 1:1 E:T ratio, intracellular flow Cells were analyzed by cytometry. Fixation / permeabilization with anti-human CD152 (C The cells were stained with TLA-4) antibody (PE, clone BNI3; Biolegend).

[0262] Expression of stimulatory or inhibitory immune checkpoint ligands on tumor cells was assessed using the following antibodies: CD80 (PE, clone 2D10; Biolgend), CD86 (BV421, clone Lone 2331 (FUN-1; BD Bioscience), PD-L1 (APC, clone 29E.2A3; Biolgend), HLA-DR (PE, clone L243 ;Biolgend), CD112 (PE, clone TX31;Biolgend), C Analysis was performed using D155 (PE, clone SKII.4; Biolgend).

[0263] To analyze the effect of TGF-β on PD-1 expression, CAR-T cells were cultured at 10 NALM irradiated in the presence of ng / ml recombinant human TGF-β1 (R&D Systems) PD-1 expression was then measured by flow cytometry. Measured.

[0264] Analysis of SMAD2 / 3 phosphorylation status by intracellular flow cytometry. SMAD2 To determine the phosphorylation status of CAR-T cells, we incubated CAR-T cells at a 1:1 E:T cell ratio with NAL The cells were incubated with M-6 cells for 4 or 24 hours. Fixation was performed with buffer (BD Bioscience), followed by Perm Buffer III (B Permeabilization was performed using LNGFR (D Bioscience). + CAR-T cell phosphorylation The oxidation state was determined by anti-human Smad2 (pS465 / pS467) / Smad3 (pS423 / pS425) antibody (PE, clone 072-670; BD Bioscience). I determined that.

[0265] Detection of induced suppressor T cells. Induction of suppressor T cells generated from CAR-T cells ( CD4 + CD25 + FOXP3 + ) after 3 days of co-culture with NALM-6 cells. The cells were analyzed by in situ staining using Foxp3 / transcription factor staining buffer (Thermofisher ) and permeabilized cells were incubated with the following antibodies: anti-CD4 (BV605, clone OKT4 ; Biolgend), anti-CD25 (FITC, clone VT-072; Biolgen d), and anti-FOXP3 (APC, clone 236A / E7; Thermofisher ) and stained.

[0266] CAR-T proliferation assay. CAR-T cells expressing the ΔLNGFR surface marker were Selection was performed using magnetic beads (Miltenyi Biotec). + CAR -T cells (1 x 10 6 100 cells (purity >95%) were irradiated every 6 days in the absence of cytokines with γ-rays. Irradiated K562-CD19-PDL1 cells (1 × 10 6 CAR-T cells were stimulated with Cell proliferation was assessed by counting cells on days 6, 12, and 18 using trypan blue exclusion. It was calculated by

[0267] In vitro cytotoxicity of CAR-T cells. The cytotoxicity of CAR-T cells was measured using Incucyto The results were determined using the teS3 live cell analysis system. NALMs constitutively expressing GFP 1 x 10 NALM-6 or NALM-6-PDL1 target cells per well in triplicate 5 pieces Cells were plated in a 96-well plate at a density of 1000 x g. + CAR-T cells 1:1, 0. The E:T ratios were 3:1 and 0.1:1, respectively. The change in GFP intensity was recorded every 2 hours as the integrated intensity of green matter (mean, mean GFP intensity × μm 2 / The ratio of relative green integrated intensities was calculated using the following formula: (total GFP integrated intensity at each time point) The intensity was calculated by (degrees / total GFP integrated intensity at the starting point)*100.

[0268] NFAT and NF-κB reporter assay. NFAT transcription factor in CAR-T cells To determine the specific activity of the molecule, 4-HT2B was incubated in the presence of human recombinant IL-2 (300 IU / mL). PBMCs were stimulated with 2 μg / ml anti-CD3 and 2 μg / ml anti-CD28 antibodies for 2 days. First, they were transduced with a lentivirus encoding the NFAT-RE×3-eGFP reporter gene. Eight days after transduction, all cells were incubated with human recombinant IL-2 (300 IU / mL). The activated cells were then restimulated with anti-CD3 and anti-CD28 antibodies for 2 days at room temperature. Split the vector into two separate wells and inoculate the vector with lentiviruses encoding different CARs (CD19-28 After 6 days, all cells in each well were transduced with NALM-6. The NFAT reporter activity in CAR-T cells was measured at 200 μg / mL. At 4 hours and 48 hours, LNGFR + CAR-T population (20% of total CD3+ cells) The gMFI value of the eGFP signal was determined within 25% of the normalized CAR-T cell population. The specific activity of NF-κB transcription factors was determined following a similar procedure, but using NF-κB-RE x 5-Measured using a lentivirus encoding the eGFP reporter gene.

[0269] Quantitative real-time PCR: 3 x 10 6 LNGFR + G28z or GBBz C AR-T cells CD19 + The cells were co-cultured with NALM-6 target cells at a 1:1 ratio for 4 hours. and LNGFR after 48 h of co-culture. + CAR-T cells are MoFlo Astrios Sorting was performed using a sorter (Beckman Coulter). + CAR- mRNA was extracted from T cells using the RNeasy mini kit (Qiagen) and analyzed by Qua ntiTect Reverse Transcription Kit (Qiagen) cDNA was reverse transcribed using a CFX96 Real-Time PCR system. PCR detection system (Biorad) and SYBR Green real-time PCR mass spectrometry The reaction was carried out using Termix (TOYOBO) according to the SYBR protocol. Primer sequences for detecting RNA include SEQ ID NOs: 268 and 269. Primer sequences for detection include SEQ ID NOs: 270 and 271. Primer sequences include SEQ ID NOs: 272 and 273. Primer sequences for detection of IL4 The row contains SEQ ID NOs: 274 and 275. The primer sequences for detection of IL17A are The primer sequence for detecting CD25 is SEQ ID NO: 278 and 279. Primer sequences for detecting CTLA4 include SEQ ID NOs: 280 and 28 Primer sequences for the detection of FOXP3 include SEQ ID NOs: 282 and 283. Primer sequences for the detection of TGF-beta 1 include SEQ ID NOs: 284 and 285. Primer sequences for the detection of TGFBR1 include SEQ ID NOs: 286 and 287. TGF Primer sequences for the detection of BR2 include SEQ ID NOs:288 and 289.

[0270] The amount of target mRNA was normalized to the endogenous reference 18s rRNA: ΔCt(assay Ct (target gene) = Ct (target gene) - Ct (18s rRNA). Apply the comparative Ct method as follows: Equation:2 -ΔΔCt =2 ∧ - based on (ΔCt [stimulated] - ΔCt [unstimulated]) , the relative fold change of target mRNA compared to the unstimulated condition was analyzed.

[0271] Animal experiments. All procedures described herein were performed using the Institutional Animal Care and Use System at KAIST. Approved by the committee. CD19 + NSG mice to establish hematological cancer models At 4-6 weeks of age, the mice express EGFP-fused firefly luciferase and human PDL1. It is operated like a 1x10 6 CD19 + NALM-6 leukemia cells (NALM6- GL-PDL1 cells) were intravenously injected. CAR-T cells were injected intravenously using the procedure described above. On day 4 post-transduction, CAR - Selecting T cells NALM6-GL-PDL1 was isolated and expanded for an additional 6 days before injection into mice. Five days after cell injection, 2.5 or 1 × 10 6 CAR-T cells were intravenously injected into mice. Bioluminescence imaging of NALM6-GL-PDL1 in mice was performed using Xenogen IV. The signal was monitored with IS Spectrum and recorded in Living Image software. Radiance (photons / second) in the area of ​​interest using a Perkin Elmer For solid tumor models, NSG mice were injected with 5 × 10 6 IM-9 thin cells (CD19 + PD-L1 + CD155 + ) was injected subcutaneously. + CAR-T thin The cells were cultured 14 days after tumor cell injection (approximately 150–300 mm 3 (tumor volume) in mice intravenously Tumors were monitored weekly by caliper measurement and the volume was (length × width) 2 ) / 2 was estimated by

[0272] Example 1: Two-in-one function by expressing CART19 and shRNA targeting PD-1 Methods for constructing and evaluating functional vectors This example demonstrates the use of a two-in-one mouse model expressing CART19 and an shRNA that inhibits PD-1. This paper describes methods for constructing and evaluating vectors with two functions.

[0273] Dual-function lentivirus encoding both CAR and shRNA expression cassettes To generate viral vectors, shRNA (linking sequence; TTCAAGAGA, end sequence; TTTTT) and a Pol III promoter (mU6, hU6, or hH1). Synthesize an shRNA expression cassette containing the nucleotide sequence upstream of the central polypurine tract (cPPT). The CAR was subcloned into an LV vector encoding the EF-1α promoter. Therefore, 4-1BB-based CART19 was targeted to PD-1 by the mU6 promoter. We constructed a lentiviral vector that spontaneously expresses shRNA targeting the target gene (Figure 1A and 1B). B).

[0274] Three shRNA candidates were used to select shRNAs targeting PD-1. shPD-1#1 significantly increased CAR expression (Figure 1C), homeostatic proliferation (Figure 1D), differentiation status, and C PD-1 expression (Fig. 1F) without affecting the D4 / CD8 composition (Fig. 1F). The expression level of shRNA was significantly higher than that of Pol III protease inhibitors. It has been reported that this may vary depending on the type of motor (Mol. Ther. ,2006,Irvin SYChen). Therefore, the mU for PD-1 expression The effects of the mU6, hU6, or hH1 promoter were evaluated. mU6 and hu6 had similar KDs. hH1 showed efficacy, but hH2 was even less effective (Figure 2).

[0275] The dual-function lentiviral vector constructed here is capable of expressing PD-1. Therefore, the PD-1 KD modified C was used in the following examples. It can be used to generate AR-T cells.

[0276] Example 2: Method for producing PD-1 KD CAR-T cells and in vitro evaluation This example describes the method for producing PD-1 KD-modified CAR-T cells and their in vitro evaluation. It is listed.

[0277] Peripheral blood mononuclear cells were cultured using Ficoll-Paque Plus (GE Healthcare e) These cells were isolated from whole blood samples of healthy donors by density gradient centrifugation. 4 μg / ml in the presence of 300 IU / ml human recombinant IL-2 (BMIKOREA) Plate-bound anti-CD3 antibody (clone OKT3; Bio X cell) and 2 μ The cells were stimulated with 1 μg / ml of soluble anti-CD28 antibody (clone CD28.2; Bio X cell). For recombinant lentivirus production, 2.5 mL of growth medium was added 24 hours before transfection. Culture medium (10% FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1 mM 6x10 in DMEM (supplemented with M sodium pyruvate) 5 6 293T cells The cells were seeded in a well plate. 10 μl of Lipofectamine 2000 (Ther Using mofisher, packaging vectors (pMDL, pRev, pMDG Two days after transfection, the lentils were transfected with a mixture of .1) and transfer vector. The virus-containing culture supernatant was collected and centrifuged at 1800 rpm for 5 minutes. The cells were mixed with the viral supernatant in the presence of protamine sulfate (1 μg / ml) and centrifuged at 1000 × g. The mixture was centrifuged for 90 minutes and incubated overnight at 37°C. The next day, the culture supernatant was aspirated and diluted with 10% F BS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate The mixture was then mixed with fresh RPMI-1640 supplemented with 55 μM sodium and 55 μM β-mercaptoethanol. 1 × 10 transduced T cells were 6 10% heat-inactivated at a density of less than 10 cells / mL Supplemented with activated FBS, 2 mM L-glutamine, and 1% penicillin / streptomycin. RPMI-1640, containing human recombinant IL-2 (300 IU / mL), T cells The cells were cultured in medium, which was replenished every 2–3 days.

[0278] The in vitro lytic and proliferative activities of shPD-1 CAR-T cells were examined. m-6-PDL1 or K562-CD19-PDL1 to CD19 + PDL1 + target cell First, we used the IncuCyte real-time imaging system. Long-term lytic activity was investigated. When CAR-T cells were mixed with NALM-6 cells, WT (s hGFP) CAR-T cells and PD-1 KD (shPD-1) CAR-T cells had similar lytic activity. However, when CAR-T cells were cultured with NALM-6-PDL1 cells, When compared with the shPD-1 CAR-T cells, the lytic activity of the shGFP CAR was surprisingly higher. Unexpectedly, BBz containing no shRNA cassette showed effective lytic activity (Fig. 3A). CAR-T cells and shGFP CAR-T cells were also found to have similar cytotoxicity. This suggests that shRNA expression itself has little effect on CAR-T cell activity. This suggests that it does not have

[0279] Next, we investigated the proliferative activity of CAR-T cells repeatedly exposed to CD19 antigen and PD-L1. shPD-1 CAR-T cells expressed shGFP C in the absence of exogenous IL-2. 4-5 times higher T cell response to K562-CD19-PDL1 targets than AR-T cells Costimulatory ligands promote optimal T cell proliferation and tumor proliferation (Figure 3B). It is known that it is not rare for tumor cells to express co-stimulatory ligands in target cells. To investigate whether expression of endothelin affects cell proliferation upon repeated stimulation, a representative We investigated whether the costimulatory ligand CD80 was expressed on target cells. D19 expressed CD80 but not Nalm-6 (Fig. 3C). Overexpression of NALM-6-PDL1 (NALM-6-PDL1-CD80) inhibits repeated stimuli. CD80 was found to enable CAR-T cells to proliferate under stimulation. It may promote CART proliferation under repeated in vitro stimulation (Figure 3B). Cell-intrinsic PD-1 disruption is associated with CD19-specific CAR activation upon repeated stimulation with CAR and PDL1. This demonstrates that it contributes surprisingly effectively to improving the function of RT cells in vitro.

[0280] In this example, the two-in-one vector described in Example 1 was used. The generated PD-1 KD-modified CAR-T cells showed improved in vitro performance compared with WT CAR-T cells. The therapeutic potential of the compound has been demonstrated by demonstrating a surprising level of enhanced lytic and proliferative activity in the cells. However, CD19 in the following examples + PDL1 + in a mouse model bearing hematologic tumors It was further evaluated in an in vivo model.

[0281] Example 3: CD19 using PD-1 KD CAR-T cells + In vivo blood cancer Treatment method This example demonstrates the use of PD-1 KD CAR-T cells to target CD19 + Blood cancer in vivo The treatment method is described.

[0282] CD19 + To establish a hematological cancer model, NSG mice (4-6 weeks old) were treated with EGF. 1x10 engineered to express P-fused firefly luciferase and human PDL1 6 pieces CD19 +NALM-6 leukemia cells (NALM6-GL-PDL1 cells) were injected intravenously. CAR-T cells were isolated from whole blood samples of healthy donors according to the procedure described above. Four days after transduction, CAR - T cells were sorted and further purified prior to injection into mice. Five days after injection of NALM6-GL-PDL1 cells, 1 × 10 6 pieces CAR-T cells were intravenously injected into mice. Bioluminescence imaging was monitored with Xenogen IVIS Spectrum and signals The images were analyzed using Living Image software (Perkin Elmer). The radiance was quantified as photons / second in the area of ​​the shPD-1 CAR-T cells. Mice treated with shGFP CAR-T cells showed a strikingly uniform tumor burden compared to shGFP CAR-T cells. In addition, the promoters expressing the two types of shRNAs had similar anti- It was found that shRNA expression itself surprisingly did not affect the antitumor effect. It was offered.

[0283] To investigate the effect of cell-intrinsic PD-1 disruption in CAR-T cells on cytokine production in vivo. Peripheral blood from individual mice was obtained 24 and 72 hours after CAR-T injection. Plasma was collected from peripheral blood by centrifugation at 300 × g for 5 minutes at room temperature. Cytokine levels in the rats were measured using a human Th1 / Th2 cytokine kit according to the manufacturer's instructions. The cell-specific PD-1 expression of CAR-T cells was analyzed using a CT scan (BD Bioscience). The necrosis unexpectedly reduced cytokine production in vivo (Figure 5A).

[0284] Effect of cell-intrinsic PD-1 disruption on CAR-T cell proliferation in vivo To examine the effect, spleens from individual mice were obtained 3 or 20 days after CAR-T injection. CAR-T cells (live cells / dead cells) ― CD3 + ) or NALM-6-PDL1 (live cells / dead cells ― GFP + The ratio of cell-intrinsic PD-1 disruption was assessed by flow cytometry. unexpectedly delayed the in vivo expansion of CAR-T cells ( Fig. 5B ).

[0285] obtained by the dual-function vector system described herein. CAR-T cell-intrinsic PD-1 disruption inhibits CD19 + PDL1 + CD19 specificity for tumors The results showed an unexpectedly high increase in the in vivo antitumor effect of allogeneic CAR-T cells. The PD-1 KD CAR-T cells were further used in the methods described in the following examples.

[0286] Example 4: Evaluation of the costimulatory molecule environment in PD-1 signaling This example demonstrates the role of costimulatory molecules including CD28 and 4-1BB in PD-1 signaling. It describes the child's environmental assessment.

[0287] Cell-intrinsic PD-1 disruption is associated with CD28 / CD3ζ or 4-1BB / CD3ζ CAR- We investigated how G28z and GBBz affect T cell function (Figure 6A). P28z and PBBz CAR-T cells were first generated. The PD-1 expression level was significantly higher in G28z CAR-T cells than in G28z CAR-T cells (Figure 6B). The PD-1 levels of P28z were higher than those of GBBz CAR-T cells, and P28z PD-1 levels were higher than those of PBBz The differential PD-1 protein levels were also higher (Fig. 7A). Each costimulatory domain inhibits the activity of factors involved in PD-1 transcription. We hypothesized that these factors may express different potencies for PD-1 transcription. NFAT and NF-κB are highly involved in PD-1 transcription, and SMAD2 / 3 are involved in tumor microenvironment. It is involved in PD-1 transcription in the presence of exogenous TGF-β, a major immunosuppressive factor in the environment. (Cancer Discov., 2016, Benjamin, V. Park). NFAT or NF-κB activity was examined during CAR stimulation. T-RE ×3)-eGFP or classical NF-κB response element (NF-κB RE ×5) )-eGFP reporter lentiviral vectors were constructed. The BRE was shown to mediate eGFP induction (Figures 8A and 8B). The transduced T cells were restimulated and further transduced with G28z or GBBz. Transduced NFAT reporter CAR-T cells (G28z-NFAT) were transduced with GBBz-NF NFAT activity was slightly higher than that of AT (Fig. 9A), indicating that NFAT target genes The mRNA levels of α-glucan were also higher in G28z (Fig. 9B). G28z-transduced NF-κB reporter CAR-T cells (G28z-NF-κB) NFAT activity was similar compared to BB-NF-κB (Figure 10). To examine the activity of SMAD2 / 3 in T cells, TGF-β stimulated G28z during CAR stimulation. or GBBz CAR-T cells. Phosphorylation of BBz CART was slightly higher (Fig. 11A), and PD-1 phosphorylation was significantly higher with TGF-β treatment. The degree of increase in expression was greater in G28z than in GBBz (G28z 2.39±0.0 31, GBBz 1.61 + 0.034 fold change) were found (Figure 11B). We investigated whether this difference was due to differences in the expression levels of TGF-β signaling components. TGF-β and TGF-β receptor 1 (TGFBR1) showed no significant differences between the two CARTs. TGF-β receptor 2 (TGFBR2) was expressed at even higher levels in CD28z. It was found to be expressed in the bell (Fig. 11C).

[0288] In this example, costimulation involving CD28 and 4-1BB in PD-1 signaling We assessed the molecular milieu. CD28 costimulation strongly induced signaling associated with PD-1 transcription. The induction may be associated with activation of NFAT and TGF-β signaling. The induction of 8-costimulation was stronger than that of 4-1BB-costimulation.

[0289] Example 5: Engineering and evaluation of PD-1 KD CAR-T cells This example describes the engineering and evaluation of PD-1 KD CAR-T cells.

[0290] CAR-T cell activation with multiple sequential antigen and immune checkpoint ligand encounters To mimic the in vivo condition, CAR-T cells were transfected with CD1 9 + PDL1 + Primary CAR-T cells were co-cultured with target cells. The CAR-T group showed surprisingly higher lytic activity than the WT CAR-T group, but CD28 There was no difference in lytic activity between the 4-1BB and 4-1BB CAR-T groups (Figure 12A). After restimulation of CAR-T cells in the eye, G28z CAR-T cells proliferate after the second stimulation After losing potency, the lytic activity of the three CAR-T cells was analyzed: PBBz and P28z CAR-T cells were more effective against repeated antigen and PD-L1 exposure than GBBz CAR-T cells. PBBz CAR-T cells showed surprisingly high lytic activity compared with P28z CAR- They showed an unexpectedly higher ability to retain lytic function than T cells (Fig. 12A). The effect was also observed in cell proliferation assays (Figure 12B). The application of the 4-1BB costimulatory domain to ART has been shown to be more effective than the CD28 costimulatory domain. This contributes to the retained cytotoxicity and proliferation capacity in vitro. PBBz CAR-T cells Repeated exposure to CD19 and PD-L1 shows delayed exhaustion.

[0291] We investigated the possible influence of immunosuppressive mechanisms on BBz CAR-T cells. Because AR-T cells were more sensitive to TGF-β than BBz CART ( Figure 1 1) To what extent TGF-β inhibits antigen-specific proliferation, GBBz C CAR-T cells were compared with G28z CAR-T cells. During stimulation, no significant difference in proliferation was observed between the two CAR-T cells, while GBB The proliferation of G28z CAR-T cells, but not z CAR-T cells, was significantly reduced. (Figure 13A).

[0292] TGF-β not only inhibits proliferation but is also deeply involved in the induction of regulatory T cells. It has been reported (JEM, 2003, WanJun Chen; Science, 2 003, Shohei Hori; Blood, 2007, Dat Q. Tran). outside Between G28z and BBz CAR-T cells in the absence of TGF-β To investigate whether Treg induction differs, G28z CAR-T cells and BBz CAR-T cells were cultured with NALM-6 target cells for 3 days. G28z CAR-T cells Two to three times higher in vitro CD4 T cell counts compared to GBBz CAR-T cells + CD25 + F OXP3 + The results showed that the expression of CD25 and FOXP3 was significantly higher in the Treg cells (Figure 13B). This coincides with the increased expression of Treg-related genes (Fig. 9B). The changes in Treg% after treatment were observed and compared. TGF-β was significantly elevated in 28z CAR-T cells. BBz CAR-T cells significantly improved Treg%. It was shown that CD28 costimulation stimulated Tregs more effectively than 4-1BB costimulation (Fig. 13B). The results showed that TGF-β has a high potential for induction of inflammatory cytokines, and that this potential is even more pronounced in the presence of TGF-β. We also investigated whether cell-specific PD-1 disruption affects Treg induction. P28z CAR-T cells and PBBz CAR-T cells were compared with G28z and GBBz. In particular, PBBz had the lowest Treg %. % (Figure 13C). Finally, PD-1 / PD-L1 signaling was activated. The proportion of Tregs in the presence of NALM-6 or NALM-6-PDL1 was examined. The change in Treg% after culture was examined. PD-1 / PDL1 signaling was CAR-T-derived. It was found that there was no significant effect on the induction of Tregs (Fig. 13C). These results suggest that the expression level of intracellular PD-1 is related to the formation of Tregs. It was concluded that Bz CAR-T cells may be surprisingly insensitive to immunosuppressive mechanisms. .

[0293] In this example, PBBz CAR-T cells were generated, which express CD19 and PD-L Repeated exposure to PBBz CAR-T cells demonstrated delayed exhaustion. These cells unexpectedly evade the tumor suppressor, thus providing further insight into their antitumor effects in vivo. It was appreciated.

[0294] Example 6: In vivo treatment of CD19+ blood cancers using PBBz CAR-T cells This example demonstrates the in vivo treatment of CD19+ hematologic cancers using PBBz CAR-T cells. The method is described.

[0295] CD19 + A hematological tumor model was established as described in Example 3. + BA In vivo treatment of G28z, GBBz, P28z, and PBBz CAR-T cells against LL cells The antitumor effects of CAR-T cells were compared between 2.5 × 10 6 In vivo imaging of mice bearing NALM6-GL-PDL1 The process is performed using Xenogen IVIS Spectrum, which measures the radiance of the target area. As shown in Figure 14, PD-1 KD CART cells ( PBBz or P28z) than WT CAR-T cells (GBBz or G28z). In particular, PBBz CAR-T cells showed superior antitumor effects compared to the other three CAR-T cells. WT CAR-T cells suppressed leukemia progression for a longer period than WT CAR-T cells. However, PD-1 KD CAR-T showed no durable antitumor response. Although the response to CAR-T cells was weak, the sustained antitumor response was significant. These results suggest that disruption of PD-1 in cells may reduce the risk of CRS. PBBz CAR-T cells showed unexpectedly superior in vivo efficacy compared with P28z. It conferred antitumor effects.

[0296] Example 7: Two-in-one with two shRNA cassettes inserted in →← or ←→ directions Method for constructing a dual vector with the functions of This example describes how to create a dual vector with two functions in one. Two types of shRNA cassettes are arranged in the ←→ direction (shTIM-3-mU6←→hU6-shP D-1) or →← direction (mU6-shTIM-3 →←shPD-1-hU6), shPD-1, shTIM-3, and CD19-CAR are expressed simultaneously.

[0297] shRNA for PD-1 (hereafter referred to as shPD-1), shRNA for TIM-3 ( The expression of shTIM-3 and CD19-CAR was controlled by the human U6 promoter (hereafter referred to as hU6 (hereafter referred to as "mU6"), mouse U6 promoter (hereafter referred to as "mU6"), and EF1-α promoter To construct a lentivirus that is regulated by the shRNA gene, both types of shRNA are expressed simultaneously. The resulting plasmid contains shRNA cassettes in the →←direction (mU6-shTIM-3 →←shP D-1-hU6) and in the ←→ direction (shTIM-3-mU6←→hU6-shPD-1). For this purpose, (1) a multicloning site ( (2) the insertion of the human U6 promoter into the mouse U6 promoter of shPD-1 (3) insertion of shTIM-3, and (4) cleavage of shTIM-3 and shPD-1. Cloning of the rearranged sites was performed.

[0298] To insert the MCS into mU6-shPD-1, we used pLV-ΔLNGFR_P2A_C 3' portion and SEQ ID NO: of D19-CAR_mU6-shPD-1 (Plasmid ID#2) Primers containing 228 and 229 were used to clone the 3' Hpa1 fragment of mU6-shPD-1. The restriction enzyme recognition site is BsgZ171-Xba1-Nde-1-Bmt1-Spe1 multiclone A PCR product (416 bp) was generated that was modified to the cloning site. was digested with BstZ17y and Hpa1 restriction enzymes, and plasmid ID#2 was digested with Hpa1 restriction enzyme. After treatment with ribosomal DNA and CIP, shPD-1-mU6-MC was synthesized using blunt-end ligation. pLV-ΔLNGFR-P2A containing the S base sequence (shRNA cassette SEQ ID NO: 223) -CD19-CAR-mU6-shPD-1_MCS (Plasmid ID#4) was prepared. .

[0299] Then, shPD-1 was expressed by the human U6 promoter instead of the mouse U6 promoter. The plasmid was constructed so that it could be expressed by the LentiCRISPR V2 plasmid. was used with primers containing SEQ ID NOs: 230 and 231 to identify a gene encoding a human U6 promoter. The PCR product was digested with Hpa1 and Spe1 restriction enzymes and then purified with Hp This was ligated with plasmid ID#4 treated with a1 and Spe1 restriction enzymes to give hU6 -pLV-ΔLNGFR_P2A_CD containing shPD-1 base sequence (SEQ ID NO: 224) 19-CAR_hU6-shPD-1_MSC (Plasmid ID#5) was prepared. 6-shTIM-3 cassette was inserted into plasmid ID#5 to generate shTIM-3 and shPD The aim of this study was to construct a plasmid that simultaneously expresses α- and β-actin.

[0300] The PCR product containing the mU6-shTIM-3 cassette was identified as plasmid ID#3 and SEQ ID NO: The PCR product was obtained using primers containing 232 and 233. After digestion with restriction enzymes, plasmid ID#5 was digested with Bmt1 and Spe1 restriction enzymes. The resulting fragments were ligated in the →← direction (mU6-shTIM-3 →←shPD-1-hU 6) pLV-ΔLNGFR_P2A_CD19-C containing the base sequence (SEQ ID NO: 220) AR mU6-shTIM-3 → ← shPD-1-hU6 (Plasmid ID#6) was prepared. did.

[0301] Two types of shRNA cassettes are arranged in the ←→ direction (shTIM-3-mU6←→hU6-shP D-1), plasmid ID#6 and SEQ ID NO: A PCR product was obtained using primers containing 234 and 235. Spe1 and Hpa1 After digestion with restriction enzymes, it was inserted into plasmid ID#4 which had been digested with the same restriction enzymes to form p LV-ΔLNGFR-P2A-CD19-CAR-shPD-1-hU6-MCS (Plastic Plasmid ID#6 and SEQ ID NOs: 236 and 237 were then prepared. PCR products were obtained using primers containing the following: After digestion with the enzymes, plasmid ID#7 and its corresponding plasmids were digested with Bmt1 and Spe1 restriction enzymes. Finally, the fragments were ligated in the ←→ direction (shTIM-3-mU6←→hU6-shP pLV-ΔLNGFR-P2A-CD19 containing the base sequence (SEQ ID NO: 221) of -CAR_shTIM-3-mU6←→hU6-shPD-1 (Plasmid ID#8) Prepared.

[0302] Two types of shRNA cassettes were prepared in this study and inserted in either the left-right or right-left direction. The lentiviral vector with two functions is the PD-1 KD modified C It can be used to generate AR-T cells.

[0303] Example 8: Method for producing PD-1 KD CAR-T cells and in vitro evaluation This example demonstrates the simultaneous expression of shPD-1, shTIM-3, and CD19-CAR. We describe a method to generate and evaluate in vitro double-KD CAR-T cells.

[0304] Use Lipofectamine to identify the plasmid ID#1 (pLV-ΔLNGFR_ P2A_CD19-CAR_mU6-shGFP), #6(pLV-ΔLNGFR_P2 A_CD19-CAR_mU6-shTIM-3→←shPD-1-hU6), and #8 (pLV-ΔLNGFR_P2A_CD19-CAR_shTIM-3-mU6←→hU 6-shPD-1), and packaging plasmids pMDL g / p and pRSVr HEK293T cells were transfected with ev and pMDG.1, and after 48 hours, lentivirus A cell culture medium containing Ficoll-paque was obtained. (PBMC) were isolated from human blood and T cells were cultured using human CD3 and CD28 targeting antibodies. One to two days after the initial activation of T cells, the previously obtained virus The prepared CAR-T cells were then transduced using 5% human plasma and human plasma. Six days after transduction, the cells were cultured in AIM-V medium containing human IL-2. Using the lect LNGFR system (miltenyibiotec, Germany) Pure CAR-T cells were obtained using a flow cytometer with an antibody targeting LNGFR. LNGFR+CAR-T cells were isolated using the following plasmids ID#1, #6, and # Cells prepared using 8 were ΔLNGFRCART19 / shGFP (or s hGFP / CART19), ΔLNGFR-CART19 / mU6-shTIM-3→← shPD-1-hU6 (or shPD-1_shTIM-3 / CART19) and ΔL Denoted as NGFR-CART19 / shTIM-3-mU6←→hU6-shPD-1 (Figure 16).

[0305] The mU6-shPD-1, mU6-TIM-3, and shPD-1-hU6 cassettes were To prepare control CAR-T cells containing each of the CAR-T cells, lipofectamine was used to prepare the CAR-T cells containing the CAR-T cells listed in Table 1. Plasmid ID#2 (pLV-ΔLNGFR_P2A_CD19-CAR_mU6-s hPD-1), #3(pLV-ΔLNGFR_P2A_CD19-CAR_mU6-sh TIM-3), and #7 (pLV-ΔLNGFR_P2A_CD19-CAR_shPD -1-hU6_MCS), and packaging plasmids pMDL g / p and pRSVr HEK293T cells were transfected with ev and pMDG.1. After 48 hours, lentil A cell culture solution containing the virus was obtained. PBMCs were isolated from human blood and T cells were cultured using human CD3 and CD28 targeting antibodies. One to two days after the initial activation of T cells, the previously obtained virus was injected into the infected cells. The prepared CAR-T cells were then transduced using 5% human plasma and The cells were cultured in AIM-V medium containing human IL-2. Six days after transduction, the cells were cultured in a MACS elect LNGFR system (miltenyibiotec, Germany) Obtain pure CAR-T cells using a flow cytometer with an LNGFR-targeting antibody LNGFR+CAR-T cells were isolated using the plasmid ID#2, #3, and The cells prepared using #7 were ΔLNGFR-CART19 / shPD-1 (or or shPD-1 / CART19), ΔLNGFR-CART19 / shTIM-3 (or or shTIM-3 / CART19) and ΔLNGFRCART19 / shPD-1-h Denoted as U6.

[0306] To measure the purity of the double KD CAR-T cells generated herein, the above-prepared The cells were subjected to flow cytometry using an antibody targeting LNGFR. As shown, approximately 80% LNGFR+ CAR-T cells were obtained.

[0307] Expression of PD-1 and TIM-3 in the double KD CAR-T cells generated herein The double KD CAR-T cells were used to measure the decrease in expression of CAR-T cells, and a sustained decrease in expression was observed. Stimulation with human CD3 and CD28 targeting antibodies for 3 days induced the expression of PD-1 and TIM-3. This was followed by flow cytometry analysis using antibodies targeting CAR, PD-1, and TIM-3. We analyzed PD-1 and TIM-3 expression in double KD CAR-T cells via a cytometry analysis. As shown in Figures 18A and 18B, two similar responses to the downregulation of PD-1 and TIM-3 expression were observed. Analysis of the effect of the type of shRNA expressed at the time of shPD-1 and shTIM-3 Low PD-1 and TIM-3 expression observed in CD19-CAR-T cells expressing CD19 The lower level indicates CD19-CAR- The degree of reduction was similar to that in T cells.

[0308] The effect on differentiation of the double KD CAR-T cells generated herein was measured. To observe the degree of differentiation of PD-1 and TIM-3 in KD CAR-T cells, CD4 Flow cytometry was performed using 5RA and CCR7 targeting antibodies. Thus, in ΔLNGFR-CART19 / shPD-1 cells subjected to repeated antigen stimulation, Terminally differentiated TEMRA (CCR7-C On the other hand, ΔLNGF, which contains shTIM-3, increases the number of T cells. R-CART19 / mU6-shTIM-3→←shPD-1-hU6 is ΔLNGFR -more TN (CCR7+CD45RA+) T cells than CART19 / shPD-1; TCM(CCR7+CD45RA-)T cells and TEM(CCR7-CD45RA-)T Similar results were observed in ΔLNGFR-CART19 / shTIM-3 cells. This is also observed in ΔLNGFR-CAR cells, suggesting that the suppression of TIM-3 expression is responsible for the ΔLNGFR-CAR The reduced differentiation potential of T19 / mU6-shTIM-3 → ← shPD-1-hU6 cells Therefore, the effect of shTIM-3 on cell differentiation is more pronounced than that of shPD-1. It can be said that it has been prioritized in terms of its impact on the It is known that cell subtypes can enhance the cancer-fighting ability of T cells, so Δ LNGFR-CART19 / mU6-shTIM-3 →←shPD-1-hU6 cells are Δ showed better in vivo anti-cancer efficacy than LNGFR-CART19 / shPD-1 cells. It is expected that this will happen.

[0309] Two CD19-CAR vectors containing shRNA cassettes in different orientations were introduced. The transduction efficiency, proliferation capacity, and survival rate of the heavy KD CAR-T cells were compared. Among the CAR-T cells generated, the →←direction (mU6-shTIM-3 →←shPD-1 -hU6) and ←→ direction (shTIM-3-mU6←→hU6-shPD-1) cassettes Flow cytometry was performed using LNTFR antibodies on cells containing the plasmid. For cell viability analysis, trypan blue staining was performed. As shown in Figures 20A to 20C, the ratio of cells containing →← cassette (ΔLNGFR -CART19 / mU6-shTIM-3 →←shPD-1-hU6) All, ←→ direction cassette (ΔLNGFR-CART19 / shTIM-3-mU6←→h Cells using U6-shPD-1 unexpectedly struggled to form transduced cells, The transduced T cells had poor proliferation and survival capacity.

[0310] This example describes a method for generating double KD CAR-T cells, in which s hPD-1, shTIM-3, and CD19-CAR are expressed simultaneously. The two-in-one function constructed herein has two shRNA cassettes inserted in the left-direction. We generated double KD CAR-T cells using a lentiviral vector capable of expressing CAR-T cells. Compared with cells using the →← cassette, cells using the →← cassette (ΔLNGFR -CART19 / mU6-shTIM-3 →←shPD-1-hU6) showed surprisingly high It demonstrated reproductive ability and viability and was used in the following examples.

[0311] Example 9: Method for generating dual vector and dual KD CAR-T cells with two functions in one law This example demonstrates the generation of a two-in-one vector and dual KD CAR-T cells. The method is described.

[0312] Two shRNAs expressed by different promoters (mU6 and hU6) To generate a dual vector with two functions, BstZ171-Xba1-Nde1- The multiple cloning site (MCS) of Bmt1-Spe1 was inserted downstream of the hU6 promoter. It was inserted into the pLV-hU6-shPD-1_ΔLNGFR-CD19-BBz vector. The second mU6-shRNA cassette fragment was subcloned into the MCS. Two-in-one technology to limit checkpoint blockade to CAR-T cells A vector was designed to encode two sRNAs using the mU6 and hU6 Pol III promoters. hRNA was expressed, suppressing the expression of two immune checkpoints in CAR-T cells. Effective siRs targeting CTLA-4, LAG-3, TIGIT, or TIM-3 To detect NA, siRNA was electroporated into CD3 / CD28 stimulated T cells. Two or more effective siRNAs were selected (Figure 21A). 4, with one for LAG-3, TIGIT, or TIM-3 KD CAR-T cells The dual-function vector converts 21-mer siRNA into shRNA format. Finally, shRNAs targeting each immune checkpoint (shCT LA-4:#1;shLAG-3:#1278;shTIGIT:#739;shTIM -3:#3) were selected, but they significantly suppressed expression and had little effect on CAR-T proliferation. (Fig. 21B-C). Finally, a dual vector with two functions in one PD-1_CTLA-4, PD-1_LAG-3, PD-1_TIGIT, or PD CAR-T cells of -1_TIM-3 were constructed (Figures 22A-22E). All RT cells proliferated less compared to single PD-1 KD CAR-T cells. These results were also observed with single KD CAR-T cells (Fig. 21B).

[0313] In this example, a dual vector with two functions in one was created. We will generate double KD CAR-T cells using dual vectors capable of targeting CAR-T cells and investigate their therapeutic potential. CD19 in the following examples + PDL1 + In vivo mouse models and solid tumor-bearing hematologic tumors Further evaluation was carried out in tumor models.

[0314] Example 10: C using dual KD CAR-T cells targeting PD-1 and TIGIT In vivo treatment of D19+ blood cancer This example illustrates the following combinations: PD-1 and CTLA-4, PD-1 and LAG-3, Two immune checkpoints, including PD-1 and TIGIT, and PD-1 and TIM-3, were identified. Treating CD19+ hematological cancers in vivo with targeted dual KD CAR-T cells It states the law.

[0315] The CD19+ hematologic tumor model was established as described in Example 3. Blood CD1 9 + PD-1_CTLA-4, PD-1_LAG-3, and PD- The antitumor effects of 1_TIGIT or PD-1_TIM-3 KD CAR-T cells were evaluated. Each CAR-T cell was administered at a dose of 1 × 10 6 Injected in a single dose PD-1_TIGIT KD CAR-T cells were significantly superior to other double KD CAR-T cells. It was found that the antitumor effect of the IFN-γ-α-glucan-1-phosphate dehydrogenase (IFN-γ-α) was surprisingly superior to that of the IFN-γ-α-glucan-1-phosphate dehydrogenase (IFN-γ-α) (Fig. 23). PD-1_TIGIT KD CAR-T cells in a thyroid tumor model are further described in the following examples. The results were further evaluated.

[0316] Example 11: Immunotherapy using dual-KD CAR-T cells targeting PD-1 and TIGIT Treatment strategies in tumor models. This example uses dual-KD CAR-T cells targeting PD-1 and TIGIT. The present application describes a method for treating cancer in a solid tumor model.

[0317] To establish solid tumor models, 4- to 6-week-old NSG mice were inoculated with 5 x 10 6 IM- 9 cells (CD19 + PD-L1 + CD155 + ) was injected subcutaneously. ~250mm 3 When the volume reached 3 × 10 6 Single dose intravenously Tumors were monitored weekly by caliper measurement and volume was determined (length × width). 2 ) / 2 As shown in a hematologic tumor model (Nalm-6-PDL1), PD -1_TIGIT KD CAR-T cells also showed the most surprising effect on the solid tumor model (IM-9). The more effective the blockade of cell-intrinsic PD-1_TIGIT, the more effective the blockade of PD-1. It has an unexpectedly higher level of antitumor effect than blockade. The present application provides the following aspects of the invention. (Aspect 1) A vector comprising: Two short hairpin Rs inhibit the expression of at least one gene that weakens immune cell function a base sequence encoding NA (shRNA); Encoding chimeric antigen receptors (CARs) or monoclonal T cell receptors (mTCRs) and a base sequence as set forth above. (Aspect 2) The expression of the two shRNAs is controlled by two different promoters. The vector according to embodiment 1, wherein the vector is regulated by (Aspect 3) The method according to embodiment 2, wherein the two promoters are promoters for RNA polymerase III. Vector on the list. (Aspect 4) 3. The method according to claim 2, wherein the two promoters are U6 promoters derived from different species. vector. (Aspect 5) 3. The method of claim 2, wherein the two promoters are oriented in different directions on the vector. Vector on the list. (Aspect 6) The gene that weakens the function of immune cells is a receptor or ligand for immune checkpoints. The vector according to certain embodiment 1. (Aspect 7) The immune checkpoint receptor or ligand is PD1, PD-L1, CTLA 4. TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM -5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD 96, MerTK, and 2B4. (Aspect 8) The genes that weaken immune cell function are FAS, CD45, PP2A, SHIP1, and S HIP2, DGK alpha, DGK zeta, Cbl-b, CD147, LRR1, TGF BR1, IL10R alpha, KLGR1, DNMT3A and A2aR The vector according to embodiment 1, selected from the group consisting of: (Aspect 9) The two shRNAs target a single gene that attenuates immune cell function, or The method according to embodiment 1, wherein the two types of shRNAs target different genes that attenuate immune cell function. Vector on the list. (Aspect 10) The vector of embodiment 1, wherein the two shRNAs target PD-1. (Aspect 11) Of the two types of shRNAs, (i) one shRNA targets PD-1 and the second (ii) one shRNA targets TIM-3, or (iii) one shRNA targets PD-1. 2. The vector of embodiment 1, wherein the first shRNA targets TIGIT and the second shRNA targets TIGIT. (Aspect 12) The base sequence encoding one of the two types of shRNA is SEQ ID NO: the second shRNA encoding the second shRNA, comprising a sequence selected from the group consisting of sequences 2 to 219; According to embodiment 1, the base sequence comprises different sequences selected from the group consisting of SEQ ID NOs: 2 to 219. Vector of. (Aspect 13) The target of the CAR or mTCR is a cancer cell, a cancer tissue, and / or a tumor microcirculation. It is a human tumor antigen that shows elevated expression in cancer cells, cancer tissues and / or tumor cells. 2. The vector of embodiment 1, wherein said antigen is a mutated form of an antigen found in the tumor microenvironment. (Aspect 14) The vector according to embodiment 1, wherein the vector comprises the nucleotide sequence of SEQ ID NO: 220 or 221. . (Aspect 15) The vector may be a plasmid vector, a lentiviral vector, an adenoviral vector, or 2. The method of claim 1, wherein the vector is a vector, an adeno-associated virus vector, or a retrovirus vector. vector. (Aspect 16) An immune cell comprising the vector of embodiment 1, wherein the expression of one or more genes is controlled by shRNA The immune cell, wherein the expression of the antibody is reduced to 40% or less compared to that in the absence of the antibody. (Aspect 17) Aspect 16, wherein the immune cells are T cells or natural killer (NK) cells of human origin The immune cell described in (Aspect 18) A pharmaceutical composition comprising the immune cell according to any one of aspects 1 to 17. (Aspect 19) for the treatment of a patient in need of immunotherapy, wherein said immune cells are originally obtained from said patient. 19. The pharmaceutical composition according to embodiment 18. (Aspect 20) The patient is a patient with a tumor necrosis factor (TNF) receptor agonist (TNF-α) that is a cytotoxic TNF-α receptor agonist (CTA ... or a tumor or cancer in which elevated or altered levels of said target are detected. The pharmaceutical composition described. (Aspect 21) an immune cell having a genetically engineered antigen receptor that specifically binds to a target antigen; Reducing or decreasing the expression in immune cells of genes that impair the function of said immune cells. and a gene disrupting agent capable of inhibiting the immune cell. (Aspect 22) The engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor ( 22. The immune cell of embodiment 21, wherein the immune cell is a TCR. (Aspect 23) 23. The immune cell of embodiment 22, wherein the engineered antigen receptor is a CAR. (Aspect 24) The CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. 24. The immune cell of embodiment 23, comprising a main. (Aspect 25)

[0033] Embodiment 24: wherein the extracellular antigen recognition domain of the CAR specifically binds to the target antigen The immune cell described in (Aspect 26) the intracellular signaling domain of the CAR is an intracellular signaling domain of the CD3 zeta (CD3ζ) chain 25. The immune cell of embodiment 24, comprising the domain. (Aspect 27) 27. The method of claim 26, wherein the intracellular signaling domain of the CAR further comprises a costimulatory molecule. Immunologic cells. (Aspect 28) The costimulatory molecules are ICOS, OX40, CD137 (4-1BB), CD27, and C 28. The immune cell of embodiment 27, selected from the group consisting of D28. (Aspect 29) 29. The immune cell of embodiment 28, wherein the costimulatory molecule is CD137(4-1BB). (Aspect 30) 29. The immune cell of embodiment 28, wherein the costimulatory molecule is CD28. (Aspect 31) 23. The immune cell of embodiment 22, wherein the engineered antigen receptor is a TCR. (Aspect 32) 32. The immune cell of embodiment 31, wherein the TCR is a monoclonal TCR (mTCR). (Aspect 33) The target antigen is present in or on the surface of cancer cells, cancer tissues, and / or the tumor microenvironment. 33. The immune cell of embodiment 31 or 32, wherein the immune cell is expressed (Aspect 34) The target antigen is 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (α -fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human Prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α-metabolite Acyl-coenzyme A racemase, ART-4 (ADP-ribosyltransferase-4) ), B7H4 (v-set domain-containing T-cell activation inhibitor 1), BAGE-1 (B-meta BCL-2 (B-cell CLL / lymphoma-2), BING-4 ( WD repeat domain 46), CA15-3 / CA27-29 (mucin 1), CA19-9 ( Cancer antigen 19-9), CA72-4 (cancer antigen 72-4), CA125 (cancer antigen 125 ), calreticulin, CAMEL (CTL-recognized antigen on melanoma), CASP-8 ( Supase 8), cathepsin B, cathepsin L, CD19 (cluster of differentiation 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, C D80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel accessory 2 ), CML28 (chronic myeloid leukemia tumor antigen 28), coactosin-like protein, collagen Gen XXIII, COX-2 (cyclooxygenase-2), CT-9 / BRD6 (cancer / testis antigen 9), Cten (c-terminal tensin-like protein), cyclin B1, cyclin Phosphorin D1, cyp-B, CYPB1 (cytochrome p450 family 1 subfamily) b Member 1), DAM-10 / MAGE-B1 (melanoma-associated antigen B1), DAM- 6 / MAGE-B2, EGFR / Her1 (epidermal growth factor receptor), EMMPRIN ( Isidine), EpCam, EphA2 (EPH receptor A2), EphA3, ErbB3 ( Erb-B2 receptor tyrosine kinase 3), EZH2 (zeste2 polycomb inhibitory complex Enhancer of fusion 2 subunits), FGF-5 (fibroblast growth factor 5), FN (F fibronectin), Fra-1 (Fos-related antigen-1), G250 / CAIX (carbonic anhydride) Enzyme 9), GAGE-1 (G antigen-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP (prostate GnT-V (gluconate kinase), gp100 (melanin kinase), Cell lineage-specific antigen GP100, GPC3 (glypican 3), HAGE (helical antigen) , HAST-2 (sulfotransferase family 1A member 1), hepsin, H er2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K -MEL, HNE (medullasin), homeobox NKX3.1, HOM-TES-1 4 / SCP-1, HOM-TES-85, HPV-E6, HPVE7, HST-2 (sil TWIN-2), hTERT, iCE (caspase 1), IGF-1R (insulin-like growth factor receptor factor-1 receptor), IL-13Ra2 (interleukin-13 receptor subunit α2 ), IL-2R (interleukin-2 receptor), IL-5 (interleukin-5), Immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205 (lysophosphatidylglycerol acyltransferase 1), KK-LC-1 (Kita Kyushu Lung Cancer Antigen-1), KM-HN-1, LAGE-1 (L antigen family member-1) , Livin, MAGE-A1, MAGE-A10, MAGE-A12, MAGEA2, MA GE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MA GE-B10, MAGE-B16, MAGE-B17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2 (melanoma antigen pharmacokinetics) Milli-L2), mammaglobin A, MART-1 / Melan-A (T-cell-1 recognized melanoma antigen), MART-2, matrix protein 22, MC1R (melanoma antigen) lanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor), mesothelin , MG50 / PXDN (peroxidasin), MMP11 (matrix metalloproteinase 11), MN / CA IX-antigen (carbonic anhydrase 9), MRP-3 (multidrug resistance-related protein Protein-3), MUC1 (mucin 1), MUC2, NA88-A (VENT-like homeobox ox2 pseudogene 1), N-acetylglucosaminyltransferase-V, Neo- PAP (Neo-poly(A) polymerase), NGEP (New gene expressed in the prostate) NMP22 (nuclear matrix protein 22), NPM / ALK (nucleophosmin ), NSE (neuron-specific enolase), NY-ESO-1, NY-ESO-B, O A1 (osteoarthritis QTL1), OFA-iLRP (carcinoembryonic antigen-immature laminin receptor OGT (O-GlcNAc transferase), OS-9 (endoplasmic reticulum protein) cutin), osteocalcin, osteopontin, p15 (CDK inhibitor 2B), p53 , PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor) Prostatic acid phosphatase inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PART-1 ( Prostate androgen-regulated transcript 1), PATE (prostate and testis expressed 1), PD EF (prostate-derived Ets factor), Pim-1 kinase (proviral integration site 1 ), Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1), POTE (expressed in the prostate, ovaries, testes, and placenta), PRAME (melanoma) antigens preferentially expressed in tumors), prostein, proteinase-3, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate-specific G-protein co-receptor), PSM, PSMA (prostate-specific membrane antigen), RAGE-1 (renal tumor carcinoma antigen) Raw material), RHAMM / CD168, RU1 (renal ubiquitous protein 1), RU2, SAGE (meat SART-1 (squamous cell carcinoma antigen recognized by T-cells-1), S ART-2, SART-3, Sp17 (sperm protein 17), SSX-1 (SSX factor Milly Member 1), SSX-2 / HOM-MEL-40, SSX-4, STAMP-1 (STEAP2 metalloreductase), STEAP, survivin, survivin-213 , TA-90 (tumor-associated antigen-90), TAG-72 (tumor-associated glycoprotein-72), TARP (TCRγ alternative reading frame protein), TGFb (transforming growth factor β), TGFbR11 (transforming growth factor β receptor 11), TGM-4 (transglutaminase 4) taminase 4), TRAG-3 (Taxol resistance-associated gene 3), TRG (T-cell receptor γ locus), TRP-1 (transient receptor potential-1), TRP-2 / 6b, T RP-2 / INT2, Trp-p8, tyrosinase, UPA (U-plasminogen activity) activating factor), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK-1, and WT1 (Wilm's tumor 1). (Aspect 35) 36. The immune cell of embodiment 35, wherein the target antigen is CD19 or CD22. (Aspect 36) 37. The immune cell of embodiment 36, wherein the target antigen is CD19. (Aspect 37) The target antigen is a cancer antigen, and the expression of the cancer antigen is detected in cancer cells, cancer tissues, and and / or a cancer antigen increased in or on the surface of the tumor microenvironment. Any of the immune cells. (Aspect 38) The target antigen is α-actinin-4 / m, ARTC1 / m, bcr / abl, beta -Catenin / m, BRCA1 / m, BRCA2 / m, CASP-5 / m, CASP-8 / m, CDC27 / m, CDK4 / m, CDKN2A / m, CML66, COA-1 / m, DEK-CAN, EFTUD2 / m, ELF2 / m, ETV6-AML1, FN1 / m, GPNMB / m, HLA-A*0201-R170I, HLA-A11 / m, HLA-A 2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LDLR-FUT , MART2 / m, ME1 / m, MUM-1 / m, MUM-2 / m, MUM-3 / m, Mi Osin class 1 / m, neo-PAP / m, NFYC / m, N-Ras / m, OGT / m , OS-9 / m, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, R BAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, TEL- AML1, TGFbRII, and TPI / m, wherein the target The antigen is a cancer antigen, and the cancer antigen is a cancer cell, a cancer tissue, and / or a tumor microenvironment. 34. The immune cell of claim 33, wherein the antigen is a mutated form of the antigen expressed in or on the surface of the immune cell. Cell. (Aspect 39) The expression of the gene that attenuates the function of the immune cell is determined by: (i) inhibiting the proliferation of said immune cells; (ii) inducing cell death of said immune cells; (iii) inhibiting the ability of said immune cells to recognize and / or be activated by said target antigen. , (iv) inducing differentiation of the immune cells into cells that do not induce an immune response against the target antigen. , (v) a decrease in the responsiveness of said immune cells to molecules that stimulate the immune response of said immune cells; and teeth (vi) increasing the response of the immune cells to molecules that suppress the immune response of the immune cells; The immune cell according to any one of aspects 21 to 38, which causes the above. (Aspect 40) The genes that weaken the function of the immune cells include PD1, PD-L1, CTLA4, and TIM 3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), L AG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, Me rTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGK alpha, DGK zeta, Cbl-b, Cbl-c, CD148, LRR1, TGFBR1, IL1 39. The method of claim 39, wherein the IL-16 is selected from the group consisting of IL-16, IL-16, IL-16A, IL-16B, IL-16C, IL-16D, IL-16E, IL-16F, IL-16H, IL-16I ... Immune cells as described. (Aspect 41) The gene that weakens the function of the immune cells is converted into a molecule that suppresses the immune response of the immune cells. 40. The immune cell of embodiment 39, wherein the immune cell enhances a response to a (Aspect 42) The gene that enhances the response of the immune cell to a molecule that suppresses the immune response of the immune cell. 42. The immune cell of embodiment 41, wherein the molecule encodes a receptor or ligand of an immune checkpoint. Cell. (Aspect 43) The immune checkpoint receptor or ligand is PD1, PD-L1, CTLA 4. TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM -5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD 96, MerTK, and 2B4. (Aspect 44) The gene disrupting agent increases the expression of the immune cells relative to the immune cells in the absence of the gene disrupting agent. The expression of genes in said immune cells that weaken the function of said immune cells is suppressed by at least 30, 40, 50, or 44. The method according to any one of embodiments 21 to 43, wherein the concentration of hydroxybenzoates is reduced by 60, 70, 80, 90, or 95%. immune cells. (Aspect 45) The gene disrupting agent inhibits the immune cells from reacting with a molecule that suppresses the immune response of the immune cells. 45. The immune cell of embodiment 44, wherein the immune cell reduces expression of a gene that enhances a response. (Aspect 46) The gene disrupting agent is a gene encoding a receptor or ligand of an immune checkpoint. 46. ​​The immune cell of embodiment 45, wherein the immune cell reduces expression of (Aspect 47) The gene disrupting agent is selected from the group consisting of PD1, PD-L1, CTLA4, TIM3, and CEACAM (C EACAM-1, CEACAM-3 or CEACAM-5), LAG3, VISTA, From BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4 47. The immune cell of embodiment 46, wherein the immune cell reduces expression of a gene selected from the group consisting of: (Aspect 48) Before the gene disrupting agent attenuates the function of the immune cells by RNA interference (RNAi). 48. The immune cell according to any one of embodiments 45 to 47, which reduces the expression of the gene. (Aspect 49) More than one gene disrupting agent inhibits the function of said immune cell in said immune cell by RNAi. 49. The immune cell of embodiment 48, wherein the expression of the gene that attenuates the ability of the immune cell to inhibit the immune cell's growth factor receptor 2 (GFR) is reduced. (Aspect 50) the gene disrupting agent targets a single gene to attenuate the function of the immune cell; or Different gene disrupting agents target different genes that impair the function of the immune cells, e.g. One gene disruptor targets a first gene, and a second gene disruptor targets a second gene. 50. The immune cell according to embodiment 49, (Aspect 51) 51. Any of embodiments 48 to 50, wherein the RNAi is mediated by short hairpin RNA (shRNA). The immune cell described above. (Aspect 52) 52. The immune cell of embodiment 51, wherein said RNAi is mediated by more than one shRNA. (Aspect 53) 53. The immune cell of embodiment 52, wherein the RNAi is mediated by two shRNAs. (Aspect 54) 54. The immune cell according to any one of aspects 52 to 53, wherein two shRNAs target PD-1. . (Aspect 55) The first shRNA targets PD-1 and the second shRNA targets TIM-3 54. The immune cell according to any one of embodiments 52 to 53. (Aspect 56) The first shRNA targets PD-1 and the second shRNA targets CTLA-4. 54. The immune cell according to any one of embodiments 52 to 53. (Aspect 57) The first shRNA targets PD-1 and the second shRNA targets LAG-3 54. The immune cell according to any one of embodiments 52 to 53. (Aspect 58) The first shRNA targets PD-1 and the second shRNA targets TIGIT 54. The immune cell according to any one of embodiments 52 to 53. (Aspect 59) Any of embodiments 51 to 58, wherein the immune cells comprise a nucleotide sequence encoding an shRNA. The immune cell described in any one of the above. (Aspect 60) 59. The method of claim 58, wherein the immune cells comprise a nucleotide sequence encoding more than one shRNA. Immune cells as described. (Aspect 61) 60. The method of claim 59, wherein the immune cells comprise a nucleotide sequence encoding two shRNAs. immune cells. (Aspect 62) The nucleotide sequence encoding the shRNA(s) is selected from SEQ ID NOs: 2 to 219 62. The method of claim 59, further comprising administering to said patient a nucleotide sequence selected from the group consisting of 238 to 267. Any of the immune cells. (Aspect 63) The nucleotide sequence encoding the shRNA(s) is / are present on a vector 63. The immune cell according to any one of embodiments 59 to 62. (Aspect 64) Any of embodiments 63, wherein expression of the different shRNAs is regulated by different promoters. The immune cell described in (Aspect 65) Embodiment 6 in which the expression of two different shRNAs is regulated by two different promoters 4. An immune cell according to claim 4. (Aspect 66) An embodiment in which the two different promoters are RNA polymerase III promoters 65. An immune cell according to claim 65. (Aspect 67) 67. The immune cell of embodiment 66, wherein the two promoters are U6 promoters. (Aspect 68) 68. The immune cell of embodiment 67, wherein the U6 promoter is derived from a different species. (Aspect 69) In any one of embodiments 65 to 68, the two promoters are oriented in different directions. Immune cells as described. (Aspect 70) The engineered antigen receptor and the gene disrupting agent(s) are then separated from the vector. 70. The immune cell according to any one of embodiments 21 to 69, wherein each of the immune cells is expressed. (Aspect 71) The engineered antigen receptor and the gene disrupting agent(s) are delivered from the same vector. 71. The immune cell of embodiment 70, wherein the immune cell is expressed. (Aspect 72) Any of embodiments 70 to 71, wherein the vector is a plasmid vector or a viral vector. The immune cell according to any one of the preceding claims. (Aspect 73) The viral vector is a lentiviral vector, an adenoviral vector, or an adenoviral vector. 73. The immune cell of embodiment 72, wherein the immune cell is a virus-associated vector. (Aspect 74) The immune cell of embodiment 73, wherein the lentiviral vector is a retroviral vector. Cell. (Aspect 75) The immune cells are selected from the group consisting of T cells and natural killer (NK) cells. 75. The immune cell according to any one of claims 21 to 74. (Aspect 76) The immune cell of embodiment 75, wherein the immune cell is a T cell. (Aspect 77) 77. The immune cell of embodiment 76, wherein the T cell is a CD4+ T cell or a CD8+ T cell. (Aspect 78) the immune cells comprising two shRNAs and a nucleotide encoding a CAR or mTCR. 78. The immune cell of any of embodiments 76 or 77, comprising the sequences on the same vector. (Aspect 79) The two shRNAs were directed in different directions to each other. 79. The immune cell of embodiment 78, wherein each of the immune cells is regulated by a promoter of ZeIII. (Aspect 80) wherein the CAR targets CD19, the first shRNA targets PD-1, and 80. The immune cell of embodiment 79, wherein the second shRNA targets TIGIT. (Aspect 81) 1. A method for producing immune cells, comprising: (1) a gene encoding a genetically engineered antigen receptor that specifically binds to a target antigen; (2) a gene disrupting agent or a gene whose expression attenuates the function of said immune cell; or a gene disrupting agent capable of reducing or reducing expression in sequentially in any order into said immune cells, This results in the expression of a genetically engineered antigen receptor, which weakens the function of the immune cells. The method further comprises producing the immune cell in which expression of a gene is reduced. (Aspect 82) The engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor ( 82. The method of embodiment 81, wherein the TCR is a TCR. (Aspect 83) 83. The method of embodiment 82, wherein the engineered antigen receptor is a CAR. (Aspect 84) The CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. 84. The method of embodiment 83, comprising: (Aspect 85)

[0039] Embodiment 84: wherein the extracellular antigen recognition domain of the CAR specifically binds to the target antigen The method described below. (Aspect 86) the intracellular signaling domain of the CAR is an intracellular signaling domain of the CD3 zeta (CD3ζ) chain 85. The method of embodiment 84, comprising a domain. (Aspect 87) 87. The method of claim 86, wherein the intracellular signaling domain of the CAR further comprises a costimulatory molecule. How to post. (Aspect 88) The costimulatory molecules are ICOS, OX40, CD137 (4-1BB), CD27, and C The method of embodiment 87, selected from the group consisting of D28. (Aspect 89) 89. The method of embodiment 88, wherein the costimulatory molecule is CD137(4-1BB). (Aspect 90) 89. The method of embodiment 88, wherein the costimulatory molecule is CD28. (Aspect 91) 83. The method of embodiment 82, wherein the engineered antigen receptor is a TCR. (Aspect 92) 92. The method of embodiment 91, wherein the TCR is a monoclonal TCR (mTCR). (Aspect 93) The target antigen is present in or on the surface of cancer cells, cancer tissues, and / or the tumor microenvironment. 93. The method according to any one of embodiments 81 to 92, wherein the polypeptide is expressed. (Aspect 94) The target antigen is 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (α -fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human Prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α-metabolite Acyl-coenzyme A racemase, ART-4 (ADP-ribosyltransferase-4) ), B7H4 (v-set domain-containing T-cell activation inhibitor 1), BAGE-1 (B-meta BCL-2 (B-cell CLL / lymphoma-2), BING-4 ( WD repeat domain 46), CA15-3 / CA27-29 (mucin 1), CA19-9 ( Cancer antigen 19-9), CA72-4 (cancer antigen 72-4), CA125 (cancer antigen 125 ), calreticulin, CAMEL (CTL-recognized antigen on melanoma), CASP-8 ( Supase 8), cathepsin B, cathepsin L, CD19 (cluster of differentiation 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, C D80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel accessory 2 ), CML28 (chronic myeloid leukemia tumor antigen 28), coactosin-like protein, collagen Gen XXIII, COX-2 (cyclooxygenase-2), CT-9 / BRD6 (cancer / testis antigen 9), Cten (c-terminal tensin-like protein), cyclin B1, cyclin Phosphorin D1, cyp-B, CYPB1 (cytochrome p450 family 1 subfamily) b Member 1), DAM-10 / MAGE-B1 (melanoma-associated antigen B1), DAM- 6 / MAGE-B2, EGFR / Her1 (epidermal growth factor receptor), EMMPRIN ( Isidine), EpCam, EphA2 (EPH receptor A2), EphA3, ErbB3 ( Erb-B2 receptor tyrosine kinase 3), EZH2 (zeste2 polycomb inhibitory complex Enhancer of fusion 2 subunits), FGF-5 (fibroblast growth factor 5), FN (F fibronectin), Fra-1 (Fos-related antigen-1), G250 / CAIX (carbonic anhydride) Enzyme 9), GAGE-1 (G antigen-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GDEP (prostate GnT-V (gluconate kinase), gp100 (melanin kinase), Cell lineage-specific antigen GP100, GPC3 (glypican 3), HAGE (helical antigen) , HAST-2 (sulfotransferase family 1A member 1), hepsin, H er2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K -MEL, HNE (medullasin), homeobox NKX3.1, HOM-TES-1 4 / SCP-1, HOM-TES-85, HPV-E6, HPVE7, HST-2 (sil TWIN-2), hTERT, iCE (caspase 1), IGF-1R (insulin-like growth factor receptor factor-1 receptor), IL-13Ra2 (interleukin-13 receptor subunit α2 ), IL-2R (interleukin-2 receptor), IL-5 (interleukin-5), Immature laminin receptor, kallikrein 2, kallikrein 4, Ki67, KIAA0205 (lysophosphatidylglycerol acyltransferase 1), KK-LC-1 (Kita Kyushu Lung Cancer Antigen-1), KM-HN-1, LAGE-1 (L antigen family member-1) , Livin, MAGE-A1, MAGE-A10, MAGE-A12, MAGEA2, MA GE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-B1, MA GE-B10, MAGE-B16, MAGE-B17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2 (melanoma antigen pharmacokinetics) Milli-L2), mammaglobin A, MART-1 / Melan-A (T-cell-1 recognized melanoma antigen), MART-2, matrix protein 22, MC1R (melanoma antigen) lanocortin 1 receptor), M-CSF (macrophage colony-stimulating factor), mesothelin , MG50 / PXDN (peroxidasin), MMP11 (matrix metalloproteinase 11), MN / CA IX-antigen (carbonic anhydrase 9), MRP-3 (multidrug resistance-related protein Protein-3), MUC1 (mucin 1), MUC2, NA88-A (VENT-like homeobox ox2 pseudogene 1), N-acetylglucosaminyltransferase-V, Neo- PAP (Neo-poly(A) polymerase), NGEP (New gene expressed in the prostate) NMP22 (nuclear matrix protein 22), NPM / ALK (nucleophosmin ), NSE (neuron-specific enolase), NY-ESO-1, NY-ESO-B, O A1 (osteoarthritis QTL1), OFA-iLRP (carcinoembryonic antigen-immature laminin receptor OGT (O-GlcNAc transferase), OS-9 (endoplasmic reticulum protein) cutin), osteocalcin, osteopontin, p15 (CDK inhibitor 2B), p53 , PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor) Prostatic acid phosphatase inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PART-1 ( Prostate androgen-regulated transcript 1), PATE (prostate and testis expressed 1), PD EF (prostate-derived Ets factor), Pim-1 kinase (proviral integration site 1 ), Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1), POTE (expressed in the prostate, ovaries, testes, and placenta), PRAME (melanoma) antigens preferentially expressed in tumors), prostein, proteinase-3, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate-specific G-protein co-receptor), PSM, PSMA (prostate-specific membrane antigen), RAGE-1 (renal tumor carcinoma antigen) Raw material), RHAMM / CD168, RU1 (renal ubiquitous protein 1), RU2, SAGE (meat SART-1 (squamous cell carcinoma antigen recognized by T-cells-1), S ART-2, SART-3, Sp17 (sperm protein 17), SSX-1 (SSX factor Milly Member 1), SSX-2 / HOM-MEL-40, SSX-4, STAMP-1 (STEAP2 metalloreductase), STEAP, survivin, survivin-213, TA-90 (tumor-associated antigen-90), TAG-72 (tumor-associated glycoprotein-72), T ARP (TCRγ alternative reading frame protein), TGFb (transforming growth factor β), TGFbR11 (transforming growth factor β receptor 11), TGM-4 (transglutaminase amine 4), TRAG-3 (Taxol resistance-associated gene 3), TRG (T-cell receptor γ locus), TRP-1 (transient receptor potential-1), TRP-2 / 6b, TR P-2 / INT2, Trp-p8, tyrosinase, UPA (U-plasminogen activator factor), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK-1, and WT1( 94. The method of embodiment 93, wherein the tumor is selected from the group consisting of Wilms' tumor 1). (Aspect 95) 96. The method of embodiment 95, wherein the target antigen is CD19 or CD22. (Aspect 96) 97. The method of embodiment 96, wherein the target antigen is CD19. (Aspect 97) The target antigen is a cancer antigen, and the expression of the cancer antigen is detected in cancer cells, cancer tissues, and / or or any of embodiments 94 to 96, wherein the antigen is increased in or on the surface of the tumor microenvironment. The method described above. (Aspect 98) The target antigen is α-actinin-4 / m, ARTC1 / m, bcr / abl, beta -Catenin / m, BRCA1 / m, BRCA2 / m, CASP-5 / m, CASP-8 / m, CDC27 / m, CDK4 / m, CDKN2A / m, CML66, COA-1 / m, DEK-CAN, EFTUD2 / m, ELF2 / m, ETV6-AML1, FN1 / m, GPNMB / m, HLA-A*0201-R170I, HLA-A11 / m, HLA-A 2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LDLR-FUT , MART2 / m, ME1 / m, MUM-1 / m, MUM-2 / m, MUM-3 / m, Mi Osin class 1 / m, neo-PAP / m, NFYC / m, N-Ras / m, OGT / m , OS-9 / m, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, R BAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, TEL- AML1, TGFbRII, and TPI / m, wherein the target The antigen is a cancer antigen, and the cancer antigen is present in cancer cells, cancer tissues, and / or the tumor microenvironment. 94. The method of embodiment 93, wherein the antigen expressed in or on the surface is a mutated form of the antigen. (Aspect 99) The expression of the gene that attenuates the function of the immune cell is determined by: (i) inhibiting the proliferation of said immune cells; (ii) inducing cell death of said immune cells; (iii) inhibiting the ability of said immune cells to recognize and / or be activated by said target antigen. , (iv) inducing differentiation of the immune cells into cells that do not induce an immune response against the target antigen. , (v) a decrease in the responsiveness of said immune cells to molecules that stimulate the immune response of said immune cells; and teeth (vi) increasing the response of the immune cells to molecules that suppress the immune response of the immune cells; The method according to any one of aspects 81 to 98, wherein the method causes the above. (Aspect 100) The genes that weaken the function of the immune cells include PD1, PD-L1, CTLA4, and TIM 3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), L AG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, Me rTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGK alpha, DGK zeta, Cbl-b, Cbl-c, CD148, LRR1, TGFBR1, IL1 99. In one embodiment, the method further comprises administering to a subject a subject selected from the group consisting of: ORA, KLGR1, DNMT3A, and A2aR. The method described. (Aspect 101) The gene that weakens the function of the immune cells is converted into a molecule that suppresses the immune response of the immune cells. 100. The method of embodiment 99, wherein the immune cell response to a medicament is enhanced. (Aspect 102) The gene that enhances the response of the immune cell to a molecule that suppresses the immune response of the immune cell. 102. The method of embodiment 101, wherein the gene encodes a receptor or ligand for an immune checkpoint. . (Aspect 103) The immune checkpoint receptor or ligand is PD1, PD-L1, CTLA 4. TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM -5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD 96, MerTK, and 2B4. (Aspect 104) the gene disrupting agent is administered to the immune cell, compared to the immune cell in the absence of the gene disrupting agent(s). and inhibiting said expression of a gene in said immune cell that attenuates the function of said immune cell for at least 30 minutes. , 40, 50, 60, 70, 80, 90, or 95% reduction in any of embodiments 81 to 103. The method according to any one of the preceding claims. (Aspect 105) The gene disrupting agent inhibits the immune cells from reacting with a molecule that suppresses the immune response of the immune cells. 105. The method of embodiment 104, wherein the expression of a gene that enhances the response is reduced. (Aspect 106) The gene disrupting agent is a gene encoding a receptor or ligand of an immune checkpoint. 106. The method of embodiment 105, wherein the expression of (Aspect 107) The gene disrupting agent is selected from the group consisting of PD1, PD-L1, CTLA4, TIM3, and CEACAM (C EACAM-1, CEACAM-3 or CEACAM-5), LAG3, VISTA, From BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4 107. The method of embodiment 106, wherein the expression of a gene selected from the group consisting of: (Aspect 108) Before the gene disrupting agent attenuates the function of the immune cells by RNA interference (RNAi). 108. The method according to any one of embodiments 105 to 107, wherein the expression of the gene is reduced. (Aspect 109) More than one gene disrupting agent inhibits the function of said immune cell in said immune cell by RNAi. 109. The method of embodiment 108, wherein the expression of the gene that attenuates the ability of the gene to be treated is reduced. (Aspect 110) The gene disrupting agent targets a single gene that impairs the function of the immune cell, or The first gene disrupting agent targets a different gene that weakens the function of the immune cell. a first gene disrupting agent targets a second gene, and a second gene disrupting agent targets a second gene, or any combination thereof. The method of embodiment 109, wherein the targeting is combined. (Aspect 111) Any of embodiments 108 to 110, wherein the RNAi is mediated by short hairpin RNA (shRNA). The method according to any one of the preceding claims. (Aspect 112) 112. The method of embodiment 111, wherein the RNAi is mediated by more than one shRNA. (Aspect 113) 113. The method of embodiment 112, wherein the RNAi is mediated by two shRNAs. (Aspect 114) 114. The method of embodiment 112 or 113, wherein two shRNAs target PD-1. (Aspect 115) The first shRNA targets PD-1 and the second shRNA targets TIM-3 114. The method of embodiment 112 or 113. (Aspect 116) The first shRNA targets PD-1 and the second shRNA targets CTLA-4. 114. The method of embodiment 112 or 113. (Aspect 117) The first shRNA targets PD-1 and the second shRNA targets LAG-3 114. The method of embodiment 112 or 113. (Aspect 118) The first shRNA targets PD-1 and the second shRNA targets TIGIT 114. The method of embodiment 112 or 113. (Aspect 119) 119. The immune cells according to any one of embodiments 111 to 118, wherein the immune cells comprise a nucleotide sequence encoding an shRNA. Any of the methods described above. (Aspect 120) embodiment 119, wherein the immune cells comprise nucleotide sequences encoding more than one shRNA The method described below. (Aspect 121) 120. The method of claim 119, wherein the immune cells comprise a nucleotide sequence encoding two shRNAs. How to post. (Aspect 122) The nucleotide sequence encoding the shRNA(s) is selected from SEQ ID NOs: 2 to 219 Any of embodiments 119 to 121, comprising a sequence selected from the group consisting of 238 to 267 The method described below. (Aspect 123) 119. The nucleotide sequence encoding the shRNA is present on a vector A method according to any one of claims 1 to 122. (Aspect 124) Embodiment 123 in which the expression of different shRNAs is regulated by different promoters A method according to any one of the preceding claims. (Aspect 125) The expression of two different shRNAs was regulated by two different promoters. The method according to embodiment 124. (Aspect 126) An embodiment in which the two different promoters are RNA polymerase III promoters 125. (Aspect 127) 127. The method of embodiment 126, wherein the two promoters are U6 promoters. (Aspect 128) 128. The method of embodiment 127, wherein the U6 promoters are derived from different species. (Aspect 129) Any of embodiments 125 to 128, wherein the two promoters are oriented in different directions. The method described above. (Aspect 130) The engineered antigen receptor and the gene disrupting agent(s) are then separated from the vector. The method according to any one of embodiments 81 to 129, wherein each of the above is expressed. (Aspect 131) The engineered antigen receptor and the gene disrupting agent(s) are delivered from the same vector. 131. The method of embodiment 130, wherein the (Aspect 132) Aspects 130 to 13, wherein the vector is a plasmid vector or a viral vector 1. The method according to any one of claims 1 to 9. (Aspect 133) The viral vector is a lentiviral vector, an adenoviral vector, or an adenoviral vector. 133. The method of embodiment 132, wherein the vector is a virus-associated virus vector. (Aspect 134) The method of embodiment 133, wherein the lentiviral vector is a retroviral vector. . (Aspect 135) The immune cells are selected from the group consisting of T cells and natural killer (NK) cells. A method according to any one of claims 81 to 134. (Aspect 136) 136. The method of embodiment 135, wherein the immune cell is a T cell. (Aspect 137) 137. The method of embodiment 136, wherein the T cells are CD4+ T cells or CD8+ T cells. (Aspect 138) The immune cells express two shRNAs and a nucleotide sequence encoding a CAR in the same vector. 138. The method of embodiment 136 or 137, comprising on a monitor. (Aspect 139) The two shRNAs were directed in different directions to each other. 139. The method of embodiment 138, wherein each of the genes is regulated by a promoter of ZeIII. (Aspect 140) wherein the CAR targets CD19, the first shRNA targets PD-1, and 140. The method of embodiment 139, wherein the second shRNA targets TIGIT. (Aspect 141) A composition comprising the immune cell according to any one of aspects 21 to 80. (Aspect 142) A pharmaceutical composition comprising the immune cells according to any one of aspects 21 to 80 and a pharmaceutically acceptable carrier. Finished product. (Aspect 143) A subject having a disease or condition requiring immunotherapy according to any of embodiments 21 to 80. or a composition according to embodiment 141 or 142. Law. (Aspect 144) The engineered antigen receptor specifically targets an antigen associated with the disease or condition. 144. The method of embodiment 143, wherein the binding (Aspect 145) 145. The method of claim 143 or 144, wherein the disease or condition is cancer, e.g., a tumor. Law. (Aspect 146) The immune cells or immunoglobulins according to any one of aspects 21 to 80 for use in treating a disease or condition. or the composition according to any one of embodiments 141 to 142. (Aspect 147) any one of embodiments 21 to 80 in the manufacture of a medicament for treating a disease or condition. or the use of a composition according to any one of embodiments 121 to 122. (Aspect 148) The engineered antigen receptor specifically targets an antigen associated with the disease or condition. 148. An immune cell or composition according to embodiment 146 which binds or the use according to embodiment 147. (Aspect 149) 149. According to embodiment 147 or 148, wherein the disease or condition is cancer, e.g., a tumor. Uses, compositions, or immune cells.

Claims

1. 1. An in vitro method for generating immune cells, comprising: (1) a gene encoding a genetically engineered antigen receptor that specifically binds to a target antigen; (2) A gene disrupting agent, wherein the gene disrupting agent or the expression thereof can reduce the expression in the immune cell of a gene that attenuates the function of the immune cell, and the gene disrupting agent comprises a first shRNA targeting PD-1 and a second shRNA targeting TIGIT, and the gene disrupting agent is introduced into the immune cell simultaneously or sequentially in any order; The in vitro method thereby produces immune cells in which a genetically engineered antigen receptor is expressed and in which expression of a gene that attenuates the function of the immune cell is reduced.

2. 1. An in vitro method for producing immune cells, comprising: (1) a gene encoding a genetically engineered antigen receptor that specifically binds to a target antigen; (2) A gene disrupting agent, wherein the gene disrupting agent or the expression thereof can reduce the expression in the immune cell of a gene that attenuates the function of the immune cell, and the gene disrupting agent comprises a first shRNA targeting PD-1 and a second shRNA targeting TIGIT, and the gene disrupting agent is introduced into the immune cell simultaneously or sequentially in any order; The in vitro method thereby produces immune cells in which a genetically engineered antigen receptor is expressed and in which expression of a gene that attenuates the function of the immune cells is reduced.

3. 3. The method of claim 1 or 2, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

4. The method of claim 3, wherein the genetically engineered antigen receptor is a CAR.

5. The method of claim 4, wherein the CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain.

6. The method of claim 5, wherein the extracellular antigen recognition domain of the CAR specifically binds to the target antigen.

7. The method of claim 5, wherein the intracellular signaling domain of the CAR comprises the intracellular domain of the CD3 zeta (CD3ζ) chain.

8. 8. The method of claim 7, wherein the intracellular signaling domain of the CAR further comprises a costimulatory molecule.

9. 9. The method of claim 8, wherein the costimulatory molecule is selected from the group consisting of ICOS, OX40, CD137 (4-1BB), CD27, and CD28.

10. The method of any one of claims 1 to 9, wherein the target antigen is expressed in or on the surface of cancer cells, cancer tissues and / or the tumor microenvironment.

11. The method according to any one of claims 1 to 10, wherein the target antigen is CD19 or CD22.

12. The method according to any one of claims 1 to 11, wherein the target antigen is a cancer antigen, and the expression of the cancer antigen is increased in or on the surface of cancer cells, cancer tissues, and / or tumor microenvironments.

13. The method of any of claims 1 to 12, wherein the gene disrupting agent reduces the expression of a gene in the immune cell that attenuates the function of the immune cell by at least 30, 40, 50, 60, 70, 80, 90, or 95% compared to the immune cell in the absence of the gene disrupting agent.

14. 14. The method of claim 13, wherein the gene disrupting agent reduces expression of a gene that enhances the response of the immune cell to a molecule that suppresses the immune response of the immune cell.

15. The method of any one of claims 1 to 14, wherein the gene disrupting agent reduces the expression of the gene that attenuates the function of the immune cell by RNA interference (RNAi).

16. The method of claim 15 , wherein the RNAi is mediated by the first shRNA and the second shRNA.

17. The method according to any one of claims 1 to 16, wherein the immune cells are selected from the group consisting of T cells and natural killer (NK) cells.

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