Enhanced immune cells using dual shRNA and compositions containing immune cells

Abstract

The present disclosure relates broadly to the field of cancer immunotherapy. For example, the present invention generally relates to immune cells comprising a genetically engineered antigen receptor that specifically binds to a target antigen and a gene disrupting agent that reduces or is capable of reducing the expression of a gene that impairs immune cell function in the immune cell.

Description

[0001] Related Applications

[0002] This application claims the benefit of Korean Patent Application No. 10-2018-0004238, filed on January 12, 2018, the disclosure of which is hereby incorporated by reference in its entirety.

[0003] References to electronically submitted sequence listings

[0004] This application incorporates by reference the sequence list submitted with this application into a text file named 14570-001-228_SEQ_LISTING.txt, which was created on January 5, 2019 and is 88,751 bytes in size. Technical Field

[0005] The present disclosure relates broadly to the field of cancer immunotherapy. For example, the present invention generally relates to immune cells, which include a genetically engineered antigen receptor that specifically binds to a target antigen and a gene disruptor that reduces or is capable of reducing the expression of a gene in the immune cell that impairs immune cell function. Background Art

[0006] By isolating T cells or NK cells (natural killer cells) from the body of a patient or donor, culturing these cells in vitro, and then introducing them back into the patient, anti-cancer therapy using immune cells is currently receiving widespread attention as a new cancer treatment method. In particular, it is reported that immune cells that have undergone a process of injecting new genetic information using viruses, etc., and then culturing in vitro have greater anti-cancer effects than cells that have not been cultured. Here, the genetic information injected into T cells is usually a chimeric antigen receptor (hereinafter referred to as CAR) or a monoclonal T cell receptor (hereinafter referred to as mTCR) that has been modified to have a high affinity for the target antigen. These modified immune cells recognize and attack cancer cells expressing the target antigen and induce cell death without being limited by their inherent antigen specificity. In 1989, Eshhar et al. first proposed a method for genetically modifying T cells using CAR and named it "T-body".

[0007] Provided herein are immune cell compositions and methods, which solve the problems of conventional simultaneous immune cell therapies noted above, wherein the problems bring huge economic burdens to patients due to their high costs, act on T cells other than CAR-T, and cause the risk of autoimmune symptoms and cytokine release syndrome. In short, for example, disclosed herein are preparation methods with high yields and low production costs. In addition, by suppressing molecules that inhibit immune cell function with higher probability and efficacy, the disclosure herein meets the technical needs of providing effective cell therapy. The technical problems that the present disclosure is intended to solve are not limited to the above-mentioned technical problems, and other unmentioned technical problems will be obvious to those of ordinary skill in the art. Summary of the invention

[0008] Provided herein are vectors, immune cells, pharmaceutical compositions comprising the immune cells, and compositions comprising the immune cells. Also provided herein are methods of producing immune cells, and methods of treatment and use of immune cells.

[0009] On the one hand, a vector is provided herein. In some embodiments, a vector is provided, which comprises a base sequence of a short hairpin RNA (shRNA) encoding two types of gene expression that inhibits the weakening of immune cell function, and a base sequence encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) (e.g., a monoclonal T cell receptor (mTCR)). In some embodiments, the target of CAR or TCR (e.g., mTCR) is a human tumor antigen, which is selected from an antigen that shows an increase in expression in cancer or a mutant form of an antigen found in cancer (e.g., cancer cells, cancer tissues and / or tumor microenvironments).

[0010] In some embodiments, the expression of the two types of shRNAs is characterized in that they are regulated by two different promoters, respectively.

[0011] In some embodiments, the two promoters are RNA polymerase III promoters. In some embodiments, the two promoters are U6 promoters, for example, U6 promoters derived from different species. In some embodiments, the two promoters are oriented in the same direction relative to each other on the vector. In some embodiments, the two promoters are oriented in different directions on the vector. For example, in a certain embodiment, the promoters are oriented head to head. In another embodiment, the promoters are oriented tail to tail.

[0012] In some embodiments, the gene that impairs immune cell function is an immune checkpoint receptor or ligand.

[0013] In some embodiments, the immune checkpoint receptor or ligand is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4.

[0014] In some embodiments, the gene that impairs immune cell function is selected from the group consisting of: FAS, CD45, PP2A, SHIP1, SHIP2, DGKα, DGKζ, Cb1-b, CD147, LRR1, TGFBR1, IL10Rα, KLGR1, DNMT3A, and A2aR.

[0015] In some embodiments, two types of shRNA are utilized, which target one or more genes that weaken immune cell function. In some embodiments, two types of shRNAs either target a single gene that weakens immune cell function, or they target different genes that weaken immune cell function. In some embodiments, two types of shRNAs target PD-1. In some embodiments comprising two types of shRNAs, one shRNA targets PD-1, and the second shRNA targets TIM-3. In some embodiments comprising two types of shRNAs, one shRNA targets PD-1, and the second shRNA targets TIGIT.

[0016] The shRNA forms a hairpin structure comprising a sense shRNA sequence and an antisense shRNA sequence. In some embodiments, the base sequence encoding the shRNA described herein comprises a sequence selected from the group consisting of SEQ ID NO: 2-219. In certain embodiments, the base sequence encoding the shRNA described herein comprises a sequence selected from the group consisting of SEQ ID NO: 2-219, wherein the sequence encodes a sense shRNA sequence. In certain embodiments, the base sequence encoding the shRNA described herein comprises a sequence selected from the group consisting of SEQ ID NO: 2-219, wherein the sequence encodes an antisense shRNA sequence.

[0017] In some embodiments, the vector comprises any one of the base sequences SEQ ID NO: 220 or 221. In some embodiments, the vector is a plasmid vector or a viral vector, such as a lentiviral vector, for example, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0018] On the other hand, provided herein is an immune cell comprising a vector expressing CAR or TCR (e.g., mTCR), and wherein the expression of the target gene of two types of shRNA is reduced to 40% or less compared to the expression of a control group of shRNA that does not express the target gene. In some embodiments, the immune cell is selected from between T cells and NK cells of human origin.

[0019] On the other hand, provided herein is a pharmaceutical composition for immunotherapy of a human patient, comprising the above-mentioned immune cells. In some embodiments, the immune cells are initially derived from the patient. In some embodiments, the patient suffers from a tumor or cancer, in which an increase or change in the level of a cancer antigen targeted by a CAR or TCR (e.g., mTCR) expressed in the cell is detected.

[0020] In another aspect, provided herein is an immune cell comprising a genetically engineered antigen receptor that specifically binds to a target antigen and a gene disrupting agent that reduces or is capable of reducing the expression of a gene in the immune cell that impairs the function of the immune cell.

[0021] In another aspect, provided herein is an immune cell comprising a genetically engineered antigen receptor that specifically binds to a target antigen and a gene disrupting agent that reduces or is capable of reducing the expression of a gene in the immune cell that impairs the function of the immune cell.

[0022] In some embodiments, the genetically engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0023] In some embodiments, the genetically engineered antigen receptor is CAR. In some embodiments, CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, the extracellular antigen recognition domain of CAR specifically binds to the target antigen.

[0024] In some embodiments, the intracellular signal transduction domain of CAR includes the intracellular domain of CD3 zeta (CD3ζ) chain. In some embodiments, the intracellular signal transduction domain of CAR also includes a costimulatory molecule. In some embodiments, the costimulatory molecule is selected from the group consisting of: ICOS, 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.

[0025] In some embodiments, the genetically engineered antigen receptor is a TCR. In some embodiments, the TCR is a monoclonal TCR (mTCR).

[0026] In some embodiments, the target antigen is expressed in or on the surface of cancer cells, cancer tissues, and / or tumor microenvironments.

[0027] In some embodiments, the target antigen is selected from the group consisting of:

[0028] 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (alpha-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α-methylacyl-CoA 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), CA 15-3 / CA 27-29 (mucin 1), CA 19-9 (cancer antigen 19-9), CA 72-4 (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 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel attachment 2), CML28 (chronic myeloid leukemia tumor antigen 28), trichometin, 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 B1), DAM-6 / MAGE-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 protein inhibitory complex 2 subunit enhancer), FGF-5 (fibroblast growth 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, GDEP (differentially expressed genes in prostate), GnT-V (gluconokinase), gp100 (melanocyte lineage-specific antigen GP100), GPC3 (glypican 3), HAGE (helical antigen), HAST-2 (sulfotransferase family 1A member 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K-MEL, HNE (medullin), 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, KIAA0205 (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- AGE-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 (melanoma antigen recognized by T cells-1), MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony stimulating factor), mesothelin, MG50 / PXDN (peroxiredoxin), MMP11 (matrix metalloproteinase 11), MN / CA1X-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 (new gene expressed in prostate), NMP22 (nuclear matrix protein 22), NPM / ALK (nuclear phosphokinin), 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 (endoplasmic reticulum lectin), osteocalcin, osteopontin, p15 (CDK inhibitor 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PART-1 (prostatic 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 interacting 1), POTE (expressed in prostate, ovary, testis and placenta), PRAME (antigen preferentially expressed in melanoma), prostaglandin, proteinase-3, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate specific G protein-coupled receptor), PSM, PSMA (prostate specific membrane antigen), RAGE-1 (renal tumor cancer antigen), RHAMM / CD168, RU 1 (renal ubiquitin 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-213, TA-90 (tumor associated antigen-90), TAG-72 (tumor associated glycoprotein-72), TARP (TCR γ alternative reading frame protein), TGFb (transforming growth factor β), TGFbR11 (transforming growth factor β receptor 11), TGM-4 (transglutaminase 4), TRAG-3 (paclitaxel 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-type plasminogen 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. In some embodiments, the target antigen is CD19.

[0029] In some embodiments, the target antigen is a cancer antigen whose expression is increased in or on the surface of cancer cells, cancer tissues, and / or tumor microenvironment.

[0030] In some embodiments, the target antigen is selected from the group consisting of:

[0031] α-actinin-4 / m, ARTC1 / m, bcr / abl, β-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-A2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LD LR-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, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, TEL-AML1, TGFbRII and TPI / m; and wherein the target antigen is a mutated form of a cancer antigen expressed on or on the surface of cancer cells, cancer tissues and / or tumor microenvironments.

[0032] In some embodiments, expression of a gene that impairs immune cell function results in one or more of the following:

[0033] i) Inhibit the proliferation of immune cells;

[0034] ii) inducing cell death of immune cells;

[0035] iii) inhibiting the ability of immune cells to recognize target antigens and / or become activated;

[0036] iv) inducing immune cells to differentiate into cells that do not induce an immune response to the target antigen;

[0037] v) reducing the reactivity of immune cells to molecules that promote an immune response in immune cells; or

[0038] vi) Increase the response of immune cells to molecules that inhibit the immune response of immune cells.

[0039] In some embodiments, the gene that impairs immune cell function is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGKα, DGKζ, Cb1-b, Cb1-c, CD148, LRR1, TGFBR1, IL10RA, KLGR1, DNMT3A and A2aR.

[0040] In some embodiments, a gene that impairs immune cell function increases the immune cell's response to a molecule that inhibits the immune cell's immune response.

[0041] In some embodiments, the gene that increases the response of an immune cell to a molecule that inhibits an immune response of the immune cell encodes an immune checkpoint receptor or ligand.

[0042] In some embodiments, the immune checkpoint receptor or ligand is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4.

[0043] In some embodiments, the gene disrupting agent reduces expression of a gene that impairs immune cell function in an immune cell by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to an immune cell in the absence of the gene disrupting agent.

[0044] In some embodiments, a gene disrupting agent reduces expression of a gene that increases the response of an immune cell to a molecule that inhibits an immune response of the immune cell.

[0045] In some embodiments, the gene disrupting agent reduces expression of a gene encoding an immune checkpoint receptor or ligand.

[0046] In some embodiments, the gene disrupting agent reduces the expression of a gene selected from the group consisting of PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK, and 2B4.

[0047] In some embodiments, the gene disrupting agent reduces the expression of genes that impair immune cell function by RNA interference (RNAi). In some embodiments, more than one gene disrupting agent reduces the expression of genes that impair immune cell function in immune cells by RNAi.

[0048] In some embodiments, the gene disrupting agents target a single gene that impairs immune cell function, or target different genes that impair immune cell function, wherein a first gene disrupting agent targets a first gene and a second gene disrupting agent targets a second gene, or any combination thereof.

[0049] In some embodiments, RNAi is mediated by short hairpin nuclear RNA (shRNA). In some embodiments, RNAi is mediated by more than one shRNA. In some embodiments, RNAi is mediated by two shRNAs.

[0050] In some embodiments, both shRNAs target PD-1. In some embodiments, 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 CTLA-4. In some embodiments, the first shRNA targets PD-1 and the second shRNA targets LAG-3. In some embodiments, the first shRNA targets PD-1 and the second shRNA targets TIGIT.

[0051] In some embodiments, the immune cell comprises a nucleotide sequence encoding an shRNA. In some embodiments, the immune cell comprises a nucleotide sequence encoding more than one shRNA. In some embodiments, the immune cell comprises a nucleotide sequence encoding two shRNAs. Unless otherwise indicated, the terms "base sequence" and "nucleotide sequence" used herein are interchangeable.

[0052] In some embodiments, the nucleotide sequence encoding the shRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 2-219 and 238-267.

[0053] In some embodiments, the nucleotide sequence encoding the shRNA is on a vector.

[0054] In some embodiments, the expression of different shRNAs is regulated by different promoters. In some embodiments, the expression of two different shRNAs is regulated by two different promoters. In some embodiments, the two different promoters are RNA polymerase III promoters. In some embodiments, the two promoters are U6 promoters. In some embodiments, the U6 promoters are from different species. In some embodiments, the two promoters are oriented in different directions from each other. For example, in a certain embodiment, the promoters are oriented in a head-to-head orientation. In another embodiment, the promoters are oriented in a tail-to-tail orientation.

[0055] In some embodiments, the genetically engineered antigen receptor and the gene disrupting agent are each expressed by a vector. In some embodiments, the genetically engineered antigen receptor and the gene disrupting agent are expressed by the same vector.

[0056] 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.

[0057] In some embodiments, the immune cell is selected from the group consisting of a T cell and a natural killer (NK) cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD4+T cell or a CD8+T cell.

[0058] In some embodiments, immune cells include nucleotide sequences encoding two shRNAs and CARs on the same vector. In some embodiments, the two shRNAs are each regulated by two different RNA polymerase III promoters, and the promoters are oriented in different directions from each other. For example, in a certain embodiment, the promoter is oriented head to head. In another embodiment, the promoter is oriented tail to tail. In some embodiments, CAR targets CD19, the first shRNA targets PD-1, and the second shRNA targets TIGIT.

[0059] In another aspect, the present invention provides a method for producing immune cells, which comprises introducing the following substances into immune cells simultaneously or sequentially in any order:

[0060] (1) a gene encoding a genetically engineered antigen receptor that specifically binds to a target antigen; and

[0061] (2) gene disruptors that reduce or are capable of reducing the expression of genes that weaken immune cell function in immune cells,

[0062] Thereby, immune cells are generated in which the genetically engineered antigen receptors are expressed and the expression of genes that impair immune cell function is reduced.

[0063] In some embodiments, the genetically engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0064] In some embodiments, the genetically engineered antigen receptor is CAR. In some embodiments, CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, the extracellular antigen recognition domain of CAR specifically binds to the target antigen.

[0065] In some embodiments, the intracellular signal transduction domain of CAR includes the intracellular domain of CD3 zeta (CD3ζ) chain. In some embodiments, the intracellular signal transduction domain of CAR also includes a costimulatory molecule. In some embodiments, the costimulatory molecule is selected from the group consisting of: ICOS, 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.

[0066] In some embodiments, the genetically engineered antigen receptor is a TCR. In some embodiments, the TCR is a monoclonal TCR (mTCR).

[0067] In some embodiments, the target antigen is expressed in or on the surface of cancer cells, cancer tissues, and / or tumor microenvironments.

[0068] In some embodiments, the target antigen is selected from the group consisting of:

[0069] 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (alpha-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α-methylacyl-CoA 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), CA 15-3 / CA 27-29 (mucin 1), CA 19-9 (cancer antigen 19-9), CA 72-4 (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 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel attachment 2), CML28 (chronic myeloid leukemia tumor antigen 28), trichometin, 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 B1), DAM-6 / MAGE-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 protein inhibitory complex 2 subunit enhancer), FGF-5 (fibroblast growth 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, GDEP (differentially expressed genes in prostate), GnT-V (gluconokinase), gp100 (melanocyte lineage-specific antigen GP100), GPC3 (glypican 3), HAGE (helical antigen), HAST-2 (sulfotransferase family 1A member 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K-MEL, HNE (medullin), homeobox NKX 3.1, 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, KIAA0205 (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, MAG E-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 family L2), Mammaglobin A, MART-1 / Melan-A (melanoma antigen recognized by T cells-1), MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony stimulating factor), mesothelin, MG50 / PXDN (peroxiredoxin), MMP11 (matrix metalloproteinase 11), MN / CA1X-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 (new gene expressed in prostate), NMP22 (nuclear matrix protein 22), NPM / ALK (nuclear phosphokinin), 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 (endoplasmic reticulum lectin), osteocalcin, osteopontin, p15 (CDK inhibitor 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PART-1 (prostatic 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 interacting 1), POTE (expressed in prostate, ovary, testis and placenta), PRAME (antigen preferentially expressed in melanoma), prostaglandin, proteinase-3, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate specific G protein-coupled receptor), PSM, PSMA (prostate specific membrane antigen), RAGE-1 (renal tumor cancer antigen), RHAMM / CD168, RU 1 (renal ubiquitin 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-213, TA-90 (tumor associated antigen-90), TAG-72 (tumor associated glycoprotein-72), TARP (TCR γ alternative reading frame protein), TGFb (transforming growth factor β), TGFbR11 (transforming growth factor β receptor 11), TGM-4 (transglutaminase 4), TRAG-3 (paclitaxel 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-type plasminogen 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. In some embodiments, the target antigen is CD19.

[0070] In some embodiments, the target antigen is a cancer antigen whose expression is increased in or on the surface of cancer cells, cancer tissues, and / or tumor microenvironment.

[0071] In some embodiments, the target antigen is selected from the group consisting of:

[0072] α-actinin-4 / m, ARTC1 / m, bcr / abl, β-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-A2 / m, HSP70-2M, KIAA0205 / m, K-Ras / m, LD LR-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, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, TEL-AML1, TGFbRII and TPI / m; and wherein the target antigen is a mutated form of a cancer antigen expressed on or on the surface of cancer cells, cancer tissues and / or tumor microenvironments.

[0073] In some embodiments, expression of a gene that impairs immune cell function results in one or more of the following:

[0074] i) Inhibit the proliferation of immune cells;

[0075] ii) inducing cell death of immune cells;

[0076] iii) inhibiting the ability of immune cells to recognize target antigens and / or become activated;

[0077] iv) inducing immune cells to differentiate into cells that do not induce an immune response to the target antigen;

[0078] v) reducing the reactivity of immune cells to molecules that promote an immune response in immune cells; or

[0079] vi) Increase the response of immune cells to molecules that inhibit the immune response of immune cells.

[0080] In some embodiments, the gene that impairs immune cell function is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGKα, DGKζ, Cb1-b, Cb1-c, CD148, LRR1, TGFBR1, IL10RA, KLGR1, DNMT3A and A2aR.

[0081] In some embodiments, a gene that impairs immune cell function increases the immune cell's response to a molecule that inhibits the immune cell's immune response.

[0082] In some embodiments, the gene that increases the response of an immune cell to a molecule that inhibits an immune response of the immune cell encodes an immune checkpoint receptor or ligand.

[0083] In some embodiments, the immune checkpoint receptor or ligand is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4.

[0084] In some embodiments, the gene disrupting agent reduces expression of a gene that impairs immune cell function in an immune cell by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to an immune cell in the absence of the gene disrupting agent.

[0085] In some embodiments, a gene disrupting agent reduces expression of a gene that increases the response of an immune cell to a molecule that inhibits an immune response of the immune cell.

[0086] In some embodiments, the gene disrupting agent reduces expression of a gene encoding an immune checkpoint receptor or ligand.

[0087] In some embodiments, the gene disrupting agent reduces the expression of a gene selected from the group consisting of PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK, and 2B4.

[0088] In some embodiments, the gene disrupting agent reduces the expression of genes that impair immune cell function by RNA interference (RNAi). In some embodiments, more than one gene disrupting agent reduces the expression of genes that impair immune cell function in immune cells by RNAi.

[0089] In some embodiments, the gene disrupting agents target a single gene that impairs immune cell function, or target different genes that impair immune cell function, wherein a first gene disrupting agent targets a first gene and a second gene disrupting agent targets a second gene, or any combination thereof.

[0090] In some embodiments, RNAi is mediated by short hairpin nuclear RNA (shRNA). In some embodiments, RNAi is mediated by more than one shRNA. In some embodiments, RNAi is mediated by two shRNAs.

[0091] In some embodiments, both shRNAs target PD-1. In some embodiments, 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 CTLA-4. In some embodiments, the first shRNA targets PD-1 and the second shRNA targets LAG-3. In some embodiments, the first shRNA targets PD-1 and the second shRNA targets TIGIT.

[0092] In some embodiments, the immune cell comprises a nucleotide sequence encoding an shRNA. In some embodiments, the immune cell comprises a nucleotide sequence encoding more than one shRNA. In some embodiments, the immune cell comprises a nucleotide sequence encoding two shRNAs.

[0093] In some embodiments, the nucleotide sequence encoding the shRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 2-219 and 238-267.

[0094] In some embodiments, the nucleotide sequence encoding the shRNA is on a vector.

[0095] In some embodiments, the expression of different shRNAs is regulated by different promoters. In some embodiments, the expression of two different shRNAs is regulated by two different promoters. In some embodiments, the two different promoters are RNA polymerase III promoters. In some embodiments, the two promoters are U6 promoters. In some embodiments, the U6 promoters are from different species. In some embodiments, the two promoters are oriented in different directions from each other. For example, in a certain embodiment, the promoters are oriented in a head-to-head orientation. In another embodiment, the promoters are oriented in a tail-to-tail orientation.

[0096] In some embodiments, the genetically engineered antigen receptor and the gene disrupting agent are each expressed by a vector. In some embodiments, the genetically engineered antigen receptor and the gene disrupting agent are expressed by the same vector.

[0097] 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.

[0098] In some embodiments, the immune cell is selected from the group consisting of a T cell and a natural killer (NK) cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD4+T cell or a CD8+T cell.

[0099] In some embodiments, immune cells include nucleotide sequences encoding two shRNAs and CARs on the same vector. In some embodiments, the two shRNAs are each regulated by two different RNA polymerase III promoters, and the promoters are oriented in different directions from each other. For example, in a certain embodiment, the promoter is oriented head to head. In another embodiment, the promoter is oriented tail to tail. In some embodiments, CAR targets CD19, the first shRNA targets PD-1, and the second shRNA targets TIGIT.

[0100] In another aspect, provided herein are compositions comprising engineered immune cells. In another aspect, provided herein are pharmaceutical compositions comprising immune cells and a pharmaceutically acceptable carrier.

[0101] On the other hand, provided herein is a method of treatment, which includes administering an immune cell or composition to a subject suffering from a disease or illness. In some embodiments, a genetically engineered antigen receptor specifically binds to an antigen, and the antigen is associated with the disease or illness. In some embodiments, the disease or illness is cancer or a tumor.

[0102] In another aspect, provided herein are immune cells or compositions for treating a disease or disorder. In another aspect, provided herein are uses of immune cells or compositions in the manufacture of a drug for treating a disease or disorder. In some embodiments, a genetically engineered antigen receptor specifically binds to an antigen associated with the disease or disorder. In some embodiments, the disease or disorder is a cancer or tumor.

[0103] Additional non-limiting embodiments are given below.

[0104] 1. A vector comprising:

[0105] A base sequence encoding two types of short hairpin RNA (shRNA) that inhibits the expression of at least one gene that weakens the function of immune cells; and

[0106] The base sequence encoding the chimeric antigen receptor (CAR) or monoclonal T cell receptor (mTCR).

[0107] 2. The vector according to embodiment 1, wherein the expression of the two types of shRNAs is characterized in that they are regulated by two different promoters, respectively.

[0108] 3. The vector according to embodiment 2, wherein both promoters are RNA polymerase III promoters.

[0109] 4. The vector according to embodiment 2, wherein the two promoters are U6 promoters derived from different species.

[0110] 5. The vector according to embodiment 2, wherein the two promoters are oriented in different directions relative to each other on the vector.

[0111] 6. The vector according to embodiment 1, wherein the gene that weakens the function of immune cells is an immune checkpoint receptor or ligand.

[0112] 7. A vector according to embodiment 6, wherein the immune checkpoint receptor or ligand is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4.

[0113] 8. The vector according to embodiment 1, wherein the gene that weakens the immune cell function is selected from the group consisting of: FAS, CD45, PP2A, SHIP1, SHIP2, DGKα, DGKζ, Cb1-b, CD147, LRR1, TGFBR1, IL10Rα, KLGR1, DNMT3A and A2aR.

[0114] 9. The vector according to embodiment 1, wherein the two types of shRNA target a single gene that impairs immune cell function, or wherein the two types of shRNA target different genes that impair immune cell function.

[0115] 10. The vector according to embodiment 1, wherein the two types of shRNA target PD-1.

[0116] 11. The vector according to embodiment 1, wherein among the two types of shRNAs, i) one shRNA targets PD-1 and the second shRNA targets TIM-3, or ii) one shRNA targets PD-1 and the second shRNA targets TIGIT.

[0117] 12. The vector according to embodiment 1, wherein, among the two types of shRNAs, the base sequence encoding one shRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 2-219, and the base sequence encoding the second shRNA comprises a different sequence selected from the group consisting of SEQ ID NOs: 2-219.

[0118] 13. A vector according to embodiment 1, wherein the target of the CAR or mTCR is a human tumor antigen that shows increased expression in cancer cells, cancer tissues and / or tumor microenvironment, or is a mutated form of an antigen found in cancer cells, cancer tissues and / or tumor microenvironment.

[0119] 14. The vector according to embodiment 1, which comprises the base sequence of SEQ ID NO: 220 or 221.

[0120] 15. The vector according to embodiment 1, wherein the vector is a plasmid vector, a lentiviral vector, an adenoviral vector, an adeno-associated vector or a retroviral vector.

[0121] 16. An immune cell comprising the vector according to embodiment 1, wherein the expression of the one or more genes is reduced to 40% or less compared to the expression in the absence of shRNA.

[0122] 17. The immune cell according to embodiment 16, wherein the immune cell is a T cell or a natural killer (NK) cell of human origin.

[0123] 18. A pharmaceutical composition comprising the immune cell according to any one of embodiments 1-17.

[0124] 19. A pharmaceutical composition according to embodiment 18 for use in treating a patient in need of immunotherapy, wherein the immune cells are initially obtained from the patient.

[0125] 20. A pharmaceutical composition according to embodiment 19, wherein the patient suffers from a tumor or cancer in which an increase or change in the target and / or target level of the CAR or mTCR expressed in immune cells is detected.

[0126] 21. An immune cell, comprising the engineered antigen receptor that specifically binds to a target antigen, and the gene disrupting agent that reduces or is capable of reducing the expression of a gene that weakens the function of the immune cell in the immune cell.

[0127] 22. The immune cell of embodiment 21, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0128] 23. The immune cell of embodiment 22, wherein the genetically engineered antigen receptor is CAR.

[0129] 24. The immune cell of embodiment 23, wherein the CAR comprises an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain.

[0130] 25. The immune cell of embodiment 24, wherein the extracellular antigen recognition domain of the CAR specifically binds to the target antigen.

[0131] 26. The immune cell of embodiment 24, wherein the intracellular signal transduction domain of the CAR comprises the intracellular domain of the CD3 zeta (CD3ζ) chain.

[0132] 27. The immune cell of embodiment 26, wherein the intracellular signal transduction domain of the CAR also comprises a co-stimulatory molecule.

[0133] 28. The immune cell of embodiment 27, wherein the co-stimulatory molecule is selected from the group consisting of: ICOS, 0X40, CD137 (4-1BB), CD27 and CD28.

[0134] 29. The immune cell of embodiment 28, wherein the co-stimulatory molecule is CD137 (4-1BB).

[0135] 30. The immune cell of embodiment 28, wherein the co-stimulatory molecule is CD28.

[0136] 31. The immune cell of embodiment 22, wherein the genetically engineered antigen receptor is a TCR.

[0137] 32. The immune cell of embodiment 31, wherein the TCR is a monoclonal TCR (mTCR).

[0138] 33. The immune cell of embodiment 31 or 32, wherein the target antigen is expressed in or on the surface of cancer cells, cancer tissues and / or tumor microenvironment.

[0139] 34. The immune cell of embodiment 33, wherein the target antigen is selected from the group consisting of:

[0140] 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (alpha-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α-methylacyl-CoA 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), CA 15-3 / CA 27-29 (mucin 1), CA 19-9 (cancer antigen 19-9), CA 72-4 (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 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel attachment 2), CML28 (chronic myeloid leukemia tumor antigen 28), trichometin, 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 B1), DAM-6 / MAGE-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 protein inhibitory complex 2 subunit enhancer), FGF-5 (fibroblast growth 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, GDEP (differentially expressed genes in prostate), GnT-V (gluconokinase), gp100 (melanocyte lineage-specific antigen GP100), GPC3 (glypican 3), HAGE (helical antigen), HAST-2 (sulfotransferase family 1A member 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K-MEL, HNE (medullin), homeobox NKX 3.1, 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, KIAA0205 (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, MAG E-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 family L2), Mammaglobin A, MART-1 / Melan-A (melanoma antigen recognized by T cells-1), MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony stimulating factor), mesothelin, MG50 / PXDN (peroxiredoxin), MMP11 (matrix metalloproteinase 11), MN / CA1X-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 (new gene expressed in prostate), NMP22 (nuclear matrix protein 22), NPM / ALK (nuclear phosphokinin), 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 (endoplasmic reticulum lectin), osteocalcin, osteopontin, p15 (CDK inhibitor 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PART-1 (prostatic 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 interacting 1), POTE (expressed in prostate, ovary, testis and placenta), PRAME (antigen preferentially expressed in melanoma), prostaglandin, proteinase-3, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate specific G protein-coupled receptor), PSM, PSMA (prostate specific membrane antigen), RAGE-1 (renal tumor cancer antigen), RHAMM / CD168, RU 1 (renal ubiquitin 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-213, TA-90 (tumor associated antigen-90), TAG-72 (tumor associated glycoprotein-72), TARP (TCR gamma alternative reading frame protein), TGFb (transforming growth factor beta), TGFbR11 (transforming growth factor beta receptor 11), TGM-4 (transglutaminase 4), TRAG-3 (paclitaxel resistance-associated gene 3), TRG (T cell receptor gamma locus), TRP-1 (transient receptor potential-1), TRP-2 / 6b, TRP-2 / INT2, Trp-p8, tyrosinase, UPA (U-type plasminogen activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK-1, and WT1 (Nephroblastoma 1).

[0141] 35. The immune cell of embodiment 35, wherein the target antigen is CD19 or CD22.

[0142] 36. The immune cell of embodiment 36, wherein the target antigen is CD19.

[0143] 37. The immune cell of any one of embodiments 34-36, wherein the target antigen is a cancer antigen whose expression is increased in or on the surface of cancer cells, cancer tissues and / or tumor microenvironments.

[0144] 38. The immune cell of embodiment 33, wherein the target antigen is selected from the group consisting of:

[0145] α-actinin-4 / m, ARTC1 / m, bcr / abl, β-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-A2 / m, HSP70-2M, KI AA0205 / 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, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, TEL-AML1, TGFbRII, and TPI / m; and

[0146] Wherein the target antigen is a cancer antigen, which is a mutated form of an antigen expressed in or on the surface of cancer cells, cancer tissues and / or tumor microenvironment.

[0147] 39. The immune cell of any one of embodiments 21-38, wherein expression of a gene that impairs immune cell function results in one or more of the following:

[0148] i) Inhibit the proliferation of immune cells;

[0149] ii) inducing cell death of immune cells;

[0150] iii) inhibiting the ability of immune cells to recognize target antigens and / or to activate;

[0151] iv) inducing immune cells to differentiate into cells that do not induce an immune response to the target antigen;

[0152] v) reducing the reactivity of immune cells to molecules that promote an immune response in immune cells; or

[0153] vi) Increase the response of immune cells to molecules that inhibit the immune response of immune cells.

[0154] 40. The immune cell of embodiment 39, wherein the gene that weakens the function of the immune cell is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGKα, DGKζ, Cb1-b, Cb1-c, CD148, LRR1, TGFBR1, IL10RA, KLGR1, DNMT3A and A2aR.

[0155] 41. The immune cell of embodiment 39, wherein the gene that weakens the function of the immune cell increases the response of the immune cell to a molecule that inhibits the immune response of the immune cell.

[0156] 42. The immune cell of embodiment 41, wherein the gene that increases the response of the immune cell to a molecule that inhibits the immune response of the immune cell encodes an immune checkpoint receptor or ligand.

[0157] 43. The immune cell of embodiment 42, wherein the immune checkpoint receptor or ligand is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4.

[0158] 44. The immune cell of any one of embodiments 21-43, wherein the gene disrupting agent reduces the expression of a gene that impairs immune cell function in the immune cell by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% compared to an immune cell in the absence of the gene disrupting agent.

[0159] 45. The immune cell of embodiment 44, wherein the gene disrupting agent reduces the expression of a gene that increases the immune cell's response to a molecule that inhibits the immune response of the immune cell.

[0160] 46. ​​The immune cell of embodiment 45, wherein the gene disrupting agent reduces the expression of a gene encoding an immune checkpoint receptor or ligand.

[0161] 47. The immune cell of embodiment 46, wherein the gene disruptor reduces the expression of a gene selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4.

[0162] 48. The immune cell of any one of embodiments 45-47, wherein the gene disrupting agent reduces the expression of a gene that impairs immune cell function by RNA interference (RNAi).

[0163] 49. The immune cell of embodiment 48, wherein more than one gene disrupting agent reduces the expression of a gene that impairs immune cell function in the immune cell through RNAi.

[0164] 50. The immune cell of embodiment 49, wherein the gene disrupting agent targets a single gene that impairs immune cell function, or wherein different gene disrupting agents target different genes that impair immune cell function, for example, wherein a first gene disrupting agent targets a first gene and a second gene disrupting agent targets a second gene.

[0165] 51. The immune cell of any one of embodiments 48-50, wherein the RNAi is mediated by short hairpin RNA (shRNA).

[0166] 52. The immune cell of embodiment 51, wherein the RNAi is mediated by more than one shRNA.

[0167] 53. The immune cell according to embodiment 52, wherein the RNAi is mediated by two shRNAs.

[0168] 54. The immune cell of any one of embodiments 52-53, wherein two shRNAs target PD-1.

[0169] 55. The immune cell of any one of embodiments 52-53, wherein the first shRNA targets PD-1 and the second shRNA targets TIM-3.

[0170] 56. The immune cell of any one of embodiments 52-53, wherein the first shRNA targets PD-1 and the second shRNA targets CTLA-4.

[0171] 57. The immune cell of any one of embodiments 52-53, wherein the first shRNA targets PD-1 and the second shRNA targets LAG-3.

[0172] 58. The immune cell of any one of embodiments 52-53, wherein the first shRNA targets PD-1 and the second shRNA targets TIGIT.

[0173] 59. The immune cell of any one of embodiments 51-58, wherein the immune cell comprises a nucleotide sequence encoding a shRNA.

[0174] 60. The immune cell of embodiment 59, wherein the immune cell comprises a nucleotide sequence encoding more than one shRNA.

[0175] 61. The immune cell of embodiment 59, wherein the immune cell comprises a nucleotide sequence encoding two shRNAs.

[0176] 62. An immune cell according to any one of embodiments 59-61, wherein the nucleotide sequence encoding one or more of the shRNAs comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-219 and 238-267.

[0177] 63. The immune cell of any one of embodiments 59-62, wherein the nucleotide sequence encoding one or more of the shRNAs is present on a vector.

[0178] 64. The immune cell of any one of embodiments 63, wherein the expression of different shRNAs is regulated by different promoters.

[0179] 65. The immune cell of embodiment 64, wherein the expression of two different shRNAs is regulated by two different promoters.

[0180] 66. The immune cell of embodiment 65, wherein the two different promoters are RNA polymerase III promoters.

[0181] 67. The immune cell of embodiment 66, wherein the two promoters are U6 promoters.

[0182] 68. The immune cell of embodiment 67, wherein the U6 promoter is derived from a different species.

[0183] 69. The immune cell of any one of embodiments 65-68, wherein the two promoters are oriented in different directions relative to each other.

[0184] 70. The immune cell of any one of embodiments 21-69, wherein the genetically engineered antigen receptor and one or more of the gene disrupting agents are each expressed by a vector.

[0185] 71. The immune cell of embodiment 70, wherein the genetically engineered antigen receptor and one or more of the gene disrupting agents are expressed by the same vector.

[0186] 72. The immune cell of any one of embodiments 70-71, wherein the vector is a plasmid vector or a viral vector.

[0187] 73. The immune cell of embodiment 72, wherein the viral vector is a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0188] 74. The immune cell of embodiment 73, wherein the lentiviral vector is a retroviral vector.

[0189] 75. The immune cell of any one of embodiments 21-74, wherein the immune cell is selected from the group consisting of T cells and natural killer (NK) cells.

[0190] 76. The immune cell of embodiment 75, wherein the immune cell is a T cell.

[0191] 77. The immune cell of embodiment 76, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

[0192] 78. The immune cell of any one of embodiments 76 or 77, wherein the immune cell comprises a nucleotide sequence encoding two shRNAs and a CAR or mTCR on the same vector.

[0193] 79. The immune cell of embodiment 78, wherein the two shRNAs are each regulated by two different RNA polymerase III promoters oriented in different directions from each other.

[0194] 80. The immune cell of embodiment 79, wherein the CAR targets CD19, the first shRNA targets PD-1, and the second shRNA targets TIGIT.

[0195] 81. A method for producing immune cells, comprising simultaneously or sequentially in any order introducing into the immune cells:

[0196] (1) a gene encoding a genetically engineered antigen receptor that specifically binds to a target antigen; and

[0197] (2) a gene disrupting agent, wherein the gene disrupting agent or its expression reduces or is capable of reducing the expression of a gene that weakens the function of an immune cell in an immune cell,

[0198] Thereby, immune cells are generated in which the genetically engineered antigen receptors are expressed and the expression of genes that impair immune cell function is reduced.

[0199] 82. The method of embodiment 81, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0200] 83. The method of embodiment 82, wherein the genetically engineered antigen receptor is CAR.

[0201] 84. The method of embodiment 83, wherein the CAR comprises an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain.

[0202] 85. The method of embodiment 84, wherein the extracellular antigen recognition domain of the CAR specifically binds to the target antigen.

[0203] 86. The method of embodiment 84, wherein the intracellular signaling domain of the CAR comprises the intracellular domain of the CD3 zeta (CD3ζ) chain.

[0204] 87. The method of embodiment 86, wherein the intracellular signal transduction domain of the CAR also comprises a co-stimulatory molecule.

[0205] 88. The method of embodiment 87, wherein the co-stimulatory molecule is selected from the group consisting of: ICOS, 0X40, CD137 (4-1BB), CD27 and CD28.

[0206] 89. The method of embodiment 88, wherein the co-stimulatory molecule is CD137 (4-1BB).

[0207] 90. The method of embodiment 88, wherein the co-stimulatory molecule is CD28.

[0208] 91. The method of embodiment 82, wherein the genetically engineered antigen receptor is a TCR.

[0209] 92. The method of embodiment 91, wherein the TCR is a monoclonal TCR (mTCR).

[0210] 93. The method of any one of embodiments 81-92, wherein the target antigen is expressed in or on the surface of cancer cells, cancer tissues and / or tumor microenvironments.

[0211] 94. The method of embodiment 93, wherein the target antigen is selected from the group consisting of:

[0212] 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (alpha-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α-methylacyl-CoA 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), CA 15-3 / CA 27-29 (mucin 1), CA 19-9 (cancer antigen 19-9), CA 72-4 (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 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel attachment 2), CML28 (chronic myeloid leukemia tumor antigen 28), trichometin, 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 B1), DAM-6 / MAGE-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 protein inhibitory complex 2 subunit enhancer), FGF-5 (fibroblast growth 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, GDEP (differentially expressed genes in prostate), GnT-V (gluconokinase), gp100 (melanocyte lineage-specific antigen GP100), GPC3 (glypican 3), HAGE (helical antigen), HAST-2 (sulfotransferase family 1A member 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K-MEL, HNE (medullin), homeobox NKX 3.1, 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, KIAA0205 (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, MAG E-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 family L2), Mammaglobin A, MART-1 / Melan-A (melanoma antigen recognized by T cells-1), MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony stimulating factor), mesothelin, MG50 / PXDN (peroxiredoxin), MMP11 (matrix metalloproteinase 11), MN / CA1X-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 (new gene expressed in prostate), NMP22 (nuclear matrix protein 22), NPM / ALK (nuclear phosphokinin), 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 (endoplasmic reticulum lectin), osteocalcin, osteopontin, p15 (CDK inhibitor 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PART-1 (prostatic 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 interacting 1), POTE (expressed in prostate, ovary, testis and placenta), PRAME (antigen preferentially expressed in melanoma), prostaglandin, proteinase-3, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate specific G protein-coupled receptor), PSM, PSMA (prostate specific membrane antigen), RAGE-1 (renal tumor cancer antigen), RHAMM / CD168, RU 1 (renal ubiquitin 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-213, TA-90 (tumor associated antigen-90), TAG-72 (tumor associated glycoprotein-72), TARP (TCR gamma alternative reading frame protein), TGFb (transforming growth factor beta), TGFbR11 (transforming growth factor beta receptor 11), TGM-4 (transglutaminase 4), TRAG-3 (paclitaxel resistance-associated gene 3), TRG (T cell receptor gamma locus), TRP-1 (transient receptor potential-1), TRP-2 / 6b, TRP-2 / INT2, Trp-p8, tyrosinase, UPA (U-type plasminogen activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK-1, and WT1 (Nephroblastoma 1).

[0213] 95. The method of embodiment 95, wherein the target antigen is CD19 or CD22.

[0214] 96. The method of embodiment 96, wherein the target antigen is CD19.

[0215] 97. The method of any one of embodiments 94-96, wherein the target antigen is a cancer antigen, wherein the cancer antigen is an antigen with increased expression in or on the surface of cancer cells, cancer tissues and / or tumor microenvironments.

[0216] 98. The method of embodiment 93, wherein the target antigen is selected from the group consisting of:

[0217] α-actinin-4 / m, ARTC1 / m, bcr / abl, β-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-A2 / m, HSP70-2M, KI AA0205 / 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, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, TEL-AML1, TGFbRII, and TPI / m; and

[0218] Wherein the target antigen is a cancer antigen, wherein the cancer antigen is a mutated form of an antigen expressed in or on the surface of cancer cells, cancer tissues and / or tumor microenvironment.

[0219] 99. The method of any one of embodiments 81-98, wherein the expression of the gene that impairs immune cell function results in one or more of the following:

[0220] i) Inhibit the proliferation of immune cells;

[0221] ii) inducing cell death of immune cells;

[0222] iii) inhibiting the ability of immune cells to recognize target antigens and / or become activated;

[0223] iv) inducing immune cells to differentiate into cells that do not induce an immune response to the target antigen;

[0224] v) reducing the reactivity of immune cells to molecules that promote an immune response in immune cells; or

[0225] vi) Increase the response of immune cells to molecules that inhibit the immune response of immune cells.

[0226] 100. The method of embodiment 99, wherein the gene that weakens the function of immune cells is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGKα, DGKζ, Cb1-b, Cb1-c, CD148, LRR1, TGFBR1, IL10RA, KLGR1, DNMT3A and A2aR.

[0227] 101. The method of embodiment 99, wherein the gene that impairs immune cell function increases the response of the immune cell to a molecule that inhibits the immune response of the immune cell.

[0228] 102. The method of embodiment 101, wherein the gene that increases the response of immune cells to molecules that inhibit the immune response of immune cells encodes an immune checkpoint receptor or ligand.

[0229] 103. The method of embodiment 102, wherein the immune checkpoint receptor or ligand is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4.

[0230] 104. The method of any one of embodiments 81-103, wherein the gene disrupting agent reduces the expression of a gene that impairs immune cell function in an immune cell by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to an immune cell in the absence of the one or more gene disrupting agents.

[0231] 105. The method of embodiment 104, wherein the gene disrupting agent reduces the expression of a gene that increases the response of an immune cell to a molecule that inhibits an immune response of the immune cell.

[0232] 106. The method of embodiment 105, wherein the gene disrupting agent reduces the expression of a gene encoding an immune checkpoint receptor or ligand.

[0233] 107. The method of embodiment 106, wherein the gene disrupting agent reduces the expression of a gene selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4.

[0234] 108. The method of any one of embodiments 105-107, wherein the gene disrupting agent reduces the expression of a gene that impairs immune cell function by RNA interference (RNAi).

[0235] 109. The method of embodiment 108, wherein more than one gene disrupting agent reduces the expression of genes that impair immune cell function in immune cells through RNAi.

[0236] 110. The method of embodiment 109, wherein the gene disrupting agent targets a single gene that impairs immune cell function, or targets different genes that impair immune cell function, wherein a first gene disrupting agent targets a first gene and a second gene disrupting agent targets a second gene, or any combination thereof.

[0237] 111. The method according to any one of embodiments 108-110, wherein the RNAi is mediated by short hairpin RNA (shRNA).

[0238] 112. The method of embodiment 111, wherein the RNAi is mediated by more than one shRNA.

[0239] 113. The method of embodiment 112, wherein the RNAi is mediated by two shRNAs.

[0240] 114. The method of embodiment 112 or 113, wherein both shRNAs target PD-1.

[0241] 115. The method of embodiment 112 or 113, wherein the first shRNA targets PD-1 and the second shRNA targets TIM-3.

[0242] 116. The method of embodiment 112 or 113, wherein the first shRNA targets PD-1 and the second shRNA targets CTLA-4.

[0243] 117. The method of embodiment 112 or 113, wherein the first shRNA targets PD-1 and the second shRNA targets LAG-3.

[0244] 118. The method of embodiment 112 or 113, wherein the first shRNA targets PD-1 and the second shRNA targets TIGIT.

[0245] 119. The method of any one of embodiments 111-118, wherein the immune cell comprises a nucleotide sequence encoding a shRNA.

[0246] 120. The method of embodiment 119, wherein the immune cell comprises a nucleotide sequence encoding more than one shRNA.

[0247] 121. The method of embodiment 119, wherein the immune cell comprises a nucleotide sequence encoding two shRNAs.

[0248] 122. The method of any one of embodiments 119-121, wherein the nucleotide sequence encoding one or more of the shRNAs comprises a sequence selected from the group consisting of SEQ ID NOs: 2-219 and 238-267.

[0249] 123. The method of any one of embodiments 119-122, wherein the nucleotide sequence encoding the shRNA is present on a vector.

[0250] 124. The method according to any one of embodiments 123, wherein the expression of different shRNAs is regulated by different promoters, respectively.

[0251] 125. The method of embodiment 124, wherein the expression of two different shRNAs is regulated by two different promoters, respectively.

[0252] 126. The method of embodiment 125, wherein the two different promoters are RNA polymerase III promoters.

[0253] 127. The method of embodiment 126, wherein the two promoters are U6 promoters.

[0254] 128. The method of embodiment 127, wherein the U6 promoter is derived from a different species.

[0255] 129. The method of any one of embodiments 125-128, wherein the two promoters are oriented in different directions relative to each other.

[0256] 130. The method of any one of embodiments 81-129, wherein the genetically engineered antigen receptor and one or more of the gene disrupting agents are each expressed by a vector.

[0257] 131. The method of embodiment 130, wherein the genetically engineered antigen receptor and one or more of the gene disrupting agents are expressed by the same vector.

[0258] 132. The method according to any one of embodiments 130-131, wherein the vector is a plasmid vector or a viral vector.

[0259] 133. The method of embodiment 132, wherein the viral vector is a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0260] 134. The method of embodiment 133, wherein the lentiviral vector is a retroviral vector.

[0261] 135. The method of any one of embodiments 81-134, wherein the immune cells are selected from the group consisting of T cells and natural killer (NK) cells.

[0262] 136. The method of embodiment 135, wherein the immune cell is a T cell.

[0263] 137. The method of embodiment 136, wherein the T cells are CD4+ T cells or CD8+ T cells.

[0264] 138. The method of embodiment 136 or 137, wherein the immune cell comprises a nucleotide sequence encoding two shRNAs and a CAR on the same vector.

[0265] 139. The method of embodiment 138, wherein the two shRNAs are each regulated by two different RNA polymerase III promoters oriented in different directions relative to each other.

[0266] 140. The method of embodiment 139, wherein the CAR targets CD19, the first shRNA targets PD-1, and the second shRNA targets TIGIT.

[0267] 141. A composition comprising the immune cell of any one of embodiments 21-80.

[0268] 142. A pharmaceutical composition comprising the immune cell of any one of embodiments 21-80 and a pharmaceutically acceptable carrier.

[0269] 143. A method of treatment comprising administering the immune cell of any one of embodiments 21-80 or the composition of embodiment 141 or 142 to a subject having a disease or condition in need of immunotherapy.

[0270] 144. The method of embodiment 143, wherein the genetically engineered antigen receptor specifically binds to an antigen, and the antigen is associated with the disease or disorder.

[0271] 145. The method of embodiment 143 or 144, wherein the disease or the disorder is cancer, e.g., a tumor.

[0272] 146. The immune cell of any one of embodiments 21-80 or the composition of embodiments 141-142, for use in treating a disease or disorder.

[0273] 147. Use of the immune cell of any one of embodiments 21-80 or the composition of embodiments 121-122 in the manufacture of a medicament for treating a disease or disorder.

[0274] 148. The immune cell or composition of embodiment 146 or the use of embodiment 147, wherein the genetically engineered antigen receptor specifically binds to an antigen associated with the disease or disorder.

[0275] 149. The use, composition or immune cell of embodiment 147 or embodiment 148, wherein the disease or disorder is cancer, e.g., a tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0276] Figures 1A-1E .Cell-intrinsic PD-1 production blocks CAR-T cells. ( Figure 1A ) Schematic diagram of the two-in-one CAR vector. Figure 1B -C) Analysis of LNGFR and CAR expression on day 4 after transduction. ( Figure 1D ) CAR-T cells were sorted by using LNGFR magnetic beads and 2×10 5 / ml inoculated with the cells. Cumulative CAR T cell counts were assessed by trypan blue staining. Figure 1E ) LNGFR+CAR T cells were mixed with γ-ray-irradiated NALM-6 in the absence of exogenous cytokines.

[0277] Figure 2 .The effect of Pol III promoter type on cell-intrinsic PD-1 blockade.

[0278] Figure 3A-3B .In vitro cytotoxicity and proliferation upon PD-L1 stimulation with CD19 and PD-1 blockade. Figure 3A )LNGFR+CAR T cells were mixed with live NALM-6 or NALM-6-PDL1 at an E:T ratio of 1:1, 0.3:1, or 0.1:1. Figure 3B ) LNGFR+CAR T cells were mixed with γ-ray-irradiated NALM-6-PDL1, NALM-6-PDL1-CD80, or K562-CD19-PDL1 at an E:T ratio of 1:1 in the absence of exogenous cytokines.

[0279] Figure 4 . Antitumor effects of CAR-T cells with cell-intrinsic PD-1 blockade in vivo.

[0280] Figure 5A .Reduced in vivo cytokine production upon cell-intrinsic PD-1 disruption by CAR-T cells. Figure 5B Delayed in vivo expansion of CAR-T cells with cell-intrinsic PD-1 disruption was demonstrated.

[0281] Figure 6A-6B .Function of CD28 / CD3ζ or 4-1BB / CD3ζ CAR-T cells in cell-intrinsic PD-1 destruction. ( Fig. 6A ) Schematic diagram of G28z, GBBz, P28z and PBBz vectors. Figure 6B ) Flow cytometric analysis showing LNGFR expression in transduced T cells 4 days after transduction.

[0282] Figure 7A-7B .PD-1 expression levels when co-stimulated with CD28 or 4-1BB. ( Fig. 7A )LNGFR+CAR T cells were incubated with γ-irradiated NALM-6 or K562-CD19 in the absence of exogenous cytokines. On day 3 after incubation, PD-1 expression of LNGFR+CART cells was analyzed. ( Figure 7B ) Quantitative real-time PCR results using PD-1 primers.

[0283] Figures 8A-8B .Establishment of NFAT or NF-κB reporter system. ( Fig. 8A ) Schematic diagram of NFAT-RE 3x-eGFP and NF-κB-RE 5x-eGFR receptor reporter vectors. ( Figure 8B ) Fold change of reporter activity calculated using eGFP gMFI of LNGFR+CAR T cells.

[0284] Figures 9A-9B .Activated NFAT signaling in CD28 co-stimulation, but not in 4-1BB co-stimulation. ( Fig. 9A ) Reporter-transduced T cells were restimulated and transduced with G28z or GBBz. Fig. 9B ) The mRNA levels of NFAT target genes in CART cells were evaluated by qPCR.

[0285] Fig.10 .CD28 and 4-1BB co-stimulate activated NF-κB signaling in both.

[0286] Figures 11A-11C.The TGF-β signaling intensity of G28z CART is slightly higher than that of BBz CART. ( Fig.11A ) Phosphorylated SMAD2 / 3 in CAR-T cells was analyzed by intracellular flow cytometry after incubation with NALM-6 at a 1:1 E:T cell ratio for 4 or 24 hours. ( Fig. 11B ) Flow cytometry analysis showed PD-1 expression in G28z and GBBz CAR T cells in the presence of 10 ng / ml of recombinant human TGF-β1. ( Fig. 11C ) The mRNA levels of TGF-β1, TGFBR1, and TGFBR2 in CAR T cells were evaluated by qPCR.

[0287] Figures 12A-12B .PBBz CAR T cells maintained cytotoxicity and proliferation capacity in vitro under repeated CD19 and PD-L1 stimulation. ( Fig. 12A ) Cytotoxicity. Top panel, after LNGFR magnetic sorting, 12-day primary LNGFR+CAR T cells were mixed with NALM-6-PDL1 at an E:T ratio of 1:1, 0.3:1, and 0.1:1. Bottom panel, LNGFR+CART cells were stimulated with γ-ray-irradiated K562-CD19-PDL1 at an E:T ratio of 1:1. ( Fig. 12B ) In the absence of exogenous cytokines, LNGFR+CAR T cells were repeatedly stimulated by mixing with γ-irradiated NALM-6-PDL1-CD80 or K562-CD19-PDL1 at an E:T ratio of 1:1.

[0288] Figures 13A-13C . were less sensitive to TGF-β-mediated dysfunction and had low levels of CAR-derived regulatory T cells generated in vitro in PBBz CAR T cells. ( Fig.13A )TGF-β-mediated inhibition of cell proliferation. Fig. 13B )Targeting Treg-induced TGF-β sensitivity. Fig. 13C )The effect of PDL 1 / PD-1 on Treg induction.

[0289] Fig.14 .Sustained inhibition of leukemia progression by PBBz CAR T cells.

[0290] Fig.15 .Composition diagram of two types of vectors encoding two types of shRNA (one of which inhibits the expression of PD-1 and the second inhibits the expression of TIM-3) and the CD19 CAR expression cassette.

[0291] Fig.16.Diagram of the CAR-T cell preparation process, in which ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6 cells and ΔLNGFR-CART19 / shTIM-3-mU6←→hU6-shPD-1 cells are prepared and separated as described in this article.

[0292] Fig.17 .From contains Fig.15 Flow cytometry data of CAR-T cells expressing the indicated vectors (ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6 cells and ΔLNGFR-CART19 / shTIM-3-mU6→←hU6-shPD-1 cells).

[0293] Figures 18A-18B . Fig.18A . Flow cytometry data of ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6 cells and ΔLNGFR-CART19 / shTIM-3-mU6→←hU6-shPD-1 cells generated using the method in Example 8. Fig.18B .Expression of PD-1 and TIM-3 in CAR-T cells.

[0294] Fig.19 .Flow cytometry data, in which CAR-T cells were repeatedly stimulated with target cells, followed by confirmation of cell differentiation using CD45RA and CCR7 antibodies.

[0295] Figures 20A-20C .Evaluation of CAR-T cells produced using the method in Example 8. Fig. 20A : Transduction efficiency. Fig. 20B : Proliferation ability. Fig. 20C : Survival ability.

[0296] Figures 21A-21C . Selection of shRNA targeting CTLA-4, LAG-3, TIGIT and TIM-3. Fig.21A : 2-day CD3 / CD28 stimulated T cells were electroporated with 21-mer siRNA targeting CTLA-4, LAG-3, TIGIT and TIM-3. siRNA-mediated knockdown efficiency was confirmed on day 2 after transfection. Shaded area: Based on the sequence of the selected siRNA, a two-in-one vector expressing CAR and shRNA was constructed. The shaded area indicates the initially selected siRNA. Fig. 21B : T cells were transduced with a two-in-one vector containing CTLA-4, LAG-3, TIGIT, or TIM-3 shRNA and sorted using LNGFR magnetic beads. 5After inoculation with 100 μg / ml, LNGFR+CAR T cell counts were measured every 3 days. Fig. 21C : Tim3 expression (%).

[0297] Figures 22A-22E .Generation of CART cells with dual immune checkpoint disruption. Fig.22A ) Schematic diagram of the dual two-in-one vector. Fig. 22B : Analysis of LNGFR+ T cells % on day 4 after dual 2-in-1 transduction. ( Fig. 22C ) double KD (knockdown) CAR-T cells were sorted and 2×10 5 / ml for inoculation. The cumulative CAR T cell count was determined by trypan blue staining. Figures 22D-22E ) LNGFR+CAR T cells were analyzed for LAG-4, PD-1, TIGIT, or TIM-3 expression 3 days after co-culture with γ-ray-irradiated NALM-6 or K562-CD19. The expression of CTLA-4 was analyzed by intracellular flow cytometry.

[0298] Fig.23 .In vivo treatment of CD19+ blood cancers using dual KD CAR-T cells targeting two immune checkpoints.

[0299] Fig.24 .Use of dual KD CAR-T cells targeting PD-1 and TIGIT to treat cancer in solid tumor models. DETAILED DESCRIPTION

[0300] The features of the present disclosure are specifically set forth in the appended claims. A better understanding of the features and benefits of the present disclosure will be obtained by reference to the following detailed description of illustrative embodiments, in which the principles of the present disclosure are utilized. In order to facilitate a comprehensive understanding of the disclosure set forth herein, some terms are defined below.

[0301] Briefly, in one aspect, disclosed herein is a vector comprising: a base sequence encoding two types of short hairpin RNAs (shRNAs) that inhibit the expression of one or more genes that weaken immune cell function (including immune checkpoint receptors and ligands), and a base sequence encoding an antigen receptor such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR) (e.g., a monoclonal T cell receptor (mTCR)); an immune cell comprising a genetically engineered antigen receptor that specifically binds to a target antigen and one or more gene disruptors that reduce or are capable of reducing the expression of one or more genes that weaken immune cell function in the immune cell; a method for producing the immune cell; a composition or pharmaceutical composition comprising the immune cell, for example, for immunotherapy of a human patient; and a treatment method comprising administering the immune cell to a subject suffering from a disease or disorder. Since the immune cells, compositions or pharmaceutical compositions contain one or more gene disrupting agents, for example, encoding two shRNAs that reduce the expression of two immune checkpoint molecule genes that can be activated by cancer cells to weaken the function of immune cells, it is possible to eliminate serious systemic adverse reactions (such as cytokine release syndrome or autoimmune symptoms) that may be caused by the use of individual inhibitors of these genes, as well as to alleviate the burden of increased treatment costs due to expensive concurrent treatments, while providing more effective cell therapy than when only one shRNA is expressed.

[0302] 1. General Technology

[0303] The techniques and procedures described or referenced herein include those commonly understood and / or commonly used by those skilled in the art using conventional methods, such as the widely used methods described in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition 2012); Current Protocols in Molecular Biology (Ausubel et al., ed., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010) and Antibody Engineering Volumes 1 and 2 (Kontermann and Dübel, eds., 2nd edition, 2010 Molecular Biology of the Cell (6th edition, 2014).

[0304] 2. Definition

[0305] Unless otherwise described, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. For the purpose of interpreting this specification, the following description of the terms will apply, and whenever appropriate, terms used in the singular will also include the plural, and vice versa. All patents, applications, published applications, and other publications are incorporated herein by reference in their entirety. If any description of the terms conflicts with any document incorporated herein by reference, the description of the terms described below shall prevail.

[0306] The terms used in this disclosure are only used to explain specific embodiments, and are not intended to limit the scope of the present invention. Unless otherwise clearly indicated in the context, singular expressions include plural expressions. It should be understood that the present invention is not limited to specific methods, schemes and reagents described herein, etc., and therefore can be changed. The terms used herein are only to describe specific embodiments, and are not intended to limit the scope of the present invention, and the scope of the present invention is only limited by the claims.

[0307] As used herein, the articles "a", "an", and "the" are used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, "an element" means one element or more than one element.

[0308] The use of alternatives (eg, "or") should be understood to mean one, two, or any combination of the alternatives.

[0309] The term "and / or" should be understood to mean one or both of the two options.

[0310] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% with respect to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0311] Throughout the specification, references to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "another embodiment," or "another embodiment," or combinations thereof, mean that a particular characteristic, structure, or feature described in conjunction with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the aforementioned phrases in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, in one or more embodiments, the particular characteristics, structures, or features may be combined in any suitable manner.

[0312] "Construct" refers to a macromolecule or molecular complex comprising a polynucleotide to be delivered to a target cell in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct capable of delivering or transferring exogenous genetic material to a target cell, where the exogenous genetic material can be replicated and / or expressed. As used herein, the term "vector" includes a construct to be delivered. A vector can be a linear or circular molecule. A vector can be integrated or non-integrated. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

[0313] The "two-in-one vector" described herein is a vector comprising a base sequence of a short hairpin RNA (shRNA) encoding one or more genes expressing one or more genes that inhibit the weakening of immune cell function, and a base sequence encoding a chimeric antigen receptor (CAR) or a T cell receptor (e.g., a monoclonal T cell receptor (mTCR)). The "double two-in-one vector" described herein is a vector comprising a base sequence of a short hairpin RNA (shRNA) encoding two types of genes expressing the inhibition of weakening immune cell function, and a base sequence encoding any one of a chimeric antigen receptor (CAR) and a T cell receptor (e.g., a monoclonal T cell receptor (mTCR)). The double two-in-one vector described herein is a form of a two-in-one vector.

[0314] "RNAi" (also known as post-transcriptional gene silencing (PTGS), quelling, or co-suppression) is a post-transcriptional gene silencing process in which RNA molecules inhibit gene expression in a sequence-specific manner (usually by destroying specific mRNA molecules). The active component of RNAi is a short / small double-stranded RNA (dsRNA), called a small interfering RNA (siRNA), which typically contains 15-30 nucleotides (e.g., 19-25, 19-24, or 19-21 nucleotides) and a 2 nucleotide 3' overhang, and matches the nucleic acid sequence of the target gene. These short RNA species can be naturally produced in vivo by Dicer-mediated cleavage of larger dsRNAs, and they are functional in mammalian cells. DNA expression plasmids can be used to stably express the siRNA duplexes or dsRNAs of the present invention in cells and achieve long-term inhibition of target gene expression. On the one hand, the sense and antisense strands of the siRNA duplexes are typically connected by a short spacer sequence, resulting in the expression of a stem-loop structure known as a short hairpin RNA (shRNA). The hairpin is recognized and cleaved by Dicer, thereby producing a mature siRNA molecule.

[0315] The term "shRNA" refers to an RNA molecule in which some of the self-complementary sequences produce a dense hairpin structure with its stem. The RNA molecule can have a length of about 80bp. When shRNA is expressed in a cell, it is processed into small interfering RNA (siRNA) through a series of steps for guiding gene silencing. In short, when shRNA is expressed, it is processed into pre-shRNA by the Drosha complex in the cell, then transported outside the nucleus, where it is further processed into siRNA by Dicer, then single-stranded and loaded by RISC (RNA-induced silencing complex) complex. Here, the antisense strand of siRNA acts as a guide that the RISC complex combines with the mRNA of the target gene, and when the RISC complex combined in this way cuts mRNA, gene silencing occurs. Since the shRNA in the target gene allows for continuous and specific gene silencing of a specific gene, in order to inhibit the target gene, it is included in the vector.

[0316] The term "promoter" refers to the upstream region of a gene that participates in the start of gene transcription. The above two types of shRNA also cause promoter-regulated expression. Here, the expression characteristics of the two types of shRNA may be that they are regulated by two different promoters, respectively. If repeated inserts are used for cloning with the same base sequence, it is very likely that correct cloning cannot be performed due to the binding between these same base sequences, resulting in recombination or deletion. The promoter may be an RNA polymerase I promoter, an RNA polymerase II promoter, or an RNA polymerase III promoter, depending on which RNA polymerase binds to the promoter and starts transcription. The above two promoters are characterized in that they are RNA polymerase III promoters (hereinafter referred to as pol III promoters). The pol III promoter can be accurately transcribed from the 5' end to the 3' end without connecting a cap at the 5' end of the RNA or connecting a poly (A) tail at the 3' end, and the RNA is transcribed by the promoter regulation. The types of Pol III promoters include, but are not limited to, U6 promoters, H1 promoters, and 7SK promoters, etc.

[0317] As used herein, the term "G28z" refers to a construct comprising a shGFP expression cassette, a CD28 co-stimulatory domain, and a CD3 zeta domain ( Fig. 6A ). More specifically, in the term "G28z", "G" represents shGFP; "28" represents CD28; and "z" represents CD3ζ. Following the same pattern, as used herein, the term "P28z" refers to a construct comprising a shPD-1 expression cassette, a CD28 costimulatory domain, and a CD3ζ domain ( Fig. 6A ), wherein "P" represents shPD-1, "28" represents "CD28", and "z" represents CD3ζ. As used herein, the term "GBBz" refers to a construct comprising an shGFP expression cassette, a 4-1BB costimulatory domain, and a CD3ζ domain ( Fig. 6A ), wherein "G" represents shGFP; "BB" represents 4-1BB; "z" represents CD3ζ. As used herein, the term "PBBz" refers to a construct comprising an shPD-1 expression cassette, a 4-1BB costimulatory domain, and a CD3ζ domain ( Fig. 6A ), where "P" represents shPD-1; "BB" represents 4-1BB; and "z" represents CD3ζ.

[0318] "CAR" is generally a group of polypeptides, which, when present on immune cells, cause immune cells to be specific to target cells (usually cancer cells), while causing signal transduction in cells. CAR includes at least an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain that recognize the following target antigens, wherein the intracellular signal transduction domain is derived from the following promotion molecules or costimulatory molecules. The group comprising polypeptides can be connected, or they can be in the form of being connected by a switch, which is dimerized by stimulation. The promotion molecule can be the ζ chain of the above-mentioned TCR. "CD19 CAR" is a CAR targeting CD19 cancer antigen.

[0319] As used herein, the term "T cell receptor (TCR)" refers to a protein receptor on T cells composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells, the TCR is composed of gamma and delta (γ / δ) chains. In certain embodiments, the TCR can be modified on any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0320] As used herein, the term "monoclonal T cell receptor (mTCR)" refers to a T cell receptor (TCR) that is genetically modified to specifically target a specific antigen. It may also be referred to as antigen-specific TCR. It is reported that T cells with mTCR effects are used for immunotherapy of viral infection and cancer, such as adoptive T cell therapy. In some aspects, retroviral transfer of chimeric single-chain antibody constructs (scFv) has been used as a strategy for producing T cells with determined antigen specificity. In most cases, chimeric single-chain antibody constructs are connected to the intracellular signaling domain of FcR-γ or CD3ζ to trigger T cell effector functions. CD3ζ domains have been combined with signaling domains of costimulatory molecules such as CD28, 4-1BB or OX40. Monoclonal T cell receptors (mTCRs) and their use in cancer therapy are described in Stauss et al., 2007, Molecular Therapy, 15(10): 1744-50, Zhang and Morgan, 2012, Advanced 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.

[0321] As used herein, the term "ΔLNGFR" refers to LNGFR (low affinity nerve growth factor receptor) without the cytoplasmic domain used to purify cells in which such insertion has occurred.

[0322] "Immune cells" herein may be characterized as being selected from, but not limited to, lymphocytes, such as killer T cells, helper T cells, γδ T cells and B cells, natural killer cells, mast cells, eosinophils, basophils; phagocytic cells include macrophages, neutrophils and dendritic cells. T cells include CD4+ T cells and CD8+ T cells.

[0323] As used herein, the terms "T lymphocytes" and "T cells" are used interchangeably and refer to the main types of leukocytes that complete maturation in the thymus and have various roles in the immune system (including the recognition of specific exogenous antigens in the body and the activation and inactivation of other immune cells). T cells can be any T cells, such as cultured T cells, such as primary T cells, or T cells from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells obtained from mammals. T cells can be CD3+ cells. T cells can be any type of T cells and can be at any stage of development, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor infiltrating lymphocytes (TIL), memory T cells, naive T cells, regulatory T cells, γδT cells (γδT cells), etc. Other types of helper T cells include Th3 (Treg), Th17, Th9 or Tfh cells. Other types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells), etc. T cells may also refer to genetically engineered T cells, such as T cells modified to express T cell receptors (TCR) or chimeric antigen receptors (CAR). T cells may also be differentiated from stem cells or progenitor cells.

[0324] "CD4+T cells" refer to a T cell subset that expresses CD4 on its surface and is associated with a cell-mediated immune response. They are characterized by a secretory profile after stimulation, which may include the secretion of cytokines such as IFN-γ, TNF-α, IL2, IL4, and IL10. "CD4" is a 55-kD glycoprotein that was originally defined as a differentiation antigen on T lymphocytes, but is also present in other cells including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin epigenetic family and is considered to be a joint recognition element in the MHC (major histocompatibility complex) class II restricted immune response. On T lymphocytes, they define a subset of helper cells / inducer cells.

[0325] "CD8+ T cells" refers to a subpopulation of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is a joint recognition element in the major histocompatibility complex class I restricted interaction.

[0326] As used herein, the term "NK cell" or "natural killer cell" refers to a peripheral blood lymphocyte subpopulation defined by the expression of CD56 or CD16 and the absence of a T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and refer to a subpopulation of NK cells with a phenotype of CD3- and CD56+, which express at least one of NKG2C and CD57, and optionally CD16, but lack the expression of one or more of the following: PLZF, SYK, FceRγ and EAT-2. In some embodiments, the isolated subpopulation of CD56+NK cells includes the expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activation and inhibitory KIR, NKG2A and / or DNAM-1. CD56+ can be dim or bright expression.

[0327] As used herein, the term "immune checkpoint" refers to molecules that are present in the immune system and are able to turn the immune response on or off. Originally, they were safety devices that regulate over-activation of immune cells, which can lead to cell death or autoimmune responses. These immune checkpoint molecules can be broadly divided into stimulatory immune checkpoint molecules that increase the immune response and inhibitory immune checkpoint molecules that inhibit the immune response. For example, immune checkpoint receptors and ligands can be selected from the group consisting of: 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), and PD-L1 (programmed death ligand 1). containing-3)), CEACAM (carcinoembryonic antigen-related cell adhesion molecule, including three subtypes CEACAM-1, CEACAM-3 or CEACAM-5), LAG3 (lymphocyte activation gene 3), VISTA (V-domain Ig inhibitor of T cell activation), BTLA (B lymphocyte and T lymphocyte attenuator), 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), and can be selected, for example, between PD1 and TIM3.

[0328] The terms "culturing" or "cell culture" refer to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Cell culture medium," "culture medium" (in each case the singular "culture medium"), "supplements" and "culture medium supplements" refer to the nutrient components of a cultured cell culture. The terms "culturing" or "maintaining" refer to the maintenance, propagation (growth) and / or differentiation of cells outside of a tissue or body, such as in a sterile plastic (or (coated plastic) cell culture dish or flask. "Culturing" or "maintaining" may utilize a culture medium as a source of nutrients, hormones and / or other factors that aid in the propagation and / or maintenance of the cells.

[0329] The "pharmaceutical composition" described herein for immunotherapy of human patients comprises immune cells. As is self-evident, in addition to cells, other pharmaceutically acceptable salts, carriers, excipients, vehicles and other additives that can further enhance the immune response may be added to the pharmaceutical composition, so a detailed explanation thereof will be omitted.

[0330] The term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein to refer to a human subject.

[0331] The term "treating" or "treat" refers to inhibiting, eliminating, alleviating and / or ameliorating the symptoms, severity of symptoms and / or frequency of symptoms of the disease being treated. As used herein, the terms "treat", "treatment" and "treating" also refer to a reduction or improvement in the progression, severity and / or duration of a disease or disorder resulting from the administration of one or more therapies.

[0332] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material or composition (such as a T cell) as described herein that is effective to achieve a specific biological result. Such results may include, but are not limited to, cancer inhibition as determined by any suitable method in the art.

[0333] "Administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body into a patient's body, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0334] 3. Two-in-one vectors targeting one or more immune checkpoints

[0335] Tumor cells express various immune checkpoints, for example, checkpoint ligands. Therefore, even if one immune checkpoint is inhibited, it may be difficult to expect to obtain a sustained effect of CAR-T by activating other immune checkpoints. Combinations of monoclonal antibodies are mainly used to inhibit multiple immune checkpoints, and their anti-tumor effects are constantly reported (J Clin Invest., 2015, Chauvin JM; PNAS, 2010, Curran MA; Blood, 2018, Wierz M; Cancer cell. 2014, Johnston RJ). However, it is well known that therapeutic antibodies can induce systemic excessive immune responses. In addition, CAR-T therapy is also associated with life-threatening cytokine release syndrome (CRS) and neurotoxicity (Nat Rev Clin Oncol, 2017, Neelapu SS), which suggests that the combination of CAR-T therapy and antibody therapy can maximize the potential for side effects. In addition, conventional parallel immune cell therapy imposes a greater economic burden on patients due to its high cost, and they also act on T cells other than CAR-T, and bring the risk of autoimmune symptoms and cytokine release syndrome. The present invention is made to solve the above-mentioned problems.

[0336] In one embodiment, provided herein is a two-in-one vector comprising: a base sequence encoding one or more types of short hairpin RNA (shRNA) that inhibits the expression of genes that weaken the function of immune cells, and a base sequence encoding a chimeric antigen receptor (CAR) and a T cell receptor (such as a monoclonal T cell receptor (mTCR)).

[0337] The vector can be selected from DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector. For example, lentiviral vector and retroviral vector can insert the gene into the genomic DNA of the cell, so that the gene is stably expressed. In some embodiments, for example, a two-in-one lentiviral vector, for example, a dual two-in-one vector, can be used to introduce the gene on the vector into the cell genome.

[0338] In some embodiments, a vector is provided that contains a base sequence encoding two types of short hairpin RNA (shRNA) that inhibits the expression of genes that weaken the function of immune cells, and a base sequence encoding any one of a chimeric antigen receptor (CAR) and a T cell receptor (e.g., a monoclonal T cell receptor (mTCR)).

[0339] In some embodiments, the expression of two types of shRNA is characterized in that they are regulated by two different promoters, respectively. In some embodiments, the two promoters are RNA polymerase III promoters. In some embodiments, the two promoters are U6 promoters derived from different species. In some embodiments, the two promoters are oriented in different directions on the vector. For example, in a certain embodiment, the promoter is oriented head to head. In another embodiment, the promoter is oriented tail to tail. In some embodiments, the gene that weakens the function of immune cells is an immune checkpoint receptor or ligand.

[0340] In some embodiments, the immune checkpoint receptor or ligand is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4. In some embodiments, the gene that weakens the function of immune cells is selected from the group consisting of: FAS, CD45, PP2A, SHIP1, SHIP2, DGKα, DGKζ, Cb1-b, CD147, LRR1, TGFBR1, IL10Rα, KLGR1, DNMT3A and A2aR.

[0341] In some embodiments, the two types of shRNAs either target different parts of a single gene that weakens immune cell function, or target different genes that weaken immune cell function. In some embodiments, the two types of shRNAs target different parts of PD-1. In some embodiments, the two types of shRNAs target PD-1 and TIM-3, respectively. In some embodiments, the base sequences encoding the two types of shRNAs include different sequences selected from the group consisting of SEQ ID NO: 2-219.

[0342] In some embodiments, the target of the CAR or TCR (e.g., mTCR) is a human tumor antigen selected from a cancer antigen that is increased in cancer or a mutated form of a cancer antigen found in cancer.

[0343] In some embodiments, the vector comprises any one of the base sequences SEQ ID NO: 220 or 221. In some embodiments, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, and retroviral vector.

[0344] 3.1 RNA interference and short hairpin RNA

[0345] RNAi (also known as post-transcriptional gene silencing (PTGS), quelling or co-suppression) is a post-transcriptional gene silencing process in which RNA molecules inhibit gene expression (usually by destroying specific mRNA molecules) in a sequence-specific manner. The active component of RNAi is a short / small double-stranded RNA (dsRNA), called small interfering RNA (siRNA), which usually contains 15-30 nucleotides (e.g., 19-25, 19-24 or 19-21 nucleotides) and 2 nucleotide 3' overhangs, and matches the nucleic acid sequence of the target gene. These short RNA species can be naturally produced in vivo by the cleavage of larger dsRNA mediated by Dicer, and they are functional in mammalian cells. DNA expression plasmids can be used to stably express siRNA duplexes or dsRNAs described herein in cells and achieve long-term inhibition of target gene expression. On the one hand, the sense and antisense strands of the siRNA duplex are usually connected by a short spacer sequence, resulting in the expression of a stem-loop structure known as short hairpin RNA (shRNA). The hairpin is recognized and cut by Dicer to produce a mature siRNA molecule.

[0346] As used herein, short hairpin RNA (shRNA) is an RNA molecule in which some of the self-complementary sequences produce a tight hairpin structure with its stem. The length of the shRNA molecules described herein can be about 40 to 120 nucleotides long, for example, about 70 to 90 nucleotides in length. In an exemplary embodiment, the length of the shRNA can be 80 nucleotides. shRNA is modeled after microinterfering RNA (miRNA), an endogenous trigger of the RNAi pathway (Lu et al., 2005, Advances in Genetics 54: 117-142, Fewell et al., 2006, Drug Discovery Today 11: 975-982). When shRNA is expressed in a cell, it is processed into small interfering RNA (siRNA) through a series of steps for guiding gene silencing. In brief, when shRNA is expressed, it is processed into pre-shRNA by the Drosha complex in the cell and then transported out of the nucleus, where it is further processed into siRNA by Dicer, then single-stranded and loaded by RISC (RNA-induced silencing complex). Here, the antisense strand of siRNA acts as a guide for the RISC complex to bind to the mRNA of the target gene, and when the RISC complex bound in this way cuts the mRNA, gene silencing occurs. Since shRNA in the target gene allows for sustained and specific gene silencing of a specific gene, it is included in the vector in order to inhibit the target gene.

[0347] Naturally expressed small RNA molecules, called microRNAs (miRNAs), trigger gene silencing by regulating the expression of mRNAs. RISC containing miRNAs targets mRNAs that show perfect sequence complementarity with nucleotides 2-7 in the 5' region of the miRNA, which are called seed regions, and other base pairs in its 3' region. MiRNA-mediated downregulation of gene expression can be caused by the breakage of the target mRNA, the translation inhibition of the target mRNA, or mRNA attenuation. The miRNA targeting sequence is usually located in the 3'UTR of the target mRNA. A single miRNA can target more than 100 transcripts from different genes, and one mRNA can be targeted by different miRNAs.

[0348] siRNA duplexes or dsRNA targeting specific mRNA can be designed and synthesized in vitro and introduced into cells to activate the RNAi process. Elbashir et al. demonstrated that 21-nucleotide siRNA duplexes (called small interfering RNA) can achieve potent and specific gene knockdown in mammalian cells without inducing immune response (Elbashir SM et al., Nature, 2001, 411, 494-498). Since the initial report, post-transcriptional gene silencing caused by siRNA has rapidly become a powerful tool for mammalian cell gene analysis and is likely to produce novel therapeutic agents.

[0349] In U.S. Pat. Nos. 9,169,483 and 9,181,544 and International Patent Publication No. WO2015179525, each of which is incorporated herein by reference in its entirety, RNAi molecules designed to target nucleic acid sequences encoding polyglutamine repeat proteins that cause poly-glutamine expansion diseases such as Huntington's disease are described. U.S. Pat. Nos. 9,169,483 and 9,181,544 and International Patent Publication No. WO2015179525 each provide an isolated RNA duplex comprising a first RNA strand (e.g., 15 consecutive nucleotides) and a second RNA strand (e.g., complementary to at least 12 consecutive nucleotides of the first strand), wherein the RNA duplex is about 15 to 30 base pairs in length. The first RNA and the second RNA strand can be operably linked by an RNA loop (about 4 to 50 nucleotides) to form a hairpin structure, which can be inserted into an expression cassette. Non-limiting examples of loop portions include SEQ ID NOs: 9-14 of U.S. Pat. No. 9,169,483 (the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of RNA chains that can be used (whether in full or partial sequence) to form RNA duplexes include SEQ ID NOs: 1-8 of U.S. Pat. No. 9,169,483 and SEQ ID NOs: 1-11, 33-59, 208-210, 213-215 and 218-221 of U.S. Pat. No. 9,181,544, the contents of each of which are incorporated herein by reference in their entirety. Non-limiting examples of RNAi molecules include SEQ ID NOs: 1-8 of U.S. Pat. No. 9,169,483, SEQ ID NOs: 1-11, 33-59, 208-210, 213-215 and 218-221 of U.S. Pat. No. 9,181,544, and SEQ ID NOs: 1, 6, 7 and 35-38 of International Patent Publication No. WO2015179525, the contents of each of which are incorporated herein by reference in their entirety.

[0350] In vitro synthesized siRNA molecules can be introduced into cells to activate RNAi. When exogenous siRNA duplexes are introduced into cells, similar to endogenous dsRNA, they can be assembled to form RNA-induced silencing complexes (RISC), a multi-unit complex that interacts with an RNA sequence complementary to one of the two chains of the siRNA duplexes (i.e., antisense strand). In the process, the sense strand (or passenger strand) of siRNA is lost from the complex, and the antisense strand (or guide strand) of siRNA is matched with its complementary RNA. In particular, the target of the RISC complex comprising siRNA is an mRNA presenting perfect sequence complementarity. Then, the gene silencing mediated by siRNA occurs by cutting, releasing and degrading the target.

[0351] Compared with the use of single-stranded (ss)-siRNA (e.g., antisense RNA or antisense oligonucleotides), siRNA duplexes consisting of a sense strand homologous to the target mRNA and an antisense strand complementary to the target mRNA have greater advantages in terms of target RNA destruction efficiency. In many cases, it requires a higher concentration of β-siRNA to achieve effective gene silencing efficacy of the corresponding duplex.

[0352] There are guiding principles for designing siRNA in the prior art. These guiding principles generally suggest producing a double-stranded region of 19 nucleotides targeting the region in the gene to be silenced, a symmetrical 3' overhang of 2-3 nucleotides, a 5'-phosphate and a 3'-hydroxyl. Other rules that may govern siRNA sequence preferences include, but are not limited to, (i) A / U at the 5' end of the antisense strand; (ii) G / C at the 5' end of the sense strand; (iii) at least five A / U residues at the 5' end of the antisense strand; and (iv) the absence of any GC segment longer than 9 nucleotides. Based on this consideration, in combination with the specific sequence of the target gene, it is easy to design efficient siRNA molecules necessary for inhibiting the expression of mammalian target genes.

[0353] As provided herein, a two-in-one vector includes a base sequence encoding one or more types of short hairpin RNA (shRNA) that inhibits the expression of one or more genes that weaken the function of immune cells, and a base sequence encoding any one of a chimeric antigen receptor (CAR) or a T cell receptor (e.g., a monoclonal T cell receptor (mTCR)).

[0354] In some embodiments, the base sequence encodes one type of shRNA that inhibits the expression of genes that weaken immune cell function. In some embodiments, the base sequence encodes two types of shRNA that inhibit the expression of two genes that weaken immune cell function, wherein the vector may be referred to as a "dual two-in-one vector". In other embodiments, the base sequence encodes more than two shRNAs that inhibit the expression of more than two genes that weaken immune cell function.

[0355] In some embodiments, two or more types of shRNA are characterized in that they target a single gene that weakens the function of immune cells, such as different parts of a single gene. For example, two or more types of shRNA can target PD-1, such as different parts of PD-1. In other embodiments, two or more types of shRNA may be characterized in that they target different genes that weaken the function of immune cells, such as targeting PD-1 and TIM-3.

[0356] In an exemplary embodiment, the base sequences encoding two or more types of shRNA are characterized in that they comprise different sequences selected from SEQ ID NOs 2 to 219 and 238 to 267, for example, they may comprise different sequences 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 the group consisting of SEQ ID NOs 2 to 12, 70 to 75 and 266 to 267.

[0357] In some embodiments, the expression of the two types of shRNAs is characterized in that they are regulated by two different promoters, respectively, to minimize recombination or deletion artifacts during the cloning process.

[0358] In some embodiments, the promoter can be an RNA polymerase I promoter, an RNA polymerase II promoter, or an RNA polymerase III promoter, depending on which RNA polymerase binds to the promoter and begins transcription. The above two promoters may be characterized in that they are RNA polymerase III promoters (hereinafter referred to as pol III promoters). The pol III promoter can be accurately transcribed from the 5' end to the 3' end without connecting a cap at the 5' end of the RNA or connecting a poly (A) tail at the 3' end, and the RNA is regulated and transcribed by the promoter. The types of pol III promoters include, but are not limited to, U6 promoters, H1 promoters, and 7SK promoters, etc. The two promoters contained in the vector may be different and may be selected from pol III promoters including the above three types. If promoters of the same type are selected, they may be from different species. For example, the two promoters may be U6 promoters, for example, U6 promoters from different species, such as U6 promoters from humans and mice. Since the transcripts produced by the U6 promoter are retained in the nucleus, it is judged that this will be able to cause the Drosha complex present in the nucleus to promote the process of shRNA processing into pre-shRNA.

[0359] In some embodiments, two or more promoters may be characterized in that they are oriented in a different direction on the vector than other promoters. For example, in a certain embodiment, the promoters are oriented in a head-to-head (→←) orientation. In another embodiment, the promoters are oriented in a tail-to-tail (←→) orientation. In a dual two-in-one vector, being oriented in different directions on the vector means that when the corresponding shRNA whose expression is regulated by the two promoters is transcribed, the direction in which the RNA polymerase moves is oriented in different directions on a single nucleic acid molecule. In an exemplary embodiment, the two promoters may be in a →← direction ( Fig.15 A). In another exemplary embodiment, the two promoters may be in the ←→ direction ( Fig.15 B) For example, the two promoters can be in a →← orientation on the vector.

[0360] In some embodiments, the expression of the target gene of one of the multiple types of shRNAs is reduced to about 90% or less of the control group, for example, the expression of the target gene is reduced to about 80% or less, about 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 of the control group.

[0361] Typically, shRNA is designed to have a sequence with high homology to a portion of the mRNA sequence of its target gene (hereinafter referred to as a sense shRNA base sequence), a sequence capable of producing a sharp hairpin, and a sequence complementary to a sequence with high homology (hereinafter referred to as an antisense shRNA base sequence). The non-covalent bonds between the self-complementary parts form a stem cell structure, and when the shRNA is expressed and processed in the cell, the antisense shRNA base sequence acts as a guide for the mRNA of the target gene during gene silencing. For example, the base sequence of the box used for shRNA expression herein may include a NNNNNNNNNNNNNNNNNNNNNNN (21 bases)-loop sequence-NNNNNNNNNNNNNNNNNNNNNN (19 bases) structure. In one embodiment, a segment of 21 bases encodes a sense shRNA base sequence, and a segment of 19 bases is complementary or substantially complementary to a segment of 21 bases, and encodes an antisense shRNA base sequence. In another embodiment, the 19-base segment encodes a sense shRNA base sequence, and the 21-base segment is complementary or substantially complementary to the 19-base segment and encodes an antisense shRNA base sequence. Thus, when expressed, the resulting RNA forms a stem and loop structure. In certain embodiments, such sense or antisense shRNA base sequences of target genes (of human origin) that may be included in the cassette are selected from the group consisting of SEQ ID NOs: 1-219. In a specific embodiment, the base sequence of the cassette for shRNA expression herein may be selected from the group consisting of SEQ ID NOs: 220-224.

[0362] In some embodiments, the entire shRNA base sequence may be located at the 3' end of the mouse or human U6 promoter, and TTTTT necessary for terminating transcription of the U6 promoter may be located at the 3' end of all shRNA base sequences.

[0363] In some embodiments, in addition to the sequences described herein, the nucleic acid sequences of the corresponding shRNA may also include nucleic acid sequences that show at least 50%, particularly at least 70%, more particularly at least 80%, even more particularly at least 90%, and most particularly at least 95% sequence homology with these sequences. This is because, in the case of siRNA (small interfering RNA), especially shRNA processed into siRNA in cells, it is reported that a certain degree of mutation, especially mutation at the 5' end is tolerated, resulting in normal knockdown of the target gene, and mutations that make siRNA and shRNA have a structure similar to that of miRNA that plays a role in gene silencing are more effective in inducing knockdown of the target gene. In addition, in the use of the vector, it is self-evident to those skilled in the art that variants within the vector, that is, additions, modifications or deletions of base sequences that may occur during the cloning process of introducing a specific sequence into the vector, or changes to components or introduction of components (to improve the ease of use of the vector and achieve the desired degree of gene expression).

[0364] Genes that weaken immune cell function have a variety of modes of action. Examples include inhibiting the proliferation of immune cells or causing cell death, reducing responses to molecules that immune cells need to react with to be activated, inhibiting the expression of genes necessary for immune cells to recognize targets of response, and causing differentiation into different types of immune cells to perform different functions rather than causing an immune response to a specific target. Representative examples include, but are not limited to, molecules associated with immune checkpoints, which will be explained below.

[0365] In some embodiments, the gene that weakens the function of immune cells may be characterized in that it is an immune checkpoint receptor or ligand. Immune checkpoints are molecules present in the immune system that can turn on or off immune responses. They can be considered as safety devices that regulate excessive activation of immune cells, which can lead to cell death or autoimmune responses. These immune checkpoint molecules can be broadly divided into stimulatory immune checkpoint molecules that increase immune responses and inhibitory immune checkpoint molecules that inhibit immune responses. It is reported that many cancer cells escape the immune system by activating inhibitory immune checkpoint signals, especially inhibitory immune checkpoint receptors and ligands on immune cells. Therefore, immune cell therapies targeting specific cancers can become effective by invalidating this escape effect of cancer, and this can be achieved by inhibiting the activation of inhibitory immune checkpoint receptors and their ligands or by reducing their expression. For example, the immune checkpoint receptors and ligands can be selected from the group consisting of: 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-3), containing-3)), CEACAM (carcinoembryonic antigen-related cell adhesion molecule, including three subtypes CEACAM-1, CEACAM-3 or CEACAM-5), LAG3 (lymphocyte activation gene 3), VISTA (V-domain Ig inhibitor of T cell activation), BTLA (B lymphocyte and T lymphocyte attenuator), 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), and can be selected, for example, between PD1, TIM3 and TIGIT.

[0366] In other embodiments, the gene that weakens the function of immune cells may be characterized in that its encoding can promote AICD (activation-induced cell death), acting as a receptor for a negative regulator of T lymphocytes, which has been activated by repeated stimulation of TCR such as FAS (also known as CD95, APO-1 or apoptosis antigen-1). In some embodiments, the gene that weakens the function of immune cells may be characterized in that its encoding inhibits the signal activation of TCR, for example, the factor may be selected from CD45, PP2A, SHP1, SHP2, DGKα, DGKζ, Cb1-b, Cb1-c and CD148. In some embodiments, the gene that weakens the function of immune cells may be characterized in that its encoding inhibits CAR and / or TCR (e.g., mTCR inhibited by 4-1BB signals) The protein (e.g., LRR1 (leucine-rich repeat protein 1)) of the efficacy, wherein 4-1BB is a co-stimulatory molecule of TCR described herein. In some embodiments, the gene that weakens the function of immune cells may be characterized in that it encodes a receptor that inhibits T cells (whose ligand is a cytokine), such as TGFBR1 (transforming growth factor beta receptor 1) and IL10Rα (IL10R subunit α). In some embodiments, the gene that weakens the function of immune cells may be characterized in that it encodes a receptor that inhibits the proliferation ability and cytotoxicity of T cells and NK cells, such as KLGR1 (killer cell lectin-like receptor G1). In some embodiments, the gene that weakens the function of immune cells may be characterized in that it encodes a regulatory factor associated with the methylation of new DNA (the regulatory factor is reported to inhibit the exhaustion of T cells when it is knocked out (KO)), such as TNMT3a (DNA methyltransferase 3α). In some embodiments, the gene that weakens the function of immune cells may be characterized in that it encodes an adenosine receptor that is over-existing in the tumor microenvironment and can inhibit the cytotoxicity and cytokine production ability of T cells when activated, such as A2aR (adenosine receptor subtype A2a).

[0367] In some embodiments, the gene that impairs immune cell function may be characterized in that it is selected from the group consisting of: FAS, CD45, PP2A, SHP1, SHP2, DGKα, DGKζ, Cb1-b, Cb1-c, CD148, LRR1, TGFBR1, IL10RA, KLGR1, DNMT3A and A2aR.

[0368] 3.2 Chimeric antigen receptors (CARs) and T cell receptors, such as monoclonal T cell receptors (mTCRs).

[0369] As provided herein, a two-in-one vector includes a base sequence encoding one or more types of short hairpin RNA (shRNA) that inhibits the expression of one or more genes that weaken the function of immune cells, and a base sequence encoding any one of a chimeric antigen receptor (CAR) or a T cell receptor (e.g., a monoclonal T cell receptor (mTCR)).

[0370] CAR is usually a group of polypeptides, which, when present on immune cells, cause the immune cells to be specific to target cells (usually cancer cells) and simultaneously cause signal transduction in the cells. CAR comprises at least an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain that recognize the following target antigens, wherein the intracellular signal transduction domain is derived from a promoting molecule or a costimulatory molecule.

[0371] The structure of CAR commonly used in clinical applications currently includes a single-chain variable fragment domain (hereinafter referred to as scFv) specific to the antigen, a spacer domain that regulates the distance between scFv and the cell membrane, a transmembrane domain, and an intracellular signaling domain (hereinafter referred to as ISD). ISD also contains a co-stimulatory domain (CD28, CD137 or OX40) that contributes to the proliferation and longevity of one or more T cells in vivo, and a TCR signaling domain (CD3zeta, CD3ζ) that contributes to T cell activation. T cells modified to express CAR prepared in this way can be activated by recognizing cancer cells that express target antigens with high specificity, effectively inducing the death of such cancer cells, while exponentially proliferating in vivo, and surviving for a long time. For example, when CAR-T cells (CART-19) prepared to target CD19 (a B cell-specific antigen) are administered to patients with B cell leukemia, it is reported that the cells proliferate to 1,000 to 10,000 times and survive in vivo for several years. Therefore, in clinical trials of patients with advanced acute lymphoblastic leukemia (B-ALL) who were refractory to conventional chemotherapy, CART-19 showed a 90% complete response, which led to a rare case of licensing to global pharmaceutical companies at the early stage of investigator-initiated clinical trials. In 2017, it became the first CAR-T cell therapy approved by the US FDA, and since then, the second CAR-T has also been approved.

[0372] On the surface of immune cells such as T cells, there are immune checkpoint receptors, such as CTLA-4 (cytotoxic T lymphocyte-associated protein-4) or PD-1 (programmed cell death protein-1). These receptors were originally safety devices that regulated T cell overactivation and cell death, or autoimmune response triggering. However, it is reported that cancer cells, especially solid cancers, use it to avoid immune surveillance of T cells. For example, if cancer cells express PD-L1 (programmed death ligand 1) on the surface, T cells expressing PD-1 and its receptor recognize cancer cells and are activated, but will soon be exhausted by activation inhibition signals from PD-1. In order to prevent the inhibition of T cell activity by signals from these immune checkpoint receptors, monoclonal antibodies against CTLA4 or PD-1 that inhibit signal transmission by targeting immune checkpoint receptors have been developed. Therapies that improve the overall immune function of T cells by blocking immune checkpoints using these immune checkpoint receptor inhibitors have also shown efficacy against various solid cancers.

[0373] Since CAR-T cells are ultimately a therapy that relies on the cytotoxicity of activated T cells, the presence of an immunosuppressive environment around CAR-T cells is a major obstacle to their therapeutic effect. In fact, unlike their therapeutic effects in B-cell leukemia, CAR-T prepared for solid tumors rarely show promising therapeutic effects. This is believed to be because solid tumors, unlike blood cancers, produce an immunosuppressive tumor microenvironment to inhibit the activity and proliferation of CAR-T cells. In addition, even in B-cell blood cancers, it has been reported that, unlike almost 90% of acute lymphoblastic leukemia (ALL) patients who respond to therapy with CART-19, the therapeutic effect is relatively low in lymphoma patients (20-50% response) or chronic lymphoblastic leukemia patients (CLL, about 20% response).

[0374] In addition, it is reported that PD-L1 and other immunosuppressive ligands are expressed in the tumor microenvironment formed by lymphoma, according to which the function of T cells in cancer tissues is exhausted. In addition, it is reported that T cells obtained from CLL patients have been substantially exhausted, in which immune checkpoint receptors such as PD-1, CD160 and CD244 are highly expressed.

[0375] Preclinical trial results reportedly show that the simultaneous use of anti-CTLA or anti-PD-1 inhibitory antibodies and CAR-T cells to restore this reduced CAR-T cell activity can improve the anti-cancer effect, and clinical trials using these combinations are currently underway. However, the problem with the parallel treatment of such antibodies and CAR-T cells is that the antibodies distributed throughout the body affect not only CAR-T cells, but also all other T cells present in the body, potentially leading to serious systemic adverse reactions such as cytokine release syndrome and autoimmune symptoms. Another problem that has been pointed out is the increased cost of treatment due to the simultaneous use of expensive antibody therapy and cell therapy.

[0376] Therefore, recently, attempts have been made to regulate gene expression in cells to allow inhibition of immune checkpoints of CAR-T cells. International patent application publication WO2016 / 069282 discloses a composition and method for producing modified T cells, wherein the modified T cells have a nucleic acid capable of downregulating the expression of endogenous genes (selected from a group consisting of TCRα chain, TCRβ chain, β-2 microglobulin and FAS), and the nucleic acid also comprises a nucleic acid encoding a modified T cell receptor (TCR) (which has affinity for surface antigens on target cells) or an electroporated nucleic acid encoding a chimeric antigen receptor (CAR). The disclosure points out that gene scissors such as CRISPR / Cas9 can be used to knock out the expression of endogenous genes, but the method for preparing CAR-T cells disclosed in the patent disclosure is quite complicated and has problems of low yield and high production cost.

[0377] At the same time, International Patent Publication WO2015 / 090230 discloses a single type of short hairpin RNA (shRNA) that inhibits molecules that inhibit T cell function in addition, which can be used for cells expressing CAR. Due to the high cost burden of cell therapy, patients face various burdens when they fail. However, a single type of shRNA may not effectively inhibit the activity of such target molecules. In some embodiments, a group comprising a polypeptide may be attached. In some embodiments, a group comprising a polypeptide may be in a form in which they are combined by a switch, and the switch is dimerized by stimulation. In some embodiments, CAR may be a fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain. In another embodiment, the CAR fusion protein may further include a leader sequence at the N-terminus, and the leader sequence may be cut off during the process in which CAR is expressed and anchored in the cell membrane.

[0378] TCR described herein, such as mTCR, may include a chain selected from α, β, γ and δ chains. In some embodiments, the chain is capable of recognizing the following target antigens, CD3 and ζ chains, and other costimulatory molecules, wherein the costimulatory molecules can be selected from ICOS, OX40, CD137 (4-1BB), CD27 or CD28.

[0379] In some embodiments, the retroviral transfer of chimeric single-chain antibody constructs (scFv) can be used to produce TCR, such as mTCR with determined antigen specificity. In other embodiments, chimeric scFv constructs can be connected to the intracellular signaling domain of FcR-γ or CD3ζ to trigger T cell effector functions. In CD3ζ domains, it can be combined with the signaling domain of costimulatory molecules, wherein antibody binding can trigger effector T cell functions, and costimulatory signals are also transmitted. In other embodiments, costimulatory molecules can be selected from CD28, 4-1BB and OX40.

[0380] In some embodiments, the two-in-one vector may include a CD3 ζ domain, wherein the chain of a TCR (e.g., mTCR) can form a complex through non-covalent bonds with CD3 and zeta (ζ) chains. When an antigen is recognized through the antigen recognition site of the chain, the CD3 and ζ chains send signals to the cytoplasm of immune cells on which such a TCR complex is expressed, inducing functional activation.

[0381] In some embodiments, the intracellular signal transduction domain may further comprise one or more functional signal transduction domains derived from co-stimulatory molecules. In some embodiments, the promoting molecule may be the zeta chain of the TCR described above.

[0382] CAR and TCR (e.g., mTCR) are cell surface receptors. In some embodiments, the target of CAR or TCR (e.g., mTCR) may be characterized in that it is a cancer antigen whose expression in cancer specifically increases. In some embodiments, the target of CAR or TCR (e.g., mTCR) may be characterized in that it is a cancer antigen present in a mutated form in cancer.

[0383] In some embodiments, the target of a CAR or TCR (e.g., mTCR) can be a human tumor antigen whose expression is increased in the cancer to be treated. For example, the target can be selected from 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (alpha-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α-methylacyl-CoA racemase, ART-4 (ADP ribosyltransferase-4), B7H4 (T cell activation inhibitor 1 containing v-set domain), BAGE-1 (B melanoma antigen-1), BCL-2 (B cell CLL / lymphoma-2), BING-4 (WD repeat domain 46), CA 15-3 / CA 27-29 (mucin 1), CA 19-9 (cancer antigen 19-9), CA 72-4 (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 19), CD20, 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), hairy 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 B1), DAM-6 / MAGE-B2, EGFR / Her1 (epidermal growth factor receptor), EMMPRIN (basigin), EpCam, EphA2 (EPH receptor A2), EphA3, ErbB3 (Erb-B2 receptor tyrosine kinase 3), EZH2 (enhancer of zeste2 polycomb inhibitory complex 2 subunit), FGF-5 (fibroblast growth 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, GDEP (differentially expressed genes in prostate), GnT-V (gluconate kinase), gp100 (melanocyte lineage-specific antigen GP100), GPC3 (phosphatidylinositol proteoglycan 3), HAGE (helical antigen),HAST-2 (sulfotransferase family 1A member 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K-MEL, HNE (medullin), 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, KIAA0205 (lysophosphatidylglycerol acyltransferase 1), KK-LC-1 (Kitakyushu lung cancer antigen-1), KM-HN-1, LAGE-1 (L antigen family member-1), Livin, M AGE-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, MA GE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2 (melanoma antigen family L2), Mammaglobin A, MART-1 / Melan-A (melanoma antigen recognized by T cells-1), MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony stimulating factor), mesothelin, MG50 / PXDN (peroxiredoxin), MMP 11 (matrix metalloproteinase 11), MN / CA1X-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 (new gene expressed in prostate), NMP22 (nuclear matrix protein 22), NPM / ALK (nucleophosmin), NSE (neuron-specific enolase), NY-ESO-1, NY-ESO-B, OA1 (osteoarthritis QTL 1), OFA-iLRP (carcinoembryonic antigen immature laminin 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 inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PART-1 (prostatic 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 interacting 1), POTE (expressed in prostate, ovary, testis and placenta), PRAME (antigen preferentially expressed in melanoma), prostaglandin, proteinase-3, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate Specific G protein-coupled receptor), PSM, PSMA (prostate-specific membrane antigen), RAGE-1 (renal tumor antigen), RHAMM / CD168, RU1 (renal ubiquitin 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-213, TA-90 (tumor-associated antigen-90), TAG-72 (tumor-associated glycoprotein-72 ), TARP (TCRγ alternate reading frame protein), TGFb (transforming growth factor β), TGFbR11 (transforming growth factor β receptor 11), TGM-4 (transglutaminase 4), TRAG-3 (paclitaxel 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-type plasminogen activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK-1 and WT1 (Wilms tumor 1), or a mutated form of a human tumor antigen found in the cancer to be treated, which is selected from α-actinin-4 / m, ARTC1 / m, bcr / abl, β-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, ET V6-AML1, FN1 / m, GPNMB / m, HLA-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, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, TEL-AML1, TGFbRII, and TPI / m. For example, the target antigen can be selected between CD19 or CD22.

[0384] 3.3 Components of the two-in-one carrier

[0385] As provided herein, a two-in-one vector includes a base sequence encoding one or more types of short hairpin RNA (shRNA) that inhibits the expression of one or more genes that weaken the function of immune cells, and a base sequence encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) (e.g., a monoclonal T cell receptor (mTCR)).

[0386] In some embodiments, the two-in-one vector may include a sequence encoding a factor that promotes insertion of the sequence into the host cell genome. In some embodiments, the sequence is located at either end or both ends of the vector gene. In some embodiments, the sequence is an LTR (long terminal sequence).

[0387] In some embodiments, the two-in-one vector may include a domain encoding a protein for purification of cells in which the above insertion has occurred. In some embodiments, the domain is a ΔLNGFR domain, wherein ΔLNGFR is LNGFR (low affinity nerve growth factor receptor) without a cytoplasmic domain for purification of cells in which the above insertion has occurred.

[0388] In some embodiments, the two-in-one vector may comprise a promoter that induces expression of ΔLNGFR and CAR characterized by sustained expression, such as Fig.22A The EF1α promoter of the induction ΔLNGFR and CD19 CAR expression shown. In another embodiment, ΔLNGFR and CD19 CAR are first transcribed into a form in which they are present on a single mRNA. In such embodiments, two or more cistrons are present on the same mRNA, and IRES (internal ribosome entry site) can be used therebetween to cause the expression of two cistrons. However, IRES is too long, and it is reported that the expression efficiency of the downstream cistron is reduced. In some embodiments, components other than IRES (e.g., P2A (2a peptide)) can be used to overcome such disadvantages, wherein during translation, the ribosome passes through and does not form a peptide bond at the C-terminus of P2A, thereby allowing the downstream gene to be expressed later.

[0389] Some exemplary two-in-one vectors are FIG. 1 , FIG. 6 , Fig.14 and Fig.22A The two-in-one vector shown in , the vector includes any one of the base sequences SEQ ID 220 or 221. An exemplary vector containing the base sequences of SEQ ID 220 and 221 may have Figure 1A and 1B Table 1 lists some exemplary plasmids.

[0390] Table 1. Exemplary plasmids.

[0391]

[0392] In some embodiments, in addition to the sequences described herein, the base sequences contained in the above-mentioned vectors and the nucleic acid sequences of the corresponding shRNA may contain nucleic acid sequences showing at least 50%, particularly at least 70%, more particularly at least 80%, even more particularly at least 90%, and most particularly at least 95% sequence homology with these sequences. This is because, in the case of siRNA (small interfering RNA), especially shRNA processed into siRNA in cells, it is reported that a certain degree of mutation, especially mutation at the 5' end is tolerated, resulting in normal knockdown of the target gene, and mutations that make siRNA and shRNA have a structure similar to that of miRNA that plays a role in gene silencing more effectively induce knockdown of the target gene. In addition, in the use of the vector, it is self-evident to those skilled in the art that variants within the vector, that is, additions, modifications or deletions of base sequences that may occur during the cloning process of introducing a specific sequence into the vector, or changes to components or introduction of components (to improve the ease of use of the vector and achieve the desired degree of gene expression).

[0393] 4. Generation and evaluation of CAR-T cells targeting one or more immune checkpoints

[0394] As provided herein, a two-in-one vector comprises a base sequence encoding one or more types of short hairpin RNA (shRNA) that inhibits the expression of genes that weaken immune cell function, and a base sequence encoding any one of a chimeric antigen receptor (CAR) and a T cell receptor (TCR) (e.g., a cloned T cell receptor (mTCR)). The vector can be used to produce immune cells that inhibit the expression of genes that weaken immune cell function. In some embodiments, the vector is selected from the group consisting of: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, and retroviral vector.

[0395] On the one hand, the immune cells described herein are characterized in that they contain the above-mentioned vectors and express CAR or TCR, such as mTCR, and the expression of the target gene of one or more types of shRNA is reduced. In some embodiments, the expression of the target gene of one of the multiple types of shRNA is reduced to about 90% or less of the control group, such as the expression of the target gene is reduced to about 80% or less of the control group, about 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. In some embodiments, immune cells are selected from between T cells and NK cells of human origin.

[0396] In another aspect, methods are provided herein. In some embodiments, a method for producing an immune cell is provided, comprising simultaneously or sequentially in any order introducing into the immune cell: (1) a gene encoding a genetically engineered antigen receptor that specifically binds to a target antigen; and (2) a gene disrupting agent that reduces or is capable of reducing the expression of a gene that weakens the function of an immune cell in an immune cell, thereby producing an immune cell in which the genetically engineered antigen receptor is expressed and the expression of a gene that weakens the function of an immune cell is reduced.

[0397] In some embodiments, the genetically engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the genetically engineered antigen receptor is a CAR. In some embodiments, CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain.

[0398] In some embodiments, the extracellular antigen recognition domain of the CAR specifically binds to a target antigen.

[0399] In some embodiments, the intracellular signal transduction domain of CAR comprises the intracellular domain of CD3 zeta (CD3ζ) chain. In some embodiments, the intracellular signal transduction domain of CAR also comprises a costimulatory molecule.

[0400] In some embodiments, the costimulatory molecule is selected from the group consisting of ICOS, 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.

[0401] In some embodiments, the target antigen is expressed on the cell surface of cancer cells, cancer tissues and / or tumor microenvironments. In some embodiments, the target antigen is a cancer antigen or a mutant form of a cancer antigen that is expressed increased in cancer cells, cancer tissues and / or tumor microenvironments.

[0402] In some embodiments, the cancer antigen whose expression increases in cancer cells, cancer tissues and / or tumor microenvironments is selected from the group consisting of: 5T4 (trophoblast glycoprotein), 707-AP, 9D7, AFP (alpha-fetoprotein), AlbZIP (androgen-induced bZIP), HPG1 (human prostate-specific gene-1), α5β1-integrin, α5β6-integrin, α-methylacyl-CoA 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), CA 15-3 / CA27-29 (mucin 1), CA 19-9 (cancer antigen 19-9), CA 72-4 (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 19), CD20, CD22, CD25, CD30, CD33, CD4, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen SG8), CLCA2 (chloride channel attachment 2), CML28 (chronic myeloid leukemia tumor antigen 28), trichometin, 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 B1), DAM-6 / MAGE-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 protein inhibitory complex 2 subunit enhancer), FGF-5 (fibroblast growth 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, GDEP (differentially expressed genes in prostate), GnT-V (gluconokinase), gp100 (melanocyte lineage-specific antigen GP100), GPC3 (glypican 3), HAGE (helical antigen), HAST-2 (sulfotransferase family 1A member 1), hepsin, Her2 / neu / ErbB2 (Erb-B2 receptor tyrosine kinase 2), HERV-K-MEL, HNE (medullin), homeobox NKX 3.1, 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, KIAA0205 (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, MAG E-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 family L2), Mammaglobin A, MART-1 / Melan-A (melanoma antigen recognized by T cells-1), MART-2, matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF (macrophage colony stimulating factor), mesothelin, MG50 / PXDN (peroxiredoxin), MMP11 (matrix metalloproteinase 11), MN / CA1X-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 (new gene expressed in prostate), NMP22 (nuclear matrix protein 22), NPM / ALK (nuclear phosphokinin), 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 (endoplasmic reticulum lectin), osteocalcin, osteopontin, p15 (CDK inhibitor 2B), p53, PAGE-4 (P antigen family member-4), PAI-1 (plasminogen activator inhibitor-1), PAI-2, PAP (prostatic acid phosphatase), PART-1 (prostatic 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 interacting 1), POTE (expressed in prostate, ovary, testis and placenta), PRAME (antigen preferentially expressed in melanoma), prostaglandin, proteinase-3, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSGR (prostate specific G protein-coupled receptor), PSM, PSMA (prostate specific membrane antigen), RAGE-1 (renal tumor cancer antigen), RHAMM / CD168, RU 1 (renal ubiquitin 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-213, TA-90 (tumor associated antigen-90), TAG-72 (tumor associated glycoprotein-72), TARP (TCR gamma alternative reading frame protein), TGFb (transforming growth factor beta), TGFbR11 (transforming growth factor beta receptor 11), TGM-4 (transglutaminase 4), TRAG-3 (paclitaxel resistance-associated gene 3), TRG (T cell receptor gamma locus), TRP-1 (transient receptor potential-1), TRP-2 / 6b, TRP-2 / INT2, Trp-p8, tyrosinase, UPA (U-type plasminogen activator), VEGF (vascular endothelial growth factor A), VEGFR-2 / FLK-1, and WT1 (Nephroblastoma 1).

[0403] In some embodiments, the target antigen is CD19 or CD22. In some embodiments, the target antigen is CD19.

[0404] In some embodiments, the mutant form of the tumor antigen is selected from the group consisting of α-actinin-4 / m, ARTC1 / m, bcr / abl, β-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- 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, p53 / m, Pml / RARa, PRDX5 / m, PTPRX / m, RBAF600 / m, SIRT2 / m, SYTSSX-1, SYT-SSX-2, TEL-AML1, TGFbRII, and TPI / m

[0405] In some embodiments, expression of a gene that impairs immune cell function results in one or more of the following: i) inhibiting the proliferation of immune cells; ii) inducing cell death of immune cells; iii) inhibiting the function of immune cells to recognize target antigens and / or acquire molecules necessary for activation; iv) inducing immune cells to differentiate into different types that perform different functions rather than causing an immune response to the target antigen; v) reducing the response of immune cells to molecules that promote the immune response of immune cells; or vi) increasing the response of immune cells to molecules that inhibit the immune response of immune cells.

[0406] In some embodiments, the gene that weakens immune cell function is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK, 2B4, FAS, CD45, PP2A, SHP1, SHP2, DGKα, DGKζ, Cb1-b, Cb1-c, CD148, LRR1, TGFBR1, IL10RA, KLGR1, DNMT3A and A2aR. In some embodiments, the gene that weakens immune cell function increases the reaction of immune cells to molecules that inhibit the immune response of immune cells. In some embodiments, the gene encoding immune checkpoint receptor or ligand that increases the reaction of immune cells to molecules that inhibit the immune response of immune cells.

[0407] In some embodiments, the immune checkpoint receptor or ligand is selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG 3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4.

[0408] In some embodiments, compared with immune cells without gene disruptors, gene disruptors reduce the expression of genes that weaken immune cell function in immune cells by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In some embodiments, gene disruptors reduce the expression of genes that increase immune cells and molecules that inhibit the immune response of immune cells. In some embodiments, gene disruptors reduce the expression of genes encoding immune checkpoint receptors or ligands. In some embodiments, gene disruptors reduce the expression of genes selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3 or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, CD96, MerTK and 2B4.

[0409] In some embodiments, gene disruptors reduce the expression of genes that weaken immune cell function by RNA interference (RNAi). In some embodiments, more than one gene disruptors reduce the expression of genes that weaken immune cell function in immune cells by RNAi. In some embodiments, gene disruptors target different parts of a single gene that weakens immune cell function, target different genes that weaken immune cell function, or any combination thereof. In some embodiments, RNAi is mediated by short hairpin nuclear RNA (shRNA). In some embodiments, RNAi is mediated by more than one shRNA.

[0410] In some embodiments, RNAi is mediated by two shRNAs. In some embodiments, the two shRNAs target different parts of PD-1. In some embodiments, the two shRNAs target PD-1 and TIM-3, respectively. In some embodiments, 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.

[0411] In some embodiments, the base sequence encoding the shRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1-219 and 238-267.

[0412] In some embodiments, the expression of different shRNAs is regulated by different promoters. In some embodiments, the expression of two different shRNAs is regulated by two different promoters. In some embodiments, the two different promoters are RNA polymerase III promoters. In some embodiments, the two promoters are U6 promoters derived from different species. In some embodiments, the two promoters are oriented in different directions from each other.

[0413] In some embodiments, the genetically engineered antigen receptor and the gene disruptor are each expressed by a vector. In some embodiments, the genetically engineered antigen receptor and the gene disruptor are expressed by the same vector. In some embodiments, the vector is selected from the group consisting of: DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector. In some embodiments, the vector is a lentiviral vector.

[0414] Immune cells are characterized in that they are selected from lymphocytes, such as killer T cells, helper T cells, γδT cells and B cells, natural killer cells, mast cells, eosinophils, basophils; phagocytic cells include macrophages, neutrophils and dendritic cells. T cells include CD4+T cells and CD8+T cells. In some embodiments, B cell lymphoma is diffuse large B cell lymphoma (DLBCL), primary mediastinal B cell lymphoma (PMBL), Hodgkin lymphoma (HL), non-Hodgkin lymphoma, mediastinal gray zone lymphoma or nodular sclerosis HL. In some embodiments, T cell lymphoma is anaplastic large cell lymphoma (ALCL), peripheral T cell lymphoma unspecified type (PTCL-NOS) or angioimmunoblastic T cell lymphoma (AITL). In a preferred embodiment, immune cells can be selected from human-derived T cells or T lymphocytes and natural killer (NK) cells. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD4+ T cell or a CD8+ T cell.

[0415] In some embodiments, immune cells are produced by cells originally derived from a subject. In some embodiments, the subject may be a human. In some embodiments, the human may be a healthy donor. In other embodiments, the subject may be characterized as having a tumor or cancer in which an increase or change in the level of a cancer antigen targeted by a CAR or TCR (e.g., mTCR) expressed in a cell is detected. In some embodiments, cells may be generally generated and expanded using methods such as those described in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 and U.S. Patent Application Publication No. 20060121005.

[0416] In some embodiments, CAR-T cells are produced. In other embodiments, the production of CAR-T cells includes the step of providing peripheral blood monoclonal cells. In other embodiments, peripheral blood monoclonal cells can be separated from whole blood samples. In some embodiments, the generation of CAR-T cells described herein includes the step of stimulating peripheral blood monoclonal cells using antibodies. For example, the agent providing the primary stimulation signal is an anti-CD3 antibody or its antigen-binding fragment, and the agent providing the costimulatory signal is an anti-CD28 antibody or its antigen-binding fragment. In some embodiments, the generation of CAR-T cells described herein includes the step of transducing CAR, wherein CAR can target any target described herein and any other known target of CAR, for example, CAR can be a CD19 CAR targeting CD19. In other embodiments, the CAR-T cells produced are separated.

[0417] Conditions suitable for T cell culture include a suitable culture medium (e.g., minimal essential medium or RPMI medium 1640 or, X-vivo 15 (Lonza)), which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, interferon-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, human plasma protein powder (plasmanate) and reducing agents, such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, to which amino acids, sodium pyruvate and vitamins are added, or serum-free or supplemented with an appropriate amount of serum (or plasma) or a set of determined hormones, and / or a certain amount of one or more cytokines sufficient to grow and expand T cells. Antibiotics, such as penicillin and streptomycin, are only included in experimental cultures and not in cell cultures to be infused into subjects. The target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2). Several stimulation cycles may also be required so that the culture time of T cells can be 60 days or longer.

[0418] T cells exposed to different stimulation times can show different characteristics. For example, typical blood or apheresis peripheral blood mononuclear cell products have a helper T cell group (TH, CD4+) greater than cytotoxic or inhibitory T cell groups (TC, CD8+). The T cell group mainly composed of TH cells before about 8-9 days is produced by stimulating CD3 and CD28 receptors in vitro, and after about 8-9 days, the T cell group contains an increasingly large TC cell group. Therefore, according to the purpose of treatment, it can be advantageous to infuse a T cell group mainly composed of TH cells to a subject. Similarly, if an antigen-specific TC cell subset has been isolated, it can be beneficial to expand the subset to a greater extent. In addition, in addition to CD4 and CD8 markers, other phenotypic markers also have significant changes, but are reproducible to a large extent in the cell expansion process. Therefore, this reproducibility makes it possible to have the ability to customize activated T cell products for specific purposes.

[0419] Various assays can be used to evaluate CAR-T cells, such as, but not limited to, the ability to expand after antigen stimulation, the ability to maintain the expansion of T cells without restimulation, and the anti-cancer activity in appropriate in vitro and animal models. The assays will be described in further detail below.

[0420] In some embodiments, Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).

[0421] In some embodiments, the in vitro expansion of CAR+T after antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4+ and CD8+T cells is stimulated with αCD3 / αCD28 aAPC, and then transduced with a lentiviral vector expressing GFP under the control of the promoter to be analyzed. Exemplary promoters include CMV IE gene, EF-1a, ubiquitin C, or phosphoglycerol kinase (PGK) promoter. GFP fluorescence is assessed by flow cytometry in CD4+ and / or CD8+T cell subsets on the 6th day of culture. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464(2009). Alternatively, a mixture of CD4+ and CD8+T cells is stimulated with magnetic beads coated with αCD3 / αCD28 on day 0, and transduced with CAR on day 1 using a bicistronic lentiviral vector that expresses CAR and eGFP using a 2A ribosomal skipping sequence. After washing, the culture is re-stimulated with, for example, K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and anti-CD28 antibodies (K562-BBL-3 / 28). 100 IU / ml of exogenous IL-2 is added to the culture every other day. Green GFP+T cells are counted by flow cytometry using bead-based counting. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464(2009). CAR+T cell expansion that persists in the absence of restimulation can also be measured.

[0422] Assessment of cell proliferation and cytokine production has been previously described, for example, in Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, CAR-mediated proliferation was assessed in microtiter plates by mixing washed T cells with target cells such as 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. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies were added to cultures containing KT32-BBL cells and used as positive controls for stimulating T cell proliferation, as these signals support long-term CD8+ T cell ex vivo expansion. CountBright was used according to the manufacturer's instructions. TM Fluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry were used to count T cells in culture. Using T cells engineered with a lentiviral vector expressing a CAR connected to eGFP-2A, CAR+T cells were identified by GFP expression. The expression of CD4+ and CD8+ on T cells was also detected simultaneously with specific monoclonal antibodies (BD Biosciences). According to the manufacturer's instructions, human TH1 / TH2 cytokine cell counting bead array kit (BD Biosciences, SanDiego, CA) was used to measure cytokines in the supernatant collected 24 hours after re-stimulation. Fluorescence was evaluated using a FACScalibur flow cytometer, and the data were analyzed according to the manufacturer's instructions.

[0423] This can be done, for example, by the methods described herein in the Examples, or by standard 51 Cytotoxicity can be assessed using the Cr-release assay (Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, target cells (e.g., BHK or CHO cells) are loaded with 51Cr (e.g., NaCr04, New England Nuclear, Boston, MA) for 2 h at 37°C with frequent agitation, washed twice in complete RPMI, and plated into microtiter plates. Effector T cells are mixed with target cells in complete RPMI in wells at different effector cell:target cell (E:T) ratios. Additional wells containing only culture medium (spontaneous release, SR) or 1% triton-X100 detergent solution (total release, TR) are also prepared. After incubation at 37°C for 4 h, the supernatant of each well is collected. The released 51Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, MA).51 Each condition was performed at least three times, and the percentage of lysis was calculated using the following formula: % lysis = (ER-SR) / (TR-SR), where ER represents the average release of each experimental condition. 51 Cr. Alternative cytotoxicity assays, such as flow-based cytotoxicity assays, may also be used.

[0424] Other assays, including those described in the Examples section herein and known in the art, can also be used to evaluate the CAR-T cells generated herein.

[0425] In some embodiments, the immune cells produced herein may participate in an immune response to a disease in which an antigen targeted by two types of shRNA and CAR or TCR (e.g., mTCR) is expressed. In other embodiments, immune cells may be used to provide a pharmaceutical composition for immunotherapy for human patients. In some embodiments, the pharmaceutical composition may show a therapeutic effect on the target disease without the need for immune checkpoint inhibitors, which may cause serious adverse reactions and additionally bring a high cost burden to the patient.

[0426] In some embodiments, immune cells can be used as immune cell therapeutic agents; such immune cells are generally used to treat cancer, but are not limited thereto. In other embodiments, in order to make these immune cells recognize cancer, they are modified to express cell surface receptors targeting cancer antigens.

[0427] In another aspect, provided herein is a composition comprising the above-mentioned engineered immune cells.

[0428] 5. Therapy using CAR-T cells targeting one or more immune checkpoints

[0429] As provided herein, two-in-one vectors include base sequences of short hairpin RNA (shRNA) encoding one or more types of inhibition of gene expression that weakens immune cell function, and base sequences encoding any one of chimeric antigen receptor (CAR) and T cell receptor (TCR) (e.g., monoclonal T cell receptor (mTCR)). Using a vector according to one embodiment, immune cells can be produced, wherein the immune cells have reduced immune checkpoint receptor expression, and they express CAR or TCR, such as mTCR specific to a target molecule. The immune cells can be used to provide a pharmaceutical composition for immunotherapy.

[0430] By introducing the immune cells described herein into subjects suitable for adoptive immunotherapy, a variety of diseases can be improved. In some embodiments, the CAR-T cells provided are used for allogeneic adoptive cell therapy. Also provided herein are therapeutic uses of the compositions described herein, including introducing the compositions into subjects suitable for adoptive cell therapy, wherein the subject suffers from an autoimmune disease; a hematological malignancy; a solid tumor; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0431] Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymphomas, non-Hodgkin's lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndrome. Examples of solid cancers include, but are not limited to, brain cancer, prostate cancer, breast cancer, lung cancer, colon cancer, uterine cancer, skin cancer, liver cancer, bone cancer, pancreatic cancer, ovarian cancer, testicular cancer, bladder cancer, kidney cancer, head cancer, neck cancer, stomach cancer, cervical cancer, rectal cancer, laryngeal cancer, and esophageal cancer. Examples of various autoimmune diseases include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), certain forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, 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, Syndrome, systemic lupus erythematosus, certain forms of thyroiditis, certain forms of thyroiditis, certain forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease). Examples of viral infections include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (Kaposi's sarcoma-associated herpes virus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella zoster virus), adenovirus, lentivirus, BK polyoma virus-associated disorders.

[0432] Acute leukemia is characterized by the rapid proliferation of immature blood cells. This crowding makes it impossible for the bone marrow to produce healthy blood cells. Acute forms of leukemia can occur in children and young adults. In fact, it is the most common cause of death in American children than any other type of malignant disease. Acute leukemia requires immediate treatment because the malignant cells develop and accumulate rapidly, then spill into the bloodstream and spread to other organs of the body. Central nervous system (CNS) involvement is rare, although the disease occasionally causes cranial nerve palsy. Chronic leukemia is characterized by excessive accumulation of relatively mature but still abnormal blood cells. Usually taking months to years to develop, these cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Chronic leukemia mostly occurs in the elderly, but in theory it can occur in any age group. However, acute leukemia must be treated immediately, while chronic leukemia is sometimes monitored for a period of time before treatment to ensure the maximum effect of the treatment. In addition, the disease is classified as lymphocytic or lymphoblastic, which indicates that the cancer develops in a type of bone marrow cell that normally gives rise to lymphocytes, and myeloid or myeloid, which indicates that the cancer develops in a type of bone marrow cell that normally gives rise to red blood cells, certain types of white blood cells, and platelets (see Lymphoid vs. Myeloid).

[0433] Acute lymphocytic leukemia (also called acute lymphoblastic leukemia, or ALL) is the most common type of leukemia in young children. This disease also affects adults, especially those aged 65 and older. Chronic lymphocytic leukemia (CLL) most often affects adults over the age of 55. It sometimes occurs in younger people but almost never affects children. Acute myeloid leukemia (also called acute myeloid leukemia, or AML) is more common in adults than in children. This type of leukemia was previously called "acute nonlymphocytic leukemia" Chronic myeloid leukemia (CML) occurs mainly in adults. Very rarely, children can also develop this disease.

[0434] Lymphoma is a type of cancer that originates in lymphocytes (a type of white blood cell in the vertebrate immune system). There are many types of lymphoma. According to the National Institutes of Health, lymphoma accounts for about 5% of all cancer cases in the United States, and Hodgkin lymphoma in particular accounts for less than 1% of all cancer cases in the United States. Because the lymphatic system is part of the body's immune system, patients with weakened immune systems, such as those due to HIV infection or taking certain medications or medicines, also have a higher incidence of lymphoma.

[0435] In the 19th and 20th centuries, the disease was called Hodgkin's disease because it was discovered by Thomas Hodgkin in 1832. In layman's terms, lymphomas are broadly divided into Hodgkin lymphoma and non-Hodgkin lymphoma (all other types of lymphoma). The scientific classification of lymphoma types is more detailed. While older classifications referred to histiocytic lymphomas, these are considered to be of the B, T, or NK cell lineage in newer classifications.

[0436] When the pharmaceutical composition is administered to a patient suffering from cancer in which the target molecule of CAR or TCR (e.g., mTCR) is expressed, the pharmaceutical composition is able to recognize the cancer and have immune activity without activating genes that weaken the function of immune cells against cancer cells, and there are no problems such as exhaustion due to the resulting inhibition of activated signal transduction.

[0437] In some embodiments, the above-mentioned pharmaceutical composition comprising immune cells can more effectively inhibit the expression of immune checkpoint receptors, while maximizing the efficacy of anti-cancer immune cell therapy in which chimeric antigen receptors can play a role. In other embodiments, when cells that inhibit the expression of immune checkpoint receptors as described above are used in pharmaceutical compositions, it is possible to eliminate severe systemic adverse reactions, such as cytokine release syndrome or autoimmune symptoms (the adverse reactions may be caused by the use of a separate inhibitor of immune checkpoint receptors), and the burden caused by the increased cost of treatment due to the simultaneous use of expensive antibody therapy and cell therapy.

[0438] As it goes without saying, in addition to cells, other pharmaceutically acceptable salts, carriers, excipients, vehicles and other additives that can further enhance immune response may be added to the pharmaceutical composition, and thus a detailed explanation thereof will be omitted.

[0439] In some embodiments, the pharmaceutical composition comprises dual CAR-T cells targeting two immune checkpoints as described herein. For example, the dual immune checkpoint can be selected from the group consisting of: 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-3), CEACAM (carcinoembryonic antigen-related cell adhesion molecule, including three subtypes CEACAM-1, CEACAM-3 or CEACAM-5), LAG3 (lymphocyte activation gene 3), VISTA (V-domain Ig inhibitor of T cell activation), BTLA (B lymphocyte and T lymphocyte attenuator), 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), and can be selected, for example, between PD1 and TIM3.

[0440] In some embodiments, the type of targeted immune check affects the anti-tumor effect of the pharmaceutical composition. For example, a pharmaceutical composition targeting PD-1 and TIM3 can show different levels of anti-tumor effects from a pharmaceutical composition targeting PD-1 and TIGIT. In other embodiments, the difference in the anti-tumor effect is not predictable from the knowledge of the anti-tumor effect of other drugs targeting the same immune checkpoint (for example, the other drugs may include antibodies targeting immune checkpoints). In some embodiments, targeting two immune checkpoints can produce a surprisingly high anti-tumor effect. In an exemplary embodiment (Example 10), CAR-T cells targeting PD-1 and TIGIT are compared with other dual KD CAR-T cells targeting PD-1 and CTLA-4, PD-1 and LAG-3, and a combination of PD-1 and TIM-3, showing a surprisingly superior anti-tumor effect.

[0441] On the one hand, the compositions described herein can be provided in unit dosage form, wherein each dosage unit, such as injection, contains a predetermined amount of a composition alone or in appropriate combination with other active agents. As used herein, the term unit dosage form refers to a physically discrete unit suitable as a single dose for human and animal subjects, each unit containing a predetermined amount of a composition alone or in combination with other active agents (in appropriate cases, in combination with a pharmaceutically acceptable diluent, carrier or vehicle) calculated in an amount sufficient to produce the desired effect. The specifications of the novel unit dosage form of the cell or composition described herein depend on the specific pharmacodynamics associated with the pharmaceutical composition in a specific subject.

[0442] In some embodiments, the preferred pharmaceutical dosage form of cell or composition as described herein can be determined based on the content of the present disclosure and the general knowledge of formulation technology and according to the expected route of administration, delivery method and target dose. Regardless of the method of administration, the effective dose can be calculated according to the patient's body weight, surface area or organ size. The calculation of the appropriate dosage for therapy is determined using the various dosage forms described in this specification and additional purification is carried out every day in this area, and is included in the scope of work carried out every day in this area. Suitable dosage can be determined by using appropriate dose-response data.

[0443] The pharmaceutical compositions described herein can be used alone or in combination with other known agents for treating cancer. Whether delivered alone or in combination with other drugs, the pharmaceutical compositions described herein can be delivered by various routes and delivered to various parts in mammals (especially humans) to achieve specific effects. Those skilled in the art will recognize that, although more than one route can be used for administration, a specific route can provide a more direct and more effective response than another route. For example, for the treatment of melanoma, intradermal delivery can be advantageously used relative to inhalation. Local or systemic delivery can be completed by administration, and the administration includes applying the preparation to or instilling it into the body cavity, inhaling or blowing an aerosol, or introducing it parenterally, including intramuscular, intravenous, intraportal, intrahepatic, intraperitoneal, subcutaneous or intradermal administration. Exemplary administration routes for subjects include intravenous (IV) injection and local (intratumoral, intraperitoneal) administration. In some embodiments, the pharmaceutical composition can be administered to solid tumors by infusion.

[0444] In some embodiments, in addition to the genetically engineered immune cells provided herein, additional therapeutic agents may be used together with these effector cells in a combined therapy, wherein the therapeutic agent package targets an antibody or antibody fragment of an antigen associated with the disorder, disease, or indication. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, antibodies suitable for combination therapy as an additional therapeutic agent for the administered genetically engineered immune cells include, but are not limited to, anti-CD20 (rituximab, veltuzumab, ofatumumab, ublituximab, calutuzumab, atezolizumab), anti-HER2 (trastuzumab, pertuzumab), anti-CD52 (alemtuzumab), anti-EGFR (cetuximab), anti-GD2 (dinutuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab); and humanized or Fc-modified variants or fragments thereof, or functional equivalents and biosimilars thereof.

[0445] Ideally, an effective amount or sufficient number of isolated transduced T cells are present in the composition and introduced into a subject such that a long-term, specific anti-tumor response is established to reduce the size of a tumor or eliminate the growth or regeneration of a tumor that would not otherwise occur without such treatment. Ideally, the amount of transduced T cells reintroduced into a subject results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% reduction in tumor size when compared to otherwise identical conditions in which the transduced T cells are not present.

[0446] Therefore, the amount of transduced T cells administered should take into account the route of administration and should allow a sufficient number of transduced T cells to be introduced to obtain the desired therapeutic response. In addition, the amount of each active agent contained in the composition described herein (e.g., the amount of each cell to be contacted or the amount per specific body weight) may vary in different applications. In general, the concentration of transduced T cells should ideally be sufficient to provide at least about 1×10 6 About 1×10 9 transduced T cells, or even more ideally, about 1 × 10 7 About 5×10 8 Although more than 5 × 10 transduced T cells may be used, for example,8 cells, or less than, for example, less than 1×10 7 Any suitable amount of cells may be administered. The dosing regimen may be based on well-established cell-based therapies (see, e.g., Topalian and Rosenberg, 1987; U.S. Pat. No. 4,690,915), or an alternative continuous infusion strategy may be employed.

[0447] These values ​​provide a general guide to the range of transduced T cells to be used by practitioners when optimizing the methods described herein. Enumerating such ranges herein in no way excludes the use of higher or lower amounts of components, which may be warranted in a particular application. For example, actual dosages and schedules may vary depending on whether the composition is administered in conjunction with other pharmaceutical compositions, or depending on individual differences in pharmacokinetics, drug disposition, and metabolism. Those skilled in the art can easily make any necessary adjustments according to the urgency of a particular situation.

[0448] Any composition described herein may be included in a kit. In some embodiments, CAR T cells are provided in a kit, and the kit may also include reagents suitable for amplifying cells, such as culture medium, aAPC, growth factors, antibodies (e.g., for sorting or characterizing CART cells) and / or plasmids encoding CAR or transposase.

[0449] In non-limiting examples, a chimeric receptor expression construct, one or more reagents for producing a chimeric receptor expression construct, cells for transfecting the expression construct, and / or one or more instruments for obtaining allogeneic cells for transfecting the expression construct (such instruments may be syringes, pipettes, forceps, and / or any such medically approved instruments).

[0450] In some embodiments, an expression construct for eliminating endogenous TCR α / β expression, one or more reagents for producing the construct, and / or a CAR are provided in a kit. + T cells. In some embodiments, an expression construct encoding one or more zinc finger nucleases is included. In some aspects, the kit includes reagents or devices for cell electroporation.

[0451] The kit may include one or more compositions described herein in appropriate equal parts or reagents for producing compositions described herein. The components of the kit may be packaged in an aqueous medium or in a lyophilized form. The container device of the kit may include at least one vial, test tube, flask, bottle, syringe or other container device, in which the components may be placed, and in certain embodiments, appropriately equal parts. When there is more than one component in the kit, the kit generally also includes a second, third or other additional container, and the additional components may be placed in the container alone. However, the combination of various components may be included in a vial. The kit described herein generally also includes a device for accommodating a chimeric receptor construct and any other closed reagent container for commercial sale. Such containers may include, for example, injection or blow molding plastic containers in which the desired vials are maintained.

[0452] On the one hand, provided herein is a pharmaceutical composition comprising the above-mentioned immune cells and a pharmaceutically acceptable carrier. On the other hand, provided herein is a pharmaceutical composition for immunotherapy of human patients, comprising the above-mentioned immune cells. In some embodiments, the immune cells are initially derived from the patient. In some embodiments, the patient suffers from a tumor or cancer, in which an increase or change in the level of a cancer antigen targeted by a CAR or TCR (eg, mTCR) expressed in the cell is detected.

[0453] In another aspect, methods are provided herein. In some embodiments, a method of treatment is provided, comprising administering the above-mentioned immune cell or the above-mentioned composition to a subject suffering from a disease or illness. In some embodiments, a genetically engineered antigen receptor specifically binds to an antigen, and the antigen is associated with the disease or illness. In some embodiments, the disease or illness is cancer or a tumor.

[0454] In another aspect, immune cells and compositions are provided herein. In some embodiments, the above immune cells and compositions are provided for use in treating a disease or disorder.

[0455] On the other hand, the present invention provides the use of immune cells or compositions. In some embodiments, the use of the above-mentioned immune cells or compositions in the manufacture of a medicament for the treatment of a disease or illness is provided. In some embodiments, the genetically engineered antigen receptor specifically binds to an antigen, and the antigen is associated with the disease or illness. In some embodiments, the disease or illness is cancer or a tumor.

[0456] The above description of the present invention is intended to be exemplary, and those of ordinary skill in the art will appreciate that the present invention can be easily modified into some other forms without changing the technical ideas or basic features of the present invention. Therefore, the embodiments described above should be understood to be exemplary rather than limiting in all respects. For example, each component described as integrated can be executed separately, and similarly, the component described as executed separately can be executed in an integrated manner.

[0457] The scope of the present invention is indicated by the appended claims, and all modifications or changed forms derived from the meaning and scope of the claims and concepts equivalent thereto should be construed as being included in the scope of the present invention.

[0458] Sequence Listing

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[0469] Example

[0470] The following describes embodiments related to the present invention. In most cases, alternative techniques can be used. The embodiments are intended to be illustrative, rather than limiting the scope of the present invention.

[0471] General approach

[0472] Cell lines and culture. Nalm-6, Nalm-6GL (expressing GFP and firefly luciferase), K562, K562-CD19, IM-9, Raji, Daudi cell lines were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and 2mM L-glutamine and 1% penicillin / streptomycin at 37°C in a humidified incubator with 5% CO2. Lenti-X TM The 293T cell line was purchased from Takara and maintained in DMEM supplemented with 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. + PD-L1 + Cell lines, K562-CD19 or NALM-6-G1 cells were transduced with lentivirus encoding human PD-L1 (NM_014143.3). The Nalm-6-PDL1-CD80 cell line was generated by transducing Nalm-6-PDL1 cells with lentivirus encoding human CD80 (NM_005191.3).

[0473] Plasmid construction. The construction of the pLV-CD19-BBz vector containing anti-CD19 scFv (FMC63), CD8α hinge and transmembrane regions, and the cytoplasmic domains of 4-1BB (CD137) and CD3ζ has been described previously (PNAS, 2016, MaJSY). To generate pLV-CD19-28z, the cytoplasmic domain of 4-1BB was replaced with the sequence of the human CD28 co-stimulatory domain. In order to detect and purify transduced CAR-T cells, the ΔLNGFR (CD271 with truncated cytoplasmic domain) sequence was amplified from the pMACs-ΔLNGFR (Milteny Biotec) vector and inserted into the front of the CAR-transgene through the P2A sequence to generate pLV-ΔLNGFR-CD19-28z or P1V-ΔLNGFR-CD19-bBz.

[0474] In order to generate a two-in-one LV vector encoding both CAR and shRNA expression cassettes, an shRNA expression cassette containing shRNA (linker sequence; termination sequence. TTTTT) and Pol promoter (mU6, hU6 or hH1) was synthesized and subcloned into the LV vector encoding CAR upstream of the central polypurine tract (cPPT). In order to generate a dual two-in-one vector expressing two shRNAs through different promoters (mU6 and hU6), the BstZ171-Xba1-Nde1-Bmt1-Spe1 MCS sequence was inserted into the pLV-hU6-shPD-1_ΔLNGFR-CD19-BBz vector located downstream of the hU6 promoter. The second mU6-shRNA cassette fragment was subcloned into the MCS.

[0475] To create the reporter vector, the NFAT RE x3 sequence derived from the pGL2_NFAT-Luc reporter gene (addgene #10959) was amplified by PCR. NF-kB-re5x (5'-GGGAATTTCC-3') and miniP sequences (IDT Technologies) were synthesized. These reporter fragments were used to replace the EF-1a promoter of the pLV-eGFP vector to generate pLV-NFAT-RE3x-eGFP or pLV-NF-kB-RE 5x-eGFP reporter vectors.

[0476] Select siRNA or shRNA sequence. Before synthesis, use BLOCK-iT TM Candidate sequences of 21-mer siRNAs specific for inhibitory immune checkpoints (CTLA-4, LAG-3, TIGIT, and TIM-3) were designed by RNAi Designer or Sfold programs. RNA oligomers. siRNAs targeting CTLA-4 were selected from the group consisting of SEQ ID NO.255-260. siRNAs targeting LAG-3 were selected from the group consisting of SEQ ID NO.244-254. siRNAs targeting TIGIT were selected from the group consisting of SEQ ID NO.238-243. siRNAs targeting TIM-3 were selected from the group consisting of SEQ ID NO.261-264. (IDT Technologies). To analyze the expression dynamics of immune checkpoints, PBMCs were stimulated with Dynabeads human T activator CD3 / CD28 (Thermofisher) or 4 μg / ml anti-CD3 antibody and 2 μg / ml anti-CD28 antibody in the presence of human recombinant IL-2. The expression levels of immune checkpoints were analyzed for 12 days (day 3, day 6, and day 12). To select the best siRNA sequence, 2 days after stimulation, Transfection system (Thermofisher) was used to electroporate PBMCs with siRNA oligomers. On the second day after transfection, the knockdown efficiency of siRNA was measured by flow cytometry. Based on their efficiency, 2-3 siRNA sequences were selected for each immune checkpoint and converted into shRNA format to generate dual 2-in-1 lentiviral vectors. To verify the shRNA-mediated knockdown efficiency, lentiviral-transduced T cells were stimulated with γ-irradiated K562-CD19 cells at a ratio of 1:1 for 3 days, and the expression levels of their immune checkpoints were analyzed by flow cytometry.

[0477] Flow cytometry. The expression level of anti-CD19 CAR was analyzed by anti-mouse F(ab′)2 antibody conjugated with AF 647 (115-606-072, Jackson ImmunoResearch) or rhCD19-Fc conjugated with biotin (CD9-H5259, ACRO Biosystems) coupled to streptavidin conjugated with AF 647 (405237, Biolegend). ΔLNGFR expression was analyzed by anti-CD271 antibody conjugated with APC or conjugated with FITC (ME20.4-1.H4; Mitenyi Biotec). The expression of immune checkpoints in CAR-T cells was measured by conventional flow cytometry using the following antibodies: PD-1 (PE, clone J105; Thermofisher), TIM-3 (PE, clone 344823; R&D systems), LAG-3 (PE, clone 7H2C65; Biolgend), TIGIT (PE, clone MBSA43; Thermofisher).

[0478] After 3 days of incubation with irradiated NALM-6 or K562-CD19 cells at an E:T ratio of 1:1, CTLA-4 expression on CAR-T cells was analyzed by intracellular flow cytometry. TM Cells were fixed / permeabilized with PD-L1 antibody (BD Bioscience) and then stained with anti-human CD152 (CTLA-4) antibody (PE, clone BNI3; Biolegend).

[0479] The expression of stimulatory or inhibitory immune checkpoint ligands on tumor cells was analyzed using the following antibodies: CD80 (PE, clone 2D10; Biolgend), CD86 (BV421, clone 2331 (FUN-1); BD Bioscience), PD-L1 (APC, clone 29E.2A3; Biolgend), HLA-DR (PE, clone L243; Biolgend), CD112 (PE, clone TX31; Biolgend), CD155 (PE, clone SKII.4; Biolgend).

[0480] To analyze the effect of TGF-β on PD-1 expression, CAR-T cells were stimulated with irradiated NALM-6 cells in the presence of 10 ng / ml recombinant human TGF-β1 (R&D systems) for 3 days before measuring PD-1 expression by flow cytometry.

[0481] The phosphorylation status of SMAD2 / 3 was analyzed by intracellular flow cytometry. To determine the phosphorylation status of SMAD2 / 3, CAR-T cells were incubated with NALM-6 cells at a 1:1 E:T cell ratio for 4 h or 24 h. The cells were fixed with lysis / fixation buffer (BD Bioscience) and then permeabilized with Perm buffer III (BD Bioscience). LNGFR was determined using anti-human Smad2 (pS465 / pS467) / Smad3 (pS423 / pS425) antibodies (PE, clone O72-670; BD Bioscience) + Phosphorylation status on CAR-T cells.

[0482] Detection of induced regulatory T cells. After 3 days of co-culture with NALM-6 cells, regulatory T cells (CD4 + CD25 + FOXP3 + ) were induced by . Cells fixed and permeabilized with Foxp3 / transcription factor staining buffer (Thermofisher) were stained with the following antibodies: anti-CD4 (BV605, clone OKT4; Biolgend), anti-CD25 (FITC, clone VT-072; Biolgend), and anti-FOXP3 (APC, clone 236A / E7; Thermofisher).

[0483] CAR-T proliferation assay. CAR-T cells expressing ΔLNGFR surface markers were sorted by magnetic beads (Miltenyi Biotec). +CAR-T cells (1×10 6 ; >95% purity) were cultured every 6 days with γ-irradiated K562-CD19-PDL1 cells (1×10 6 ) for stimulation. The fold expansion of CAR-T cells was calculated by cell counting on days 6, 12, and 18 using the trypan blue exclusion method.

[0484] In vitro cytotoxicity of CAR-T cells. The cytotoxicity of CAR-T was determined by using the Incucyte S3 live cell analysis system. NALM-6 or NALM-6-PDL1 target cells constitutively expressing GFP were cultured in triplicate at 1×10 5 The cells were plated in 96-well plates at a density of 10 cells / well. LNGFR was added to each well at an E:T ratio of 1:1, 0.3:1, or 0.1:1. + CAR-T cells. The real-time changes of GFP intensity in each well were recorded every 2 hours as the green object integrated intensity (average GFP intensity x μm 2 The percentage of the relative green object integrated intensity was calculated by the following formula: (total integrated GFP intensity at each time point / total integrated GFP intensity at the start time point)*100.

[0485] NFAT and NF-κB reporter assay. To determine the specific activity of NFAT transcription factors in CAR-T cells, PBMCs were first stimulated with 4 μg / ml anti-CD3 and 2 μg / ml anti-CD28 antibodies for 2 days by transduction with lentivirus encoding the NFAT-RE x3-eGFP reporter gene in the presence of human recombinant IL-2 (300 IU / mL). Eight days after transduction, total cells were stimulated again with anti-CD3 and anti-CD28 antibodies for 2 days in the presence of human recombinant IL-2 (300 IU / mL). The activated cells were divided into two separate wells and transduced with different CAR-encoding lentiviruses (CD19-28z or CD19-BBz). After 6 days, the total cells in each well were co-incubated with NALM-6 cells at a 1:1 ratio. At the 24 and 48 h time points, the LNGFR + The gMFI value of the eGFP signal within the CAR-T population (20-25% of total CD3+ cells) was used to determine the reporter activity of NFAT in CAR-T cells. The specific activity of NF-κB transcription factor in CAR-T cells was determined in a similar manner, but using a lentivirus encoding the NF-κB-RE x5-eGFP reporter gene.

[0486] Quantitative real-time PCR. 3×10 6 LNGFR +G28z or GBBz CAR-T and CD19 + NALM-6 target cells were co-cultured at a ratio of 1:1. After 4 and 48 hours of co-culture, LNGFR was sorted using a MoFlo Astrios sorter (Beckman Coulter). + CAR-T cells were sorted. LNGFR was extracted using RNeasy mini kit (Qiagen). + The genes of CAR-T cells were reverse transcribed into cDNA using the QuantiTect reverse transcription kit (Qiagen). Quantitative real-time PCR was performed using the SYBR protocol using the CFX96 real-time polymerase chain reaction detection system (Biorad) and SYBR Green real-time PCR premix (TOYOBO). The primer sequences for detecting 18s rRNA included SEQ ID NOs: 268 and 269. The primer sequences for detecting PDCD1 included SEQ ID NOs: 270 and 271. The primer sequences for detecting IL2 included SEQ ID NOs: 272 and 273. The primer sequences for detecting IL4 included SEQ ID NOs: 274 and 275. The primer sequences for detecting IL17A included SEQ ID NOs: 276 and 277. The primer sequences for detecting CD25 included SEQ ID NOs: 278 and 279. The primer sequences for detecting CTLA4 included SEQ ID NOs: 280 and 281. The primer sequences for detecting FOXP3 included SEQ ID NOs: 282 and 283. The primer sequences for detecting TGF-β1 include SEQ ID NOs: 284 and 285. The primer sequences for detecting TGFBR1 include SEQ ID NOs: 286 and 287. The primer sequences for detecting TGFBR2 include SEQ ID NOs: 288 and 289.

[0487] The amount of target mRNA was normalized to the endogenous reference 18s rRNA: ΔCt(sample)=Ct(target gene)-Ct(18srRNA). The comparative Ct method was applied to analyze the relative fold change of target mRNA compared to the unstimulated condition based on the following formula: -ΔΔCt =2Λ-(ΔCt[stimulated]-ΔCt[unstimulated]).

[0488] Animal experimentation. All procedures described in this article were approved by the Institutional Animal Care and Use Committee of KAIST. + In the blood cancer model, NSG mice (4 to 6 weeks old) were injected intravenously with 1x10 6 CD19 +NALM-6 leukemia cells engineered to express firefly luciferase fused to EGFP and human PDL1 (NALM6-GL-PDL1 cells). CAR-T cells were prepared from whole blood samples of healthy donors according to the above procedure. On day 4 after transduction, CAR-T cells were sorted and further expanded for 6 days before injection into mice. Five days after injection of NALM6-GL-PDL1 cells, mice were intravenously infused with 2.5 or 1×10 6 CAR-T cells. Bioluminescence imaging of NALM6-GL-PDL1 in mice was monitored using Xenogen IVIS Spectrum, and the signal was quantified as the radiation intensity (photons / second) in the target area using Living Image software (Perkin Elmer). For solid tumor models, NSG mice were subcutaneously injected with 5×10 6 IM-9 cells (CD19 + PD-L1 + CD155 + After injection of tumor cells (tumor volume is about 150-300mm 3 ) 14 days after ovarian cancer, mice were intravenously infused with LNGFR+CAR-T cells. Tumors were monitored weekly by caliper measurement and by (length × width 2 ) / 2 to estimate the volume.

[0489] Example 1: Methods for generating and evaluating a two-in-one vector expressing CART19 and shRNA targeting PD-1

[0490] This example describes methods for generating and evaluating a two-in-one vector expressing CART19 and shRNA that inhibits PD-1 expression.

[0491] To generate a two-in-one lentiviral vector encoding both CAR and shRNA expression cassettes, an shRNA expression cassette containing shRNA (linker sequence; TTCAAGAGA, termination sequence; TTTTT) and Pol III promoter (mU6, hU6 or hH1) was synthesized and subcloned into a LV vector encoding CAR upstream of the central polypurine region (cPPT). A lentiviral vector was constructed that spontaneously expresses 4-1BB-based CART19 through the EF-1α promoter and expresses shRNA targeting PD-1 through the mU6 promoter ( Figure 1A and Figure 1B ).

[0492] To select shRNA targeting PD-1, three shRNA candidates were used. shPD-1#1 showed surprisingly potent inhibitory effects on PD-1 expression ( Figure 1F ), without affecting CAR expression ( Figure 1C), steady-state amplification ( Figure 1D ), differentiation status and CD4 / CD8 composition. Figure 1F ). It has been reported that the expression level of shRNA can be different depending on the type of Pol III promoter (MolTher, 2006, Irvin SYChen). Therefore, the effect of mU6, hU6 or hH1 promoter on PD-1 expression was evaluated. mU6 and hu6 had similar KD efficacy, but hH1 was lower ( Figure 2 ).

[0493] The two-in-one lentiviral vector generated herein showed surprisingly effective inhibition of PD-1 expression and can therefore be used to generate PD-1 KD-modified CAR-T cells in the following examples.

[0494] Example 2: Methods for generating and evaluating PD-1KD CAR-T cells in vitro

[0495] This example describes methods for generating and evaluating PD-1 KD-modified CAR-T cells in vitro.

[0496] Peripheral blood mononuclear cells were isolated from whole blood samples of healthy donors by Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation. These cells were stimulated with 4 μg / ml plate-bound anti-CD3 antibody (clone OKT3; Bio X cell) and 2 μg / ml soluble anti-CD28 antibody (clone CD28.2; BioX cell) in the presence of 300 IU / ml human recombinant IL-2 (BMIKOREA). To produce recombinant lentivirus, 6×10 cells / mL in 2.5 mL growth medium (DMEM supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate) were added 24 h before transfection. 5 293T cells were seeded in 6-well plates. Using 10 μl Lipofectamine 2000 (Thermofisher), the cells were transfected with a mixture of packaging vectors (pMDL, pRev, pMDG.1) and transfer vectors. Two days after transfection, the culture supernatant containing the lentivirus was collected and centrifuged at 1800 rpm for 5 minutes. In the presence of protamine sulfate (1 μg / ml), the activated T cells were mixed with the viral supernatant, centrifuged at 1000 × g for 90 minutes, and incubated at 37 ° C overnight. The next day, the culture supernatant was aspirated and replaced with fresh PRMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 55 μM β-mercaptoethanol. The transduced T cells were cultured at a rate of less than 1×10 6T cells were cultured at a density of 10 cells / mL in RPMI-1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 1% penicillin / streptomycin. T cell culture medium contained recombinant IL-2 (300 IU / mL) and was replenished every 2-3 days.

[0497] The in vitro lytic and proliferation activities of shPD-1CAR-T cells were studied. Nalm-6-PDL1 or K562-CD19-PDL1 was used as CD19 + PDL1 + Target cells. First, the long-term lytic activity was examined using the IncuCyte real-time imaging system. When CAR-T cells were mixed with NALM-6 cells, WT (shGFP) CART 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, shPD-1 CAR-T cells showed more effective lytic activity than shGFP CAR ( Figure 3A ). It was also found that, unexpectedly, BBz w / o shRNA cassette CAR-T cells and shGFP CAR-T cells retained similar cytotoxicity, indicating that shRNA expression itself had little effect on the activity of CAR-T cells.

[0498] Next, the proliferation activity of CAR-T cells repeatedly exposed to CD19 antigen and PD-L1 was evaluated. In the absence of exogenous IL-2, shPD-1CAR-T cells achieved 4 to 5 times the T cell proliferation activity against K562-CD19-PDL1 target as shGFP CAR-T cells ( Figure 3B ). It is known that co-stimulatory ligands contribute to optimal T cell expansion and are frequently expressed in tumor cells. To examine whether the expression of co-stimulatory ligands in target cells affects cell proliferation upon repeated stimulation, we examined whether CD80, a representative co-stimulatory ligand, is expressed in target cells. K562-CD19 expressed CD80 but not Nalm-6 ( Figure 3C ). It was found that NALM-6-PDL1 (NALM-6-PDL1-CD80) overexpressing CD80 can make CAR-T cells proliferate under repeated stimulation. CD80 may promote CART proliferation under repeated in vitro stimulation ( Figure 3B ). Overall, we demonstrated that cell-intrinsic PD-1 disruption surprisingly effectively contributed to the improved in vitro function of CD19-specific CAR-T cells upon repeated CAR and PDL1 stimulation.

[0499] In this example, the PD-1 KD-modified CAR-T cells generated using the two-in-one vector described in Example 1 showed surprisingly enhanced levels of in vitro lytic and proliferative activity compared to WT CAR-T cells, so in the following examples, their therapeutic potential was further evaluated in an in vivo model of a mouse model bearing CD19+PDL1+ hematological tumors.

[0500] Example 3: Method for treating CD19+ blood cancer in vivo using PD-1 KD CAR-T cells

[0501] This example describes the use of PD-1 KD CAR-T cells to treat CD19 in vivo + Methods for blood cancer.

[0502] To establish CD19 + In the blood cancer model, NSG mice (4 to 6 weeks old) were injected intravenously with 1x10 6 CD19 + NALM-6 leukemia cells engineered to express firefly luciferase fused to EGFP and human PDL1 (NALM6-GL-PDL1 cells). CAR-T cells were prepared from whole blood samples of healthy donors according to the above procedure. On day 4 after transduction, CAR - T cells were further expanded for 6 days and then injected into mice. 5 days after the injection of NALM6-GL-PDL1 cells, 1×10 6 CAR-T cells were infused intravenously into mice. Bioluminescent imaging of NALM6-GL-PDL1 in mice was monitored using Xenogen IVIS Spectrum, and the signal was quantified as the radiation intensity (photons / second) in the target area using Living Image software (Perkin Elmer). Mice treated with shPD-1CAR-T cells had a surprisingly uniform reduction in tumor burden compared to shGFP CAR-T cells ( Figure 4 ). In addition, it was found that the two types of shRNA expression promoters had similar anti-tumor effects, and shRNA expression itself surprisingly did not affect the anti-tumor effect.

[0503] The effect of cell-intrinsic PD-1 disruption by CAR-T cells on in vivo cytokine production was examined. 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. In vivo cytokine levels were analyzed using a human Th1 / Th2 cytokine kit (BD Bioscience) according to the manufacturer's instructions. Cell-intrinsic PD-1 disruption by CAR-T cells unexpectedly reduced in vivo cytokine production ( Figure 5A ).

[0504] To examine the effect of cell-intrinsic PD-1 disruption of CAR-T cells on the in vivo expansion of CAR-T cells, spleens of individual mice were harvested 3 or 20 days after CAR-T injection. CAR-T cells (live / dead) were assessed by flow cytometry. - CD3 + ) or NALM-6-PDL1 (live / dead - GFP + ) percentage. Cell-intrinsic PD-1 disruption unexpectedly delayed the in vivo expansion of CAR-T cells ( Figure 5B ).

[0505] The CAR-T cell-intrinsic PD-1 destruction obtained by the two-in-one vector system described herein showed an unexpectedly high level increase in the in vivo anti-tumor effect of CD19-specific CAR-T cells against CD19+PDL1+ tumors. Therefore, PD-1KD CAR-T cells were further used in the methods of the following examples.

[0506] Example 4: Assessment of the milieu of co-stimulatory molecules in PD-1 signaling

[0507] This example describes the assessment of the milieu of co-stimulatory molecules, including CD28 and 4-1BB, in PD-1 signaling.

[0508] We investigated how cell-intrinsic PD-1 disruption affects the function of CD28 / CD3ζ or 4-1BB / CD3ζ CAR-T cells ( Fig. 6A ). First, G28z GBBz, P28z, and PBBz CAR-T cells were generated. These cells were similarly transduced ( Figure 6B ). The expression level of PD-1 in G28zCAR-T cells was higher than that in GBBz CAR-T cells, and the PD-1 level of P28z was higher than that of PBBz ( Fig. 7A ). It was also found that this different PD-1 protein level originated from the transcription level ( Figure 7B). It is assumed that each co-stimulatory domain exhibits different intensities on the activation of factors involved in PD-1 transcription. Among the transcription factors involved in PD-1 transcription, NFAT and NF-κB are significantly involved in PD-1 transcription, and SMAD2 / 3 is involved in PD-1 transcription in the presence of exogenous TGF-β, which is the main immunosuppressive factor in the tumor microenvironment (Cancer Discov., 2016, Benjamine V. Park). The activity of NFAT or NF-kB was studied during CAR stimulation. NFAT response element (NFAT-RE x3)-eGFP or classical NF-κB response element (NF-κB RE x5)-eGFP reporter lentiviral vectors were constructed. The results showed that NFAT or NF-kBRE-mediated eGFP induction ( Fig. 8A and Figure 8B ). The reporter-transduced T cells were restimulated and additionally transduced with G28z or GbZ. It was found that G28z-transduced NFAT reporter CAR-T cells (G28z-NFAT) had slightly higher levels of NFAT activity compared to GbZ-NFAT ( Fig. 9A ), and the mRNA levels of NFAT target genes were also higher in G28z ( Fig. 9B However, G28z-transduced NF-κB reporter CAR-T cells (G28z-NF-κB) were similar to GBB-NF-κB in NFAT activity ( Fig.10 ). To investigate the SMAD2 / 3 activity of each CAR-T cell, TGF-β was treated into G28z or GBBz CAR-T cells during CAR stimulation. It was found that SMAD2 / 3 phosphorylation of G28z CART was slightly higher than that of BBz CART ( Fig.11A ), and G28z increased PD-1 expression by TGF-β treatment to a greater extent than GBBz (fold change of 2.39±0.031 for G28z and 1.61±0.034 for GBBz) ( Fig. 11B ). We investigated whether this difference was caused by differences in the expression levels of TGF-β signaling components. We found that TGF-β and TGF-β receptor 1 (TGFBR1) did not differ significantly between the two CARTs, but TGF-β receptor 2 (TGFBR2) was expressed at higher levels in CD28z ( Fig. 11C ).

[0509] In this embodiment, the environment of costimulatory molecules (including CD28 and 4-1BB) in PD-1 signaling was evaluated. CD28 costimulation strongly induces signals associated with PD-1 transcription, which may be associated with the activation of NFAT and TGF-β signaling. The induction of CD28 costimulation is stronger than that of 4-1BB costimulation.

[0510] Example 5: Engineering and evaluation methods of PD-1KD CAR-T cells

[0511] This example describes the engineering and evaluation methods of PD-1 KD CAR-T cells.

[0512] To mimic the in vivo conditions of CAR-T cells with multiple sequential antigen and immune checkpoint ligand encounters, CAR-T cells were incubated with CD19 in the absence of exogenous cytokines. + PDL1 + In primary CAR-T cells, the PD-1KD CAR-T group showed surprisingly higher lytic activity than the WT CAR-T group, but there was no difference in lytic activity between the CD28-based and 4-1BB-based CAR-T groups ( Fig. 12A ). After the second restim CAR-T cells, G28z CAR-T cells lost their ability to proliferate after the second stimulation, so the lytic activity of the 3 CAR-T cells was analyzed. Compared with GBBz CAR-T cells, PBBz and P28z CAR-T cells had surprisingly high lytic activity upon repeated antigen and PD-L1 exposure. Compared with P28z CAR-T cells, PBBz CAR-T cells showed an unexpectedly higher ability to maintain lytic function ( Fig. 12A This trend was also observed in cell proliferation assays ( Fig. 12B ). Overall, compared with the CD28 costimulatory domain, the application of the 4-1BB costimulatory domain to PD-1KD CART helps to maintain cytotoxicity and proliferation capacity in vitro. After repeated CD19 and PD-L1 exposure, PBBz CAR-T cells showed delayed exhaustion.

[0513] The possible effects of immunosuppressive mechanisms on BBz CAR-T cells were examined. CD28 CAR-T cells are more sensitive to TGF-β than BBz CART (Figure 11), so GBBz CAR-T cells were compared with G28z CAR-T cells in terms of how much their antigen-specific proliferation would be inhibited by TGF-β. Although no significant difference in proliferation was observed between the two CAR-T cells during CAR stimulation in the absence of TGF-β, the proliferation of G28z CAR-T cells, but not GBBz CAR-T cells, was significantly reduced ( Fig.13A ).

[0514] It has been reported that TGF-β not only inhibits proliferation but is also deeply involved in the induction of regulatory T cells (JEM, 2003, WanJun Chen; Science, 2003, Shohei Hori; Blood, 2007, Dat Q. Tran). To examine whether Treg induction differs between G28z and BBz CAR-T cells in the absence of exogenous TGF-β, G28z and BBz CAR-T cells were cultured with NALM-6 target cells during 3 days. + CD25 + FOXP3 + Treg% was 2 to 3 times that of GBBz CAR-T cells ( Fig. 13B This result is consistent with the increased expression of Treg-related genes such as CD25 and FOXP3 ( Fig. 9B ). Next, the changes in Treg% after TGF-β treatment were observed and compared. The results showed that TGF-β significantly increased the Treg% of 28z CAR-T cells, but did not affect the Treg% of BBz CAR-T cells ( Fig. 13B ). It was concluded that CD28 co-stimulation has a greater potential to induce Tregs than 4-1BB co-stimulation, and its potential is further enhanced if TGF-β is present. It was also investigated whether cell-intrinsic PD-1 disruption affects Treg induction. The Treg% of P28z and PBBz CAR-T cells were 2-3 times lower than that of G28z and GBBz cells, respectively. In particular, PBBz had the lowest Treg% ( Fig. 13C Finally, we examined the Treg% under conditions of activated PD-1 / PD-L1 signaling. We examined the changes in Treg% after co-culture with NALM-6 or NALM-6-PDL1. We found that PD-1 / PDL1 signaling did not significantly affect the induction of CAR-T-derived Tregs ( Fig. 13C ), indicating that intracellular PD-1 expression levels are associated with Treg formation. The conclusion is that PBBz CAR-T cells may be surprisingly insensitive to immunosuppressive mechanisms.

[0515] In this example, PBBz CAR-T cells were generated and showed delayed exhaustion upon repeated CD19 and PD-L1 exposure. PBBz CAR-T cells also unexpectedly avoided immunosuppressive mechanisms. Therefore, the in vivo anti-tumor effects of these cells were further evaluated.

[0516] Example 6: Method for treating CD19+ blood cancer in vivo using PBBz CAR-T cells This example describes a method for treating CD19+ blood cancer in vivo using PBBz CAR-T cells.

[0517] CD19 was established as described in Example 3 + Hematologic tumor models. Comparison of G28z, GBBz, P28z, and PBBz CAR-T cells targeting CD19 + In vivo antitumor effect of B-ALL cells. On day 4 after target cell infusion, 2.5×10 6 A single dose of CAR-T cells was administered. In vivo imaging of NALM6-GL-PDL1-bearing mice was obtained using a heterologous Xenogen IVIS Spectrum and quantified as the radiation intensity (photons / second) in the target area. Fig.14 As shown, PD-1KD CART cells (PBBz or P28z) showed anti-tumor effects superior to WT CAR-T cells (GBBz or G28z). In particular, PBBz CAR-T cells inhibited leukemia progression longer than the other three CAR-T cells. WT CAR-T cells had a strong anti-tumor response, but no sustained anti-tumor response. On the other hand, PD-1KD CAR-T showed a weak response to the initial stage, but a sustained anti-tumor response, indicating that the destruction of PD-1 in CAR-T cells may potentially reduce the risk of CRS. PBBz CAR-T cells unexpectedly conferred an in vivo anti-tumor effect superior to P28z.

[0518] Example 7: Method for producing a dual 2-in-1 vector having two types of shRNA cassettes entering in the →← or ←→ direction

[0519] This example describes a method for generating a dual two-in-one vector in which two types of shRNA boxes enter in the ←→ direction (shTIM-3-mU6←→hU6-shPD-1) or →← direction (mU6-shTIM-3→←shPD-1-hU6) and simultaneously express shPD-1, shTIM-3 and CD19-CAR.

[0520] Lentivirus in which the expression of shRNA for PD-1 (hereinafter referred to as shPD-1), shRNA for TIM-3 (hereinafter referred to as shTIM-3) and CD19-CAR is regulated by human U6 promoter (hereinafter referred to as hU6), mouse U6 promoter (hereinafter referred to as mU6) and EF1-α promoter, respectively, was constructed. A plasmid in which two types of shRNA are simultaneously expressed was prepared so that the corresponding shRNA cassettes were placed in the →← direction (mU6-shTIM-3→←shPD-1-hU6) and the ←→ direction (shTIM-3-mU6←→hU6-shPD-1) ( Fig.15To this end, (1) a multiple cloning site (MCS) was inserted, (2) the mouse U6 promoter of shPD-1 was converted to the human U6 promoter, (3) shTIM-3 was inserted, and (4) clones such as shTIM-3 and shPD-1 were switched.

[0521] MCS was inserted into mU6-shPD-1 (3' part of pLV-ΔLNGFR_P2A_CD19-CAR_mU6-shPD-1 (plasmid ID#2)), and primers containing SEQ ID NOs. 228 and 229 were used to prepare a PCR product (416 bp), in which the Hpa1 restriction endonuclease recognition site of mU6-shPD-13' was modified to a BsgZ171-Xba1-Nde-1-Bmt1-Spe1 multiple cloning site. Afterwards, the PCR product was treated with BstZ17y and Hpa1 restriction enzymes, plasmid ID#2 was treated with Hpa1 restriction enzyme and CIP, and then pLV-ΔLNGFR-P2A-CD19-CAR-mU6-shPD-1_MCS (plasmid ID#4) containing the shPD-1-mU6-MCS base sequence (shRNA box SEQ ID NO.223) was prepared by blunt end ligation.

[0522] Afterwards, a plasmid was constructed so that shPD-1 was expressed by the human U6 promoter instead of the mouse U6 promoter. The LentiCRISPR V2 plasmid was used with primers containing SEQ ID NOs. 230 and 231 to obtain a PCR product containing the human U6 promoter. The PCR product was treated with Hpa1 and Spe1 restriction enzymes and then ligated to plasmid ID#4 treated with Hpa1 and Spe1 restriction enzymes to prepare pLV-ΔLNGFR_P2A_CD19-CAR_hU6-shPD-1_MSC (plasmid ID#5) containing the hU6-shPD-1 base sequence (SEQ ID NO.224). The purpose is to insert the mU6-shTIM-3 box into plasmid ID#5 to construct a plasmid that simultaneously expresses shTIM-3 and shPd-1.

[0523] A PCR product including the mU6-shTIM-3 cassette was obtained using plasmid ID#3 and primers containing SEQ ID NOs. 232 and 233. After the PCR product was treated with Bmt1 and Spe1 restriction enzymes, it was ligated to plasmid ID#5 treated with Bmt1 and Spe1 restriction enzymes to prepare pLV-ΔLNGFR_P2A_CD19-CAR mU6-shTIM-3→←shPD-1-hU6 (plasmid ID#6) containing a base sequence (SEQ ID NO. 220) in the →← direction (mU6-shTIM-3→←shPD-1-hU6).

[0524] To construct a plasmid in which two types of shRNA boxes are arranged in the ←→ direction (shTIM-3-mU6←→hU6-shPD-1), a PCR product was obtained using plasmid ID#6 and primers containing SEQ ID NOs. 234 and 235. After being treated with Spe1 and Hpa1 restriction enzymes, it was inserted into plasmid ID#4 treated with the same restriction enzymes to generate pLV-ΔLNGFR-P2A-CD19-CAR-shPD-1-hU6-MCS (plasmid ID#7). Thereafter, a PCR product was obtained using plasmid ID#6 and primers containing SEQ ID NOs. 236 and 237. After being treated with Bmt1 and Spe1 restriction enzymes, it was ligated to plasmid ID#7 treated with the same restriction enzymes to finally prepare pLV-ΔLNGFR-P2A-CD19-CAR_shTIM-3-mU6←→hU6-shPD-1 (plasmid ID#8) containing the base sequence (SEQ IDNO.221) in the ←→ direction (shTIM-3-mU6←→hU6-shPD-1).

[0525] In the following examples, the two-in-one lentiviral vectors generated herein in which 2 types of shRNA cassettes enter in the ←→ or →← direction can be used to generate PD-1 KD-modified CAR-T cells.

[0526] Example 8: Generation and in vitro evaluation of PD-1 KD CAR-T cells

[0527] This example describes methods for generating and in vitro evaluating dual KD CAR-T cells in which shPD-1, shTIM-3, and CD19-CAR are simultaneously expressed.

[0528] Plasmid ID #1 (pLV-ΔLNGFR_P2A_CD19-CAR_mU6-shGFP), #6 (pLV-ΔLNGFR_P2A_CD19-CAR_mU6-shTIM-3→←shPD-1-hU6) and #8 (pLV-ΔLNGFR_P2A_CD19-CAR_shTIM-3-mU6←→hU6-shPD-1) and packaging plasmids pMDL g / p, pRSVrev and pMDG.1 in Table 1 were transfected into HEK293 T cells using lipofectamine, and after 48 hours, cell cultures containing lentivirus were obtained. Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using ficoll-paque solution, and human CD3 and CD28 target antibodies were used to specifically activate T cells. After 1 to 2 days after the initial activation of T cells, they were transduced with the previously obtained virus. The prepared CAR-T cells were then cultured with AIM-V culture medium containing 5% human plasma and human IL-2. On the 6th day after transduction, pure CAR-T cells were obtained using the MACSelect LNGFR system (miltenyibiotec, Germany), and LNGFR+CAR-T cells were separated by flow cytometry using the LNGFR target antibody. In the following, cells prepared using plasmid IDs #1, #6, and #8 are represented as ΔLNGFRCART19 / shGFP (or shGFP / CART19), ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6 (or shPD-1_shTIM-3 / CART19), and ΔLNGFR-CART19 / shTIM-3-mU6←→hU6-shPD-1 ( Fig.16 ).

[0529] In order to prepare control CAR-T cells containing mU6-shPD-1, mU6-TIM-3 and shPD-1-hU6 boxes, plasmid ID #2 (pLV-ΔLNGFR_P2A_CD19-CAR_mU6-shPD-1), #3 (pLV-ΔLNGFR_P2A_CD19-CAR_mU6-shTIM-3) and #7 (pLV-ΔLNGFR_P2A_CD19-CAR_shPD-1-hU6_MCS) in Table 1 and packaging plasmids pMDL g / p, pRSVrev and pMDG.1 were transfected into HEK293T cells using lipofectamine. After 48 hours, cell culture fluid containing lentivirus was obtained. Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using ficoll-paque solution, and human CD3 and CD28 target antibodies were used to specifically activate T cells. After 1 to 2 days after the initial activation of T cells, they were transduced with the previously obtained virus. The prepared CAR-T cells were subsequently cultured with AIM-V culture medium containing 5% human plasma and human IL-2. On the 6th day after transduction, pure CAR-T cells were obtained using the MACSelect LNGFR system (miltenyibiotec, Germany), and LNGFR+CAR-T cells were separated using a flow cytometer using the LNGFR target antibody. In the following, cells prepared using plasmid IDs #2, #3, and #7 are represented as ΔLNGFR-CART19 / shPD-1 (or shPD-1 / CART19), ΔLNGFR-CART19 / shTIM-3 (or shTIM-3 / CART19), and ΔLNGFRCART19 / shPD-1-hU6, respectively.

[0530] To measure the purity of the dual KD CAR-T cells generated herein, flow cytometry was performed using the LNGFR target antibody of the cells prepared as described above. Fig.17 As shown, about 80% of LNGFR+CAR-T cells were obtained.

[0531] The dual KD CAR-T cells generated herein were measured to have decreased expression of PD-1 and TIM-3, and their expression continued to decrease. The dual KD CAR-T cells were stimulated with human CD3 and CD28 target antibodies for 3 days to induce the expression of PD-1 and TIM-3. Afterwards, the dual KD CAR-T cells were analyzed for PD-1 and TIM-3 expression by flow cytometry using CAR, PD-1, and TIM-3 target antibodies. Fig.18A and Fig.18BAs shown, analysis of the effects of the two simultaneously expressed shRNA types on the reduction of PD-1 and TIM-3 expression showed that the extent of reduction in PD-1 and TIM-3 expression observed in CD19-CAR T cells expressing both shPD-1 and shTIM-3 was similar to that in CD19-CAR T cells in which only shPD-1 or shTIM-3 was expressed.

[0532] Measurement of the effects of dual KD CAR-T cells generated in this paper on differentiation. To observe the degree of PD-1 and TIM-3 differentiation of dual KD CAR-T cells, flow cytometry was performed using CD45RA and CCR7 target antibodies. Fig.19 As shown, in ΔLNGFR-CART19 / shPD-1 cells subjected to repeated antigen stimulation, terminally differentiated TEMRA (CCR7-CD45RA+) T cells increased compared to ΔLNGFR-CART19 / shGFP. On the other hand, ΔLNGFR-CART 19 / mU6-shTIM-3→←shPD-1-hU6 with shTIM-3 added contained more TN (CCR7+CD45RA+) T cells, TCM (CCR7+CD45RA-) T cells, and TEM (CCR7-CD45RA-) T cell subtypes than ΔLNGFR-CART19 / shPD-1. Since similar results were observed for ΔLNGFR-CART19 / shTIM-3 cells, it can be said that the reduced differentiation ability of ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6 cells is caused by the inhibition of TIM-3 expression, and therefore, it can be said that the effect of ShTiM-3 takes precedence over the effect of ShPD-1 in terms of the effect on cell differentiation. It is known that less differentiated T cell subtypes can promote the improvement of T cell cancer therapeutic ability, and it is expected that ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6 cells will show better in vivo anticancer efficiency than ΔLNGFR-CART19 / shPD-1 cells.

[0533] The transduction efficiency, proliferation capacity and viability of double KD CAR-T cells into which CD19-CAR vectors containing shRNA boxes with different orientations have been introduced were compared. In the CAR-T cells produced herein, flow cytometry was performed using LNTER antibodies on cells using plasmids containing boxes in the →← direction (mU6-shTIM-3→←shPD-1-hU6) and ←→ direction (shTIM-3-mU6←→hU6-shPD-1). For the analysis of cell viability, trypan blue staining was performed, and the ratio of unstained cells was found. As Figures 20A-20CAs shown, cells using the ←→ direction cassette (ΔLNGFR-CART19 / shTIM-3-mU6←→hU6-shPD-1) had unexpected difficulties in forming transduced cells compared to cells using the →← direction cassette (ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6), and the transduced T cells had low proliferation capacity and viability.

[0534] This embodiment describes the method for producing dual KD CAR-T cells in which shPD-1, shTIM-3 and CD19-CAR are expressed simultaneously. Dual KD CAR-T cells are produced using the two-in-one lentiviral vector produced herein, the vector having 2 types of shRNA boxes entering in the →← or ←→ direction. When compared with cells using the ←→ direction box, cells using the →← direction box (ΔLNGFR-CART19 / mU6-shTIM-3→←shPD-1-hU6) show surprisingly higher proliferation capacity and viability, and are therefore used in the following embodiments.

[0535] Example 9: Method for Producing Dual Two-in-One Vectors and Dual KD CAR-T Cells

[0536] This example describes methods for generating dual 2-in-1 vectors and dual KD CAR-T cells.

[0537] To generate a dual two-in-one vector expressing two shRNAs through different promoters (mU6 and hU6), the BstZ171-Xba1-Nde1-Bmt1-Spe1 multiple cloning site (MCS) was inserted into the pLV-hU6-shPD-1_ΔLNGFR-CD19-BBz vector downstream of the hU6 promoter. The second mU6-shRNA cassette fragment was subcloned into the MCS. In order to limit the blockade of multiple immune checkpoints to CAR-T only, the dual two-in-one vector was designed to express two shRNAs through the mU6 and hU6 Pol III promoters to inhibit the expression of two immune checkpoints of CAR-T cells. In order to discover effective CTLA-4, LAG-3, TIGIT or TIM-3 targeting siRNAs, siRNAs were electroporated into CD3 / CD28 stimulated T cells. Two or more effective siRNAs were selected ( Fig.21A). A two-in-one vector for CTLA-4, LAG-3, TIGIT, or TIM-3 KD CAR-T cells was constructed by converting 21-mer siRNA into shRNA form, and finally each shRNA targeting immune checkpoint was selected (shCTLA-4: #1; shLAG-3: #1278; shTIGIT: #739; shTIM-3: #3), which significantly inhibited expression and had little effect on CAR-T expansion ( Fig. 21B -C). Finally, PD-1_CTLA-4, PD-1_LAG-3, PD-1_TIGIT or PD-1_TIM-3CAR-T cells were constructed by dual two-in-one vectors ( Figures 22A-22E Compared with single PD-1 KD CAR-T cells, all dual KDCAR-T cells expanded less ( Fig. 22C These results were also observed in single KD CAR-T cells ( Fig. 21B ).

[0538] In this example, a dual 2-in-1 vector was generated. Dual KD CAR-T cells were generated using the dual 2-in-1 vector and in the presence of CD19 + PDL1 + Their therapeutic potential was further evaluated in in vivo mouse models of hematological tumors and in solid tumor models in the Examples below.

[0539] Example 10: Method for in vivo treatment of CD19+ blood cancer using dual KD CAR-T cells targeting PD-1 and TIGIT

[0540] This example describes a method for in vivo treatment of CD19+ blood cancer using dual KD CAR-T cells targeting two immune checkpoints, including the following combinations: PD-1 and CTLA-4, PD-1 and LAG-3, PD-1 and TIGIT, and PD-1 and TIM-3.

[0541] CD19 was established as described in Example 3 + Hematologic tumor models. PD-1_CTLA-4, PD-1_LAG-3, PD-1_TIGIT, or PD-1_TIM-3KD CAR-T cells targeting blood CD19 + Antitumor effect of B-ALL model. On the 5th day after target cell infusion, 1×10 6 A single dose of each CAR-T cell was injected. It has been found that PD-1_TIGITKD CAR-T cells showed surprisingly superior anti-tumor effects compared to other dual KD CAR-T cells ( Fig.23). Therefore, in the following examples, PD-1_TIGITKD CAR-T cells were further evaluated in solid tumor models.

[0542] Example 11: Therapeutic approach using dual KD CAR-T cells targeting PD-1 and TIGIT in solid tumor models

[0543] This example describes a method for treating cancer using dual KD CAR-T cells targeting PD-1 and TIGIT in solid tumor models.

[0544] To establish a solid tumor model, 4- to 6-week-old NSG mice were injected subcutaneously with 5 × 10 6 IM-9 cells (CD19 + PD-L1 + CD155 + Once the tumor volume reaches 150-250mm 3 , let ×10 6 A single dose of CAR-T cells was injected intravenously. Tumors were monitored weekly by caliper measurement and (length × width 2 ) / 2 to estimate the volume. As shown in the blood tumor model (Nalm-6-PDL1), PD-1_TIGITKD CAR-T cells were also most surprisingly effective against the solid tumor model (IM-9) ( Fig.24 ). Cell-intrinsic PD-1_TIGIT blockade has an unexpectedly higher level of anti-tumor effect than PD-1 blockade. Sequence Listing <110> CUROCELL CO. LTD. 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TIM-3 for RNA interference <400> 15 ggaattcgct cagaagaaa 19 <210> 16 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 16 ggaccaaact gaagctatat t 21 <210> 17 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 17 agaactttgg tttcctttaa t 21 <210> 18 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 18 atgaaaggga tgtgaattat t 21 <210> 19 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 19 tcttatcttc ggcgctttaa t 21 <210> 20 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 20 cttatcttcg gcgctttaat t 21 <210> twenty one <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> twenty one ttatcttcgg cgctttaatt t 21 <210> twenty two <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> twenty two gaggagccca atgagtatta t 21 <210> twenty three <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> twenty three aggagcccaa tgagtattat t 21 <210> twenty four <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> twenty four atagatccaa ccaccttatt t 21 <210> 25 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 25 atgtcattgc ctctgtattt a 21 <210> 26 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 26 tgtcattgcc tctgtattta a 21 <210> 27 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 27 accaccatgc ccagctaatt t 21 <210> 28 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 28 tgttgagatt taggcttatt t 21 <210> 29 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 29 gaccaaactg aagctatatt t 21 <210> 30 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 30 aggccttcag caatctatat t 21 <210> 31 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 31 ggccttcagc aatctatatt a 21 <210> 32 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 32 gagtggtccc taaacttaaa t 21 <210> 33 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 33 agtggtccct aaacttaaat t 21 <210> 34 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 34 gtggtcccta aacttaaatt t 21 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIM-3 for RNA interference <400> 35 ctaacacaaa tatccacat 19 <210> 36 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAG-3 for RNA interference <400> 36 tcagcagccc agtccaaata a 21 <210> 37 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAG-3 for RNA interference <400> 37 cagcagccca gtccaaataa a 21 <210> 38 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAG-3 for RNA interference <400> 38 tcaacgtctc catcatgtat a 21 <210> 39 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAG-3 for RNA interference <400> 39 caacgtctcc atcatgtata a 21 <210> 40 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAG-3 for RNA interference <400> 40 ctggagacaa tggcgacttt a 21 <210> 41 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAG-3 for RNA interference <400> 41 ctcagcagcc cagtccaaat a 21 <210> 42 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAG-3 for RNA interference <400> 42 agcagcccag tccaaataaa c 21 <210> 43 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CTLA-4 for RNA interference <400> 43 gggatcaaag ctatctatat a 21 <210> 44 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CTLA-4 for RNA interference <400> 44 ggatcaaagc tatctatata a 21 <210> 45 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CTLA-4 for RNA interference <400> 45 ggcaacggaa cccagattta t 21 <210> 46 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CTLA-4 for RNA interference <400> 46 tgaagaagag agtccatatt t 21 <210> 47 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CTLA-4 for RNA interference <400> 47 ttggatgcgg aacccaaatt a 21 <210> 48 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CTLA-4 for RNA interference <400> 48 agcatcactt gggattaata t 21 <210> 49 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CTLA-4 for RNA interference <400> 49 tgatgtgggt caaggaatta a 21 <210> 50 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CTLA-4 for RNA interference <400> 50 agcgagggag aagactatat t 21 <210> 51 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CTLA-4 for RNA interference <400> 51 tttacgtatg agacgtttat a 21 <210> 52 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting BTLA for RNA interference <400> 52 gctcctgtat agtttacttc c 21 <210> 53 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting BTLA for RNA interference <400> 53 ggaaattaac ctggttgatg c 21 <210> 54 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting BTLA for RNA interference <400> 54 gcaccaacag aatatgcatc c 21 <210> 55 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting BTLA for RNA interference <400> 55 gctcaacagg atgtcaaata a 21 <210> 56 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting BTLA for RNA interference <400> 56 gcatcttgct gttcttcttta c 21 <210> 57 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting BTLA for RNA interference <400> 57 gcatttgtgg acaacttatg t 21 <210> 58 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD160 for RNA interference <400> 58 ggaacgcgac taaacttaat c 21 <210> 59 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD160 for RNA interference <400> 59 gatgttcacc ataagccaag t 21 <210> 60 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD160 for RNA interference <400> 60 gcaagatgag tctgactatg g 21 <210> 61 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD160 for RNA interference <400> 61 ggcacagaga agaatgcaac a 21 <210> 62 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD160 for RNA interference <400> 62 gggaagagat gctaaatata c 21 <210> 63 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD160 for RNA interference <400> 63 gcaaatcagt gtaatccttg a 21 <210> 64 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting VISTA for RNA interference <400> 64 gcaacttctc catcaccatg c 21 <210> 65 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting VISTA for RNA interference <400> 65 gcaaagatgc accatccaac t 21 <210> 66 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting VISTA for RNA interference <400> 66 gcggatggac agcaacattc a 21 <210> 67 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting VISTA for RNA interference <400> 67 ggacacttct gagtatgaag c 21 <210> 68 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting VISTA for RNA interference <400> 68 gggaaccaca atgcacgaaa g 21 <210> 69 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting VISTA for RNA interference <400> 69 ggtgctttcc aacacacttt c 21 <210> 70 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIGIT for RNA interference <400> 70 gcttctggcc atttgtaatg c 21 <210> 71 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIGIT for RNA interference <400> 71 gggagtactt ctgcatctat c 21 <210> 72 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIGIT for RNA interference <400> 72 gctgcatgac tacttcaatg t 21 <210> 73 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIGIT for RNA interference <400> 73 taacgtggat cttgatcata a 21 <210> 74 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIGIT for RNA interference <400> 74 ggagacatac acaggccttc a 21 <210> 75 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TIGIT for RNA interference <400> 75 gcatttgggc cttgatctac c 21 <210> 76 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting 2B4 for RNA interference <400> 76 gacaggttgc aaggcagttc t 21 <210> 77 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting 2B4 for RNA interference <400> 77 gggagtgcct cttcagttac a 21 <210> 78 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting 2B4 for RNA interference <400> 78 ggacgaggag gttgacatta a 21 <210> 79 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting 2B4 for RNA interference <400> 79 gaggagaaag aggaaggaga a 21 <210> 80 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting 2B4 for RNA interference <400> 80 gcccttcctt caatagcact a 21 <210> 81 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting 2B4 for RNA interference <400> 81 ggttgactgc atttctagac t 21 <210> 82 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PD-L1 for RNA interference <400> 82 gcatttgctg aacgcattta c 21 <210> 83 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PD-L1 for RNA interference <400> 83 gctgcactaa ttgtctattg g 21 <210> 84 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PD-L1 for RNA interference <400> 84 ggatccagtc acctctgaac a 21 <210> 85 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PD-L1 for RNA interference <400> 85 gcacatcctc caaatgaaag g 21 <210> 86 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PD-L1 for RNA interference <400> 86 ggattctcaa cctgtggttt a 21 <210> 87 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PD-L1 for RNA interference <400> 87 ggtgcttggt ctcctctata a 21 <210> 88 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-1 for RNA interference <400> 88 gcacagtact cctggcttat c 21 <210> 89 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-1 for RNA interference <400> 89 gcaacaggac cacagtcaag a 21 <210> 90 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-1 for RNA interference <400> 90 gcaacaccac cctcagcata a 21 <210> 91 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-1 for RNA interference <400> 91 gcctgttcag agcactcatt c 21 <210> 92 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-1 for RNA interference <400> 92 gcagtaatgc cttctcctat t 21 <210> 93 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-1 for RNA interference <400> 93 gcaccttggt gcttagctag a 21 <210> 94 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-3 for RNA interference <400> 94 gcttccatct atgaggaatt g 21 <210> 95 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-3 for RNA interference <400> 95 gccagagaac cagctataag t 21 <210> 96 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-3 for RNA interference <400> 96 gggtccctga tgaatatctg g 21 <210> 97 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-3 for RNA interference <400> 97 ggcttgcagg gaaagtgaat g 21 <210> 98 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-3 for RNA interference <400> 98 gcttgcaggg aaagtgaatg g 21 <210> 99 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-3 for RNA interference <400> 99 agcttccatc tatgaggaat t 21 <210> 100 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-5 for RNA interference <400> 100 ggaatccaga acgaattaag t 21 <210> 101 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-5 for RNA interference <400> 101 ggctgattga tgggaacatc c 21 <210> 102 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-5 for RNA interference <400> 102 ggactacagt caagacaatc a 21 <210> 103 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-5 for RNA interference <400> 103 ggaccctcac tctattcaat g 21 <210> 104 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-5 for RNA interference <400> 104 gctactggcc gcaataattc c 21 <210> 105 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CEACAM-5 for RNA interference <400> 105 gctctttgta tgacagaata c 21 <210> 106 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD96 for RNA interference <400> 106 ggtccaaggt caccaataag a 21 <210> 107 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD96 for RNA interference <400> 107 gcaaccatac gatagaaata g 21 <210> 108 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD96 for RNA interference <400> 108 ggttctgaaa tttcctcaac a 21 <210> 109 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD96 for RNA interference <400> 109 gcaagatatc cagctacatc t 21 <210> 110 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD96 for RNA interference <400> 110 gcaactcacc ctcttccatc t 21 <210> 111 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD96 for RNA interference <400> 111 gctgcattcc ctaagataat t 21 <210> 112 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAIR1 for RNA interference <400> 112 gggacagtag atccacatac a 21 <210> 113 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAIR1 for RNA interference <400> 113 ggacaacagt cacaatgagc a 21 <210> 114 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAIR1 for RNA interference <400> 114 ggctgttgat gttctagaga g 21 <210> 115 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAIR1 for RNA interference <400> 115 gcagacaagg ccacagtcaa t 21 <210> 116 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAIR1 for RNA interference <400> 116 ggaggtttct aaccagcatc c 21 <210> 117 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LAIR1 for RNA interference <400> 117 gccttgagac tgtgctatac a 21 <210> 118 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting KLRG1 for RNA interference <400> 118 ccaagcccag aatgactatg g 21 <210> 119 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting KLRG1 for RNA interference <400> 119 gccttcttgt tcttgccttg t 21 <210> 120 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting KLRG1 for RNA interference <400> 120 gcttctgact gcagttcttc t 21 <210> 121 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting KLRG1 for RNA interference <400> 121 gctggatgaa atatggtaac c 21 <210> 122 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting KLRG1 for RNA interference <400> 122 ggaaatgagc ctgctccaag t 21 <210> 123 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting KLRG1 for RNA interference <400> 123 ggattggtct gaggaacaat t 21 <210> 124 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting FAS for RNA interference <400> 124 gccaagaagg gaaggagtac a 21 <210> 125 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting FAS for RNA interference <400> 125 gccaattcca ctaattgttt g 21 <210> 126 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting FAS for RNA interference <400> 126 ggttctcatg aatctccaac t 21 <210> 127 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting FAS for RNA interference <400> 127 gctggagtca tgacactaag t 21 <210> 128 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting FAS for RNA interference <400> 128 gcctggtttg gagatactaa c 21 <210> 129 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting FAS for RNA interference <400> 129 ggaaccacct aaagaacttc c 21 <210> 130 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-b for RNA interference <400> 130 ccgcgttgat ttaaagaaag a 21 <210> 131 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-b for RNA interference <400> 131 gcctgataca tatcagcatt t 21 <210> 132 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-b for RNA interference <400> 132 gcggaattgg aatttcttag c 21 <210> 133 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-b for RNA interference <400> 133 gcacgactac agaaatatag c 21 <210> 134 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-b for RNA interference <400> 134 tttccggtta agttgcactc g 21 <210> 135 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-b for RNA interference <400> 135 gcctggatct aattcagaaa g 21 <210> 136 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LRR1 for RNA interference <400> 136 gaaagccact gttcggttaa a 21 <210> 137 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LRR1 for RNA interference <400> 137 tcctgtggatctgtctaa g 21 <210> 138 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LRR1 for RNA interference <400> 138 ggctcataga ggctgtaatg t 21 <210> 139 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LRR1 for RNA interference <400> 139 gggcttgtcc gagttgatat g 21 <210> 140 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LRR1 for RNA interference <400> 140 aagatttgga taccgcaaaa a 21 <210> 141 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting LRR1 for RNA interference <400> 141 gtggtactga agcacctatt a 21 <210> 142 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TGFBR1 for RNA interference <400> 142 gcttgttcag agaacaattg c 21 <210> 143 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TGFBR1 for RNA interference <400> 143 ggagattgtt ggtacccaag g 21 <210> 144 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TGFBR1 for RNA interference <400> 144 gcagctaggc ttacagcatt g 21 <210> 145 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TGFBR1 for RNA interference <400> 145 ggtcctttct gtgcactatg a 21 <210> 146 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TGFBR1 for RNA interference <400> 146 ggtggtagct aaagaacatt c 21 <210> 147 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting TGFBR1 for RNA interference <400> 147 ggacctgtct acaggtgatc t 21 <210> 148 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DNMT3A for RNA interference <400> 148 gccaaggtca ttgcaggaat g 21 <210> 149 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DNMT3A for RNA interference <400> 149 gcagaacaag cccatgattg a 21 <210> 150 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DNMT3A for RNA interference <400> 150 cccaaggtca aggagattat t 21 <210> 151 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DNMT3A for RNA interference <400> 151 gcagaagtgc cggaacattg a 21 <210> 152 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DNMT3A for RNA interference <400> 152 gcgtcacaca gaagcatatc c 21 <210> 153 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DNMT3A for RNA interference <400> 153 ccggctcttc tttgagttct a 21 <210> 154 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting A2aR for RNA interference <400> 154 cagaacgtca ccaactactt t 21 <210> 155 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting A2aR for RNA interference <400> 155 ccatgctagg ttggaacaac t 21 <210> 156 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting A2aR for RNA interference <400> 156 ccaagtggcc tgtctctttg a 21 <210> 157 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting A2aR for RNA interference <400> 157 ggtgtctatt tgcggatctt c 21 <210> 158 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting A2aR for RNA interference <400> 158 agaccttccg caagatcatt c 21 <210> 159 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting A2aR for RNA interference <400> 159 tccttagcca tgagctcaag g 21 <210> 160 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-c for RNA interference <400> 160 gaaagtactg tggacacatg t 21 <210> 161 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-c for RNA interference <400> 161 gcacgtgtcc atcttcgagt t 21 <210> 162 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-c for RNA interference <400> 162 ggccaacact cctcaagaac t 21 <210> 163 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-c for RNA interference <400> 163 ggcttctacc tctacccaga t 21 <210> 164 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-c for RNA interference <400> 164 ggccatggac tccacatttg a 21 <210> 165 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting Cbl-c for RNA interference <400> 165 ggccgtgagt atctaccagt t 21 <210> 166 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKα for RNA interference <400> 166 gccagaagac aagttagaat t 21 <210> 167 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKα for RNA interference <400> 167 gctctggaag ttccagtata t 21 <210> 168 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKα for RNA interference <400> 168 ggaaatgatc tggctcgatg c 21 <210> 169 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKα for RNA interference <400> 169 gcaaagatcc tcaaggattt a 21 <210> 170 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKα for RNA interference <400> 170 ggatgcctct attgctcatc g 21 <210> 171 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKα for RNA interference <400> 171 gctatggtac ttcgaatttg c 21 <210> 172 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKζ for RNA interference <400> 172 ggaagggatt ccagcagaag t 21 <210> 173 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKζ for RNA interference <400> 173 gcaaggagat tgtggccatc a 21 <210> 174 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKζ for RNA interference <400> 174 gggcatcctt caagaggaag t 21 <210> 175 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKζ for RNA interference <400> 175 gcaaagatca tccagtcttt c 21 <210> 176 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKζ for RNA interference <400> 176 gctggagatg taccgcaaag t 21 <210> 177 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting DGKζ for RNA interference <400> 177 gcacaggatg agatttatat c 21 <210> 178 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting MerTK for RNA interference <400> 178 ggaagaccac atacaggaaa c 21 <210> 179 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting MerTK for RNA interference <400> 179 gcattggtgt ttcctgcatg a 21 <210> 180 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting MerTK for RNA interference <400> 180 gcacacggtt gggtagatta t 21 <210> 181 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting MerTK for RNA interference <400> 181 gggtctgtaa tggaaggaaa t 21 <210> 182 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting MerTK for RNA interference <400> 182 gcatgttgcg agatgacatg a 21 <210> 183 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting MerTK for RNA interference <400> 183 gcagaccgag tctacacaag t 21 <210> 184 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP1 for RNA interference <400> 184 gcaccatcat ccacctcaag t 21 <210> 185 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP1 for RNA interference <400> 185 ggtcacccac atcaaggtca t 21 <210> 186 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP1 for RNA interference <400> 186 gggcaagaac cgctacaaga a 21 <210> 187 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP1 for RNA interference <400> 187 ggaacaaatg cgtcccatac t 21 <210> 188 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP1 for RNA interference <400> 188 gcctggactg tgacattgac a 21 <210> 189 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP1 for RNA interference <400> 189 gaacctgcac actaagaaca a 21 <210> 190 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP2 for RNA interference <400> 190 gcagatccta cctctgaaag g 21 <210> 191 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP2 for RNA interference <400> 191 gcaatgacgg caagtctaaa g 21 <210> 192 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP2 for RNA interference <400> 192 ggaactgaaa tacgacgttg g 21 <210> 193 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP2 for RNA interference <400> 193 gcgtgttagg aacgtcaaag a 21 <210> 194 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP2 for RNA interference <400> 194 gctcatgact atacgctaag a 21 <210> 195 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting SHP2 for RNA interference <400> 195 ggagagaacg gtctggcaat a 21 <210> 196 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PP2A for RNA interference <400> 196 gcagctgaac gagaaccaag t 21 <210> 197 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PP2A for RNA interference <400> 197 ggagactgtg actcttcttg t 21 <210> 198 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PP2A for RNA interference <400> 198 ggaatgccaa cgtttggaaa t 21 <210> 199 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PP2A for RNA interference <400> 199 ggatcgttta caggaagttc c 21 <210> 200 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PP2A for RNA interference <400> 200 gcctttgtat gtggaagtat a 21 <210> 201 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting PP2A for RNA interference <400> 201 gcctgctgta tttatagtaa c 21 <210> 202 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD45 for RNA interference <400> 202 gctgcacatc aaggagtaat t 21 <210> 203 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD45 for RNA interference <400> 203 gctggaaata ctctggttag a 21 <210> 204 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD45 for RNA interference <400> 204 ggagcttgtt gaaagggatg a 21 <210> 205 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD45 for RNA interference <400> 205 gcagaatact ggccgtcaat g 21 <210> 206 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD45 for RNA interference <400> 206 ggttatgttg tcaagctaag g 21 <210> 207 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD45 for RNA interference <400> 207 gcagaaccca aggaattaat c 21 <210> 208 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD148 for RNA interference <400> 208 ggtgtaacat cacaggctta c 21 <210> 209 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD148 for RNA interference <400> 209 gccatagagt tcaggacaaa t 21 <210> 210 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD148 for RNA interference <400> 210 gcaattctcg ggtagaaata a 21 <210> 211 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD148 for RNA interference <400> 211 gctggcttca ccaacattac c 21 <210> 212 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD148 for RNA interference <400> 212 gggagcaaat catctgcatt c 21 <210> 213 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting CD148 for RNA interference <400> 213 gcgactcaaa tatcaccttg a 21 <210> 214 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting IL-10Rα for RNA interference <400> 214 gctcctgagg tatggaatag a 21 <210> 215 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting IL-10Rα for RNA interference <400> 215 gcaaatgaca catatgaaag c 21 <210> 216 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting IL-10Rα for RNA interference <400> 216 ggtctaaaga ggagtgcatc t 21 <210> 217 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting IL-10Rα for RNA interference <400> 217 gggctatttg aaacaggatc c 21 <210> 218 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting IL-10Rα for RNA interference <400> 218 gctgtaggaa tggaagcttc a 21 <210> 219 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Partial shRNA sequence included in shRNA targeting IL-10Rα for RNA interference <400> 219 gcttgtgttt gctgcttaatg t 21 <210> 220 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> mU6-shTIM-3-><-shPD-1-hU6 <400> 220 gttaacaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata 60 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta 120 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta 180 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct 240 ttatatatct tgtggaaagg acgaaacacc gcctgtggtt ctattatatt atttcaagag 300 aataatataa tagaaccaca ggtttttgac tagtcaaaaa ggaattcgct cagaagaaat 360 ctcttgaatt tcttctgagc gaattccaaa caaggctttt ctccaaggga tatttatagt 420 ctcaaaacac acaattactt tacagttagg gtgagtttcc ttttgtgctg ttttttaaaa 480 taataattta gtatttgtat ctcttataga aatccaagcc tatcatgtaa aatgtagcta 540 gtattaaaaa gaacagatta tctgtctttt atcgcacatt aagcctctat agttactagg 600 aaatattata tgcaaattaa ccggggcagg ggagtagccg agcttctccc acaagtctgt 660 gcgagggggc cggcgcgggc ctagagatgg cggcgtcgga tcgctagcca tatgtctaga 720 gtatac 726 <210> 221 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> shTIM-3-mU6<-->hU6-shPD-1 <400> 221 gttaaccaaa aacctgtggt tctattatat tattctcttg aaataatata atagaaccac 60 aggcggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag 120 ttacggtaag catatgatag tccattttaa aacataattt taaaactgca aactacccaa 180 gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat 240 taattctaat tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg 300 gaaataggcc ctcttcctgc ccgaccttac tagtgatccg acgccgccat ctctaggccc 360 gcgccggccc cctcgcacag acttgtggga gaagctcggc tactcccctg ccccggttaa 420 tttgcatata atatttccta gtaactatag aggcttaatg tgcgataaaa gacagataat 480 ctgttctttt taatactagc tacattttac atgataggct tggatttcta taagagatac 540 aaatactaaa ttattatttt aaaaaacagc acaaaaggaa actcacccta actgtaaagt 600 aattgtgtgt tttgagacta taaatatccc ttggagaaaa gccttgtttg gaattcgctc 660 agaagaaatt caagagattt cttctgagcg aattcctttt tggctagcca tatgtctaga 720 gtatac 726 <210> 222 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> shRNA cassette base sequence <220> <221> misc_feature <222> (323)..(343) <223> n is a, c, g or t <220> <221> misc_feature <222> (353)..(373) <223> n is a, c, g or t <400> 222 gttaacgatc cgacgccgcc atctctaggc ccgcgccggc cccctcgcac agacttgtgg 60 gagaagctcg gctactcccc tgccccggtt aatttgcata taatatttcc tagtaactat 120 agaggcttaa tgtgcgataa aagacagata atctgttctt tttaatacta gctacatttt 180 acatgatagg cttggatttc tataagagat acaaatacta aattattatt ttaaaaaaca 240 gcacaaaagg aaactcaccc taactgtaaa gtaattgtgt gttttgagac tataaatatc 300 ccttggagaa aagccttgtt tgnnnnnnnn nnnnnnnnnn nnnttcaaga gannnnnnnn 360 nnnnnnnnnn nnntttttgg ttaac 385 <210> 223 <211> 409 <212> DNA <213> Artificial Sequence <220> <223> mU6-shPD-1 MCS base sequence <400> 223 gttaacgatc cgacgccgcc atctctaggc ccgcgccggc cccctcgcac agacttgtgg 60 gagaagctcg gctactcccc tgccccggtt aatttgcata taatatttcc tagtaactat 120 agaggcttaa tgtgcgataa aagacagata atctgttctt tttaatacta gctacatttt 180 acatgatagg cttggatttc tataagagat acaaatacta aattattatt ttaaaaaaca 240 gcacaaaagg aaactcaccc taactgtaaa gtaattgtgt gttttgagac tataaatatc 300 ccttggagaa aagccttgtt tgcctgtggt tctattatat tatttcaaga gaataatata 360 atagaaccac aggtttttga ctagtgctag ccatatgtct agagtatac 409 <210> 224 <211> 328 <212> DNA <213> Artificial Sequence <220> <223> hU6-shPD-1 base sequence <400> 224 gttaacaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata 60 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta 120 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta 180 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct 240 ttatatatct tgtggaaagg acgaaacacc gcctgtggtt ctattatatt atttcaagag 300 aataatataa tagaaccaca ggtttttg 328 <210> 225 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> CD22-CAR <400> 225 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu Glu Asp Ala Phe Asp 100 105 110 Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 130 135 140 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 145 150 155 160 Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr Leu Asn Trp Tyr Gln 165 170 175 Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile Tyr Ala Ala Ser Ser 180 185 190 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Arg Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Phe Ala Thr 210 215 220 Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln Thr Phe Gly Gln Gly 225 230 235 240 Thr Lys Leu Glu Ile Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 245 250 255 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 260 265 270 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 275 280 285 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 290 295 300 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 305 310 315 320 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 325 330 335 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 340 345 350 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 355 360 365 Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 370 375 380 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 385 390 395 400 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 405 410 415 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 420 425 430 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 435 440 445 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 450 455 460 Leu Pro Pro Arg 465 <210> 226 <211> 486 <212> PRT <213> Artificial Sequence <220> <223> CD19-CAR <400> 226 Met Ala Leu Pro Val Thr Al...

Claims

1. A vector comprising: A base sequence encoding two types of short hairpin RNA (shRNA) that inhibits the expression of at least one gene that weakens immune cell function, wherein the first shRNA inhibits the expression of programmed death protein 1 (PD-1), and the second shRNA inhibits the expression of T cell immune receptor with Ig and ITIM domains (TIGIT), and A base sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen recognition domain that binds to a target antigen associated with cancer, wherein the target antigen is CD19, and wherein the cancer is a CD19-positive blood cancer. 2 . The vector according to claim 1 , wherein the expression of the two types of shRNAs is characterized in that they are regulated by two different promoters, respectively.

3. The vector according to claim 2, wherein the two promoters are RNA polymerase III promoters. The vector according to claim 2 , wherein the two promoters are U6 promoters derived from different species. The vector according to claim 2 , wherein the two promoters are oriented in different directions relative to each other on the vector.

6. The vector according to claim 1, wherein the gene that weakens the function of immune cells is an immune checkpoint receptor or ligand.

7. The vector according to claim 1, wherein in the two types of shRNA, the base sequence encoding the first shRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 2-11, and the base sequence encoding the second shRNA comprises a different sequence selected from the group consisting of SEQ ID NOs: 70-75.

8. The vector of claim 1, wherein the target of the CAR is a human tumor antigen that shows increased expression in cancer cells, cancer tissues and / or tumor microenvironment, or is a mutated form of an antigen found in cancer cells, cancer tissues and / or tumor microenvironment.

9. The vector according to claim 1, wherein the vector comprises the base sequence of SEQ ID NO: 220 or 221.

10. The vector according to claim 1, wherein the vector is a plasmid vector, a lentiviral vector, an adenoviral vector, an adeno-associated vector or a retroviral vector. 11 . An immune cell comprising the vector of claim 1 , wherein expression of the gene is reduced to 40% or less compared to expression in the absence of the shRNA. 12 . The immune cell according to claim 11 , wherein the immune cell is a T cell or a natural killer (NK) cell of human origin.

13. A pharmaceutical composition comprising the immune cell according to any one of claims 1-12.

14. The pharmaceutical composition according to claim 13, for use in treating a patient in need of immunotherapy, wherein the immune cells are initially obtained from the patient.

15. The pharmaceutical composition of claim 14, wherein the patient suffers from a tumor or cancer in which an increase or change in the target of the CAR and / or the target level expressed in the immune cells is detected.

16. An immune cell comprising a chimeric antigen receptor (CAR) that specifically binds to a target antigen, and a gene disruptor that reduces or is capable of reducing the expression of a gene that weakens the function of the immune cell in the immune cell, wherein the gene disruptor comprises a first shRNA that inhibits the expression of programmed death protein 1 (PD-1) and a second shRNA that inhibits the expression of a T cell immune receptor (TIGIT) having Ig and ITIM domains, and wherein the CAR comprises an extracellular antigen recognition domain that binds to a target antigen associated with cancer, wherein the target antigen is CD19, and wherein the cancer is a CD19-positive blood cancer.

17. An immune cell as described in claim 16, wherein the CAR comprises an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain.

18. The immune cell of claim 17, wherein the intracellular signaling domain of the CAR comprises the intracellular domain of the CD3 zeta (CD3ζ) chain.

19. An immune cell as described in claim 18, wherein the intracellular signal transduction domain of the CAR further comprises a co-stimulatory molecule.

20. The immune cell of claim 19, wherein the co-stimulatory molecule is selected from the group consisting of ICOS, OX40, CD137 (4-1BB), CD27, and CD28.

21. The immune cell of claim 20, wherein the co-stimulatory molecule is CD137 (4-1BB).

22. The immune cell of claim 20, wherein the co-stimulatory molecule is CD28.

23. The immune cell of any one of claims 16-22, wherein expression of the gene that impairs immune cell function results in one or more of the following: i) inhibiting the proliferation of the immune cells; ii) inducing cell death of the immune cells; iii) inhibiting the ability of the immune cells to recognize the target antigen and / or to activate; iv) inducing the immune cells to differentiate into cells that do not induce an immune response to the target antigen; v) reducing the reactivity of the immune cell to a molecule that promotes an immune response of the immune cell; or vi) increasing the reactivity of the immune cells with molecules that inhibit the immune response of the immune cells.

24. The immune cell of claim 23, wherein the gene that impairs immune cell function increases the response of the immune cell to a molecule that inhibits the immune response of the immune cell.

25. The immune cell of claim 24, wherein the gene that increases the response of the immune cell to a molecule that inhibits the immune response of the immune cell encodes an immune checkpoint receptor or ligand.

26. The immune cell of claim 16, wherein the gene disrupting agent reduces expression of a gene that impairs immune cell function in the immune cell by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% compared to an immune cell in the absence of the gene disrupting agent.

27. The immune cell of claim 26, wherein the gene disrupting agent reduces expression of a gene that increases the response of the immune cell to a molecule that inhibits an immune response of the immune cell.

28. The immune cell of claim 27, wherein the gene disrupting agent reduces expression of a gene encoding an immune checkpoint receptor or ligand.

29. The immune cell of claim 28, wherein the base sequence encoding the first shRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 2-11, and the base sequence encoding the second shRNA comprises a different sequence selected from the group consisting of SEQ ID NOs: 70-75.

30. The immune cell of any one of claims 28-29, wherein the nucleotide sequence encoding the shRNA is present on a vector.

31. The immune cell of claim 30, wherein the expression of different shRNAs is regulated by different promoters.

32. The immune cell of claim 31, wherein the expression of the two different shRNAs is regulated by two different promoters.

33. An immune cell as described in claim 32, wherein the two different promoters are RNA polymerase III promoters.

34. The immune cell of claim 33, wherein the two promoters are U6 promoters.

35. The immune cell of claim 34, wherein the U6 promoter is derived from a different species.

36. An immune cell as described in any one of claims 31-34, wherein the two promoters are oriented in different directions from each other.

37. An immune cell as described in claim 16, wherein the CAR and the gene disruptor are each expressed by a vector.

38. An immune cell as described in claim 37, wherein the CAR and the gene disruptor are expressed by the same vector.

39. The immune cell of any one of claims 37-38, wherein the vector is a plasmid vector or a viral vector.

40. The immune cell of claim 39, wherein the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

41. The immune cell of claim 40, wherein the lentiviral vector is a retroviral vector.

42. The immune cell of claim 16, wherein the immune cell is selected from the group consisting of T cells and natural killer (NK) cells.

43. The immune cell of claim 42, wherein the immune cell is a T cell.

44. The immune cell of claim 43, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

45. An immune cell as described in any one of claims 43 or 44, wherein the immune cell comprises a nucleotide sequence encoding two shRNAs and a CAR on the same vector.

46. ​​The immune cell of claim 45, wherein the two shRNAs are each regulated by two different RNA polymerase III promoters oriented in different directions from each other.

47. A method for producing immune cells, the method comprising simultaneously or sequentially in any order introducing into the immune cells: (1) A gene encoding a chimeric antigen receptor (CAR) that specifically binds to a target antigen, wherein The CAR comprises an extracellular antigen recognition domain that binds to a target antigen associated with cancer, wherein the target antigen is CD19, and wherein the cancer is a CD19-positive blood cancer; and (2) a gene disrupting agent, wherein the gene disrupting agent or its expression reduces or is capable of reducing the expression of a gene that weakens immune cell function in an immune cell, wherein the gene disrupting agent comprises a first shRNA that inhibits the expression of programmed death protein 1 (PD-1) and a second shRNA that inhibits the expression of T cell immune receptor with Ig and ITIM domains (TIGIT), Thereby, immune cells are generated in which the CAR is expressed and the expression of genes that impair the function of the immune cells is reduced.

48. A method as claimed in claim 47, wherein the CAR comprises an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain.

49. A method as claimed in claim 48, wherein the intracellular signaling domain of the CAR comprises the intracellular domain of the CD3 zeta (CD3ζ) chain.

50. The method of claim 49, wherein the intracellular signaling domain of the CAR further comprises a co-stimulatory molecule.

51. The method of claim 50, wherein the co-stimulatory molecule is selected from the group consisting of ICOS, OX40, CD137 (4-1BB), CD27, and CD28.

52. The method of claim 51, wherein the co-stimulatory molecule is CD137 (4-1BB).

53. The method of claim 51, wherein the co-stimulatory molecule is CD28.

54. The method of any one of claims 47-53, wherein expression of the gene that impairs immune cell function results in one or more of the following: i) inhibiting the proliferation of the immune cells; ii) inducing cell death of the immune cells; iii) inhibiting the ability of the immune cells to recognize the target antigen and / or become activated; iv) inducing the immune cells to differentiate into cells that do not induce an immune response to the target antigen; v) reducing the reaction of the immune cells to molecules that promote the immune response of the immune cells; or vi) increasing the reactivity of the immune cells with molecules that inhibit the immune response of the immune cells.

55. The method of claim 54, wherein the gene that impairs immune cell function increases the response of the immune cell to a molecule that inhibits the immune response of the immune cell.

56. The method of claim 55, wherein the gene that increases the response of the immune cell to a molecule that inhibits the immune response of the immune cell encodes an immune checkpoint receptor or ligand.

57. The method of claim 47, wherein the gene disrupting agent reduces expression of a gene that impairs immune cell function in 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.

58. The method of claim 57, wherein the gene disrupting agent reduces expression of a gene that increases the response of the immune cell to a molecule that inhibits an immune response of the immune cell.

59. The method of claim 58, wherein the gene disrupting agent reduces expression of a gene encoding an immune checkpoint receptor or ligand.

60. The method of any one of claims 57-59, wherein the base sequence encoding the first shRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 2-11, and the base sequence encoding the second shRNA comprises a different sequence selected from the group consisting of SEQ ID NOs: 70-75.

61. The method of claim 60, wherein the nucleotide sequence encoding the shRNA is present on a vector. The method of claim 61 , wherein the expression of different shRNAs is regulated by different promoters, respectively. The method of claim 62 , wherein the expression of the two different shRNAs is regulated by two different promoters, respectively.

64. The method of claim 63, wherein the two different promoters are RNA polymerase III promoters.

65. The method of claim 64, wherein the two promoters are U6 promoters.

66. The method of claim 65, wherein the U6 promoter is derived from a different species.

67. The method of any one of claims 63-66, wherein the two promoters are oriented in different directions relative to each other.

68. The method of claim 47, wherein the CAR and the gene disrupting agent are each expressed by a vector.

69. The method of claim 68, wherein the CAR and the gene disrupting agent are expressed by the same vector.

70. The method of any one of claims 68-69, wherein the vector is a plasmid vector or a viral vector.

71. The method of claim 70, wherein the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

72. The method of claim 71, wherein the lentiviral vector is a retroviral vector.

73. The method of claim 47, wherein the immune cells are selected from the group consisting of T cells and natural killer (NK) cells.

74. The method of claim 73, wherein the immune cell is a T cell.

75. The method of claim 74, wherein the T cells are CD4+ T cells or CD8+ T cells.

76. A method as described in claim 74 or 75, wherein the immune cell comprises a nucleotide sequence encoding two shRNAs and a CAR on the same vector.

77. The method of claim 76, wherein the two shRNAs are each regulated by two different RNA polymerase III promoters oriented in different directions from each other.

78. A composition comprising the immune cell of any one of claims 16-46.

79. A pharmaceutical composition comprising the immune cell of any one of claims 16-46 and a pharmaceutically acceptable carrier.

80. Use of the immune cell of any one of claims 16-46 or the composition of claim 78 or the pharmaceutical composition of 79 in the preparation of a medicament for treating a subject with a CD19-positive blood cancer in need of immunotherapy, wherein the CD19-positive blood cancer is acute lymphoblastic leukemia.

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