Multiplex genome editing of immune cells to enhance functionality and resistance to suppressive environment

Abstract

To provide compositions and methods related to cancer immunotherapy particularly including engineered immune cells.SOLUTION: Provided herein are methods for producing immune cells with disruption of multiple genes. Further provided are methods for inserting a chimeric antigen receptor into a locus of a gene of an immune cell.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application is a sequel to U.S. Provisional Patent Application No. 62 / 772,406, filed November 28, 2018. This provisional application claims priority to US Provisional Patent Application No. 2005 / 0133636, filed on May 13, 2005, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to the fields of immunology, cell biology, molecular biology and medicine. In particular, the present invention relates to multiplex editing of immune cells and methods of use thereof. [Background technology]

[0003] Cellular immunotherapy has great potential for treating cancer. When applied alone, immunotherapeutic approaches have been shown to be effective in treating the majority of malignancies, especially solid tumors. The reason for this limited success is that The low expression of tumor antigens in the tumors reduces the detection of tumor cells by the immune system. Inhibitory receptors that induce inactivation (e.g., PD1, NKG2A, TIGIT or CISH) and suppressing the immune response and inhibiting the expression of ligands for tumor cells. Substances that promote proliferation and survival (e.g., transforming growth factor-β (TGFβ ) and adenosine) (e.g., regulatory T cells or myeloid-derived suppressor Thus, cell immunotherapy is a promising approach to treat cancer. There is an unmet need for improved methods. Summary of the Invention

[0004] The present disclosure provides compositions and methods relating to cancer immunotherapy, particularly involving engineered immune cells. Specific embodiments are directed to the lack of expression of one, two or more genes. relates to certain immune cells that have been engineered by humans to reduce their expression, In specific cases, cells with such modifications may have non-naturally occurring modifications, such as antigen receptors. The non-native immune cells also express one or more heterologous proteins, including proteins of In certain cases, the introduction of a heterologous antigen receptor results in reduced or no expression. The gene expression vector is inserted or removed at the genomic locus of the gene to be deleted.

[0005] In one embodiment, the present disclosure provides a method for disrupting at least two genes in an immune cell. The present invention provides an in vitro method for detecting a NKG2A- or SIGLE-related gene that is involved in the expression of the NKG2A- or SIGLE-related gene. C-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD 96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIR PA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL2 1R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7 and In certain embodiments, the number of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 Or six or more genes are disrupted. The disruption of the genes is simultaneous, i.e., in the same method step. The method may include introducing a guide RNA (gRNA) for the target gene into immune cells.

[0006] The methods include, for example, a combination of specific genes, such as: (a) NKG2A and CISH, (b) NKG2A and TGFBRII, (c) CISH and TGFBRI I, (d) TIGIT and FOXO1, (e) TIGIT and TGFBRII, (f ) CD96 and FOXO1, (g) CD96 and TGFBRII, (h) FOXO1 and TGFBRII, (i) CD96 and TIGIT, (j) CISH and TIG IT, (k) TIM3 and CISH, (l) TIM3 and TGFBRII, (m) F OXO1 and TGFBRII, (n) TIM3 and TIGIT, (o) SIGLEC 7 and CISH, (p)SIGLEC7 and TGFBRII, (q)CD47 and CISH, (r) CD47 and TGFBRII, (s) SIRPA and CISH, ( (t) SIRPA and TGFBRII, (u) CD47 and TIGIT, (v) CD4 7 and SIRPA, (w) A2AR and CISH, (x) A2AR and TGFBR II, (y) ADAM17 and CISH, (z) TGFBRII and ADAM17, (a) A2AR and TIGIT, (b) SHP1 and CISH, (c) CISH and (d) SHP1 and TGFBRII, (e) SHP1 and TIG The method may include (f) knockdown of SHP1 and TIM3. 1) NKG2A, CISH and TGFBRII, (2) TIGIT, FOXO1 and TGFBRII, (3) TGFBRII, CD96 and TIGIT, (4) TGFBR 2, CISH and TIGIT, (5) TIM3, CISH and TGFBRII, (6 )CD96, FOXO1 and TGFBRII, (7)TGFBRII, TIM3 and TIGIT, (8) SIGLEC7, CISH and TGFBRII, (9) CD47, CISH and TGFBRII, (10)SIRPA, CISH and TGFBRII, (11) TGFBRII, CD47 and TIGIT, (12) TGFBRII, CD4 7 and SIRPA, (13) A2AR, CISH and TGFBRII, (14) TG FBRII, CISH and ADAM17, (15)TGFBRII, TIM3 and T IGIT, (16) TGFBRII, A2AR and TIGIT, (17) SHP1, C ISH and TGFBRII, (18)TGFBRII, CISH and SHP1, (1 9) TGFBRII, SHP1 and TIGIT, or (20) TGFBRII, SH Any of the above subgroups may include knockdown of P1 and TIM3. For example, the subgroups a to j1 may be combined with a second subgroup such as Any one of the subgroups may be combined with any one or more of the other subgroups a to j1. or one or more of subgroups a to j1 are not included in any of the other subgroups 1 to 23 or any one or more of subgroups 1 to 23 may be combined with It may be combined with any one or more of the other subgroups 1 to 23.

[0007] In some embodiments, the method comprises using an RNA-guided endonuclease such as Cas9. The introduction of an RNA-guided endonuclease into the cell may further include the step of: A nucleic acid (e.g., mRNA) encoding an RNA-guided endonuclease is added to the immune cells. The method may include introducing the

[0008] In certain embodiments, the immune cells are T cells, NK cells, B cells, macrophages, In an alternative case, the immune cell is a CAR T cell. In some embodiments, the immune cell is not a T cell, such as not expressing one or more chimeric antibodies. Expressing a carcinoma antigen receptor (CAR) and / or one or more T cell receptors (TCR) Immune cells are engineered to be virus-specific, such as virus-specific T cells. The T cells may be regulatory T cells. The B cells may be regulatory B cells. In some embodiments, the stem cells are mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPS). In certain embodiments, the T cells are CD8 + T cells, CD4 + T cells or moth The immune cells are derived from peripheral blood, umbilical cord blood, bone marrow, or a mixture of these. In some embodiments, the cord blood may be isolated from two or more individual cord blood units. These are pooled together.

[0009] In some embodiments, the introducing step comprises transfecting or transducing. For example, the introduction may be performed by electroporation more than once, for example, two or three electroporations. In some embodiments, the first group of CRISPR gR NA is introduced in the first electroporation, and the second group of CRISPR g RNA is introduced in a second electroporation. , the first group of CRISPR gRNAs is different from the second group of CRISPR gRNAs. In certain embodiments, the first and / or second group of CRISPR gRNAs comprise: In some embodiments, the CRISPR gRNA comprises one, two, three, or four or more CRISPR gRNAs. Two CRISPR gRNAs were introduced in the first electroporation. Two different CRISPR gRNAs were then introduced in the second round of electroporation. In a specific embodiment, a group of CRISPR gRNAs are introduced into a group of gRNAs. A, at least two of which target different genes, and in certain embodiments , each gRNA in the group targets a different gene.

[0010] In certain embodiments, the method comprises the steps of: detecting NKG2A, CD47, TGFβR2 and CIS. H;NKG2A, CISH, TGFβR2 and ADORA2;NKG2A, TGFβR 2 and CISH; TIGIT, CD96, CISH and ADORA2; or ADA The method includes disrupting M17, TGFβR2, NKG2A, and SHP1.

[0011] In some embodiments, the disruption results in enhanced anti-tumor cytotoxicity of immune cells, in vivo augmentation In certain embodiments, the method results in improved proliferation, in vivo persistence and / or function. of immune cells express IFN-γ, CD107 and / or TNF-α compared to those without modification. In some embodiments, the immune cells increase secretion of α in the absence of modification. Increased production of perforin and / or granzyme B compared with

[0012] In additional embodiments, the above methods involve introducing a CAR and / or a TCR into an immune cell. Steps (e.g., introducing nucleic acids encoding CAR and / or TCR into immune cells) In some embodiments, the nucleic acid further comprises a vector, such as a retroviral vector. In certain embodiments, the vector is an expression vector, such as AAV6. In some embodiments, the vector is an adenovirus-associated vector. 2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD 5, CD7, and combinations thereof. In a particular embodiment, the vector further comprises an inhibitory gene sequence. The CAR further comprises a guide RNA for the inhibitory gene. The CAR is flanked by homology arms for the inhibitory gene. In some embodiments, a vector containing a CAR sequence may be introduced. and C at the locus of an inhibitory gene in an immune cell (e.g., an exon of an inhibitory gene). The AR is inserted and the CAR is under the control of the endogenous promoter of the inhibitory gene. In certain embodiments, introduction of the vector further disrupts expression of an inhibitory gene.

[0013] In another embodiment, the immune cells (e.g., For example, an immune cell of the disclosed embodiments is provided, the immune cell comprising a nucleic acid sequence for each gene. and introducing a CRISPR guide RNA (gRNA) into the immune cells. At least two of the genes are NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, A DORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, S HIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, I CAM1, CD95, CD80, CD86, IL10R, CD5, CD7 and their In some embodiments, three, four, five or more of the above are selected from the group consisting of: Usually six or more genes are disrupted.

[0014] In certain embodiments, the immune cell is a T cell, a NK cell, a B cell, or a stem cell. In some embodiments, the immune cells are The vaccines are engineered to express the T cell receptor (TCR). The T cells may be virus-specific, such as virus-specific T cells. The T cells may be regulatory T cells. The cells can be regulatory B cells. In some embodiments, the stem cells are mesenchymal stem cells ( MSC) or induced pluripotent stem (iPS) cells. In certain embodiments, the T cells are D8 + T cells, CD4 + T cells or gamma-delta T cells. The immune cells are In some embodiments, the umbilical cord blood may be isolated from two or more units of umbilical cord blood or bone marrow. The samples are pooled from individual cord blood units.

[0015] In certain embodiments, the methods involve targeting a particular group of genes (e.g., NKG2A, CD47, TGFβR2 and CISH;NKG2A, CISH, TGFβR2 and ADORA2 NKG2A, TGFβR2 and CISH; TIGIT, CD96, CISH and A or ADAM17, TGFβR2, NKG2A, and SHP1) The method includes the step of:

[0016] In some embodiments, the disruption results in enhanced anti-tumor cytotoxicity of immune cells, in vivo augmentation In certain embodiments, the method results in improved proliferation, in vivo persistence and / or function. immune cells have increased secretion of IFN-γ, CD107 and / or TNFα In some embodiments, the immune cells express perforin and / or granzymes. It increases the production of B.

[0017] In some embodiments, the cells are engineered to express a CAR and / or a TCR. The CAR is engineered, for example, by inserting it into the locus of an endogenous inhibitory gene of the cell. The loci of the inhibitory genes may be NKG2A, SIGLEC-7, LAG3, TIM 3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR 3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADA M17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD9 5, CD80, CD86, IL10R, CD5, CD7, and combinations thereof In some embodiments, the CAR is selected from the group consisting of an endogenous promoter of an inhibitory gene. In certain embodiments, the CAR is under the control of a CRISPR-mediated gene. By child editing, it is inserted into the locus of an inhibitory gene.

[0018] In some embodiments, the CAR is an F(ab')2, Fab', Fab, Fv, and In certain embodiments, the antigen-binding domain is selected from the group consisting of: CAR is a marker that targets CD19, CD319 (CS1), ROR1, CD20, carcinoembryonic antigen, and alpha. afetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, Atypical p53, mutant ras, HER2 / Neu, ERBB2, folate-binding protein, HI V-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp 41, GD2, CD5, CD123, CD23, CD30, CD56, c-Met, Meso Thelin, GD3, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSP G4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47 , CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99 and combinations thereof. In certain embodiments, the CAR is selected from the group consisting of CD3ξ, CD28, OX40 / CD134, 4- 1BB / CD137, FcεRIγ, ICOS / CD278, ILRB / CD122, I L-2RG / CD132, DAP12, CD70, CD40 and their combinations In some embodiments, the signaling domain comprises at least one selected from the group consisting of: In the present study, immune cells are transduced with one or more heterologous cytokines (e.g., IL-7, IL-2, I In certain conditions, the IL-15, IL-12, IL-18, and IL-21 are included. In these cases, CAR binds membrane-bound non-secreted TNF-alpha mutants or inducible caspases. and further suicide genes such as Ze9.

[0019] At least one CAR and / or TCR, at least one inhibitory gene sequence, etc. Further provided herein is an expression vector encoding at least one gRNA. In some embodiments, the inhibitory gene sequence is NKG2A, SIGLEC- 7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96 , ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA , SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R , ICAM1, CD95, CD80, CD86, IL10R, CD5 and CD7 In certain embodiments, the gRNA is derived from an inhibitory gene selected from the group consisting of: In some embodiments, the vector is a viral vector, such as an AAV vector. CAR is a vector that is flanked by homology arms to an inhibitory gene. A host cell engineered to express the vector of the above embodiment (e.g., Also provided herein are cells of the form of a T cell, a NK cell, or a combination thereof. , B cells or stem cells.

[0020] Also provided herein are pharmaceutical compositions comprising the populations of immune cells of the disclosed embodiments. Another embodiment is a method for treating an immune-related disorder, an infection, and / or a cancer, comprising administering to a subject the disclosed A composition comprising a population of cells of the embodiments is provided.

[0021] In a further embodiment, a method of treating a disease or disorder in a subject is provided, the method comprising: The method includes administering to the subject an effective amount of immune cells of the disclosed embodiments. In some embodiments, the disease or disorder is an infectious disease, cancer (e.g., a solid cancer or a hematological malignancy), The immune-related disorder is, for example, an autoimmune disorder, graft-versus-host disorder, or a combination of these. The primary disease may be an allograft rejection or an inflammatory condition. In some embodiments, the primary disease may be an immune-related disorder. The harm is an inflammatory state in which immune cells inherently express glucocorticoid receptors. In certain embodiments, the immune cells are autologous or allogeneic to the recipient individual. It is of a different lineage.

[0022] In an additional embodiment, the method further comprises administering to the individual receiving the immune cells at least a second treatment. In some embodiments, the at least a second therapeutic agent is administered to the patient. includes chemotherapy, immunotherapy, surgery, radiation therapy, hormonal therapy or biological therapy. In certain embodiments, the immune cells and / or at least a second therapeutic agent are administered intravenously. intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesional, percutaneously, subcutaneously The administration may be intravenously, regionally, or by direct injection or perfusion.

[0023] Another embodiment provides a method for engineering immune cells to express a CAR, The method uses CRISPR gRNA to target the CAR to an inhibitory gene in the immune cell. In some embodiments, the CAR is inserted into the offspring locus. (e.g., retroviral vectors, plasmids, lentiviral vectors, adenovirus vectors, The vector is encoded by a vector associated with a virus (e.g., a viral vector, an adenovirus-associated virus vector, etc.). In certain embodiments, the viral vector is an adenovirus-associated vector, such as AAV6. It is.

[0024] In some embodiments, the vector is an NKG2A, SIGLEC-7, LAG3 , TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA 2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1 , ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1 , CD95, CD80, CD86, IL10R, CD5, CD7 and combinations thereof The present invention further includes an inhibitory gene sequence, such as an inhibitory gene sequence selected from the group consisting of: In some embodiments, the CRISPR gRNA is directed to an inhibitory gene. In certain embodiments, the CAR is flanked by homology arms to an inhibitory gene. In certain embodiments, the CAR is any portion of an inhibitory gene (e.g., The CAR is inserted into the locus of the inhibitory gene in the exon In a specific embodiment, the CAR can be under the control of an endogenous promoter of the gene. It disrupts gene expression.

[0025] In some embodiments, the CAR is CD19, CD319 (CS1), ROR1, C D20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor Tumor antigens, melanoma-associated antigens, mutant p53, mutant ras, HER2 / Neu, ERBB2 , folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 E Envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R alpha, Upper chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEG FR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAF One or more selected from the group consisting of FR, BCMA, CD99 and combinations thereof In certain embodiments, the CAR targets tumor-associated antigens such as CD3ξ, CD28, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278 , ILRB / CD122, IL-2RG / CD132, DAP12, CD70 and CD 40. In some embodiments, the vector encoding the CAR is an antibody that encodes a cytokine (e.g., IL-7, I IL-2, IL-15, IL-12, IL-18, IL-21 or a combination thereof) In an alternative embodiment, the cytokine is a vector encoding a CAR. In certain embodiments, the expression construct encoding the CAR is on a separate vector. The construct may be a transcription factor that encodes a suicide gene (e.g., inducible caspase 9 or membrane-bound non-secreted TNF-α). fa variants).

[0026] At least one inhibitory gene in an immune cell, such as an immune cell produced by the method. Further provided herein is an immune cell having a CAR inserted therein. populations of immune cells (e.g., T cells, B cells, NK cells, NKT cells, macrophages, Also provided herein are compositions comprising the stem cell populations, populations including mixtures thereof.

[0027] Another embodiment provides a composition comprising a population of cells of the above embodiment, and in certain embodiments In this state, the population may be used for the treatment of any type of medical condition, including at least immune-related disorders. , for the treatment of infectious diseases and / or cancer, etc.

[0028] Further embodiments provide a method of treating a disease or disorder in a subject, the method comprising: In some aspects, the method further comprises administering to the subject an effective amount of an immune cell of the above embodiments. In one embodiment, the disease or disorder is an infectious disease; cancer (e.g., a solid cancer or a hematological malignancy); and / or immune-related disorders. Immune-related disorders, in some cases, include autoimmune disorders. The disorder may be graft-versus-host disease, allograft rejection and / or inflammatory conditions. In an embodiment, the immune-related disorder is an inflammatory condition and the immune cells are In certain embodiments, the immune cells are essentially free of expression of the recipient's own antigen. It may be autologous or allogeneic to the body.

[0029] In additional embodiments, the methods further comprise administering to the individual at least a second therapeutic agent. In some embodiments, the at least second therapeutic agent includes chemotherapy, immunotherapy, or a combination thereof. Therapeutic methods include chemotherapy, surgery, radiation therapy, hormone therapy, or biological therapy. In the method, the immune cells and / or at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumoral, intramuscular, endoscopic, intralesional, percutaneous, subcutaneous, regional, or The immune cells and at least a second therapeutic agent are administered by direct injection or perfusion. , may be administered simultaneously or at different times, and they may be administered at different When administered at the same time, or simultaneously but not in the same formulation The two agents may or may not be administered by the same route.

[0030] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, from this detailed description, those skilled in the art will appreciate that various modifications and variations within the spirit and scope of the present disclosure are possible. Because various modifications and variations will become apparent, the detailed description and specific examples are provided to illustrate, but not to limit the scope of the present disclosure. are shown, but it should be understood that they are given by way of example only.

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention is understood to be encompassed by the following claims in conjunction with the detailed description of specific embodiments presented herein. The present invention may be better understood by reference to one or more of these drawings. [Brief description of the drawings]

[0032] [Figure 1] CRISPR / Cas9 mediates efficient disruption of multiple genes (NKG2A, CD47, TGFBR2 and CISH) in NK cells. For this gene set, NKG2A and CD47 were knocked out in the first electroporation, and CISH and TGFBR2 were targeted in the second electroporation. For both electroporation rounds, knockout efficiency was successfully verified using PCR and flow cytometry. The red (right peak) and blue (left peak) histograms in the flow panel represent the expression of proteins before and after CRISPR KO, respectively.

[0033] [Diagram 2] Validation of multiplex gene editing in NK cells using another set of genes (TIGIT (T), CD96 (C), CISH (CH), Adenosine (A)). In this gene set, TIGIT and CD96 were knocked out in the first electroporation, and CISH and Adenosine were targeted in the second electroporation. For both electroporation rounds, knockout efficiency was successfully verified using PCR and flow cytometry. The red (right peak) and blue (left peak) histograms in the flow panel represent the expression of proteins before and after CRISPR KO, respectively.

[0034] [Diagram 3]Disruption of multiple genes (NKG2A, CD47, TGFBR2 and CISH) in NK cells enhances their function against target tumor cells. After stimulation with target cell lines, secretion of IFN-γ, TNFα and CD107 was increased. Flow cytometry analysis of IFN-γ, TNFα and CD107 production was performed using various NK cells (edited vs. Cas9 only) co-stimulated with target cell lines in the presence of Brefeldin A for 5 hours.

[0035] [Figure 4A] Disruption of multiple genes (NKG2A, CD47, TGFBR2 and CISH) in NK cells enhances antitumor cytotoxicity. Cytotoxic activity of gene-edited NK cells versus Cas9-only NK cells was measured by 51Cr release assay against K562. [Figure 4B] Disruption of multiple genes (NKG2A, CD47, TGFBR2 and CISH) in NK cells enhances antitumor cytotoxicity. pSMAD activity was measured by flow cytometry 30 minutes after recombinant TGF-B treatment (50ng / ml). Addition of exogenous TGF-β did not induce pSMAD activation in KO CAR-NK cells.

[0036] [Diagram 5] As shown by CyTOF analysis, NK cells lose expression of CD16 and CD62L upon cytokine stimulation or target recognition.

[0037] [Figure 6] Knocking out ADAM17 in NK cells prevents shedding of CD16 and CD62L.

[0038] [Figure 7] Knocking out ADAM17 in NK cells improves ADCC and cytotoxicity against K562 targets.

[0039] [Figure 8] FACS-based screening of SHP1 knockout efficiency in NK cells after 72 h.

[0040] [Figure 9] Disruption of SHP1 in NK cells enhances antitumor effects. NK cells were co-cultured with K562 or Raji cells at a 1:1 ratio for 4 hours. After incubation, the cells were stained with Annexin V and analyzed for live and dead cells. K562 cells are sensitive to NK cell killing, and Raji cells are resistant to NK cell killing.

[0041] [Figure 10A] Disruption of SHP1 in NK cells enhances antitumor effects. NK cells were co-cultured with K562 or Raji cells at a 2:1 ratio for 5 h. [Figure 10B] Disruption of SHP1 in NK cells enhances antitumor efficacy. Percent lysis, IFNγ, TNFα and CD107a, and percentage of live or dead cells at various effector:target ratios are shown.

[0042] [Figure 11] Disruption of SHP1 in NK-CAR cells enhances antitumor efficacy as assessed by apoptosis assays.

[0043] [Figure 12A] FACS-based NKG2A knockout efficiency on day 7. [Figure 12B] Disruption of NKG2A in expanded NK cells enhances antitumor efficacy. [Figure 12C] Disruption of NKG2A in NK-CAR cells enhances the antitumor effect against Raji targets.

[0044] [Figure 13]Another set of genes was used to validate the approach: TIGIT (T), CD96 (C), CISH (CH) and adenosine (ADORA2A) (A). For this set of genes, TIGIT and CD96 were knocked out in one set of NK cells during the first round of electroporation. For the second round of knockout in TIGIT and CD96 KO cells, CISH and adenosine (ADORA2A) were targeted. For both rounds of electroporation, the knockout efficiency was successfully validated using PCR and flow cytometry. The red (right peak) and blue (left peak) histograms in the flow panels represent the expression of the proteins before and after CRISPR KO, respectively.

[0045] [Figure 14] Disruption of multiple genes in NK cells enhances antitumor efficacy. To assess this, knockout cells of multiple genes (NKG2A, CISH, TGFBRII and adenosine (ADORA2A)) and cells electroporated with cas9 alone were used for 5 hours along with K562 (NK-sensitive) and Raji (NK-resistant) cells as controls. NK cell function was assessed by flow cytometry, and increased TNFα, IFNγ and CD107a were observed in the KO cells upon stimulation with the target cell line.

[0046] [Figure 15] Disruption of multiple genes in NK cells enhances antitumor effects. To evaluate this, knockout cells of multiple genes (NKG2A, CISH, TGFBRII) and cells electroporated with cas9 alone were used as controls. NKG2A expression was confirmed by flow cytometry. Addition of exogenous TGF-β did not induce pSMAD activation in KO CAR-NK cells.

[0047] [Figure 16]Disruption of multiple genes (NKG2A, TGFβR2 and CISH) in NK-CAR cells enhances the antitumor effect.

[0048] [Figure 17] KO of TGFβR2 protects NK-CAR cells from the inhibitory effects of TGFβ.

[0049] [Figure 18] Multiplex gene editing is reproducible with different NK-CAR constructs and against different targets.

[0050] [Figure 19] Multiplex gene editing of multiple inhibitory genes maintains NK structure and protects NK cells from TFGβ-induced exhaustion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] In certain embodiments, the present disclosure provides a method for the production of human Immune cells (e.g., T cells, NK cells, CAR-transduced T cells or CAR-expressing Two or more genes (e.g., genes (e.g., adenosine triphosphate (ATP)) in transduced NK cells) The expression of NKG2A, TIGIT and / or CISH was The genes listed in Table 1 above were simultaneously knocked down (or knocked out). These immune cells are derived from peripheral blood or umbilical cord blood or their It may result from a combination.

[0052] This study demonstrated that low expression of these proteins improved the function of T cells and NK cells, and inhibited the innate immune system. It has been demonstrated that the cytotoxicity correlates with proliferation and persistence in vivo. Lategy also stimulated T cells, NK cells, NKT cells and iNKT cells with TGFβ and Protects against the immunosuppressive tumor microenvironment that is primarily driven by adenosine. The method can be used to generate a variety of adoptive cell therapy products (e.g., NK cells, T cells ( virus-specific T cells and regulatory T cells), B cells (e.g., regulatory B cells) , CAR-transduced NK cells, CAR-T cells, as well as TCR-engineered It can improve the efficacy of T and NK cells, iNKT cells, and NKT cells. The products for adoptive cell therapy include, for example, cancer (e.g., hematological malignancies or solid malignancies) These compounds can be used to treat a variety of diseases ranging from chronic inflammation to infections and immune disorders.

[0053] In certain embodiments, the immune cells express at least one CAR. There have been several advances in R technology over the past few years. It has shown impressive clinical results in patients with follicular leukemia and lymphoma, and was awarded two CAR T products have been approved by the FDA. CAR-transduced T cells have become We have taken the lead in numerous preclinical studies, as well as Phase I / II and C studies led by the applicants. The AR NK trial also demonstrated the efficacy of CAR-NK cells against cancer. Despite this, CARs are still mostly delivered to T cells or The NK cells are transduced with the viral vector, which randomly integrates into the DNA of the cells. It does not integrate, resulting in clonal proliferation, oncogenesis, altered expression of the transgene, or transcriptional silencing. Therefore, the insertion of the CAR into a specific DNA locus may result in immunization. It would be beneficial to find a way to target it.

[0054] Thus, in one embodiment, the present disclosure provides a method for the production of a gene encoding a gene for a cancer cell using CRISPR / Cas9, comprising: The locus of a particular gene (e.g., the locus of an inhibitory gene or checkpoint protein) The present invention provides a method for inserting a CAR into a gene locus. The insertion may optionally place the CAR under the control of the promoter of the gene, if desired. It can also be used to inhibit the expression of a gene while at the same time inhibiting its expression. The method uses AAV6 vectors and CRISPR / Cas9 technology to express an inhibitory gene ( For example, NKG2A, CISH, PD-1, TIGIT, TIM3, SHP1 or TG at the locus of genes such as those listed in Table 1, including but not limited to FβR2 Insertion of the CAR at the locus of the inhibitory gene can lead to the insertion of a CAR that R expression is under the control of checkpoint promoters, and apoptosis is observed in the tumor microenvironment. It also allows the expression of β-lactamase inhibitors (e.g., β-lactamase inhibitors) to be upregulated while also suppressing the inhibitory effect of checkpoint molecules (e.g., This is useful when applying CAR therapy to solid tumors. Here, upregulation of checkpoint molecules has a detrimental effect on the success of CAR therapy. Therefore, it is desirable to use a CAR insertion method that has a high safety profile. to generate adoptive cell therapy (e.g., T cells, B cells, NK, NKT or iNKT cells). Further methods for determining whether a I. Definition

[0055] As used herein, "essentially free" with respect to a particular component means that Certain components are not intentionally formulated into the composition and / or are not intended to be contaminants. "Amended" is used herein to mean absent even in trace amounts. Therefore, the total amount of a particular component resulting from any unintentional inclusion of a composition is , less than 0.05%, preferably less than 0.01%. Standard analytical methods can be used to determine Most preferred are compositions in which the amount of the component is undetectable.

[0056] As used herein, "a" or "an" can mean one or more. When used in a paragraph, the words "a" or "an" may be used in conjunction with the word "including." As used herein, "another" can mean one or more than one. can mean at least a second or more. "Having", "including", "containing" g)" and "comprising" are interchangeable and a person skilled in the art will understand It is understood that these terms are open ended terms. In the present disclosure, an embodiment may, for example, "consist essentially of" one or more sequences of the present disclosure. Some embodiments of the present invention may include one or more of the methods disclosed herein. may consist of or consist essentially of elements, method steps and / or methods Any of the methods or compositions described herein may be used in combination with other methods described herein. It is contemplated that the present invention may be practiced with any method or composition of matter disclosed herein. Particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As used herein, the terms "or" and "or" are used interchangeably herein. and "and / or" refer to the combination or exclusion of multiple elements. For example, "x, y and / or z" means "x" only and "y" Only "z"; "x, y and z", "(x and y) or z", "x or (y and It can refer to "x and z" or "x or y or z". It is expressly contemplated that certain features may be specifically excluded from the embodiments.

[0057] Use of the term "or" in the claims refers to either or, unless expressly indicated to refer to alternatives only. or used to mean "and / or" unless the alternatives are mutually exclusive. However, the present disclosure supports the definition referring to only the alternative and "and / or." As used herein, "another" means at least a second or more. The terms "about," "substantially," and "approximately" generally refer to The deviation means a value plus or minus 5%.

[0058] Throughout this specification, the terms "one embodiment," "an embodiment," "particular embodiment," "particular embodiment," "particular "Related embodiments," "certain embodiments," "additional embodiments," or "further embodiments" Reference to an "embodiment" or combination thereof is made with respect to that embodiment. It is understood that a particular feature, structure, or characteristic is included in at least one embodiment of the present disclosure. Accordingly, the appearance of the foregoing phrases in various places throughout this specification means: Not all of the above necessarily refer to the same embodiment. , structures or features may be combined in any suitable manner in one or more embodiments. .

[0059] "Immune disorder," "immune-related disorder," or "immune-mediated disorder" means a disorder in which an immune response is involved in the pathogenesis of the disease. Immune-mediated disorders are disorders that play a key role in the development or progression of the disease. These include autoimmune disorders, allograft rejection, graft-versus-host disease, and inflammatory conditions and allergies. - status can be mentioned.

[0060] An "immune response" refers to the reaction of the immune system, such as B cells or T cells or innate immune cells, to a stimulus. In one embodiment, the response is specific to a particular antigen ("antibody"). “antigen-specific response”).

[0061] As used herein, the term "inhibitory gene" refers to a gene whose product inhibits one or more directly or indirectly detrimental to the activity, proliferation and / or persistence of any type of immune cell. This refers to genes that

[0062] An "autoimmune disease" is a condition in which the immune system reacts to antigens that are part of the normal host (i.e., self-antigens). ) resulting in an immune response (e.g., a B cell response or a T cell response) to the Autoantigens can be derived from host cells or from commensal organisms. (e.g., caused by microorganisms that normally colonize mucosal surfaces (known as commensals)) It could come.

[0063] As used herein, the term "engineered" refers to a cell, a nucleic acid, a polypeptide, a vector, It refers to entities created by human hands, such as the . In some cases, engineered entities are synthetic and contain non-naturally occurring elements or or elements configured for use in the present disclosure.

[0064] "Treating" a disease or condition means alleviating the signs or symptoms of the disease. and executing a protocol that may include administering one or more drugs to the patient for the purpose of alleviating The desired effect of treatment may include slowing the progression of the disease, reversing the disease state, or Relief includes alleviation, and remission or improvement in the prognosis. Relief refers to the reduction or amelioration of the symptoms of a disease or condition that is present. This can occur before as well as after the signs or symptoms appear. Or "treatment" can mean "preventing" a disease or undesirable condition or the "prophylaxis" thereof. Additionally, "treat" or "treatment" may include any condition that requires complete relief of a sign or symptom. Protocols that do not require any specific treatment, do not require any cure, and in particular have only a marginal effect on the patient. Includes

[0065] The term "therapeutic effect" or "therapeutically effective" as used throughout this application means It refers to anything that enhances or improves the well-being of a subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer can include, for example, reducing tumor size, reducing the invasiveness of tumors, Treatment of cancer may include reducing the rate of growth of a cancer cell, or preventing metastasis. It may also refer to an extension of survival time.

[0066] "Subject" and "patient" refer to a human or non-human subject, e.g., a primate, mammal, or It refers to a vertebrate animal. In certain embodiments, the subject is a human.

[0067] As used herein, a "mammal" is a suitable subject for the methods of the present invention. A mammal may be any member of the higher vertebrate class Mammalia, including humans, and may be a live-born They may be characterized by birth, body hair, and the mammary glands in females, which secrete milk to feed their young. In general, mammals are characterized by their ability to maintain a constant body temperature despite variations in climatic conditions. Examples of mammals include humans, cats, dogs, cows, mice, rats, horses, goats, sheep and and chimpanzees. The mammals are referred to as "patients" or "subjects" or "individuals." This may happen.

[0068] The phrase "pharmacologically or pharmacologically acceptable" refers to a compound that is suitable for administration to an animal, such as a human. and a molecular entity that does not produce adverse, allergic or other untoward reactions when administered to a subject. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients is described in the present disclosure. In addition, for administration to animals (e.g., humans), the preparation is required by the FDA Office of Biological Standards. It is understood that the materials must meet the sterility, pyrogenicity, general safety and purity standards prescribed by the do.

[0069] As used herein, a "pharmaceutically acceptable carrier" is a compound known to those skilled in the art. As stated above, any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, food saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous Solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectables organic esters, e.g., ethyl oleate), dispersion media, coatings, surfactants, acids antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents and inert gas), isotonicity agent, absorption retardant, salt, drug, drug stabilizer, gel, binder, excipient, disintegrant, Lubricants, sweeteners, flavoring agents, colorants, flow and nutritional supplements, and similar materials; The pH and exact concentration of the various components in the pharmaceutical composition is adjusted according to known parameters.

[0070] As used herein, a "disruption" of a gene refers to the absence of the disruption in the gene product. The expression level of one or more genes encoded by a gene of interest in a cell is compared. This refers to the absence or reduction of expression of a gene product. Exemplary gene products include: The destruction of the gene includes the mRNA and protein products encoded by the gene. In some cases it is transient or reversible, in others it is permanent. The disruption is due to some In some cases, the destruction of a functional or full-length protein or mRNA. In some embodiments herein, the activity or function of a gene is distinct from its expression. Gene disruption is generally achieved by artificial means, i.e., the introduction of chemical compounds, molecules, or complexes. or the addition or introduction of a composition and / or a nucleic acid of the gene or the gene It is induced by disruption of the nucleic acid associated with the gene, e.g. at the DNA level. Exemplary methods include gene silencing, knockdown, knockout, and and / or gene disruption techniques such as gene editing. Antisense technologies (e.g., RNAi, siRNA, shRNA, and and / or ribozymes), and, for example, by inducing cleavage and / or homologous recombination. These include gene editing techniques that result in targeted gene inactivation or disruption. Examples include insertions, mutations and deletions. Disruptions are usually caused by a gene that is encoded by Block and / or completely suppress expression of the normal or "wild type" product that is upregulated by the Examples of such gene disruptions include insertions of genes or parts of genes, Shift and missense mutations, deletions, knock-ins and knock-outs (whole gene Such disruptions can include deletions of the coding region, e.g., one or more exons. In some cases, the insertion of a stop codon may result in a full-length product, a functional product, or Such a disruption prevents transcription of the gene, making it unable to produce any product. in promoters or enhancers or other regions that affect transcriptional activation Gene disruption can also occur by targeted gene inactivation via homologous recombination. These include gene targeting, including transcription. II. Multiplex gene editing

[0071] In certain embodiments, the present disclosure provides a method for the detection of multiplexed immune cells of any type. Regarding gene editing, CRISPR has been used to edit two or more genes (e.g., 3 , 4, 5, 6, 7, 8, 9, 10 or more genes) These genes include those listed in Table 1, e.g., NK cell receptor A (NKG2A), sialic acid-binding Ig-like lectin 7 (SIGLEC- 7, CD328), lymphocyte activation 3 (LAG3), T cell immunoglobulin mucin family Member 3 (TIM3, CD366, HAVCR2), cytokine-inducible SH2-containing Proteins (CISH, CIS-1, SOCS), Forkhead box O1 (FO XO1), transforming growth factor beta receptor 2 (TGFβR2), Ig and and ITIM domain-containing T cell immunoreceptor (TIGIT), CD96, adenosine Receptor 2A (ADORA2), nuclear receptor subfamily 3 group C member 1 (NR3C1), programmed cell death 1 (PD1), programmed cell death 1 ligand 1 (PD L-1), programmed cell death 1 ligand 2 (PDL-2), CD47, signal-regulating protein Spirulin alpha (SIRPA), SH2 domain-containing inositol 5-phosphatase 1 (SHIP1), ADAM metallopeptidase domain 17 (ADAM17), ribosomal RPS6 (receptor protein S6), eukaryotic translation initiation factor 4E-binding protein 1 (4EBP 1) CD25, CD40, interleukin 21 receptor (IL21R), intercellular Interleukin-21 receptor 1 (ICAM1), CD95, CD80, CD86, and The inhibitory genes may be selected from the group consisting of IL10R, CD5, CD7, or other inhibitory genes. Gene editing could make it possible to disrupt the expression of multiple genes simultaneously. .

[0072] In some embodiments, the gene disruption results in a disruption in the gene. (e.g., knockouts, insertions, missense or frameshift mutations, e.g., both Allelic frameshift mutations, deletions of all or part of a gene, e.g., one or more exons This can be achieved by gene deletion (or partial deletion, and / or knock-in) of the gene. For example, The disruption is specifically designed to target the sequence of the gene or a portion thereof. , zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases DNA-binding targeted nucleases such as TALENases, and CRISPR association Sequence-specific nucleases, including RNA-guided nucleases such as Cas, are The targeting may be effected by a nuclease or a targeted nuclease.

[0073] In some embodiments, the disruption is transient or reversible, and the expression of the gene In other embodiments, the disruption is not reversible or transient, For example, it is persistent.

[0074] In some embodiments, gene disruption occurs in the gene, usually in a targeted manner. by the induction of one or more double-strand breaks and / or one or more single-strand breaks in In some embodiments, double-stranded or single-stranded cleavage is induced by nucleases, e.g. In some embodiments, this is accomplished by an endonuclease, such as a gene-targeting nuclease. In such cases, the cleavage is induced in the coding region of the gene, e.g., an exon. For example, in some embodiments, the induction occurs in a coding region, e.g., a first exon. The nucleotide sequence may occur near the N-terminal portion of the second or subsequent exon.

[0075] The immune cells are then either injected with guide RNA and CRISPR enzyme or with CRISPR enzyme. In some embodiments, the cells can be provided with one, two, or three mRNA encoding the IL-1 ... One, three, four, five or more guide RNAs can be introduced simultaneously. For example, Cells were injected with one, two or three guide RNAs during the first electroporation. Then, one, two or three may be introduced during a second electroporation, etc. Additional guide RNAs may further be introduced.

[0076] In some embodiments, gene disruption is achieved by antisense methods (e.g., RNA interference ( RNAi), small interfering RNA (siRNA), short hairpin (shRNA) and / or This is achieved by using ribozymes, which selectively regulate gene expression. siRNA technology involves the nucleotide substitution of mRNA transcribed from a gene. A double-stranded RNA fragment having a sequence homologous to the nucleotide sequence and a sequence complementary to that sequence. siRNA is generally used to target the mRNA transcribed from a gene. Multiple RNAs that are homologous / complementary to one region or that are homologous / complementary to different regions In some embodiments, the siRNA may comprise a polysynaptic A molecule. It is included in the Tronic construct.

[0077] In some embodiments, the disruption specifically binds to or hybridizes with the gene. DNA targeting molecules (e.g., DNA binding proteins or DNA binding nucleic acids) that or a complex, compound or composition comprising the same. In the present invention, the DNA targeting molecule comprises a DNA binding domain, e.g., a zinc finger protein. DNA binding domains of ZFPs, DNA of transcription activator-like proteins (TALs) Binding domain or DNA binding domain of TAL effector (TALE), cluster CRISPR DNA-binding domains, or the DNA binding domain of a meganuclease. The binding domains of the CRISPR system can be, for example, naturally occurring zinc fingers or or via engineering (changing one or more amino acids) the recognition helix region of the TALE protein. The engineered DNA binding tag can be engineered to bind to a given nucleotide sequence. Zinc finger proteins (or TALEs) are non-naturally occurring proteins. Rational criteria for design include application of substitution rules and the use of existing ZFPs and and / or in a database that stores information about TALE designs and binding data. Examples of such algorithms include computer algorithms for processing information.

[0078] In the case of CRISPR-mediated destruction, the guide RNA and endonuclease are or by any means known in the art that allows delivery to the inside of an intracellular compartment. Thus, agents / chemicals and / or molecules (tasks) that can be introduced into immune cells and used Non-limiting examples of delivery vehicles include liposomes, polymeric capsules, and nucleic acids. ria, chemical carriers, lipoplexes, polyplexes, dendrimers, nanoparticles, enzymes fusion, natural endocytosis or phagocytosis pathways, as well as electroporation In a specific embodiment, electroporation is used. A guide RNA and an endonuclease or a nucleic acid encoding an endonuclease. Introduce the following.

[0079] In one exemplary specific method, a method for CRISPR knockout of multiple genes is provided. The method may include the isolation of immune cells, such as NK cells, from umbilical cord blood or peripheral blood. The cells are isolated and cultured on culture plates with irradiated feeder cells, for example at a ratio of 1:2. The cells can then be seeded in the presence of IL-2, such as at a concentration of 200 IU / mL. The gRNA and Cas9 can be electroporated under the following conditions. After 1-3 days, NK cells are isolated and the feeder cells are removed. The NK cells can then be transduced with a CAR construct. The NK cells can then be The cells may be subjected to a second CRISPR Cas9 knockout for an additional gene. After troporation, NK cells are seeded with feeder cells for, for example, 5 to 9 days. obtain.

[0080] [Table 1]

[0081] [Table 2]

[0082] In some embodiments, the immune cells of the present disclosure have altered expression of two or more genes. In some embodiments, the alteration of gene expression is such that the gene Creating a disruption in a gene (e.g., a knockout, insertion, missense mutation, or fragment) Frameshift mutations, e.g., biallelic frameshift mutations, in all or part of the gene Deletion, e.g., deletion and / or knock-in of one or more exons or portions thereof In a specific embodiment, alteration of gene expression is achieved by transfection of a gene or The DNA-binding targeting nucleic acid is specifically designed to be targeted to a portion of the sequence. a nuclease, e.g., an RNA-guided nuclease such as a CRISPR-associated nuclease (Cas) The nucleases are sequence-specific or targeted, including It can be done.

[0083] In some embodiments, alteration of the expression, activity and / or function of a gene is In some embodiments, the gene is disrupted by disrupting its expression. but expression in the absence of genetic modification or introduction of a component resulting in the modification At least 10, 20, 30 or 40% or about 10, 20, 30 or is reduced by 40%, usually at least 50, 60, 70, 80, 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99 or 100% or about 50, 60, 7 0, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 10 It will be modified to have a 0% reduction.

[0084] In some embodiments, the alteration is transient or reversible, and may be genetically modified, if desired. Expression of the gene is restored at a later time. In other embodiments, the alteration is reversible or transient. Not gender, but permanence, for example.

[0085] In some embodiments, the genetic alteration is an alteration of a gene, usually in a targeted manner. by induction of one or more double-strand breaks and / or one or more single-strand breaks in the In some embodiments, double-stranded or single-stranded cleavage is induced by the action of a nuclease, e.g. For example, this can be achieved by endonucleases such as gene-targeting nucleases. In embodiments, the cleavage is induced in a coding region, e.g., an exon, of a gene. For example, in some embodiments, the induction is by cloning a coding region, e.g., the first expression vector. exon, occurring near the N-terminal portion of the second or subsequent exons.

[0086] In some embodiments, the double-stranded or single-stranded break is caused by non-homologous end joining (NHEJ). or undergo repair via cellular repair processes such as homology-directed repair (HDR). In some embodiments, this repair process is error-prone and may result in the complete deletion of the gene. Disruption of a gene that can result in a knockout, e.g., a frameshift mutation, e.g., For example, in some embodiments, the disruption results in a frameshift mutation of the allele. In some embodiments, the disruption includes inducing a mutation and / or an insertion. In some embodiments, insertions, deletions, transpositions, etc. The presence of a gene locus, frameshift mutations and / or premature stop codons may The expression, activity and / or function of the progeny is disrupted.

[0087] In some embodiments, the modification is an RNA-guided endonuclease (RGE N) via one or more DNA-binding nucleic acids. For example, clustered regularly interspaced short palindromic repeats (CRISPR) and CR This can be done using ISPR-associated (Cas) proteins. "Cas" refers to a CRISPR-associated ("Cas") gene (a gene that contains a sequence encoding a Cas gene). tracr (transactivating CRISPR) sequences (e.g., tracrRNA or is an active partial tracrRNA), tracr mate sequence (endogenous CRISPR system In the context of the system, "direct repeats" and those processed by tracrRNA Partial direct repeats), guide sequences (in the context of the endogenous CRISPR system) (also referred to as "spacer" in Sequences and transcripts and other elements involved in the expression of or directing the activity of the transcripts. This is a general term for all

[0088] CRISPR / Cas nuclease or CRISPR / Cas nuclease system The guide RNA is a non-coding RNA molecule that binds to DNA in a sequence-specific manner, and the nuclease Cas proteins (e.g., Caspase proteins) with ase functions (e.g., two nuclease domains) One or more elements of the CRISPR system may include type I, type II, type III, type IV ... The CRISPR system may be derived from a type I or type III CRISPR system, for example an endogenous CRISPR system. Streptococcus pyogenes, which is a member of the It could come.

[0089] In some embodiments, the Cas nuclease and gRNA (cRNA specific for the target sequence) A gene encoding a tracrRNA (containing a fusion of a rRNA with a given tracrRNA) is introduced into a cell. The target site at the 5' end of the gRNA is targeted to the Cas nuclease by complementary base pairing. The enzyme is targeted to its target site, e.g., a gene. The target site is adjacent to the protospacer. Based on the position immediately 5' of the PAM motif (PAM) sequence (usually NGG or NAG) In this regard, gRNAs can be guided to correspond to the target DNA sequence. The first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11 or By modifying 10 nucleotides, the desired sequence is targeted. The SPR system is an element that promotes the formation of CRISPR complexes at the site of the target sequence. Generally, a "target sequence" is a sequence that is complementary to a guide sequence. This refers to the sequence designed to hybridize the target sequence with the guide sequence. promotes the formation of the CRISPR complex. Perfect complementarity is not necessary, as long as there is sufficient complementarity to promote the formation of the ISPR complex. Not necessary.

[0090] The CRISPR system generates a double-stranded break (DSB) at the target site, followed by In another embodiment, the disruption or alteration may be induced by a "nickase" A Cas9 variant considered to be a cytoplasmic nucleotide variant is used to nick a single strand at the target site. For example, to improve specificity, paired nickases can be used, Each nickase is guided by a different pair of gRNAs targeting the same sequence, and the nicks are generated simultaneously. In another embodiment, when the 5' overhang is introduced, it inhibits gene expression. In order to affect transcription, catalytically inactive Cas9 acts as a transcription repressor or activator. is fused to a heterologous effector domain.

[0091] The target sequence may be any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as within an organelle of a cell. Generally, a target sequence is used for recombination into a targeted locus that contains the target sequence. A sequence or template that can be used is referred to as an "edited template" or an "edited polynucleotide" or an "edited sequence." In some embodiments, the exogenous template polynucleotide is referred to as an editing template. In some embodiments, the recombination is homologous recombination.

[0092] Typically, in the context of endogenous CRISPR systems, the CRISPR complex (which binds to the target sequence) (containing a guide sequence that hybridizes with and complexes with one or more Cas proteins) at or near the target sequence (e.g., 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) One or both strand breaks occur. All or part of the wild-type tracr sequence (e.g. For example, about 20 nucleotides, about 26 nucleotides, about 32 nucleotides of the wild-type tracr sequence tide, about 45 nucleotides, about 48 nucleotides, about 54 nucleotides, about 63 nucleotides nucleotide, about 67 nucleotides, about 85 nucleotides or more, or about 20 nucleotides more than about 26 nucleotides, more than about 32 nucleotides, more than about 45 nucleotides, more than about 48 nucleotides More than about 54 nucleotides, more than about 63 nucleotides, more than about 67 nucleotides , more than about 85 nucleotides or more) The cr sequence may also be any combination of all or part of the tracr mate sequence operably linked to the guide sequence. or by hybridization along at least a portion of the tracr sequence to a , which may form part of a CRISPR complex. The tracr sequence hybridizes to the CR Participate in the formation of the ISPR complex; sufficient complementarity to the tracr mate sequence (e.g. , when optimally aligned, there are at least 5 0%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity).

[0093] Expression of one or more elements of the CRISPR system results in expression at one or more target sites. The formation of the CRISPR complex is directed in the CRISPR system. One or more vectors driving expression of these elements can be introduced into the cell. Elements can be delivered to cells as proteins and / or RNA, e.g., Cas enzymes. , a guide sequence linked to a tracr mate sequence, and a tracr sequence, The regulatory elements may be operably linked to separate regulatory elements on separate vectors. Two or more elements expressed from different regulatory elements are expressed in a single vector. may be combined, where one or more additional vectors are included in the first vector. The vector provides any component of the CRISPR system that does not contain a restriction endonuclease. The vector may include one or more insertion sites (also called "cloning sites"), such as a cleavage enzyme recognition sequence. In some embodiments, one or more insertion sites may be included in one or more vectors. Multiple different guides are located upstream and / or downstream of one or more sequence elements. When sequences are used, a single expression construct can be used to target multiple different corresponding target sequences in a cell. It can be used to target CRISPR activity against

[0094] The vector contains an enzyme coding sequence that encodes a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, C As7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas1 0, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa 5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr 3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Cs x14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf l, Csf2, Csf3, Csf4, their homologs or modified versions These enzymes are known; for example, the Cas9 enzyme of S. pyogenes. The amino acid sequence of the protein is listed in the SwissProt database under the accession number Q9 Can be seen as 9ZW2.

[0095] The CRISPR enzyme is Cas9 (e.g., S. pyogenes or S. pneum The CRISPR enzyme can be located at the location of the target sequence (e.g., from the target Directs cleavage of one or both strands of a target sequence (within the target sequence and / or within the complementary strand of the target sequence) The vector may encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme. The mutated CRISPR enzyme is capable of binding to one or more of the target polynucleotides that contain the target sequence. For example, the RuCas9 from S. pyogenes lacks the ability to cleave both strands. An aspartic acid to alanine substitution (D10A) in the vC I catalytic domain Converting Cas9, a nuclease that cuts one strand, into a nickase (that cuts one strand) In some embodiments, the Cas9 nickase binds to a guide sequence, e.g., a DNA A is combined with two guide sequences that target the sense and antisense strands of the target, respectively. This combination allows both strands to be nicked and the NHE Used to induce J or HDR.

[0096] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is a eukaryotic The codons are optimized for expression in a particular cell, such as a eukaryotic cell. A living organism (e.g., a human, a mouse, a rat, a rabbit, a dog, or a non-human primate, including, but not limited to, any of these) The cells may be cells of any organism (including but not limited to mammals) or cells derived from those organisms. Codon optimization refers to the modification of at least one amino acid sequence of a native sequence while maintaining the native amino acid sequence. The codon is then converted to a more frequently or most frequently used codon in the host cell's genes. The nucleic acid sequence can be modified to enhance expression in a host cell of interest by replacing the It refers to the process of modifying a gene by modifying the amino acid sequence of the gene. Codon bias (differences in codon usage among organisms) can affect the The translation efficiency of mRNA often correlates with the translation efficiency of the target gene, which is, among other things, It depends on the characteristics of the codon to be translated and the availability of specific transfer RNA (tRNA) molecules. It is believed that the predominantly selected tRNA in cells is usually the one most important for peptide synthesis. Therefore, it is based on codon optimization. Thus, genes can be tailored for optimal gene expression in a given organism.

[0097] Generally, a guide sequence is a target polynucleotide sequence that hybridizes with a target sequence. A target polynucleotide that directs sequence-specific binding of the CRISPR complex to the target sequence. It is any polynucleotide sequence that has sufficient complementarity to the sequence. In the embodiment, the degree of complementarity between the guide sequence and the corresponding target sequence is determined by suitable alignment. When optimally aligned using the 3D alignment algorithm, the alignment yielded approximately 50%, 60%, 75%, and 80%. 0%, 85%, 90%, 95%, 97%, 99% or more Exceeds.

[0098] Optimal alignment can be performed using any suitable algorithm for aligning sequences. A non-limiting example of such an algorithm may be the Smith-Watt algorithm. erman algorithm, Needleman-Wunsch algorithm, Burro Algorithms based on the WS-Wheeler Transform (e.g., Burro ws Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign(Novocraft Technologies,EL AND(Illumina,San Diego,Calif.)、SOAP(soap (available at www.genomics.org.cn) and Maq (maq.sour (Available at ceforge.net).

[0099] The CRISPR enzyme is part of a fusion protein that contains one or more heterologous protein domains. The CRISPR enzyme fusion protein may be any additional protein sequence, and and optionally, a linker sequence may be included between any two domains. Examples of protein domains that can be fused to the gene include epitope tags, reporter genes, and the like. Sequence, as well as the following activities: methylase activity, demethylase activity, transcription activation activity, transcription Inhibitory activity, transcription termination factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity These include, but are not limited to, protein domains having one or more of the following: Non-limiting examples of tag tags include histidine (His) tags, V5 tags, FLAG tags, and the like. tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag and thiol tag Examples of reporter genes include the redoxin (Trx) tag. -5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol Muphenicol acetyltransferase (CAT) Beta-galactosidase, beta Taglucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and Autofluorescent proteins include, but are not limited to, blue fluorescent proteins (BFPs) CRISPR enzymes bind to DNA or other cellular molecules. It may be fused to a gene sequence encoding a protein or a fragment of a protein, These proteins include maltose binding protein (MBP), S-tag, and Lex A. DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions and These include, but are not limited to, herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein containing a CRISPR enzyme are not defined. The invention is described in US20110059502, which is incorporated herein by reference. . III. Insertion of CAR and / or TCR at the locus of an inhibitory gene

[0100] In some embodiments, the present disclosure provides methods for detecting the expression of specific genes at loci in immune cells. The insertion of CAR and / or TCR. CAR and / or TCR are Gene locus (e.g., NKG2A, Siglec7, LAG3, TIM3, CISH , FOXO1, TGFBR2, TIGIT, CD96, adenosine receptor 2A, NR 3C1, PD1, PDL-1, PDL-2, CD47, SIRPa, SHIP1, ADA M17, pS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95 , CD80, CD86, IL10R, CD5, CD7, and combinations thereof The gene may be inserted into a gene selected from the group consisting of:

[0101] One or more CARs and / or TCs in any of the methods disclosed herein The insertion of R can be site-specific. For example, one or more CARs and / or TCRs can be , adjacent to or near the promoter. One or more transgenes are inserted adjacent to the exons of a gene (e.g., an inhibitory gene) The insertion may be made near an exon of the gene or within an exon of the gene. To knock in CAR and / or TCR while simultaneously disrupting gene expression, In another example, one or more CARs and / or TCRs can be used to transfect genes. Inserted adjacent to an intron, near an intron of a gene, or within an intron of a gene The CAR and / or TCR can be introduced into the adeno-associated virus (AAV) virus. It can be introduced by a vector and integrated into a targeted genomic location. In some cases, rAAV vectors have been used to insert transgenes at specific locations. For example, in some cases, CAR and / or T CR was transduced by rAAV or AAV vectors to target NKG2A, Siglec7, and LAG 3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, adenosy receptor 2A, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRP a, SHIP1, ADAM17, pS6, 4EBP1, CD25, CD40, IL21R , ICAM1, CD95, CD80, CD86, IL10R, CD5 or CD7 genes may be integrated into at least a portion of

[0102] The modification of a targeted locus in a cell is carried out by introducing DNA into the cell. where the DNA has homology to the target locus. The vector may contain a car gene, thereby allowing the selection of cells containing the integrated construct. The complementary DNA in the targeting vector is complementary to the chromosomal DNA at the target locus. The marker gene can be a complementary DNA sequence, a 3' recombination arm and a 5' recombination arm. Multiple loci within a cell can be targeted. The recombinant vector has recombination arms specific to one or more target loci so that the gene modification can be performed in one step. The homology arms may be about 0.2 kb to about 5 kb in length (e.g., For example, approximately 0.2kb, 0.4kb, 0.6kb, 0.8kb, 1.0kb, 1.2kb, 1.4kb, 1.6kb, 1.8kb, 2.0kb, 2.2kb, 2.4kb, 2.6k b, 2.8kb, 3.0kb, 3.2kb, 3.4kb, 3.6kb, 3.8kb, 4. 0kb, 4.2kb, 4.4kb, 4.6kb, 4.8kb to about 5.0kb in length) could be.

[0103] In one method, the guide RNA is a region of the locus of an inhibitory gene (e.g., to target the promoter, exon or intron adjacent regions of a gene The guide RNA can be designed to target an exon (e.g., the first, second or third exon) of the inhibitory gene. The guide RNA may target the 5' end of the AAV vector repair marker (the third exon). The AAV vector can be included in a vector that contains a self-cleaving 2A peptide, such as the P2A peptide. The CAR cassette and guide RNA sequence may be , which may be flanked by homology arms to an inhibitory gene. AAV vector and Cas9 (e.g., Cas9 mRNA) are introduced (e.g., electroporation) troporation) can be performed. IV. Immune cells

[0104] Certain embodiments of the present disclosure are directed to having multiple gene knockouts and / or or immune cells engineered to have a knock-in of a CAR at the locus of an inhibitory gene. These immune cells include T cells (e.g., regulatory T cells, CD4 + T cells, CD 8 + T cells or gamma-delta T cells), NK cells, invariant NK cells, NKT Cells, B cells, stem cells (e.g., mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) ) cells. Those immune cells can be virus-specific and can express a CAR. In some embodiments, the cells may express a single Cells or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells , eosinophils and / or basophils. Methods for producing and manipulating immune cells, and Methods of using and administering these cells for adoptive cell therapy are also provided herein, In that case, the cells may be autologous or allogeneic. The cells may be used as an immunotherapy, such as to target cancer cells.

[0105] The immune cells may be isolated from a subject, particularly a human subject. A subject of interest (e.g., a subject suspected of having a particular disease or condition, a subject who is also diagnosed with a particular disease subjects suspected of having a predisposition to a particular disease or condition, or for the treatment of a particular disease or condition. The immune cells can be obtained from a subject (e.g., a subject undergoing treatment with a cellular immune system). The cells can be harvested from any location where the cells can be harvested, including blood, umbilical cord blood, spleen, breast The isolated immune cells include, but are not limited to, lymph nodes, glands, and bone marrow. , may be used directly or may be stored for a period of time, such as by freezing.

[0106] The immune cells may be enriched / purified from any tissue in which they are present, and their Tissues include blood (including blood collected by a blood bank or umbilical cord blood bank); Spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and via biopsy procedures These include, but are not limited to, tissues obtained by enriching, isolating and / or isolating immune cells. Alternatively, the tissues / organs to be purified may be from both living and non-living subjects. In certain embodiments, the non-living subject may be an organ donor. In the present invention, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood, or a mixture thereof. In some embodiments, the immune cells isolated from the cord blood are CD4 positive or C It has high immunomodulatory activity as measured by suppression of D8 positive T cells. In this way, immune cells seek high levels of immunoregulatory capacity in pooled blood, especially in pooled blood. Pooled blood is isolated from pooled cord blood. , 6, 7, 8, 9, 10 or more sources (e.g., donor subjects) .

[0107] The population of immune cells is administered to a subject in need of treatment or to a patient suffering from a disease associated with low immune cell activity. The cells can be obtained from a subject who is afflicted and therefore in need of treatment. Alternatively, the population of immune cells may be autologous to the subject. The immune cell population can be obtained from peripheral blood, histocompatibility-matched donors. Umbilical cord blood, bone marrow, spleen, or any other organ in which immune cells are present in the subject or donor The immune cells may be collected from a pool of subjects and / or donors, For example, it can be isolated from pooled umbilical cord blood.

[0108] When the immune cell population is obtained from a donor different from the subject, the donor is preferably are allogeneic, provided that the cells obtained are capable of being introduced into the subject. Allogeneic donor cells are matched to the recipient in terms of human leukocyte antigen (HLA) identity. It may or may not be compatible. AT cells

[0109] In some embodiments, the immune cell is a T cell. Several basic approaches for cell induction, activation and expansion have been developed over the past 20 years. These include autologous cells such as tumor-infiltrating lymphocytes (TILs); DCs, lymphocytes, artificial antigen-presenting cells (APCs), or T cell ligands and activating antibodies using ELISA-coated beads, or cells isolated by capture of the target cell membrane Ex vivo activated T cells; naturally expressing anti-host tumor T cell receptors (TCRs) and tumors that exhibit antibody-like tumor-recognition capabilities known as "T-bodies." Genetically reprogrammed to express tumor-reactive or chimeric TCR molecules These include "redirected" non-tumor specific autologous or allogeneic cells. These approaches can be used in the methods described herein to prepare T cells. and has been the source of numerous protocols for immunization.

[0110] In some embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord, or lymphatic system. In some embodiments, the cells are human cells. The cells are usually primary cells, e.g., cells isolated directly from a subject, and / or are cells isolated from a subject and frozen. In some embodiments, the The cells may include one or more subsets of T cells or other cell types (e.g., the entire T cell population, CD4 + cells, CD8 + Cells, and their subpopulations, e.g., function, activation state, maturity, maturity, differentiation potential, expansion, recirculation, localization and / or persistence, antigen specificity, antigen level The type of scepter, its presence in a particular organ or compartment, and the marker or or a subset of cells defined by their cytokine secretion profile and / or degree of differentiation. With respect to the subject being treated, the cells may be allogeneic cells and / or In some embodiments, such as in existing techniques, the cells may be pluripotent and and / or are multipotent (e.g., stem cells, such as induced pluripotent stem cells (iPSCs)). In some embodiments, the method includes isolating cells from a subject, subjecting them to the method of the present invention. Preparing, treating, culturing and / or manipulating as described herein, and freezing Reintroducing them into the same patient, either before or after storage, is included.

[0111] T cells (e.g., CD4 + and / or CD8 + T cell subtypes and subsets The group includes Naive T(T N ) cells, effector T cells (T EFF ), memory -T cells and their subtypes (e.g., stem cell memory T (TSC M ), Central Molly T. M ), Effector Memory T (T EM ) or terminally differentiated effectors memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, endogenous and acquired Adaptive regulatory T (Treg) cells, helper T cells (e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular hepatitis par T cells), alpha / beta T cells and delta / gamma T cells.

[0112] In some embodiments, one or more of the T cell populations are identified by a specific marker, such as a surface marker. The cells were enriched for either marker-positive or specific marker-negative cells, and In some cases, such markers are specific to certain T cell populations. are absent or expressed at relatively low levels on the However, certain other T cell populations (e.g., memory cells) may be present or may be involved in the expression of the T cell. It is a marker that is expressed at relatively high levels.

[0113] In some embodiments, the T cells are non-T cells (e.g., B cells, monocytes or other Negative selection of markers (e.g., CD14) expressed on white blood cells (leukocytes) In some embodiments, CD4 + or CD8 + Selection process By using CD4 + Helper T cells and CD8 + Cytotoxic T cells are isolated Such CD4 + and CD8 + A population may consist of one or more naive, memory and and / or expressed or to a relatively high degree on a subpopulation of effector T cells Further subpopulations can be sorted by positive or negative selection of the markers identified. It can be tinged.

[0114] In some embodiments, CD8 + T cells express surface markers associated with each subpopulation. By antigen-based positive or negative selection, naive, central Further enrichment of memory, effector memory and / or central memory stem cells In some embodiments, the central memory T (T C M ) Cell enrichment may have beneficial effects, such as improving long-term survival, expansion, and / or engraftment after administration. This is done to increase the likelihood of survival in such subpopulations, which in some embodiments It is particularly robust.

[0115] In some embodiments, the T cells are autologous T cells. A sample is obtained from the patient to obtain a suspension of single cells. The suspension of single cells may be cultured in any suitable medium. in a suitable manner, e.g. mechanically (e.g. gentleMACS TM Dissociation The tumor was decongested using a 30-μm CT scanner (Miltenyi Biotec, Auburn, Calif.). can be obtained either by concentration or enzymatically (e.g., collagenase or DNase). Single cell suspensions from enzymatic digests of tumors were cultured in interleukin-2 (IL-2). do.

[0116] The cultured T cells can be pooled and rapidly expanded over a period of about 10 to about 14 days. Rapid expansion increases the number of antigen-specific T cells by at least about 50-fold (e.g., 50, 60 , 70, 80, 90 or 100 fold or more). More preferably, the increase is about 1 Rapid expansion over a period of 0 to about 14 days results in an increase of at least about 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900 or more) increase.

[0117] Expansion can be accomplished by any of a number of methods known in the art. For example, T cells can be produced by inducing feeder lymphocytes and interleukin-2 (IL-2). or interleukin-15 (IL-15), with IL-2 being preferred. In the presence of nonspecific T cell receptor stimulation, the T cell receptor stimulation was performed with approximately 30 ng / ml of mouse monoclonal anti-CD3 antibody. OKT3 (Ortho-McNeil®, Raritan, NJ) Alternatively, the T cells may be treated with T cell growth factor (e.g., 300 IU / ml of IL-2 or IL-15 (IL-2 is preferred) Cancer antigens (including antigenic parts thereof, such as epitopes, or cells) are cultured in peripheral blood mononuclear cells (PBMs). C) can be easily expanded by stimulating it in vitro, and the cancer antigen is Depending on the vector, for example, a human leukocyte antigen A2 (HLA-A2) binding peptide can be expressed. The in vitro induced T cells express HLA-A2 and They are rapidly expanded by restimulation with the same cancer antigen pulsed against the presenting cells. Alternatively, the T cells may be, for example, irradiated autologous lymphocytes or irradiated HLA-A 2 + They can be restimulated with allogeneic lymphocytes and IL-2.

[0118] The autologous T cells are then subjected to T cell growth factors that promote the proliferation and activation of the autologous T cells. Suitable T cell growth factors include, for example, interleukin-1 (IL-1), interleukin-2 (IL-2), and IL-1. Suitable modification methods include IL-2, IL-7, IL-15 and IL-12. Methods are known in the art. See, e.g., Sambrook et al., Molecular Biology, 1999, 144:1311-1323. lar Cloning:A Laboratory Manual,3 rd ed., Cold Spring Harbor Press,Cold Spring Har bor, NY 2001; and Ausubel et al., Current P rotocols in Molecular Biology,Greene Pub lishing Associates and John Wiley & Sons In certain embodiments, the modified autologous T cells are T cells. Expresses high levels of growth factors. T cell growth factor coding sequences (e.g., the coding sequence for IL-12) The promoter sequence (i.e., promoter sequence) is a promoter that, when operably linked to a T cell growth factor coding sequence, promotes high level expression. Like promoters, these are readily available in the art. B.NK cells

[0119] In some embodiments, the immune cells are natural killer (NK) cells. K cells are involved in the differentiation and proliferation of various tumor cells, virus-infected cells, and cells in the bone marrow and thymus. A subpopulation of lymphocytes that have spontaneous cytotoxicity against some normal cells. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus. In humans, it is detected by specific surface markers such as CD16, CD56, and CD8. NK cells can be expressed by T cell antigen receptors, the pan-T marker CD3, or surface immunoglobulins. Does not express the globulin B cell receptor.

[0120] In certain embodiments, NK cells are isolated from human peripheral blood by methods well known in the art. Mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hE SC), induced pluripotent stem cells (iPSC), bone marrow or umbilical cord blood. In certain embodiments, the NK cells are obtained by the method described previously. The previously reported method of expanding NK cells ex vivo (Spanholtz et al., 2011; Shah et al., 2013) and expanded it. In this method, CB mononuclear cells were isolated by Ficoll density gradient centrifugation and were then subjected to immunofluorescence assays to detect IL-2 and human IL-1. Culture the cells in a bioreactor together with antigen-presenting cells (aAPC). After 7 days, the cells were cultured. The culture is depleted of any cells expressing CD3 and re-cultured for an additional 7 days. Perform CD3 depletion of cells and CD56 + / CD3 - The percentage of cells or NK cells Another method uses umbilical cord blood and characterizes it to measure CD34 + Cell isolation, etc. and by culturing in a medium containing SCF, IL-7, IL-15 and IL-2. CD56 + / CD3 - NK cells are obtained by differentiation into cells.

[0121] In a specific embodiment, the NK cells are expanded at some point during the preparation. In some cases, NK cell expansion involves converting umbilical cord blood-derived mononuclear cells (MNCs) into antigen-presenting cells. (APC) and IL-2; and stimulating those cells in the presence of APC and restimulating the NK cells with 50 mM acetylcholine to generate expanded NK cells, at least in some cases. In the present invention, the method is carried out in a bioreactor. The stimulation step converts MNCs into NK cells. The restimulation step may or may not include the presence of IL-2. In certain embodiments, the method may include removing or removing any medium components during the stimulation step. In certain embodiments, the method includes the step of adding less than 5 days, e.g., 14 days).

[0122] In certain embodiments, NK cells are expanded by ex vivo methods. The method has been expanded to include the steps of (a) obtaining a starting population of mononuclear cells (MNCs) from umbilical cord blood; (b) stimulating the MNC in the presence of antigen-presenting cells (APCs) and IL-2; and (c) restimulating the cells with APCs to generate expanded NK cells. The method includes performing the method in a bioreactor and following good manufacturing practice. The stimulation in step (b) is in accordance with Good Manufacturing Practice (GMP). Thus, MNCs can be directed to NK cells. In certain embodiments, the method includes during step (b). In certain embodiments, the method does not include the removal or addition of any medium components. It is carried out in less than 15 days (e.g., 14 days).

[0123] In some embodiments, the methods include the step of determining whether one or more specific markers, such as CD3, are involved. In certain embodiments, the depletion step further comprises the steps of: In some embodiments, the cells are subjected to CD4+ 3. Remove from the bioreactor for depletion and return to the bioreactor for step (c) can be put in.

[0124] In certain embodiments, the step of obtaining a starting population of MNCs from umbilical cord blood comprises the steps of: In the presence of human serum albumin (HSA), DNAse and / or magnesium chloride In certain embodiments, the starting population of MNCs is extracted from the cord blood. The step of obtaining the above-mentioned comprises thawing the umbilical cord blood in the presence of dextran and / or DNase. In a specific embodiment, the cord blood is purified by subjecting the cord blood to a process comprising the steps of: In certain embodiments, the cord blood is washed in the presence of 100 to 300 mM sodium chloride. The suspension is suspended in the presence of magnesium chloride at a concentration of 100 mM, particularly 200 mM. In some embodiments, the obtaining step comprises performing Ficoll density gradient centrifugation to obtain mononuclear cells ( The process includes obtaining a MNC.

[0125] In certain embodiments, the bioreactor is a gas permeable bioreactor. In certain embodiments, the gas permeable bioreactor is a G-Rex100M or G In some embodiments, the stimulation in step (b) is performed with 3 to 5 L of medium. (e.g., 3, 3.5, 4, 4.5 or 5 L).

[0126] In some embodiments, the APCs are gamma irradiated. In this study, APCs were engineered to express membrane-bound IL-21 (mbIL-21). In one embodiment, the APC expresses IL-21, IL-15 and / or IL-2. In some embodiments, MNCs and APCs are cultured at a ratio of 1:2. In some embodiments, IL-2 is administered at a concentration of 50 to 200 IU / mL (e.g., In certain embodiments, IL-2 is supplemented every 2-3 days. will be done.

[0127] In certain embodiments, step (b) is carried out for 6 to 8 days (e.g., 7 days). In some embodiments, step (c) is carried out for 6 to 8 days (e.g., 7 days). In an embodiment, step (c) does not include dividing the cells. In the specific case, IL-2 is provided twice during step (c), and in the specific case, another No medium components are added or removed.

[0128] In some embodiments, the method comprises the step of: In certain embodiments, the method includes the use of less than 10 bioreactors. Including use.

[0129] In specific embodiments, the NK cells are at least 500-fold, 800-fold, 1000-fold, Magnified 200x, 1500x, 2000x, 2500x, 3000x or 5000x In certain embodiments, the NK cells are cultured in a bioreactor to produce a static medium. More than 1,000 times more NK cells are produced compared to somatic culture.

[0130] In certain embodiments, the method does not involve human leukocyte antigen (HLA) matching. In some embodiments, the starting population of NK cells is not obtained from a haploidentical donor. stomach.

[0131] In some embodiments, the expanded NK cells are NK cells expanded from peripheral blood. In certain embodiments, the expanded NK cells have a higher antitumor activity than the expanded NK cells. The cells exhibited increased expression of one or more cell cycle genes, one or more They overexpress cell division genes and / or one or more DNA replication genes. In one embodiment, the expanded NK cells are compared to NK cells expanded from peripheral blood. In some embodiments, the expanded NK cells are exhausted and have a higher proliferation potential. In certain embodiments, the exhaustion does not exhibit impairment in perforin, granzymes, CD5 7. Detected by measuring the expression of KLRG1 and / or PD1. In some embodiments, the expanded NK cells express perforin and / or granulocyte immunoglobulin (NK)-dependent NK cell proliferation and / or proliferation. In certain embodiments, the expanded NK cells are highly expressing CD5 7, low or no expression of KLRG1 and / or PD1;

[0132] In some embodiments, the expanded NK cells comprise a clinically relevant dose. In certain embodiments, the cord blood is frozen cord blood. In certain embodiments, the frozen cord blood is One or more infectious diseases (e.g., Hepatitis A, Hepatitis B, Hepatitis C, Trypanosoma) cruzi, HIV, human T-lymphotropic virus, syphilis, Zika virus, etc. In some embodiments, the cord blood is 3, 4, 5, 6, 7 or 8 units It is pooled cord blood from individual cord blood units etc.

[0133] In some embodiments, the NK cells are autologous NK cells, e.g., to the recipient itself. In certain embodiments, NK cells are not NK cells, e.g., to the recipient itself. , but not allogeneic NK cells.

[0134] In some embodiments, the APC is a universal antigen presenting cell (uAPC). In certain embodiments, the uAPCs are characterized by (1) CD48 and / or CS1 (CD31 9), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 41 In some embodiments, the u The APCs express CD48. In certain embodiments, the uAPCs express CS1. In certain embodiments, the uAPCs express CD48 and CS1. In this manner, uAPCs express endogenous HLA class I, II and / or CD1d molecules. In certain embodiments, the uAPCs are essentially free of ICAM-1 (CD5 4) and / or LFA-3 (CD58). is further defined as an aAPC derived from a leukemia cell, such as K562 cell. C. Stem cells

[0135] In some embodiments, the immune cells of the present disclosure are induced pluripotent stem cells (PSCs), The stem cells may be mesenchymal stem cells (MSCs) or hematopoietic stem cells (HSCs).

[0136] As used herein, pluripotent stem cells are commonly abbreviated as iPS cells or iPSCs. Any cell other than a germ cell can be derived from an iPSC. For example, cell types such as keratinocytes, fibroblasts, myocytes, The cells may be blood cells, mesenchymal cells, liver cells or stomach cells. There is no limit to the age of the animals that can be harvested. Undifferentiated progenitor cells (including somatic stem cells) and final differentiated Even mature cells that have been transformed may be used as a source of somatic cells in the methods disclosed herein. It is possible.

[0137] Somatic cells can be reprogrammed to generate iPS cells using methods known to those of skill in the art. In the present study, nuclear reprogramming factors are used to generate pluripotent stem cells from somatic cells. In one embodiment, Klf4, c-Myc, Oct3 / 4, Sox2, Nanog and At least three or at least four of Lin28 and Lin29 are used. In the IL-16, Oct3 / 4, Sox2, c-Myc and Klf4 are used, or Oct t3 / 4, Sox2, Nanog and Lin28 are used.

[0138] Once derived, iPSCs can be cultured in a medium sufficient to maintain their pluripotency. In certain embodiments, indeterminate conditions may be used. For example, pluripotent cells To maintain the stem cells in an undifferentiated state, The cells may be cultured on medium exposed to fibroblast feeder cells. In this study, the cells were cultured in mouse models that had been treated with irradiation or antibiotics to terminate cell division. They are cultured in the presence of fetal fibroblasts as feeder cells. Alternatively, pluripotent cells TESR TM Medium or E8 TM / Essential 8 TM Media feeder They can be cultured and maintained in an essentially undifferentiated state using a well-defined, cell-independent culture system. V. Genetically Engineered Antigen Receptors

[0139] The immune cells of the present disclosure include those that express an antigen receptor (e.g., an engineered TCR, a CAR, a chimeric receptor, These cells express a number of different receptors, including cytokines, chemokine receptors, and combinations of these. For example, immune cells can be engineered to express C-cells with antigen specificity for cancer antigens. The cells are modified to express AR and / or TCR. CRs (e.g., against various antigens) can be added to immune cells. In this study, immune cells were targeted to CRISPR-mediated expression of CAR or IFN-γ at the locus of an inhibitory gene. They are engineered to express a CAR or a TCR by knock-in of the CAR or TCR.

[0140] Suitable modification methods are known in the art. See, e.g., Heemskerk et al. ., 2008 and Johnson et al., 2009. Using the method, cells can be transduced to express a TCR with antigen specificity for a cancer antigen. do.

[0141] Pairing of the retrovirally transduced TCR chain with the endogenous TCR chain results in As an option to overcome the long-term problems of autoreactivity caused by Using electroporation of RNA encoding the Rα and β (or γ and δ) chains, Such alternative pairings can be used in transient transfection strategies. Even in TEGs, the introduced TCR α and β chains are expressed transiently Therefore, the autoreactive T cells that may be generated will eventually disappear after a while. When expression of the introduced TCR α and β chains is reduced, only normal autologous T cells are expressed. This is because full-length TCR chains are introduced by stable retroviral transduction. This is not the case when the TCR chains introduced are not lost and the patient is constantly self-reactive. There will be responsiveness.

[0142] In some embodiments, the cells encode one or more antigen receptors. One or more nucleic acids introduced via genetic engineering, and the genetically engineered versions of such nucleic acids In some embodiments, the nucleic acids are heterologous, i.e. , which are not normally present in cells or samples derived from cells (e.g., in other organisms or is obtained from a cell, which may be, for example, a cell that has been engineered and / or such a cell (not normally found in the organism from which it is derived). In some embodiments, these nucleic acids are non-naturally occurring, such as nucleic acids not found in nature (eg, chimeras).

[0143] In some embodiments, the CAR comprises an extracellular antigen-recognizing molecule that specifically binds to an antigen. In some embodiments, the antigen is expressed on the cell surface. In some embodiments, the CAR is a TCR-like CAR. The antigen is delivered to the cell in the context of major histocompatibility complex (MHC) molecules, as well as to the TCR. Processed peptide antigens that are recognized on the surface (e.g., of intracellular proteins) peptide antigen).

[0144] Exemplary antigen receptors, including CARs and recombinant TCRs, and their Methods for manipulating receptors and for introducing those receptors into cells Examples of the compounds are described in International Patent Application Publication Nos. WO200014257 and WO2013126. 726, WO2012 / 129514, WO2014031687, WO2013 / 16 6321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Opening number US2002131960, US2013287748, US201301493 37, U.S. Patent Nos. 6,451,995, 7,446,190, and 8,252, No. 592, No. 8,339,645, No. 8,398,282, No. 7,446,1 No. 79, No. 6,410,319, No. 7,070,995, No. 7,265,20 No. 9, No. 7,354,762, No. 7,446,191, No. 8,324,353 Nos. 8,479,118 and 8,479,118, and in European Patent Application No. EP2537416. and / or Sadelain et al., 2013; Dav ila et al.,2013;Turtle et al.,2012;Wu et al. In some embodiments, the gene is a genotype that is characterized by its specificity and includes those described by et al., 2012. Genetically engineered antigen receptors are described in U.S. Pat. No. 7,446,190. CARs such as those described in International Patent Application Publication No. WO / 2014055668A1 This includes those that are currently being investigated. A. Chimeric antigen receptor

[0145] In some embodiments, the CAR comprises: a) one or more intracellular signaling domains; a) a main domain, b) a transmembrane domain, and c) an extracellular domain that contains the antigen-binding region.

[0146] In some embodiments, the engineered antigen receptor includes an activating or stimulatory receptor. CARs, costimulatory CARs (see WO2014 / 055668) and / or is an inhibitory CAR (iCAR, see Fedorov et al., 2013) These CARs generally bind to one or more intracellular signals. In some embodiments, the nucleic acid is linked to a nucleic acid signaling component via a linker and / or a transmembrane domain. Such molecules contain an extracellular antigen (or ligand) binding domain linked by a cytoplasmic ligand (C-G) linker. , usually through natural antigen receptors, together with costimulatory receptors. Receptor-mediated signaling and / or costimulatory receptor-mediated signaling alone To imitate or mimic.

[0147] Certain embodiments of the present disclosure include an intracellular signaling domain, a transmembrane domain, and and an extracellular domain that contains one or more signaling motifs. Polypeptides (including humanized CAR (hCAR) to reduce immunogenicity) are co-administered In certain embodiments, the CAR comprises a nucleic acid encoding It may recognize epitopes that include spaces shared between one or more antigens. In one embodiment, the binding region is a complementarity determining region of a monoclonal antibody, In another embodiment, the antibody may comprise a variable region, and / or an antigen-binding fragment thereof. The specificity of the peptides (e.g., cytokines) that bind to the receptors is derived from the do.

[0148] Human CAR nucleic acids are human genes used to enhance cellular immunotherapy in human patients. In a specific embodiment, the present invention provides a full-length cCAR gene. The DNA or coding region of the antigen-binding region or domain is a specific human monoclonal antibody. V of single-chain variable fragment (scFv) derived from a monoclonal antibody H Chain and V L Chain F Fragments (see, for example, U.S. Pat. No. 7,109,304, incorporated herein by reference). The fragment may include any of the human antigen-specific antibodies. In a more specific embodiment, the fragment may be an antigen-binding domain having a different number of fragments. The fragment is sequence-optimized to human codon usage for expression in human cells. The antigen-specific scFv is encoded by

[0149] The configuration may be a multimer (e.g., a diabody or a multimer). The possibility that the variable portions of the low and heavy chains are formed by cross-pairing to diabodies The construct retained the first cysteine ​​in the hinge region from the complete deletion. The substitution is proline rather than serine, and the first cysteine ​​is truncated. There are several options available, ranging from the Fc portion to the stability of the antibody. Any protein that binds and / or dimerizes may serve this purpose. Only one of the domains, e.g., the CH2 or CH3 domain of a human immunoglobulin, Human immunoglobulins modified to improve dimerization can be used. Hinge, CH2 and CH3 regions may also be used. Hinge portion of immunoglobulin You can use just the CD8 alpha fragment. You can also use the CD8 alpha fragment.

[0150] In some embodiments, the CAR nucleic acid comprises a transmembrane domain and a modified C It contains sequences encoding other costimulatory receptors, such as the D28 intracellular signaling domain. Other costimulatory receptors include CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137) In addition to the primary signal induced by CD3ζ, human C An additional signal provided by the human costimulatory receptor inserted into the AR stimulates NK cells. This is important for the full activation of the cells and for the persistence and adoption of immunotherapy treatments in vivo. It can help improve success.

[0151] In some embodiments, the CAR is a specific cell type that is targeted by adoptive therapy. Certain antigens (or markers or ligands), such as antigens expressed in, e.g., Cancer markers and / or antigens intended to induce an attenuated response (e.g., normal cells The antigens are constructed to have specificity for antigens expressed on diseased or non-diseased cell types. Thus, the CAR typically contains one or more antigen-binding molecules (e.g., For example, one or more antigen-binding fragments, antigen-binding domains or antigen-binding portions) or In some embodiments, the antibody comprises one or more antibody variable domains and / or antibody molecules. In this case, the CAR is an antigen-binding portion of an antibody molecule (e.g., a monoclonal antibody (mAb) A single-chain antibody fragment (scF) derived from the variable heavy chain (VH) and variable light chain (VL) of v)).

[0152] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor The extracellular domain (which may be referred to as the extracellular domain containing the antigen-binding region) binds to a tumor-associated antigen or a pathogen-specific antigen. The antigens bind to pattern recognition receptors such as Dectin-1. Tumor-associated antigens include carbohydrate antigens that are recognized by tumor cells. Exemplary embodiments of tumor-associated antigens include CD1 9, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, C D56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma In certain embodiments, the antigens include, for example, related antigens, mutant p53, mutant ras, and the like. CARs are also used in combination with cytokines to improve persistence when tumor-associated antigens are scarce. For example, CARs can be expressed in combination with IL-7, IL-2, IL-15, IL-12, IL-14, IL-15, IL-16, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL- Co-expression of one or more cytokines such as IL-18, IL-21 or a combination thereof It can be realized.

[0153] The sequence of the open reading frame encoding the chimeric receptor is Origin, may be obtained from cDNA sources or may be synthetic (e.g., via PCR), or a combination thereof. Introns stabilize mRNA Depending on the size of the genomic DNA and the number of introns, It may be desirable to use DNA or a combination thereof. It may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the gene. It is possible.

[0154] The chimeric construct is then injected into immune cells either as naked DNA or in a suitable vector. It is contemplated that the vector may be introduced into the host by electroporation using naked DNA. Methods for stably transfecting cells are known in the art. See, e.g., U.S. Pat. See US Pat. No. 6,410,319. Naked DNA generally refers to DNA that has been prepared in a manner suitable for expression. The DNA encoding the chimeric receptor contained in the plasmid expression vector is called Point.

[0155] Alternatively, viral vectors (e.g., recombinant human leukocytes) can be used to introduce the chimeric construct into immune cells. retrovirus vector, adenovirus vector, adeno-associated virus vector or retrovirus vector Suitable vectors for use in accordance with the methods of the present disclosure include, but are not limited to, The vector is a non-replicative vector in immune cells. There are many known causes of HIV infection (e.g., those based on HIV, SV40, EBV, HSV or BPV). vector), the copy number of the virus maintained within the cell is sufficient to maintain the viability of the cell. It is low enough that

[0156] In some embodiments, an antigen-specific binding component or an antigen-specific recognition construct The element is linked to one or more transmembrane domains and an intracellular signaling domain. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the CAR comprises a native CAR domain. In some cases, the transmembrane domain associated with the The authors propose that such receptors be designed to minimize interactions with other members of the receptor complex. The nucleotide sequence of the nucleotide sequence is chosen to avoid binding to the transmembrane domains of the same or different surface membrane proteins. Thus, the sequences are selected or modified by amino acid substitution.

[0157] The transmembrane domain, in some embodiments, is derived from natural or synthetic sources. If naturally occurring in origin, the domain may in some embodiments be any membrane-bound protein. The transmembrane domain is derived from the alpha of the T cell receptor. a chain, beta chain or zeta chain, CD28, CD3 zeta, CD3 epsilon, CD3 gas CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22 , CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154, ICOS / CD278, GITR / CD357, NKG2D and DAP molecules The polypeptides include those derived from (i.e., contain at least the transmembrane region of) those molecules. Alternatively, the transmembrane domain is in some embodiments a synthetic transmembrane domain. In some embodiments, the synthetic transmembrane domain comprises a sparse amino acid sequence, such as leucine and valine. In some embodiments, it contains phenylalanine, tryptophan, and baicalenic residues. Phosphorus triplets may be found at each end of the synthetic transmembrane domain.

[0158] In certain embodiments, the present invention provides a method for genetically modifying immune cells, such as NK cells. The platform technologies disclosed in the specification include: (i) electroporation devices (ii) non-viral gene transfer using vectors (e.g., nucleofectors); Main (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ or other combinations) (iii) a CAR that signals through an extracellular domain of variable length; CARs, which have an extracellular domain that connects the CAR recognition domain to the cell surface, and several In the case of (iv) CAR + K56 can robustly and numerically expand immune cells Artificial antigen presenting cells (aAPCs) derived from 2 (Singh et al., 2008; Ingh et al., 2011). BT cell receptor (TCR)

[0159] In some embodiments, the genetically engineered antigen receptor includes a recombinant TCR. and / or TCRs cloned from naturally occurring T cells. "TCR" or "TCR" refers to the variable a chain and variable beta chain (TCR alpha and beta, respectively). and TCRβ) or the variable gamma and variable delta chains (TCRγ and and TCRδ), which binds to the MHC receptor and an antigen peptide. In some embodiments, the term "antibody" refers to a molecule that can specifically bind to a peptide. The TCR is of the αβ type.

[0160] TCRs, which usually exist as αβ and γδ types, generally have similar structures, but T cells expressing TCRs may differ in anatomical location or function. Generally, TCRs are expressed on the surface of T cells (or T lymphocytes). They are found on the surface of the body and usually bind to major histocompatibility complex (MHC) molecules. In some embodiments, a TCR comprises a constant domain, a transmembrane domain, and a It may also include a trafficking domain, and / or a short cytoplasmic tail (e.g., Janeway e (See, e.g., J. Med. Soc., 1997). For example, in some embodiments, each chain of the TCR is , one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, transmembrane In some embodiments, the T CR associates with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise stated, the term "TCR" includes functional TCR fragments thereof. This term should be understood to include TCRs of the αβ or γδ types. It also encompasses intact or full-length TCRs.

[0161] Thus, for purposes herein, reference to a TCR includes any TCR or functional TCR. Fragments (e.g., specific antigenic peptides bound in the MHC molecule, i.e. The antigen-binding portion of the TCR that binds to the MHC-peptide complex. The "antigen-binding portion" or antigen-binding fragment of a TCR may be expressed as a structural model of the TCR. It contains only a portion of the main domain but not the antigen that the complete TCR binds (e.g., MHC-peptide In some cases, the antigen-binding portion refers to a molecule that binds to a specific A sufficient number of TCRs are required to form a binding site for binding to a specific MHC-peptide complex. Variable domains (e.g., the variable a and variable β chains of a TCR), e.g., typically each chain contains three complementarity determining regions.

[0162] In some embodiments, the variable domains of the TCR chains associate to form loops, or immunoglobulins. It forms a complementarity determining region (CDR) similar to immunoglobulin, which allows it to recognize antigens. The peptide specificity is determined by the formation of the binding site of the TCR molecule, and the peptide Typically, as in immunoglobulins, the CDRs are separated by framework regions. The FRs are separated by a basal region (e.g., Jores et al., 1990; Cho See Thia et al., 1988; Lefranc et al., 2003 In some embodiments, CDR3 is involved in recognition of processed antigens. The CDR1 of the alpha chain interacts with the N-terminal part of the antigen peptide. It has also been shown that CDR1 of the beta chain is located in the C-terminal part of the peptide. CDR2 is believed to recognize the MHC molecule. In some embodiments, the variable region of the β chain may include an additional hypervariable (HV4) region.

[0163] In some embodiments, the TCR chain comprises a constant domain. Like the phospholipase A, the extracellular portion of the TCR chain (e.g., the a chain, the β chain) binds two immunoglobulins. A variable domain at the N-terminus (e.g., V a or Vp; usually Ka bat numbering, Kabat et al., “Sequences of P roteins of Immunological Interest, US Dep. t.Health and Human Services,Public Healt h Service National Institutes of Health, 1991,5 th ed.-based amino acids 1–116), and one adjacent to the cell membrane. The constant domain of the a , usually based on Kabat amino acids 117-259, beta chain constant domain or Cp, usually amino acids according to Kabat 117-295). For example, in some cases, the two chains may form The extracellular portion of the TCR that is expressed consists of two membrane-proximal constant domains and two CDR-containing The constant domain of the TCR domain contains a membrane-distal variable domain. They contain a short connecting sequence that forms a nucleotide bond, thereby creating a link between the two strands. In some embodiments, the TCR has two disulfide bonds in the constant domain. The TCR has an additional cysteine ​​residue in each of the α and β chains to include Good too.

[0164] In some embodiments, the TCR chain may include a transmembrane domain. In embodiments, the transmembrane domain is positively charged. The R chain contains the cytoplasmic tail. In some cases, due to its structure, the TCR It can associate with other molecules such as CD3. For example, it contains a constant domain together with a transmembrane region. The TCR, which contains the cytoplasmic tyrosine kinase, can anchor its proteins to the cell membrane and interact with the CD3 signaling machinery or can associate with the invariant subunit of the complex.

[0165] Generally, CD3 is composed of three distinct chains (γ, δ, and ε) and the ζ chain in mammals. For example, in mammals, this complex may be CD3γ. It may contain a CD3 δ chain, two CD3 ε chains, and a homodimeric CD3 ζ chain. The γ, δ and ε chains are immunoglobulins that contain a single immunoglobulin domain. The CD3γ chain, CD The transmembrane regions of the CD3δ and CD3ε chains are negatively charged, which is because these chains are The CD3γ chain, CD3 The intracellular tails of the δ and CD3 ε chains contain immunoreceptor tyrosine-based activation motifs or ITs. Each CD3 ζ chain contains three conserved motifs, whereas the CD3 ζ chain contains a single conserved motif known as the AM. In general, ITAMs are involved in the signaling function of the TCR complex. The leukocyte molecule has a negatively charged transmembrane domain and plays a key role in the transmission of signals from the TCR to the cell. The CD3 and ζ chains, together with the TCR, form the T cell receptor complex. They form complexes known as nuclei.

[0166] In some embodiments, the TCR comprises two chains, α and β (or optionally γ and δ) or may be a single chain TCR construct. In some embodiments, the TCR comprises two separate TCRs linked by, for example, a disulfide bond. In some embodiments, the heterodimer comprises a β chain or a γ chain and a δ chain. In this method, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into the cell. In some embodiments, the nucleic acid encoding the TCR is selected from publicly available TCR DNA. A sequences can be obtained from a variety of sources, for example by polymerase chain reaction (PCR) amplification. In some embodiments, the TCR may be derived from a biological source, e.g., a cell, e.g. , T cells (e.g., cytotoxic T cells), T cell hybridomas or other publicly available In some embodiments, the T cells are isolated in vivo. In some embodiments, the high affinity T cell clones can be obtained from The T cells may be isolated from the patient and the TCR may be isolated. In some embodiments, the T cells are The T cell hybridoma or clone may be cultured. In some embodiments, Therefore, TCR clones against target antigens are synthesized by cloning human immune system genes (e.g., human leukocyte antigen receptors). or HLA) engineered transgenic mice. For example, tumor antigens (e.g., Parkhurst et al., 2009 and C (see Ohen et al., 2005). In some embodiments, the phage Using display, TCRs against a target antigen are isolated (e.g., Varela- (See Rohena et al., 2008 and Li, 2005). In one embodiment, the TCR or antigen-binding portion thereof can be synthesized by knowledge of the sequence of the TCR. It can be produced synthetically. C. Antigen-presenting cells

[0167] Antigen-presenting cells, including macrophages, B lymphocytes, and dendritic cells, express specific MHC molecules. APCs internalize antigens and associate parts of the antigen with MHC molecules. The MHC is a large genetic entity that contains multiple gene loci. The MHC locus is a complex of MHC genes that are called class I and class II MHC. C encodes two major classes of membrane molecules. T helper lymphocytes generally encode T lymphocytes recognize antigens associated with MHC class II molecules, whereas T lymphocytes recognize antigens associated with MHC class I molecules. In humans, the MHC is called the HLA complex, and in mice, the H-2 complex. It is called the body.

[0168] In some cases, the therapeutic compositions and cell therapy products of the above embodiments are prepared using General information on the preparation and use of antigen-presenting systems. For specific guidance, see, e.g., U.S. Patent Nos. 6,225,042 and 6,355, Nos. 479, 6,362,001 and 6,790,662; U.S. Pat. Patent Applications Nos. 2009 / 0017000 and 2009 / 0004142; and International Patent Applications Nos. 2009 / 0017000 and 2009 / 0004142; See publication number WO2007 / 103009.

[0169] The aAPC system can include at least one exogenous assisting molecule. And combinations of auxiliary molecules can be used. The auxiliary molecules include costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include CD86, CD64 (F Adhesion molecules include, for example, IL-1, IL-21, 41BB ligand, and IL-21. For example, carbohydrate-binding proteins such as selectins that promote cell-cell or cell-matrix contacts Glycoproteins, transmembrane glycoproteins such as integrins, calcium ions such as cadherins IgG4-dependent proteins and single-pass transmembrane immunoglobulins such as intercellular adhesion molecules (ICAMs) Exemplary adhesion molecules include the phospho(Ig) superfamily proteins. , LFA-3, and ICAMs such as ICAM-1. Methods useful for the selection, cloning, preparation and expression of exemplary auxiliary molecules, including Methods, procedures and reagents are described, for example, in U.S. Pat. Nos. 6,225,042 and 6,355,4 This is exemplified in US Pat. Nos. 79 and 6,362,001. D. Antigen

[0170] Antigens targeted by genetically engineered antigen receptors have the potential to be used as therapeutic targets via adoptive cell therapy. These include antigens that are expressed in the context of the disease, condition, or cell type being targeted. These diseases and conditions include blood cancers, cancers of the immune system (e.g., lymphomas, leukemias, and / or or myelomas, e.g., B, T and myeloid leukemias, lymphomas and multiple myeloma) Proliferative, neoplastic and malignant diseases and disorders, including cancers and tumors, including In some embodiments, the antigen is a normal cell or tissue or a non-targeted On cells of the disease or condition, e.g., on tumor cells, relative to cells or non-targeted tissues. Alternatively, the gene is selectively expressed or overexpressed on the pathogenic cell. In this case, the antigen is expressed on a normal cell and / or on an engineered cell.

[0171] Any suitable antigen can be targeted in this method. The antigen may be a specific cancer cell. In some cases, they may not be associated with non-cancerous cells. Representative antigens include infectious agents, autoantigens / self antigens, and tumors. Antigenic molecules derived from tumor-associated antigens / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015), but are not limited to these. These antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA- 125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / In certain embodiments, the antigen receptors for two or more of the antigen receptors are The antigens that can be detected include CD19, EBNA, WT1, CD123, NY-ESO, and EGFRv. III, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4 , Mage-A10, TRAIL / DR4 and / or CEA, The sequences for these antigens are known in the art and are available, for example, in Gen The following are in the Accession Bank® database: CD19 (Accession Accession number NG_007275.1), EBNA (Accession number NG_002392. 2), WT1 (accession number NG_009272.1), CD123 (accession Accession number NC_000023.11), NY-ESO (Accession number NC_000 023.11), EGFRvIII (accession number NG_007726.3), M UC1 (accession number NG_029383.1), HER2 (accession number NG_007503.1), CA-125 (accession no. NG_055257.1 ), WT1 (accession number NG_009272.1), Mage-A3 (accession Accession number NG_013244.1), Mage-A4 (Accession number NG_01 3245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession no. NC_000003.12), and / or CEA (accession number NC_000019.10).

[0172] Tumor-associated antigens include, for example, prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelial cancer, ovarian cancer, liver cancer, brain cancer, bone cancer, stomach cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, Exemplary tumor-associated or tumor cell-derived antigens include: For example, MAGE1, 3 and MAGE4 (or other MAGE antigens, e.g., No. WO99 / 40188;PRAME;BAGE;RAGE , Lage (also known as NY ESO1); SAGE; and HAGE or GA Non-limiting examples of tumor antigens include melanoma, lung cancer, sarcoma, and bladder cancer. It is expressed in a wide range of tumor types, including cancer. For example, tumor-associated antigens of prostate cancer include prostate-specific membrane antigen (PSA). SMA), prostate specific antigen (PSA), prostatic acid phosphate, NKX3.1 and prostate glandular six-transmembrane epithelial antigen (STEAP) is one example.

[0173] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, and Cripto and Criptin. Additionally, tumor antigens are useful in the treatment of many cancers. Self-peptide hormones, such as full-length gonadotropin releasing hormone (GnRH) , a short 10 amino acid long peptide.

[0174] Tumor antigens include those found in cancers characterized by the expression of tumor-associated antigens such as HER-2 / neu expression. Tumor-associated antigens of interest include lineage-specific tumor antigens, e.g., Melanocyte-melanoma lineage antigen MART-1 / Melan-A, gp100, gp75, Exemplary tumors include mda-7, tyrosinase and tyrosinase-related proteins. Related antigens include p53, Ras, c-Myc, and cytoplasmic serine / threonine kinase ( e.g., A-Raf, B-Raf and C-Raf, cyclin-dependent kinases), MA GE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MA GE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GA GE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MA RT-1, MC1R, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP -2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor anti- Hara (WT1), AFP, -catenin / m, caspase-8 / m, CEA, CDK-4 / m , ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA020 5, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, T RP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPI / mbc r-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / A ML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling receptor 1 (T ACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor EGFR (especially EGFRvIII), platelet-derived growth factor receptor (P DGFR), vascular endothelial growth factor receptor (VEGFR), cytoplasmic tyrosine kinase ( For example, src family, syk-ZAP70 family), integrin-binding kinase ILK, signal transducer and activator of transcription 3 (STAT3), STATS and STATE, hypoxemia Inducers of HIF-1 and HIF-2, Nuclear Factor-kappa B (NF-B) , Notch receptors (e.g., Notch1-4), c-Met, and rapamycin Mammalian target of TOR, WNT, extracellular signal-regulated kinase (ERK) and their Regulatory subunits of PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (G250 (also known as AML1), STEAD, TEL / AML1, GD2, proteinase 3, hTER T, sarcoma translocation breakpoint nts), EphA2, ML-IAP, EpCAM, ERG(TMPRSS2 ETS Syngene), NA17, PAX3, ALK, androgen receptor, cyclin B1, Polysialic acid, MYCN, RhoC, GD3, Fucosyl GM1, Mesothelial thelian), PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLo boH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B 7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, T PTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, Is it derived from one or more of SUNC1, LRRN1 and idiotype? Or tumor antigens including any one or more thereof, but are not limited thereto.

[0175] Antigens come from genes that are mutated in tumor cells or are expressed in tumor cells compared to normal cells. Epitope regions or epitope peptides from genes that are transcribed at different levels in (e.g., telomerase enzyme, survivin, mesothelin, mutant ras, bcr / ab l rearrangement, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1 , and aberrantly expressed intronic sequences such as N-acetylglucosaminyltransferase-V. ); Immune system that generates unique idiotypes in myeloma and B-cell lymphoma Clonal rearrangement of globulin genes: epitopes derived from oncoviral processes. Tumor antigens containing the region or epitope peptide (e.g., human papillomavirus protein proteins E6 and E7); Epstein-Barr virus protein LMP2; tumor-selective Expressed non-mutated oncofetal proteins (e.g., carcinoembryonic antigen and alpha-f The protein may include fetoproteins.

[0176] In other embodiments, the antigen is a pathogenic or opportunistic pathogenic microorganism (see herein). Infectious diseases are caused by organisms (also known as infectious diseases) (e.g., viruses, fungi, parasites, and bacteria). In certain embodiments, the microorganism is derived from such a microorganism. The antigens delivered include the full-length proteins.

[0177] Exemplary pathogenic bacteria having antigens contemplated for use in the methods described herein. These include human immunodeficiency virus (HIV), herpes simplex virus (HSV), and respiratory Respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus EBV, influenza A, B and C, vesicular stomatitis virus (VSV), Vesicular stomatitis virus (VSV), polyomaviruses (e.g., BK virus and J C virus), adenovirus, methicillin-resistant Staphylococcus auricularia Staphylococcus species, including Methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus p As will be appreciated by those of skill in the art, the present invention also includes Streptococcus species, including Streptococcus neumoniae. The present invention relates to the preparation of antigens derived from these and other pathogenic microorganisms for use as antigens as described herein. The proteins, as well as the nucleotide sequences encoding those proteins, are published in and GENBANK®, SWISS-PROT® and TR They can be identified in public databases such as EMBL®.

[0178] Antigens derived from the human immunodeficiency virus (HIV) include the HIV virion structural proteins (e.g., gp120, gp41, p17, p24), proteases, reverse transcriptases, and HIV tat, rev, nef, vif, vpr and vpu are encoded by It contains one of the following proteins:

[0179] Antigens derived from herpes simplex viruses (e.g., HSV1 and HSV2) include: These include, but are not limited to, proteins expressed from HSV late genes. The genes of this group primarily code for proteins that form the virion particle. The proteins consist of five proteins that form the viral capsid (UL): UL6, UL18, UL35, UL38 and major capsid proteins UL19, UL45 and and UL27 (each of which can be used as an antigen as described herein). Other exemplary HSV proteins contemplated for use as antigens herein include The proteins include ICP27 (H1, H2), glycoprotein B (gB) and glycoprotein The HSV genome contains at least 74 genes. , each of which encodes a protein that can potentially be used as an antigen.

[0180] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, Viral antigens, glycoproteins I and III, expressed during the immediate early and early stages of viral replication , capsid protein, coat protein, low molecular weight matrix protein (lowe r matrix protein) pp65 (ppUL83), p52 (ppUL44) ), IE1 and 1E2 (UL123 and UL122), UL128 to UL150 Protein products of gene clusters (Rykman, et al., 2006), envelope Examples of polypeptides that may be used include glycoprotein B (gB), gH, gN, and pp150. As will be appreciated, the CMV proteins for use as antigens as described herein are , GENBANK®, SWISS-PROT® and TREMBL (e.g., Bennekov et al.,2004;Loewendorf et al.,2010;Mars (see Chall et al., 2009).

[0181] Epstein-Van Virus (EBV) Contemplated for Use in Certain Embodiments Antigens derived from EBV include the EBV lytic proteins gp350 and gp110, Epstein-Barr virus (EBV)-derived cytoplasmic antigens (EbV), Thayne-Van nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B , EBNA-3C, EBNA-reader protein (EBNA-LP), and latency Latently infected cytoplasmic chromatin (LMP)-1, LMP-2A and LMP-2B. Examples of EBV proteins produced during replication include those in the cloning process (e.g., Lockey et al. .,2008).

[0182] Antigens derived from Respiratory Syncytial Virus (RSV) contemplated for use herein include: Eleven proteins or their antigenic fragments encoded by the RSV genome NS1, NS2, N (nucleocapsid protein), M (matrix protein) SH, G and F (viral coat proteins), M2 (second matrix protein M2-1 (elongation factor), M2-2 (transcriptional regulation), RNA polymerase, and The phosphoprotein P may be any of the following:

[0183] Antigens derived from vesicular stomatitis virus (VSV) contemplated for use include those derived from the VSV genome The five major proteins and their antigenic fragments encoded by: Proteins (L), glycoproteins (G), nucleoproteins (N), phosphoproteins (P) and and matrix protein (M) (e.g., Ried (see er et al., 1999).

[0184] Antigens derived from influenza viruses contemplated for use in certain embodiments These include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), Matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, P B1-F2, and PB2.

[0185] Exemplary viral antigens include polypeptides of adenoviruses, polypeptides of alphaviruses, and polypeptides of alphaviruses. Polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigen Gen), Coronavirus polypeptide, Distemper virus polypeptide, Ebola virus polypeptide Virus polypeptides, enterovirus polypeptides, flavivirus polypeptides Hepatitis virus (AE) polypeptides (Hepatitis B core or surface antigen, Hepatitis C virus E1 or E2 glycoproteins, core or nonstructural proteins of herpesviruses, The polypeptides of the virus (glycoproteins of herpes simplex virus or varicella zoster virus) (including infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marsh Polypeptides of Burgviruses, Polypeptides of Orthomyxoviruses, Polypeptides of Papillomaviruses influenza virus polypeptides, parainfluenza virus polypeptides (e.g., hemagglutinin polypeptide and neuraminidase polypeptide), paramyxovirus polypeptide Tid, Parvovirus Polypeptide, Pestivirus Polypeptide, Picornavirus Polypeptide polypeptides of viruses (e.g., capsid polypeptides of polioviruses), poxviruses polypeptides of viruses (e.g., polypeptides of vaccinia virus), polypeptides of rabies virus Polypeptides (e.g., glycoprotein G of rabies virus), polypeptides of reovirus Also included are polypeptides of viruses, retroviruses, and rotaviruses. , but not limited to these.

[0186] In certain embodiments, the antigen may be a bacterial antigen. In certain other embodiments, the bacterial antigen of interest may be a secreted polypeptide. Bacterial antigens include those that have a portion of a polypeptide exposed on the outer surface of the bacterial cell. Included.

[0187] The methicillin-resistant Staphylococcus aureus (M Antigens derived from Staphylococcus species, including Staphylococcus aureus (RSA), include virulence regulators, e.g. , Agr system, Sar and Sae, Arl system, Sar homologs (Rot, M grA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and Others that may serve as antigens include the IL-1R and TcaR systems, the Srr system, and TRAP. The Staphylococcus spp. proteins include Clp proteins, HtrA, MsrR, and Aconitum spp. tase, CcpA, SvrA, Msa, CfvA and CfvB (e.g., Staphylococcus:Molecular Genetics,2008 C See aister Academic Press, Ed. Jodi Lindsay Two species of Staphylococcus aureus (N315 and Mu5) The genome of PATRIC (PATRIC:The VBI PathoSystems Resource Integration Ce The results are publicly available at: http: / / www.nature.com / articles / 101325 / 101325. As understood by the supplier, Staphylococcus spp. proteins for use as antigens are available from Gen. Bank®, Swiss-Prot® and TrEMBL®. They may also be identified in other public databases, such as the National Institute of Standards and Technology (NIS).

[0188] Streptoviruses contemplated for use in certain embodiments described herein Antigens derived from Coccus pneumoniae include pneumolysin, Psp A, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA , LytA, Pht and pilin proteins (RrgA; RrgB; RrgC). The antigenic proteins of Streptococcus pneumoniae are also are known in the art and may be used as antigens in some embodiments (e.g., Zy (See sk et al., 2000). Streptococcus pneumoniae The entire genome sequence of a virulent strain of E. moniae has been sequenced, and as will be appreciated by those of skill in the art, For use herein, S. pneumoniae proteins are listed in GENBANK (registered trademark), SWISS-PROT (registered trademark) and TREMBL (registered trademark), etc. Antigens of particular interest according to the present disclosure may also be identified in other public databases. Proteins include virulence factors and proteins predicted to be exposed on the surface of pneumococci. quality (see, e.g., Frolet et al., 2010).

[0189] Examples of bacterial antigens that can be used as antigens include polypeptides of Actinomyces sp. Polypeptides of the genus Bacillus, Polypeptides of the genus Bacteroides, Polypeptides of the genus Bordetella polypeptides of the Bartonella genus, polypeptides of the Borrelia genus (e.g., B. dorferi OspA), Brucella spp. polypeptides, Campylobacter spp. polypeptides polypeptides of Capnocytophaga spp., polypeptides of Chlamydia spp., polypeptides of Coli Polypeptides of Nebacterium sp., Coxiella sp., Dermatophilus sp. Polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia Polypeptides of the genus Erichsia, the genus Francisella, and the genus Fusobacterium Peptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g. H. influenzae type b outer membrane protein), polypeptides of Helicobacter spp. Klebsiella polypeptides, L-form bacteria polypeptides, Leptospira polypeptides polypeptides of Listeria spp., polypeptides of Mycobacterium spp., Mycoplasma Polypeptides of the genus Neisseria, Polypeptides of the genus Neorickettsia, Nocardia polypeptide, Pasteurella polypeptide, Peptococcus polypeptide polypeptides of the Peptostreptococcus genus, polypeptides of the Pneumococcus genus (i.e. pneumoniae polypeptides) (see description herein), Polypeptides of the genus Rhodeus, Pseudomonas, and Rickettsia Polypeptides of the genus Roshalimea, Polypeptides of the genus Salmonella, Polypeptides of the genus Shigella polypeptides of Staphylococcus spp., polypeptides of Group A Streptococcus (e.g., S.py Streptococcus agalactiae M protein, Group B Streptococcus agalactiae polypeptide polypeptides of Treponema spp., and Yersinia spp. (e.g., Y Examples of suitable antigens include, but are not limited to, F1 and V antigens of Pestis.

[0190] Examples of fungal antigens include polypeptides from the genus Absidia, polypeptides from the genus Acremonium, Tid, Alternaria polypeptide, Aspergillus polypeptide, Basidiovo Polypeptides of the genus Rus, Polypeptides of the genus Bipolaris, Polypeptides of the genus Blastomyces Polypeptides of Candida spp., Polypeptides of Coccidioides spp., Polypeptides of Conidiobolus spp. Polypeptides of the genus Cryptococcus, Polypeptides of the genus Curvalaria , Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum Polypeptides of the genus Mycoplasma, Polypeptides of the genus Histoplasma, Polypeptides of the genus Madurella, Polypeptides of the genus Racetia, Microsporum, and Moniliella Polypeptides of the genus Mortierella, Polypeptides of the genus Mucor, Polypeptides of the genus Paecilomyces Lipeptides, Penicillium polypeptides, Phialemonium polypeptides ium) polypeptides, Phialophora polypeptides, Prototheca polypeptides Polypeptides from the genus Pseudoalescheria, Pseudomicrodochium Polypeptides of the genus Microdochium, Polypeptides of the genus Pythium, Polypeptides of the genus Rhinospoli Polypeptides of the genus Rhizopus, polypeptides of the genus Scolecobasidium olecobasidium polypeptide, Sporothrix polypeptide, Ste Polypeptides of Stemphylium genus, Polypeptides of Trichophyton genus, Polypeptides of the genus Trichosporon and of the genus Xylohypha These include, but are not limited to, peptides.

[0191] Examples of protozoan parasite antigens include polypeptides from Babesia spp., Balantidium spp. Polypeptides of the genus Besnoitia, polypeptides of the genus Cryptosporum Polypeptides of the genus Rhydium, Eimeria, and Encephalitozoon Polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammond polypeptides Polypeptides of Hammondia, Polypeptides of Hepatozoon, Polypeptides of Isosporum Polypeptides of the genus Pora, polypeptides of the genus Leishmania, polypeptides of the phylum Microsporidia, Polypeptides of Neospora spp., Polypeptides of Nosema spp., Polypeptides of Pentatrichomonas spp. Examples of polypeptides include, but are not limited to, polypeptides of the genus Plasmodium. Examples of worm parasite antigens include the polypeptide aelurostron of Acanthocheironema sp. Polypeptides from Aelurostrongylus, Polypeptides from Hookworm Polypeptides of Angiostrongylus spp. Polypeptides of Ascaris spp. Polypeptides of Brugia spp. Polypeptides of the genus Nostomum, the genus Capillaria, the genus Caverthia ertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides soma) polypeptides, Dictyocaulus spp. polypeptides, Dioctophyme spp. Polypeptides of Dipetalonema spp., Polypeptides of Diphyllobothrium spp., Polypeptides of Diply Polypeptides of the genus Dirofilaria, Polypeptides of the genus Dracunculus polypeptides of the genus Enterobius, polypeptides of the genus Filaroides Lipeptides, polypeptides of the Haemonchus genus, Lagochilas caris polypeptides, Loa polypeptides, Mansonella polypeptides Polypeptides, Muellerius polypeptides, Nanophyets Polypeptides from Nanophyetus, Polypeptides from Nema Polypeptides of the genus Tojirus, Polypeptides of the genus Enterococcus, Polypeptides of the genus Onchocerca , Opisthorchis polypeptides, Ostertagia polypeptides, Parafilaria Polypeptides of the genus Parafilaria, Polypeptides of the genus Paragonimus, Parascaris Polypeptides of the genus Parascaris, Polypeptides of the genus Physaloptera, Proto Polypeptides from the genus Protostrongylus and the genus Setaria Lipeptide, Polypeptide of Spirocerca genus Spirometra genus polypeptides of the genus Stephanofilaria, polypeptides of the genus Strongyloides Peptides, Strongylus polypeptides, Thelazia polypeptides, Toxocalis Polypeptides of the genus Toxocara, Polypeptides of the genus Trichinella, Polypeptides of Trichostrongylus Polypeptides of the genus Trichuris, Polypeptides of the genus Uncinaria and Polypeptides of the genus Ukere Polypeptides of the genus P. falciparum (e.g., the peri-sporozoite polypeptides of P. falciparum) tide (PfCSP), sporozoite surface protein 2 (PfSSP2), liver status antigen The carboxyl terminus of (liver state antigen) 1 (PfLSA1 c terminal), and export protein 1 (PfExp-1), a polypeptide from Pneumocystis sp. Tid, Sarcocystis polypeptide, Schistosoma polypeptide, Theileria polypeptide Polypeptides, Toxoplasma polypeptides, and Trypanosoma polypeptides These include, but are not limited to,

[0192] Examples of ectoparasite antigens include fleas; ticks, including hard mites and ulcerative colitis; flies, e.g. For example, midges, mosquitoes, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, Stable flies, flies that cause myiasis, and small flying insects that bite and suck blood gnats); ants; spiders, lice; mites; and hemipteran insects, e.g., bedbugs These include polypeptides (including antigens and allergens) of the insect and assassin bug. The present invention is not limited to these. E. Suicide gene

[0193] In some cases, any of the cells of the present disclosure may be administered with heterologous cytokines, engineered receptors, or other In a specific embodiment, the recombinant protein is modified to produce one or more agents other than a peptide. In the present invention, cells such as NK cells are engineered to carry one or more suicide genes, The term "suicide gene" as used herein means a gene product that is produced when a prodrug is administered. , defined as a gene that is transformed into a compound that kills the host cell. NK cell therapy is administered to individuals receiving NK cell therapy and / or individuals who have received NK cell therapy. The body responds to one or more symptoms of one or more adverse events (e.g., cytokine release syndrome, neurotoxicity, toxicity, anaphylaxis / allergy and / or on-target / off-tumor toxicity (e.g. If you show symptoms of COVID-19 or are considered to be at risk for having one or more of the following conditions: In cases where a person is in a state of emergency (including a state of imminent death), they may be subject to the use of one or more suicide genes of any kind. The use of cytocidal genes may be part of a planned protocol for treatment or may be In some cases, cells may be used only when the need for them is recognized. Therapy targets the suicide gene or its gene product because it is no longer needed. The effect is terminated by the use of an agent that

[0194] Examples of suicide genes include engineered non-secreted (including membrane-bound) tumor necrosis factors (TNFs). NF)-alpha mutant polypeptide (PC T / US19 / 62009), and these polypeptides include TNF. The suicide gene may be targeted by delivery of an antibody that binds to the -alpha variant. An example of a drug / prodrug combination is the herpes simplex virus-thymidine kinase (HS V-tk) and ganciclovir, acyclovir or FIAU; oxidoreductase and Cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidine midylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytochrome P450 The prodrug 6-methylpurine deoxyriboside is The so-called suicide gene of E. coli that converts the purine to 6-methylpurine Purine nucleoside phosphorylase may be used. Other suicide genes include CD20 , CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome P450 Chromium p450 enzymes (CYP), carboxypeptidases (CP), carboxylesterases enzyme (CE), nitroreductase (NTR), guanine ribosyltransferase (X GRTP), glycosidase enzymes, methionine-α,γ-lyase (MET) and thymine An example is thiamine phosphorylase (TP). F. Delivery method

[0195] Those of skill in the art can use standard recombinant techniques (e.g., For example, Sambrook et al., 2001 and Ausubel et al. , 1996 (both of which are incorporated herein by reference) The vectors used are plasmids, cosmids, and , viruses (bacteriophages, animal viruses and plant viruses) and artificial chromosomes (e.g., YAC), e.g., retroviral vectors (e.g., Moloney Murine Leukemia Derived from viral vectors (MoMLV), MSCV, SFFV, MPSV, SNV, etc. lentiviral vectors (e.g., HIV-1, HIV-2, SIV, BIV Adenovirus (Ad) vectors (those derived from replication-competent, replication-competent, or replication-competent vectors) Adeno-associated virus (ADV), including production-deficient and gutless forms avirulence vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, virus vector, Epstein-Barr virus vector, herpes virus vector, vaccine vector Xenovirus vector, Harvey murine sarcoma virus vector, mouse mammary tumor virus vector, Rous sarcoma virus vector, parvovirus vector, poliovirus vector vector, vesicular stomatitis virus vector, Maraba virus vector vectors and group B adenovirus enadenotucirev vectors. Not limited to these.

[0196] In a specific embodiment, the vector is described in PCT / US19 / 62014 (in its entirety). (which is incorporated herein by reference) In such cases, a single vector can be used to express either the CAR or the TCR (its expression construct). It is constructed in a modular format to allow for interchangeability of CAR or TCR portions. The gene may encode a suicide gene and one or more cytokines. A. Viral vectors

[0197] Viral vectors encoding antigen receptors are provided in certain embodiments of the present disclosure. In constructing recombinant viral vectors, non-essential genes are usually (or non-native) protein genes or coding sequences are substituted. The markers utilize viral sequences to deliver nucleic acids and sometimes proteins into cells. It is a type of expression construct that expresses the ability of a particular virus to infect a cell or a receptor. their ability to enter cells via endocytosis mediated by IL-1 and to infect the host cell genome Their ability to integrate with and stably and efficiently express viral genes has made them These viruses are attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Viral Vectors That Can Be Used to Deliver the Nucleic Acids of Certain Embodiments of the Invention Non-limiting examples are given below:

[0198] Lentiviruses are complex retroviruses and are a common retroviral genome. In addition to gag, pol, and env, other genes with regulatory or structural functions are also Lentiviral vectors are well known in the art (see, e.g., U.S. Patent No. 6,013,636). (See US Pat. Nos. 3,516 and 5,994,136).

[0199] Recombinant lentiviral vectors are capable of infecting non-dividing cells and are capable of infecting both in vivo and ex vivo cells. The vectors may be used for gene transfer and expression of nucleic acid sequences both in vivo and in vivo. A recombinant lentivirus capable of infecting dividing cells (wherein a suitable host cell is a Two or more vectors with packaging functions, namely gag, pol and env, and transfected with rev and tat), which are incorporated herein by reference. No. 5,994,136, which is incorporated herein by reference. B. Regulatory elements

[0200] Expression cassettes included in vectors useful in the present disclosure include, inter alia, protein-encoding A eukaryotic transcription promoter operably linked to the sequence, a splice sigma including an intervening sequence Null, and transcription termination / polyadenylation sequences (5' to 3'). Promoters and enhancers that control transcription of genes encoding proteins in eukaryotic cells - Patents.com A gene is made up of multiple genetic elements. The cellular machinery is carried by each element. It is possible to collect and integrate regulatory information from different genes, often This allows for the development of complex patterns of different transcriptional regulation. The promoters used in this context include constitutive promoters and inducible promoters. and tissue-specific promoters. (i) Promoter / enhancer

[0201] The expression constructs provided herein include a promoter that drives expression of an antigen receptor. A promoter generally comprises a promoter sequence that determines the position of the start site for RNA synthesis. The best known example of this is the TATA box, but mammalian Mammalian terminal deoxynucleotidyl transferase gene promoter and Some promoters, such as those of the SV40 late genes, contain a TATA box. A discontinuous element that lacks a nucleotide sequence and overlaps the initiation site itself fixes the location of initiation. Additional promoter elements control the frequency of transcription initiation. These are located in the region 30110 bp upstream of the start site, but several promoters It has also been shown that the coding sequence contains functional elements downstream of the initiation site. To be "under the control of" a transcriptional open reading frame, the 5' end of the transcription initiation site is The "upstream" promoter is placed "downstream" (i.e., 3') of the motor. It stimulates transcription and promotes expression of the encoded RNA.

[0202] The spacing between promoter elements is often variable, and elements can be inverted or The function of the promoters is preserved even when they are separated or moved relative to each other. In the case of the promoter, the spacing between promoter elements can be up to 50 nm before activity begins to decline. Depending on the promoter, the individual elements may act cooperatively or independently. A promoter may function to activate transcription of a nucleic acid sequence. It may also be used in conjunction with the term "enhancer," which refers to a cis-acting regulatory sequence involved in It does not have to be used.

[0203] A promoter is a 5' non-coding region located upstream of a coding segment and / or exon. A nucleic acid sequence that is naturally associated with a nucleic acid sequence, such as may be obtained by isolating a code sequence. Such a promoter may be referred to as an "endogenous" promoter. Similarly, an enhancer can be located downstream or upstream of a nucleic acid sequence. It may be an enhancer that is naturally associated with the sequence. A recombinant promoter is a promoter that is not naturally associated with a particular environment. By placing the coding nucleic acid segment under the control of a heterologous promoter, certain advantages are afforded. In addition, recombinant or heterologous enhancers may be expressed in a manner similar to that found in the nucleus in their natural environment. Such promoters or enhancers are not naturally associated with a nucleic acid sequence. The promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers of other genes of interest. A promoter or enhancer isolated from any virus or prokaryotic or eukaryotic cell. sensors, as well as non-"naturally occurring" i.e., various elements of various transcription control regions. Contains promoters or enhancers that contain mutations that alter the expression of the For example, the most commonly used promoters in recombinant DNA construction are β-lactamase (penicillinase), lactose and tryptophan (trp-) Promoter systems include the following: Synthetic nucleic acid sequences of promoters and enhancers In addition to quantitatively generating the vector, recombinant cloning and / or PCR TM Starting with Using nucleic acid amplification techniques such as those described herein, a sequence may be generated in association with the compositions disclosed herein. In addition, in organelles other than the nucleus (e.g., mitochondria, chloroplasts, etc.), It is contemplated that regulatory sequences that direct the transcription and / or expression of the sequences can be used as well. .

[0204] Naturally, the D in the organelle, cell type, tissue, organ or organism selected for expression Use a promoter and / or enhancer that effectively directs expression of the NA segment. Those skilled in the art of molecular biology generally use a promoter for protein expression. We are aware of the use of combinations of promoters, enhancers, and cell types (e.g. See, for example, Sambrook et al. 1989, incorporated herein by reference. The promoters used are constitutive promoters, tissue-specific promoters, and , inducible promoters, and / or high level expression of the introduced DNA segment. Under appropriate conditions, useful promoters (e.g., recombinant proteins and / or or a promoter useful in the large-scale production of recombinant peptides. The motor may be heterologous or endogenous.

[0205] In addition, any promoter / enhancer combination (e.g., epd.isb- Eukaryotic Promote via the World Wide Web at sib.ch / r Data Base EPDB) can also be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another viable embodiment. Bacterial polymerases may be provided as part of a delivery complex or as additional genetic expression constructs. When provided with a nucleotide sequence, eukaryotic cells support transcription in the cytoplasm from certain bacterial promoters. obtain.

[0206] Non-limiting examples of promoters include early viral promoters or late viral promoters. Promoters (e.g., SV40 early or late promoters, cytomegalovirus ( CMV immediate early promoter, Rous sarcoma virus (RSV) early promoter); eukaryotic Cellular promoters (e.g., beta-actin promoter, GADPH promoter, the tarothionine promoter); and linked responsive element promoters (e.g., Cyclic AMP response element promoter (cre), serum response element promoter sre, phorbol ester promoter (TPA) and minimal TATA promoter Human growth hormone (HGH) Promoter sequences (e.g., human growth hormone promoter sequences listed in Genbank®) Mon minimal promoter, accession number X05244, nucleotides 283-3 41) or mouse mammary tumor promoter (available from ATCC, Cat. No. AT CC45007) may also be used. The targets were CMV IE, dectin-1, dectin-2, human CD11c, F4 / 80, and S M22, RSV, SV40, Ad MLP, beta-actin, MHC class I or M HC class II promoters, however, drive expression of therapeutic genes Any other promoter useful for this purpose may also be applied in the practice of the present disclosure.

[0207] In certain embodiments, the methods of the present disclosure include the use of enhancer sequences, i.e., promoter sequences. A nucleic acid sequence that increases the activity of a nucleic acid sequence in cis and in any orientation, and that Ability to act over long distances (up to several kilobases away from the target promoter) However, enhancers may also be located close to a given promoter. Enhancer function is not necessarily limited to such long distances, as enhancers can function at any stomach. (ii) Initiation signal and ligated expression

[0208] For efficient translation of the coding sequence, in the expression constructs provided in this disclosure, specific Other initiation signals may also be used. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, must be provided. It is within the skill of the art to determine this and provide the necessary signals. To ensure translation of the entire insert, the initiation codon must be It is well known that the reading frame of the coding sequence must be "in frame" with the coding sequence of the gene. The native translational control signals and initiation codons can be natural or synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements.

[0209] In certain embodiments, the use of an internal ribosome entry site (IRES) element The use of ribosomal RNA is a method for producing multigene, i.e. polycistronic, messages. IRES elements are used to regulate the ribosome function of 5' methylated cap-dependent translation. It is possible to bypass the scanning model and initiate translation at an internal site. IRES elements from two members of the poliovirus family (polio and encephalomyocarditis), IRES elements derived from mammalian messages have also been reported. can be linked to heterologous open reading frames, each separated by an IRES. Multiple open reading frames can be transcribed together, forming polycistronic sequences. Thanks to the IRES element, each open reading The frame becomes accessible to ribosomes for efficient translation. Efficiently express multiple genes using transcribing / enhancers to transcribe a single message It is also possible.

[0210] Additionally, to provide for linked or simultaneous expression of genes within the constructs provided in this disclosure, For this purpose, certain 2A sequence elements can be used. For example, To co-express genes by linking the rHEMS to form a single cistron , cleavage sequences may be used. Exemplary cleavage sequences include F2A (foot and mouth disease virus 2A) or "2A-like" sequences (eg, Thosea asigna virus 2A; T2A). (iii) point of origin of replication; In order to propagate a vector in a host cell, the vector contains one or more origins of replication. A site (often called an "ori"), e.g., a specific nucleic acid sequence at which replication begins EBV oriP or similar function in programming as described above. The nucleic acid sequence may include a nucleic acid sequence corresponding to a genetically engineered oriP that has been modified or enhanced. Alternatively, the origin of replication of other viruses that replicate extrachromosomally, as described above, or An autonomously replicating sequence (ARS) can be used. c. Selectable and screenable markers

[0211] In some embodiments, cells comprising a construct of the present disclosure express a marker vector. Such a molecule can be identified in vitro or in vivo by including it in a The marker induces an identifiable change in the cells that allows cells containing the expression vector to be easily identified. Generally, a selectable marker is one that confers a property that allows for selection. A positive selection marker is one whose presence allows for selection. A negative selectable marker is a marker whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0212] The inclusion of a drug selection marker usually facilitates cloning and identification of transformants. Helped by, for example, neomycin, puromycin, hygromycin, DHFR, G Genes that confer resistance to PT, zeocin, and histidinol are useful selection markers. A marker that confers a phenotype that allows for the identification of transformants based on the performance of a condition. In addition, colorimetric markers such as GFP and other screenable markers are available. Other types of markers such as herpes simplex virus thymidine kinase are also contemplated. enzyme (tk) or chloramphenicol acetyltransferase (CAT), etc. A screenable enzyme can be used as a negative selection marker. If so, we would also know how to use immunological markers, possibly in conjunction with FACS analysis. The markers used will be those that are expressed simultaneously with the nucleic acid encoding the gene product. Selection markers and screening Further examples of possible markers are well known to those of skill in the art. d. Other nucleic acid delivery methods

[0213] In addition to viral delivery of nucleic acids encoding antigen receptors, the following methods can be used to deliver antigen receptors to a given host: As such, additional methods of recombinant gene delivery into cells are contemplated in this disclosure.

[0214] The introduction of nucleic acids, such as DNA or RNA, into immune cells of the present disclosure can be accomplished using methods described herein. or as known to those skilled in the art, suitable for delivery of nucleic acid to transform cells. Any method may be used. Such methods include direct delivery of DNA (e.g., exon in vivo transfection, injection (including microinjection); electroporation precipitation; calcium phosphate precipitation; DEAE-dextran followed by polyethylene glycosylation use of sonophores; direct sonophoresis; liposome-mediated transfection and receptor Phototransduction-mediated transfection; microprojectile bombardment mbardment); agitation with silicon carbide fibers; Agrobacterium-mediated trait transformation; desiccation / inhibition-mediated DNA uptake, and any combination of such methods These and other techniques include, but are not limited to, the following: In particular, cells, tissues or organisms can be stably or transiently transformed. VI. Treatment Method

[0215] Embodiments of the present disclosure include methods for treating individuals for cancer, any type of infection, and any immune disorder. The individual may be administered the treatment as a first line treatment or after or following another treatment. During treatment, the treatment methods of the present disclosure may be used. Based on the stage, at least some of the needs of individuals with cancer can be tailored to the individual's needs. In some cases, immunotherapy may be tailored to the individual over the course of treatment.

[0216] In a specific case, the treatment method is as follows: 1) Any type of blood disorder To treat cancer patients with invasive tumors, T or NK cells (ex vivo expanded T or NK cells, or T or NK cells expressing CAR or TCR) (2) Adoptive cell therapy to treat cancer patients with any type of solid tumor; are NK cells (ex vivo expanded T or NK cells, or CAR or TCR (3) adoptive cell therapy using immune-compromised patients with Adoptive cell therapy using Treg and regulatory B cells (ex vivo expanded or expressing a CAR or TCR), (4) to treat patients with infectious diseases To achieve this, T or NK cells (including ex vivo expanded T or NK cells, or CARs) can be used. The present disclosure relates to adoptive cell therapy using human N Knocking down / knocking out multiple genes in K cells improves cell function. This is the first time that the antibody has been shown to contribute to the defense against inflammatory and tumor microenvironmental disorders. In this regard, this includes enhancing the function of the patient's own immune cells or adoptively transferred immune cells. There are direct implications for patient care using novel immunotherapeutic approaches. Highly functional T, NK and B cells (ex vivo expanded and cultured) for immunotherapy We provide novel approaches to generate AR or TCR engineered cells. These include: , cancer (both hematological and solid tumors) (NK cells and T cells, as well as CAR T cells and and CAR NK cells), autoimmune and alloimmune disorders (B cells, regulatory B cells and These include targeting immune cells (regulatory T cells) and treating infectious diseases (pathogen-specific T cells).

[0217] In some embodiments, the present disclosure provides a method for treating a cancer, comprising administering an effective amount of an immune cell of the present disclosure. In one embodiment, a method for immunotherapy is provided, comprising: The transfer of immune NK cell populations treats medical diseases or disorders. In certain embodiments, the transfer of immune cell populations that induce an immune response is used to treat cancer or rheumatoid arthritis. The infection is treated. Methods for treating cancer or slowing the progression of cancer in an individual are disclosed herein. Provided herein is a method comprising administering to an individual an effective amount of an antigen-specific cell therapy. The method may be applied to the treatment of immune disorders, solid cancers, hematological cancers and viral infections. .

[0218] Tumors for which the present treatment methods are useful include any malignant cell type, e.g., solid tumors or hematomas. Exemplary solid tumors include cell types found in tumors of the pancreas, colon, cecum, stomach, , brain, head, neck, ovaries, kidneys, larynx, sarcoma, lung, bladder, melanoma, prostate and breast Exemplary hematologic tumors include, but are not limited to, tumors of organs selected from the group consisting of: Liquid tumors include tumors of the bone marrow, T- or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, and Further cancers that can be treated using the methods provided herein include Examples include lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma); Peritoneal cancer, gastric cancer or stomach cancer (digestive cancer and digestive tract (including stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, and colorectal cancer , endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer , various types of head and neck cancer, and melanoma.

[0219] The cancer may specifically be, but is not limited to, cancer of the following tissue types: neoplasms , malignant;carcinoma;carcinoma, undifferentiated;giant cell and spindle cell carcinoma;small cell carcinoma;papillary carcinoma;squamous Skin cancer; lymphoid epithelial carcinoma; basal cell carcinoma; pilomatric carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gas Trinoma, malignant; Cholangiocarcinoma; Hepatocellular carcinoma; Mixed hepatocellular carcinoma-cholangiocarcinoma; Trabecular adenocarcinoma; Adenoid cyst Cancer; Adenocarcinoma in adenomatous polyp; Adenocarcinoma, Familial Coliform Polyposis; Solid tumor; Carcinoid tumor , malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic Carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary / follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortex carcinoma;endometrioid carcinoma;skin adnexal carcinoma;apocrine gland carcinoma;sebaceous gland carcinoma;cerumen adenocarcinoma;mucoepidermoid carcinoma;cyst gland Cancer; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease of the breast;Acinic cell carcinoma;Adenosquamous carcinoma Skin carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Theca cell tumor, malignant ;Granulosa cell tumor, malignant;Androblastoma, malignant;Sertoli cell carcinoma;Leydig cell carcinoma alveolar tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;brown Glomus angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Lentigo maligna Melanoma; Acral lentiginous melanoma; Nodular melanoma; Giant intrapigmented nevus malignant melanoma; Epithelioid cell melanoma Chromoma;Blue nevus, malignant;Sarcoma;Fibrosarcoma;Fibrous histiocytoma, malignant;Myxosarcoma;Liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumors, Malignant;Mullerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymoma, Malignant;Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant ;Ovarian goiter, malignant;Choriocarcinoma;Mesonephroma, malignant;Hemangiosarcoma;Hemangioendothelioma, malignant;Kaposi's hemangiopericytoma, malignant;lymphangiosarcoma;osteosarcoma;paracortical osteosarcoma;chondrosarcoma;chondroblastoma , malignant;mesenchymal chondrosarcoma;giant cell tumor of bone;Ewing sarcoma;odontogenic tumor, malignant;enamel Epithelial odontosarcoma;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma ;glioma, malignant;ependymoma;astrocytoma;protoplasmic astrocytoma;thin line fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal ;cerebellar sarcoma;ganglioneoblastoma;neuroblastoma;retinoblastoma;olfactory neurogenic tumor;meningioma, malignant;neuro Fibrosarcoma;Schwannoma, malignant;Granular cell tumor, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin; Granulomatous granuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell type, diffuse; Malignant lymphoma Lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin's lymphoma; B-cell lymphoma; Low-grade / Follicular non-Hodgkin's lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate grade / Follicular aggressive NHL; intermediate-grade diffuse NHL; aggressive immunoblastic NHL; aggressive lymphoblastic NHL NHL; high-grade small noncleaved cell NHL; bulky disease NHL; Mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglottitis globulinemia;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease; Leukemia; Lymphocytic leukemia; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myeloid Leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryoblastic myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphocytic Myeloblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia .

[0220] Certain embodiments relate to methods of treating leukemia, which is a cancer of the blood or bone marrow. is characterized by an abnormal proliferation (production by division) of blood cells, usually white blood cells (leukocytes). Leukemia is part of a broad group of diseases called blood malignancies. Leukemia is a broad term that encompasses a range of diseases. It can be classified into acute and chronic forms, both clinically and pathologically. It can be divided into three categories.

[0221] In certain embodiments of the present disclosure, the immune cells are administered to an individual in need thereof (e.g., The cells are then delivered to a patient (an individual with cancer or an infectious disease) who is then able to stimulate the immune system of that individual. The system is then boosted to attack the respective cancer or pathogenic cells. Immune cells are provided to an individual one or more times. If immune cells are provided to an individual two or more times, The time between administration should be sufficient for propagation in the individual and should be within the scope of the specific experiment. In embodiments, the time between administrations is 1, 2, 3, 4, 5, 6, 7 days or more.

[0222] Certain embodiments of the present disclosure provide methods for treating or preventing immune-mediated disorders. In one embodiment, the subject has an autoimmune disease. Non-limiting examples include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Dyson's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune egg Focal inflammation and autoimmune orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid Acne, cardiomyopathy, celiac polydermatitis s), chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Chronic rheumatoid arthritis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis pulmonary nephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 diabetes or immune-mediated diabetes , myasthenia gravis, nephrotic syndrome (e.g., minimal change disease, focal glomerulosclerosis or membranous glomerulosclerosis) Nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, Polymyalgia ulcerata, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, Sarcoidosis, scleroderma, Sjogren's syndrome, stiff man syndrome, systemic lupus erythematosus, ulcerative colitis, uveitis, vasculitis (e.g., nodular multifocal arteritis, Takayasu's arteritis, temporal arteritis / giant cell arteritis or dermatitis herpetiformis vasculitis), The present invention relates to a method for treating granulomatosis, including the treatment of granulomatosis, ... and Wegener's granulomatosis. Some examples of autoimmune diseases that can be treated with Systemic lupus erythematosus, type I diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, These include, but are not limited to, glomerulonephritis, ankylosing spondylitis, vasculitis or psoriasis. The subject may also have an allergic disorder, such as asthma.

[0223] In yet another embodiment, the subject is a recipient of a transplanted organ or stem cells. In certain embodiments, immune cells are used to prevent and / or treat rejection. In some embodiments, the subject has graft-versus-host disease or is at increased risk of developing graft-versus-host disease. GVHD can be treated with transplantation using stem cells from related or unrelated donors or transplantation with donors. GVHD is a complication that can occur with any transplant, including transplants with other organs. There are two types of GVHD: acute and chronic. Acute GVHD appears within the first 3 months after transplantation. Signs of acute GVHD include: Symptoms include a reddish rash on the hands and feet that may develop into peeling or blister-like skin. Acute GVHD can spread with subsequent growth and become more severe. It may also affect the skin and the nervous system, causing muscle cramps, nausea and diarrhea. Yellowing of the eyes (jaundice) indicates that acute GVHD is affecting the liver. Chronic GVHD can be It is graded based on its severity: Stage / Grade 1 being mild; Stage / Grade 4 is severe. Chronic GVHD develops 3 months or more after transplant. The symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD is characterized by It can also affect the mucous glands in the stomach, the salivary glands in the mouth and the glands that lubricate the stomach wall and intestines. Any of the immune cell populations disclosed in the above can be used. The present invention relates to organ transplants, such as kidney, liver, skin, pancreas, lung and / or heart, or cellular transplants. Cell grafts, such as islets, hepatocytes, myoblasts, bone marrow or hematopoietic stem cells or other stem cells. The graft may be a composite graft, for example facial tissue. Immune cells The antibody may be administered prior to, simultaneously with, or after transplantation. In this case, the immune cells are administered prior to transplantation, for example, at least 1 hour prior to transplantation, at least 12 hours prior to transplantation. shortly before, at least 1 day before, at least 2 days before, at least 3 days before, at least 4 days before, At least 5 days in advance, at least 6 days in advance, at least 1 week in advance, at least 2 weeks in advance, The drug is administered at least 3 weeks, at least 4 weeks, or at least 1 month prior to the start of the treatment. In a typical embodiment, administration of a therapeutically effective amount of immune cells occurs 3-5 days prior to transplantation.

[0224] In some embodiments, the subject receives a non-myeloablative lymphoma prior to immune cell therapy. Lymphocyte-depleting chemotherapy may be administered. The non-myeloablative lymphocyte-depleting chemotherapy may be administered in any suitable manner. The non-myeloablative therapy may be any suitable such therapy that can be administered by any suitable route. Suitable lymphodepleting chemotherapy is particularly useful when the cancer is melanoma, which may be metastatic, e.g. This may include administration of cyclophosphamide and fludarabine. An exemplary route of administration for darabine is intravenous. Similarly, any suitable dose of cyclophosphamide may be administered. Familiar and fludarabine may be administered. In certain embodiments, approximately 60 mg / kg Cyclophosphamide is administered for 2 days, then approximately 25 mg / m 2 Fludarabine It is administered for 5 days.

[0225] In certain embodiments, growth factors that promote the proliferation and activation of immune cells are The immune cell growth factor is administered to the subject either simultaneously with the immune cells or subsequent to the immune cells. The growth factor may be any suitable growth factor that promotes the proliferation and activation of immune cells. Examples of these include interleukin (IL)-2, IL-7, IL-12, IL-15, These include IL-18 and IL-21, which may be used alone or in various combinations ( For example, IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2 and IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15 or IL- 12 and IL2) can be used.

[0226] Therapeutically effective amounts of immune cells can be administered via a number of routes, including parenteral administration, e.g., intravenously. The administration may be by intraperitoneal, intramuscular, intrasternal or intraarticular injection or infusion.

[0227] A therapeutically effective amount of immune cells for use in adoptive cell therapy is For example, this is the amount of the immune system required to inhibit progression. The amount of cells or immune cells required to reverse an autoimmune or alloimmune disease or relieve symptoms caused by autoimmune diseases, such as pain and inflammation. This can be an amount that can reduce symptoms associated with inflammation, such as pain, swelling, and This may be the amount needed to moderate hyperthermia, which may reduce rejection of transplanted organs. It may also be the amount necessary to cause or prevent

[0228] The immune cell populations are administered a treatment regimen consistent with the disease to ameliorate the disease state. For example, it may be administered once or several times over one or several days, or Regular administration over a long period of time to inhibit progression of the disease and to prevent recurrence of the disease The exact dosage to be used in the formulation will depend on the route of administration and the disease or disorder. The severity of the disease will also depend on the physician's judgment and the circumstances of each patient. The therapeutically effective amount of immune cells will depend on the subject being treated, the severity and type of affliction, and the mode of administration. In some embodiments, doses that may be used in treating human subjects is at least 3.8 x 104 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 Ma or at least 3.8×10 10 immune cells / m 2 In certain embodiments, Therefore, the dose used in treating human subjects is about 3.8×10 9 ~Approx. 3.8×10 1 0 immune cells / m 2 In a further embodiment, the therapeutically effective amount of immune cells varies from about 5×10 6 cells / kg body weight~approx. 7.5×10 8 Cells / kg body weight, e.g., about 2×10 7 Thin Cell ~ approx. 5×10 8 Cells / kg body weight or approximately 5 x 10 7 cells ~ approx. 2 x 10 8 cells / kg body weight The exact amount of immune cells may vary depending on the age, weight, sex and physiological condition of the subject. Effective amounts can be readily determined by one of skill in the art based on in vitro or animal models. The dose-response curves derived from the various test systems can be extrapolated.

[0229] The immune cells are administered in combination with one or more other therapeutic agents for treating an immune-mediated disorder. Combination therapy may include one or more antimicrobial agents (e.g., antibiotics, antivirals, and and antifungal agents), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, cell, fludarabine, etoposide, doxorubicin or vincristine), immunodepleting agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants Antidiabetics (e.g., azathioprine or glucocorticoids, e.g., dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids (e.g., hydrocortisone, dexamethasone, acetaminophen or prednisone) or nonsteroidal anti-inflammatory drugs (e.g., acetylsalicylic acid, ricylic acid, ibuprofen or naproxen sodium), cytokines (e.g. interleukin-10 or transforming growth factor-beta), hormones (e.g. For example, estrogen) or a vaccine. , calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors anti-inflammatory drugs (e.g., rapamycin); mycophenolate mofetil; antibodies (e.g., CD3, antibodies that recognize CD4, CD40, CD154, CD45, IVIG or B cells); chemotherapy (e.g., methotrexate, treosulfan, busulfan); irradiation; and chemokines, interleukins or their inhibitors (e.g., BAFF, IL-2 , anti-IL-2R, IL-4, JAK kinase inhibitors) Suppressants or immune tolerance inducers may be administered. Such additional pharmaceutical agents may be administered to achieve the desired effect. The cells and agents may be administered before, during or after administration of the immune cells, depending on the individual. This administration of the substances may be by the same route or by different routes, and at the same site or at different sites. The administration may be at a A. Pharmaceutical Compositions

[0230] Immune cells (e.g., T cells, B cells, or NK cells) and a pharma- ceutically acceptable carrier. Pharmaceutical compositions and formulations comprising lia are also provided herein.

[0231] The pharmaceutical compositions and formulations as described herein have a desired degree of purity. The active ingredient (e.g., an antibody or polypeptide) may be combined with one or more optional pharma- ceutically acceptable Remington's Pharmaceutical Sciences es 22 nd edition, 2012) and freeze-dried. The pharmaceutical composition may be prepared in the form of a pharmaceutical preparation or an aqueous solution. are non-toxic to recipients at the dosages and concentrations employed and have no Examples of acids include buffers (e.g., phosphoric acid, citric acid, and other organic acids); antioxidants (aspartic acid, phosphate, and other organic acids); corbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzoyl chloride) Benzalkonium chloride;Benzethonium chloride ;Phenol alcohol, butyl alcohol or benzyl alcohol;Alkylparaben paraben (e.g., methylparaben or propylparaben); catechol; resorcinol; Cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (about 10 residues) polypeptides (less than 100 bases); proteins (e.g., serum albumin, gelatin or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycerol); lysine, glutamine, asparagine, histidine, arginine or lysine); monosaccharides, Disaccharides and other carbohydrates (including glucose, mannose or dextrin); sugars (e.g., sucrose, mannitol, trehalose); or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Z n-protein complexes); and / or non-ionic surfactants (e.g., polyethylene Exemplary embodiments of the present invention include, but are not limited to, PEGs. Pharmaceutically acceptable carriers include interstitial drug dispersants, e.g., neutral, active, soluble Hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hy ... Lurinidase glycoproteins, e.g., rHuPH20 (HYLENEX®), B axter International, Inc. Certain exemplary sHASEGPs and methods of use, including those disclosed in U.S. Patent Publication No. 200,200,211, are disclosed in U.S. Patent Publication No. 200,200,211. 5 / 0260186 and 2006 / 0104968. In embodiments, the sHASEGP can be coupled to one or more additional glycosaminoglycanases, e.g. In some cases, it is used in combination with chondroitinase. B. Combination Therapy

[0232] In certain embodiments, the compositions and methods of the present invention comprise at least one further The further treatment may include radiation therapy, surgery ( (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viruses rRNA therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy or The additional treatment may be an adjuvant or neoadjuvant therapy. This may be in the form of bunt therapy.

[0233] In some embodiments, the additional treatment comprises administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is a side effect limiting agent (e.g., treatment Agents aimed at reducing the incidence and / or severity of side effects of, e.g., In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiation therapy and surgery. In some embodiments, the additional treatment is gamma irradiation. Therapies include those targeting the PBK / AKT / mTOR pathway, HSP90 inhibitors, and tubulin inhibitors. The further treatment is an inhibitor of the present invention, an apoptosis inhibitor, and / or a chemopreventive agent. It may be one or more chemotherapeutic agents known in the art.

[0234] Immune cell therapy can be used before, during, or after further cancer treatment, such as immune checkpoint therapy. The compounds may be administered in a variety of combinations. They may be administered simultaneously, minutes apart, days, or weeks apart. In a clinical trial in which immune cell therapy is provided to a patient separately from additional therapeutic agents, the interval may range from 1 to 4 days. In embodiments, the two compounds are combined so that they can still exert a beneficial combined effect on the patient. It is common to ensure that no significant period of time passes between the points of delivery, so that In such cases, the antibody therapy and the anti-cancer therapy may be administered within about 12-24 or 72 hours of each other, preferably within 12-24 or 72 hours of each other. More specifically, it is contemplated that the two may be provided to a patient within about 6 to 12 hours of each other. In some situations, the time between each dose may range from a few days (2, 3, 4, 5, 6, or 7) to a few weeks. If days (1, 2, 3, 4, 5, 6, 7, or 8) have passed, the treatment period may be significantly extended. It may be desirable to

[0235] Various combinations can be used. In the example below, immune cell therapy is "A": Anti-cancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / A B / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / A A / A / B / A

[0236] Administration of any of the compounds or treatments of the present embodiments to a patient may be toxic to those agents. Follow general protocols for administering such compounds, taking into account any Thus, in some embodiments, a step of monitoring toxicity that may result from the combination therapy is performed. exists. 1.Chemotherapy

[0237] A wide variety of chemotherapeutic agents may be used in accordance with this embodiment. "Chemotherapeutic agent" refers to the use of drugs to treat cancer. These agents or drugs are used to refer to compounds or compositions that Their mode of activity within the cell determines, for example, whether they affect the cell cycle and They are classified according to the stage at which they affect the cell cycle. are known for their ability to directly crosslink DNA, to intercalate into DNA, or to mediate nucleic acid synthesis. Characterized based on their ability to induce chromosomal and mitotic abnormalities by affecting It can be done.

[0238] Examples of chemotherapeutic agents include alkylating agents (e.g., thiotepa and cyclophosphamide). alkyl sulfonates (e.g., busulfan, improsulfan and piposulfan); aziridines (e.g., benzodopa, carboquone, methacrylates, meturedopa and uredopa; ethyleneimine and methylamelamines (altretamine, trimethylamelamine, Ethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and and trimethylolomelamine); Setogenins (especially bullatacin and bullatacinone); camptothecins (synthetic analogs topotecan; bryostatin; kallistatin; CC-1065 (including its adazone Resin, carzelesin and bizelesin ) including synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin ficin 8); dolastatins; duocarmycins (synthetic analogues KW-2189 and CB1-TM1); eleutherobin; punk Pancratistatin; Sarcodicin tyin); spongistatin; nitrogen masterbator (e.g., chlorambucil, chlornaphazine, co Cholophosphamide, estramustine, ifosfa mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembikine (n ovembichin, phenesterine, prednisone tin, trofosfamide and uracil mustard); Nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine); antibiotics (e.g., Enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma I and and calicheamicin omega I1); dynemicin (dynemicin bisphosphonates (e.g., clodronate); esperamicin; and neocarzinostatin chromophore and related enediyne antibiotics Substance chromophore, aclacinomycin, actinomycin, autarnisin, azase Phosphorus, bleomycin, cactinomycin, carabicin, carmi Carminomycin, Carzinophilin in), chromosomycin, dactinomycin, daunorubicin, detorubicin rubicin), 6-diazo-5-oxo-L-norleucine, doxorubicin (mol Morpholino-doxorubicin, Cyanomorpholino-doxorubicin, 2-pyrrolino-doxol including doxorubicin and deoxydoxorubicin), epirubicin, esorubicin (esorubicin ubicin, idarubicin, marcellomycin, Mitomycin (e.g., mitomycin C), mycophenolic acid, nogalar nisin, olivomycin peplomycin, potfiromycin, pupromycin quelamycin, rodorubicin bicin), streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin and zorubicin; antimetabolites (e.g., methotrexate and 5-fluroxane) Orouracil (5-FU); folic acid analogs (e.g., denopterin in), pteropterin and trimetrexate); purine analogs (e.g., fludanas Rabin, 6-mercaptopurine, thiamiprine and thioglucose anine; pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine , carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine); androgens (e.g., calcineurin, dromic acid stanolone, epithiostanol, mepitiostane and testolactone); antiadrenal (a anti-adrenals) (e.g., mitotane and trilostane); folic acid supplements (e.g., For example, fluoronic acid; aceglatone; aldophosphatamine Aldoglycoside (aldophosphamide glycoside); Aminolevulin nicotinic acid; eniluracil; amsacrine; bestrabucil; bilin Santren (bisantrene); edatraxate; Defofamine; Demecolcine; Diazicon; Erformitin ( elformithine; elliptinium acet ate);epothilone;etoglucide;gallium nitrate;hydroxyurea;lentinan;lo lonidainine; maytansinoid s) (e.g., maytansine and ansamitocins); Toguazone; Mitoxantrone; Mopidanmol; Nitraeri pentostatin; phenamet; pirarubicin; rosoxant losoxantrone; podophyllinic acid cid);2-Ethylhydrazide;Procarbazine;PSK polysaccharide complex;Razoxane;Li Zoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone (tri aziquone;2,2',2”-trichlorotriethylamine;trichothecin (specifically T-2 toxin, veracrine A, roridin A and anguidine (anguidine)); urethane; vindesine; dacarbazine; mannomustine; mi Tobronitol; Mitolactol; Pipobroman; Gacytosine arabinosides ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel Taxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum Coordination complexes (e.g., cisplatin, oxaliplatin, and carboplatin); stine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine Listerine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin aminopterin; xeloda; ibandronate; irinotecan (e.g. For example, CPT-11; topoisomerase inhibitor RFS2000; difluoromethylol Nitin (DMFO); Retinoids (e.g., retinoic acid); Capecitabine; Carboplatin tin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl- Protein tansferase inhibitors, transplatinum as well as pharma- ceutically acceptable salts, acids or derivatives of any of the above. 2. Radiation therapy

[0239] Other agents that have been widely used to cause DNA damage include gamma radiation, X-rays, and / or or what is commonly known as the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam irradiation and UV irradiation are also being considered. All of these factors play a role in the regulation of DNA, the precursors of DNA, and the replication and repair of DNA. and most likely cause widespread damage to chromosome assembly and maintenance. X-ray doses range from 50 to 200 roentgens over a prolonged period (3 to 4 weeks) These range from a daily dose of 1000 to a single dose of 2000 to 6000 roentgens. The range of doses for radioisotopes varies widely and depends on the half-life of the isotope, the strength of the radiation emitted, and The time course of action depends on the type and origin of the antibody, as well as on the uptake by the neoplastic cells. 3. Immunotherapy

[0240] One of skill in the art will appreciate that additional immunotherapies may be used in conjunction or in conjunction with the methods of the above embodiments. In the context of cancer treatment, immunotherapeutics typically target cancer cells and Rely on the use of immune effector cells and molecules to destroy Rituximab (RITUXAN®) is such an example. The target can be, for example, an antibody specific for some marker on the surface of a tumor cell. Antibodies can function alone as therapeutic effectors or can recruit other cells. The antibodies can also bind to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as targeting agents. Alternatively, the effector may interact directly or indirectly with a tumor cell target. These may be lymphocytes that have surface molecules that interact with them. These include cytotoxic T cells and NK cells.

[0241] Antibody-drug conjugates (ADCs) are monoclonal antibodies covalently linked to a cytotoxic drug. This approach involves the use of MAbs against antigenic targets and can be used in combination therapy. Combining the high specificity of MAbs with highly potent cytotoxic drugs allows The researchers also developed "armed" MAbs that deliver payloads (drugs) to tumor cells bearing the antigens at the highest levels. Targeted delivery of drugs also minimizes exposure to normal tissues, thus reducing toxicity. Exemplary ADC drugs include ADCETRI S® (brentuximab vedotin) and KADCYLA® (trastuzumab Examples of these include stuzumab emtansine or T-DM1.

[0242] In one embodiment of immunotherapy, tumor cells are infected with any marker amenable to targeting. The cells must have some marker that is not present on most other cells. There are many tumor markers, any of which may be a target in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosine kinase, and tyrosine kinase. Enzyme (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen , MucA, MucB, PLAP, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is to combine anti-cancer and immune stimulatory effects. Cytokines such as IL-2, IL-4, IL-12, GM-CSF, and gamma-IFN are chemokines such as MIP-1, MCP-1, and IL-8, and FLT3 ligand, etc. There are also immune stimulating molecules, including growth factors.

[0243] Examples of immunotherapy include immunoadjuvants such as Mycobacterium b ovis, Plasmodium falciparum, dinitrochlorobenzene and and aromatic compounds; cytokine therapy, e.g., interferon alpha, beta and gamma, IL -1, GM-CSF and TNF; gene therapy, e.g., TNF, IL-1, IL-2 and p53; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside G The one or more anti-cancer therapies include anti-M2 and anti-p185. It is contemplated that the method may be used in conjunction with physical therapy.

[0244] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either enhance signals (e.g., costimulatory molecules) or block signals. Inhibitory effects that can be targeted by blockade of immune checkpoints Immune checkpoints include adenosine A2A receptor (A2AR) and B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA); Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), Indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (K IR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell Immunoglobulin and mucin domains 3 (TIM-3) and T cell activation In particular, V-domain Ig suppressor of immune cells (VISTA) is a Antibody inhibitors target the PD-1 axis and / or CTLA-4.

[0245] Immune checkpoint inhibitors include small molecules, recombinant ligands or receptors. It may be a drug or, in particular, an antibody, such as a human antibody. Known inhibitors of proteins or analogs thereof may be used, particularly chimeric and humanized forms. Alternatively, humanized antibodies may be used. As will be appreciated by those skilled in the art, alternative and / or Equivalent names may be used for certain antibodies described in this disclosure. Such alternative and / or equivalent designations are interchangeable in the context of this disclosure. For example, lambrolizumab is known by the alternative and equivalent names MK-3475 and Penicillin-resistant Staphylococcus aureus (PEA). It is also known as brolizumab.

[0246] In some embodiments, the PD-1 binding antagonist is an antagonist that binds PD-1 to its ligand. In a specific embodiment, the PD-1 The ligand binding partner is PDL1 and / or PDL2. In this study, a PDL1 binding antagonist inhibits PDL1 from binding to its binding partners. In a specific embodiment, the PDL1 binding partner is a molecule that inhibits PD-1 and In another embodiment, the PDL2 binding antagonist is a PDL2 inhibitor. In a specific embodiment, the IL-1 domain is a molecule that inhibits L2 from binding to its binding partner. The PDL2 binding partner is PD-1. Antagonists include antibodies, their antigen-binding fragments, and The antibody may be a fragment, an immunoadhesin, a fusion protein or an oligopeptide.

[0247] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., For example, a human antibody, a humanized antibody, or a chimeric antibody. The PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin. PDL1 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence) or an immunoadhesin that contains the extracellular portion of PDL2 or the PD-1-binding portion of PDL2. In some embodiments, the PD-1 binding antagonist is AMP-224. . MDX-1106-04, MDX-1106, ONO-4538, BMS-93655 8 and nivolumab, also known as OPDIVO®, may be used as an anti-PD-L1 inhibitor. -1 antibody. MK-3475, Merck3475, lambrolizumab, KEYTR Pembrolizumab, also known as UDA® and SCH-900475, is An exemplary anti-PD-1 antibody is CT-0, also known as hBAT or hBAT-1. 11 is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, inhibits P DL2-Fc fusion soluble receptor.

[0248] Another immune checkpoint that can be targeted in the methods provided herein is C Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as D152 The complete cDNA sequence of human CTLA-4 is available under Genbank accession number L15. CTLA-4 is found on the surface of T cells and binds to C on the surface of antigen-presenting cells. When it binds to CD80 or CD86, it acts as an "off" switch. A member of the immunoglobulin superfamily expressed on the surface of T cells , which transmits inhibitory signals to T cells. CTLA4 is a T cell costimulatory protein. 28, and both molecules bind to CD80 and CD86 (B7- CTLA4 binds to CTLA-1 and B7-2, which transmit inhibitory signals to T cells. CD28 transmits stimulatory signals, whereas intracellular CTLA4 transmits regulatory signals. It is also found on T cells and may be important for the function of those cells. Activation of T cells via CD28 and CD48 induces the expression of C, an inhibitory receptor for B7 molecules. TLA-4 is highly expressed.

[0249] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody ( For example, human antibodies, humanized antibodies, or chimeric antibodies), antigen-binding fragments thereof, It may be a munoadhesin, a fusion protein or an oligopeptide.

[0250] Anti-human CTLA-4 antibodies (or VH and / or VHs derived therefrom) suitable for use in the method or VL domains) can be produced using methods well known in the art. Any art-recognized anti-CTLA-4 antibody can be used. The A-4 antibody was administered ipilimumab (10D1, MDX-010, MDX-101 and Yer voy® or antigen-binding fragments and variants thereof In other embodiments, the antibody comprises the heavy chain CDRs and light chain CDRs of ipilimumab. DR or heavy chain VR and light chain VR. Thus, in one embodiment, The antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of ipilimumab and The VL region of ipilimumab contains the CDR1, CDR2 and CDR3 domains. In one embodiment, the antibody binds to the same epitope on CTLA-4 as the above-mentioned antibody. competes for and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies In another embodiment, the antibody has at least about 90% variable region identity to the antibody described above. Region of amino acid sequence identity (e.g., at least about 90%, 95%, or 99% with ipilimumab) % variable region identity). 4.Surgery

[0251] Approximately 60% of people with cancer undergo preventive, diagnostic or staging surgery, or curative surgery. undergoing some type of surgery, including definitive and palliative surgery. Definitive surgery includes: This includes surgical procedures that involve the physical removal, excision and / or destruction of all or part of the cancerous tissue. , other treatments (e.g., the treatment of this embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, etc. Tumor removal is the removal of tumors from the body. Surgical treatment includes tumor removal and / or removal of at least part of the tumor. Other procedures include laser surgery, cryosurgery, electrosurgery and microsurgery (Mohs surgery).

[0252] When some or all of the cancerous cells, tissue, or tumor is removed, a cavity may be formed in the body. Treatment is accomplished by perfusion, direct injection or local application of the area with additional anti-cancer therapy. Such treatments can be administered, for example, every 1, 2, 3, 4, 5, 6 or 7 days. or every 1, 2, 3, 4 and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9 These treatments may be repeated every 10, 11 or 12 months. It could also be a treatment. 5. Other agents

[0253] Certain aspects of the present invention may be combined with other actions to improve the therapeutic efficacy of treatment. It is contemplated that agents that can be used include those that act on the cell surface. Agents that affect receptor and gap junction upregulation, cell division The response of hyperproliferative cells to inhibitors and differentiation agents, inhibitors of cell adhesion, and apoptosis inducers These include agents that increase the sensitivity of the cells, or other biological agents. The increased signaling between cells by adding IL-1 to the adjacent hyperproliferative cell populations In other embodiments, to improve the anti-hyperproliferative efficacy of the treatment, To this end, cytostatic or differentiating agents may be used in combination with certain aspects of the present embodiments. To improve the effectiveness of this embodiment, inhibitors of cell adhesion are contemplated. Examples of adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Other agents that increase the sensitivity of hyperproliferative cells to cisplatin (e.g., antibody c225) have been used to treat may be used in combination with certain aspects of the present embodiment to improve the effectiveness of the device. It is further contemplated that: VII. Products or Kits

[0254] Also provided herein is an article of manufacture or kit comprising the immune cells. for treating or delaying the progression of cancer in an individual, or for the treatment of an individual with cancer The method may further include a package insert containing instructions for using the immune cells to enhance immune function. Any of the antigen-specific immune cells described herein may be included in the product or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be made of glass, plastic (e.g., polyvinyl chloride or polyvinyl chloride). Various materials such as olefins or metal alloys (e.g., stainless steel or Hastelloy) In some embodiments, the container holds the formulation and a label. The label is affixed to or associated with a container and the container contains The product or kit may be used in a commercial and user-friendly manner. The composition may further include other materials that are desirable from the standpoint of the present invention, such as other buffers. Includes fluid, diluent, filter, needle, syringe, and package insert with instructions for use. In some embodiments, the product may contain one or more additional agents, such as , chemotherapy and antineoplastic agents). Examples include bottles, vials, bags and syringes. EXAMPLES

[0255] VIII. Examples The following examples are included to demonstrate preferred embodiments of the invention. The techniques disclosed in the examples are techniques that the inventors have found to work well in the practice of the invention. and therefore may be considered as a preferred mode for its implementation. However, those of ordinary skill in the art will recognize that, in light of this disclosure, Many variations can be made in the specific embodiments, and these variations are within the spirit and scope of the present invention. and recognize that without departing from the scope of the present invention, the same or similar results would be obtained. It is. Example 1 – Multiplex gene editing

[0256] Simultaneously disrupting the expression of multiple genes in immune cells such as T cells and NK cells We have conducted several studies to test the effectiveness of CRISPR in the expression of different genes. The first study used CRISPR / Cas9 to test the disruption of a combination of genes. disrupts the expression of NKG2A, CD47, TGFBR2 and CISH in NK cells In this gene set, NKG2A and CD47 were detected in the first electroporation. knockout in the first electroporation, and CISH and PCR and primers were used for both rounds of electroporation. Knockout efficiency was successfully verified using flow cytometry (Figure 1).

[0257] TIGIT, CD96, CISH, adenosine (Figure 2), as well as NKG2A, CD4 7, TGFBR2 and CISH (Fig. 3). We verified a method to destroy genes. By destroying multiple genes, we were able to target tumor cells. In the presence of brefeldin A for 5 hours, the target Using various NK cells (edited vs. Cas9 only) costimulated with cell lines, IFN Flow cytometric analysis of the production of TNF-γ, TNF-α and CD107 was performed on target cells. After stimulation with the strain, there was an increase in the secretion of IFN-γ, TNFα, and CD107 (Fig. 3 ).

[0258] This function was enhanced by the NKG2A, CD47, TGFBR2 and CI in NK cells. The NK cells were then depleted by SH. 51 Measured by Cr release assay When the recombinant IgG was administered to K562 cells, it showed increased antitumor cytotoxicity against the K562 cells (Figure 4A). Thirty minutes after treatment with TGF-B (50 ng / ml), pSMAD activity was measured using a flow cytometer. NK cells were stimulated with cytokines or Upon target recognition, NK cells lose expression of CD16 and CD62L (Figure 5). Knocking out ADAM17 in cells reduces the shed expression of CD16 and CD62L. Binding of IgG1 to K562 was prevented (Figure 6) and improved ADCC and cytotoxicity against K562 targets It was also observed that (Figure 7).

[0259] Further studies have shown that disrupting SHP1 in NK cells enhances antitumor effects. NK cells were incubated with K562 / Raji cells at a 1:1 ratio for 4 hours. After incubation, the cells were stained with Annexin V to identify live and K562 cells were analyzed for sensitivity to NK cell killing and Raj i cells are resistant to killing by NK cells. Disruption of KG2A enhanced the antitumor effect against the Raji target (Figure 12).

[0260] This approach was extended to additional gene sets, namely TIGIT, CD96, CISH and and adenosine, and NKG2A, CISH, TGFBRII and adenosine The function of NK cells was evaluated by flow cytometry. Upon stimulation with target cell lines, TNFα, IFNγ and CD10 were expressed in those cells. An increase in 7a was observed (Figures 13-14).

[0261] Thus, the method involves simultaneously disrupting the expression of multiple genes in immune cells, It can be used to enhance immune cell function. Example 2 - Method

[0262] Precomplexation and electroporation of sgRNA-Cas9: One or two sgRNAs were designed for each gene and used. Reactions with as9 (PNA Bio) and 500ng of sgRNA (total of all sgRNAs) A mixture was prepared for each gene and incubated on ice for 20 minutes. 000 NK cells in T-buffer * (Neon Electroporation K It, the Invitrogen product, contains RNP complexes and cells. Resuspend in Neon Transfection S (volume should be 14ul) Electroporation using a 10ul electroporation tip using the system The electroporation conditions for NK cells were 1600V, 10ms, and 3 Pulse * The cells were then cultured in APC (1NK:2APC), SCGM medium (preferentially without antibiotics), in addition to a culture plate containing 200 IU / ml IL2, The plates were allowed to recover in a 7°C incubator.

[0263] Precomplexation of crRNA and electroporation: crRNA and tracrR The NA duplex was mixed using a pipette and a centrifuge. The mixture was then thermocycled. The plates were incubated at 95°C for 5 minutes in a centrifuge and then cooled to room temperature on the bench.

[0264] [Table 3]

[0265] [Table 4]

[0266] [Table 5]

[0267] The crRNA:tracrRNA duplex was pipetted into the Cas9 nuclease mix. The mixture was then combined with the crRNA and incubated at room temperature for 15 min. Combined.

[0268] [Table 6]

[0269] First, APC (1NK:2APC), SCGM medium (preferably without antibiotics), and Electroporation was performed by preparing culture plates containing 200 IU / mL IL-2. Preparations of 250,000 cells per well were prepared immediately prior to use. The cells were then resuspended in 7.5 ul of T buffer. The electroporation conditions were 1600 V, 1 0ms and 3 pulses * The cells were then added to a culture plate and incubated at 37°C. Allowed to recover in incubator.

[0270] NK Cell Expansion: Miltenyi NK Cell Isolation Kit (130-092-657) Isolate NK cells from umbilical cord blood or peripheral blood using the following procedure. Mix NK cells with feeder cells and 1: At a ratio of 2 (1 NK cell, 2 feeder cells) in the presence of IL2 (200 IU / ml) Place in SCGM medium. Replace the medium with IL2 every other day. On day 4, NK cells Select NK cells again using an isolation kit to remove feeder cells or to remove all Wait until day 7 for all feeder cells to die. , or perform electroporation for CRISPR-Cas9.

[0271] All of the methods disclosed and claimed herein are intended to be illustrative, but are not to be construed as limiting the present disclosure. The compositions and methods of the present invention may be implemented and performed without the need for Although the present invention has been described in terms of its scope, no particular limitation or exclusion may be made herein without departing from the concept, spirit and scope of the invention. that variations may be applied to the methods and method steps or to the order of steps described therein; More specifically, any combination of chemicals may be used as long as the same or similar results are achieved. Certain biologically and physiologically relevant agents may be substituted for the agents described herein. 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Claims

**Claim 1**: An immune cell comprising: (a) The expression of TGFBR2 and glucocorticoid receptor NR3C1 is disrupted, where the disruption of the expression includes that the expression levels of these at least two genes are removed or decreased compared to the case without expression disruption; and (b) A nucleic acid encoding a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR) comprising two or more antigen-binding domains. **Claim 2**: The immune cell according to claim 1, wherein one or more of the two or more antigen-binding domains target an antigen selected from the following group: Tumor-associated calcium signal transducer 2 (TACSTD2), tumor-associated calcium signal transducer 1 (TACSTD1), EGFR, EGFRvIII, PDGFR, VEGFR, ILK, STAT3, STAT5, STAT6, HIF-1, HIF-2, NF-κB, NOTCH1, NOTCH2, NOTCH3, NOTCH4, c-Met, mTOR, WNT, ERKs, PSMA, PR-3, MDM2, mesothelin, renal cell carcinoma 5T4, SM22-α, carbonic anhydrase I (CAI) and IX (CAIX, alias G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoint, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2-ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelin, PSCA, SLe, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGSS, SART3, STN, PAXS, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1 and idiotype, CD319 (CS1), ROR1, CD20, carcinoma embryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-related antigen, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120 and HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30, CD56, HERV-K, IL-11Rα, κ-chain, λ-chain, CSPG4, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, and combinations thereof.

3. The immune cell according to claim 2, comprising natural killer (NK) cells. **Claim 4**: The immune cell according to claim 3, wherein the expression disruption comprises introducing the following into TGFBR2 and / or NR3C1: a cell (i) one or more insertions, one or more frameshift mutations, one or more missense mutations, one or more deletions, or knock-in or knockout of part or all of the two genes, or (ii) one or more antisense nucleic acids including short interfering RNA (siRNA), short hairpin RNA (shRNA), or (iii) one or more DNA targeting molecules including zinc finger protein (ZFP), transcription activator-like protein (TAL) or its effector, or a CRISPR construct, or (iv) a combination thereof. **Claim 5**: The immune cell according to claim 4, further comprising an exogenous nucleic acid encoding a cytokine selected from the group consisting of IL-21, IL-7, IL-2, IL-15, IL-12, IL-18, and combinations thereof. **Claim 6**: A pharmaceutical composition comprising the immune cell according to claim 4 and a pharmaceutically acceptable excipient. **Claim 7**: The pharmaceutical composition according to claim 6, which is for cancer treatment. **Claim 8**: The pharmaceutical composition according to claim 7, further comprising an additional therapeutic agent. **Claim 9**: An in vitro method for producing the immune cell according to claim 4, comprising the step of introducing into the immune cell one or more nucleic acids encoding the following: (i) a CRISPR system comprising two or more guide RNAs (gRNAs) comprising sequences complementary to TGFBR2 and / or GR; (ii) (1) an expression vector encoding a CAR and / or a TCR, and (2) flanked by homologous arms complementary to TGFBR2 and / or GR; By introducing the expression vector, at the locus corresponding to TGFBR2 and / or GR in the genome of the immune cell, a CAR is inserted into the exon site, the CAR is controlled by one or more promoters of one or more of the at least two genes, and the expression of at least one gene is disrupted by the introduction, or both (i) and (ii) above. **Claim 10**: The method according to claim 9, further comprising the step of introducing the following: ​ One or more nucleic acids encoding a cytokine selected from IL-21, IL-7, IL-2, IL-15, IL-12, IL-18, or a combination thereof; And one or more nucleic acids encoding a CAR and / or TCR, comprising two or more antigen-binding domains, wherein one or more of the antigen-binding domains target an antigen selected from the following; Tumor-associated calcium signal transducer 2 (TACSTD2), tumor-associated calcium signal transducer 1 (TACSTD1), EGFR, EGFRvIII, PDGFR, VEGFR, ILK, STAT3, STAT5, STAT6, HIF-1, HIF-2, NF-κB, NOTCH1, NOTCH2, NOTCH3, NOTCH4, c-Met, mTOR, WNT, ERKs, PSMA, PR-3, MDM2, mesothelin, renal cell carcinoma-associated antigen 5T4, SM22-alpha, carbonic anhydrase I and IX (CAI, CAIX, alias G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation point, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2-ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelin, PSCA, SLe, PLAC1, GM3, BORIS, Tn, GloboH, NY-BR-1, RGSS, SART3, STN, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, regucalcin, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, Fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, idiotype, CD319 (CS1), ROR1, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, and combinations thereof.

11. Immune cells in which the expression of at least two genes selected from the following group is disrupted: CISH, NKG2A, ADORA2, CD25, CD40, CD47, CD5, CD7, CD80, CD86, 4EBP1, FOXO1, ICAM1, IL21R, PD-L1, PD-L2, RPS6, and SIRPA, wherein the disruption of the expression includes that the expression of the gene is decreased or removed as compared with immune cells without such disruption.

12. Immune cells in which the expression of at least two genes selected from the following group is disrupted: TGFBR2, NKG2A, ADORA2, CD25, CD40, CD47, CD5, CD7, CD80, CD86, 4EBP1, FOXO1, ICAM1, IL21R, PD-L1, PD-L2, RPS6, SIRPA, wherein the disruption of the expression means that the expression of the gene is decreased or removed as compared with immune cells without such disruption.

13. Immune cells in which the expression of at least two genes selected from the following group is disrupted: Glucocorticoid receptor (GR, NR3C1), NKG2A, ADORA2, CD25, CD40, CD47, CD5, CD7, CD80, CD86, 4EBP1, FOXO1, ICAM1, IL21R, PD-L1, PD-L2, RPS6, and SIRPA, wherein the disruption of the expression includes that the expression of the gene is decreased or removed as compared with immune cells without such disruption.

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