Multiplex genome editing of immune cells to enhance function and resistance to inhibitory environments

Abstract

Compositions and methods relating to cancer immunotherapy, particularly involving engineered immune cells, are provided. Provided herein are methods for generating immune cells in which multiple genes are disrupted. Further provided are methods for inserting a chimeric antigen receptor into a genetic locus of an immune cell.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 62 / 772,406, filed November 28, 2018, 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. More particularly, the present invention relates to multiplex editing of immune cells and methods of use thereof. [Background technology]

[0003] Cellular immunotherapy has great potential to treat cancer. However, most immunotherapeutic approaches, when applied alone, have limited value against most malignant tumors, especially solid tumors. The reasons for this limited success include low expression of tumor antigens on the surface of tumor cells, which reduces tumor cell detection by the immune system, expression of ligands for inhibitory receptors (e.g., PD1, NKG2A, TIGIT or CISH) that induce immune cell inactivation; and induction of cells (e.g., regulatory T cells or myeloid-derived suppressor cells) in the microenvironment that release substances (e.g., transforming growth factor-β (TGFβ) and adenosine) that suppress immune response and promote tumor cell proliferation and survival. Thus, there is an unmet need for improved methods of cellular immunotherapy. Summary of the Invention

[0004] The present disclosure provides compositions and methods for cancer immunotherapy, particularly involving engineered immune cells.Specific embodiments relate to certain immune cells that have been modified by human hands to lack or reduce the expression of one, two or more genes, and in certain cases, cells with such modifications also express one or more heterologous proteins, including non-natural proteins such as antigen receptors.Methods for producing non-natural immune cells are also included.In certain cases, the introduction of heterologous antigen receptors is performed at the genomic locus of the gene whose expression is reduced or eliminated.

[0005] In one embodiment, the present disclosure provides an in vitro method for disrupting at least two genes in an immune cell, the at least two genes being selected from the group consisting of 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, CD7, and combinations thereof. In certain embodiments, three, four, five, or six or more genes are disrupted. In specific embodiments, the disruption of two or more genes is simultaneous, such as disruption in the same method step. The method may include introducing a guide RNA (gRNA) for each gene into the immune cell.

[0006] The above method includes, for example, specific gene combinations such as: (a) NKG2A and CISH, (b) NKG2A and TGFBRII, (c) CISH and TGFBRII, (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 TIGIT, (k) TIM3 and CISH, (l) TIM3 and TGFBRII, (m) FOXO1 and TGFBRII, (n) TIM3 and TIGIT, (o) SIGLEC7 and CISH, (p) SIGLE (a) A2AR and TIGIT, (b) SHP1 and CISH, (c) CISH and TGFBRII, (d) SHP1 and TGFBRII, (e) SHP1 and TIGIT, or (f) SHP1 and TIM3 knockdown.The above methods include: (1) NKG2A, CISH and TGFBRII; (2) TIGIT, FOXO1 and TGFBRII; (3) TGFBRII, CD96 and TIGIT; (4) TGFBR2, 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) TGF The subgroups may include knockdown of TGFBRII, CD47 and TIGIT, (12) TGFBRII, CD47 and SIRPA, (13) A2AR, CISH and TGFBRII, (14) TGFBRII, CISH and ADAM17, (15) TGFBRII, TIM3 and TIGIT, (16) TGFBRII, A2AR and TIGIT, (17) SHP1, CISH and TGFBRII, (18) TGFBRII, CISH and SHP1, (19) TGFBRII, SHP1 and TIGIT, or (20) TGFBRII, SHP1 and TIM3. Any of the above subgroups may be combined with a second subgroup as disclosed above. For example, any one of subgroups a to j1 may be combined with any one or more of the other subgroups a to j1, any one or more of subgroups a to j1 may be combined with any one or more of the other subgroups 1 to 23, or any one or more of subgroups 1 to 23 may be combined with any one or more of the other subgroups 1 to 23.

[0007] In some embodiments, the method further comprises introducing an RNA-guided endonuclease, such as Cas9, into the cell. Introduction of the RNA-guided endonuclease may comprise introducing a nucleic acid (e.g., mRNA) encoding the RNA-guided endonuclease into the immune cell.

[0008] In certain embodiments, the immune cells are T cells, NK cells, B cells, macrophages, NKT cells or stem cells. In alternative cases, the immune cells are not T cells, such as not CAR T cells. In some embodiments, the immune cells are engineered to express one or more chimeric antigen receptors (CARs) and / or one or more T cell receptors (TCRs). The immune cells can be virus-specific, such as virus-specific T cells. The T cells can be regulatory T cells. The B cells can be regulatory B cells. In some embodiments, the stem cells are mesenchymal stem cells (MSCs) or induced pluripotent stem (iPS) cells. In certain embodiments, the T cells are CD8 + T cells, CD4 + The immune cells are T cells or gamma-delta T cells. The immune cells can be isolated from peripheral blood, umbilical cord blood, bone marrow or a mixture thereof. In some embodiments, the umbilical cord blood is pooled from two or more individual umbilical cord blood units.

[0009] In some embodiments, the introduction step comprises transfection or transduction. For example, the introduction comprises electroporation, which can be performed more than once, for example, two or three electroporations. In some embodiments, the first group of CRISPR gRNA is introduced in the first electroporation, and the second group of CRISPR gRNA is introduced in the second electroporation. In a specific case, the first group of CRISPR gRNA is different from the second group of CRISPR gRNA. In a specific embodiment, the first group and / or the second group of CRISPR gRNA comprises one, two, three or four or more CRISPR gRNAs. In some embodiments, two CRISPR gRNAs are introduced in the first electroporation, and two different CRISPR gRNAs are introduced in the second electroporation. In specific embodiments, a group of CRISPR gRNAs comprises a group of gRNAs, at least two of which target different genes, and in certain embodiments, each of the gRNAs in the group targets a different gene.

[0010] In certain embodiments, the method comprises disrupting NKG2A, CD47, TGFβR2 and CISH; NKG2A, CISH, TGFβR2 and ADORA2; NKG2A, TGFβR2 and CISH; TIGIT, CD96, CISH and ADORA2; or ADAM17, TGFβR2, NKG2A and SHP1.

[0011] In some embodiments, the disruption results in enhanced anti-tumor cytotoxicity, in vivo expansion, in vivo persistence and / or improved function of the immune cells. In certain embodiments, the immune cells have increased secretion of IFN-γ, CD107 and / or TNFα compared to the absence of the modification. In some embodiments, the immune cells have increased production of perforin and / or granzyme B compared to the absence of the modification.

[0012] In additional embodiments, the method further comprises introducing a CAR and / or TCR into an immune cell (e.g., introducing a nucleic acid encoding a CAR and / or TCR into an immune cell). In some embodiments, the nucleic acid is present in an expression vector, such as a retroviral vector. In certain embodiments, the vector is an adenovirus-associated vector, such as AAV6. In some embodiments, the vector further comprises an inhibitory gene sequence, such as an inhibitory gene sequence selected from the group consisting of 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, CD7, and combinations thereof. In certain embodiments, the vector further comprises a guide RNA for the inhibitory gene. The CAR may be flanked by homology arms for the inhibitory gene. In some embodiments, the CAR is inserted into the locus of the inhibitory gene (e.g., the exon of the inhibitory gene) of immune cells by introducing the vector that comprises the CAR sequence, and the CAR is under the control of the endogenous promoter of the inhibitory gene. In certain embodiments, the introduction of the vector further disrupts the expression of the inhibitory gene.

[0013] In another embodiment, an immune cell (e.g., an immune cell of the disclosed embodiments) is provided in which expression of at least two genes of the immune cell is disrupted, the immune cell being generated at least by a process comprising introducing a CRISPR guide RNA (gRNA) for each gene into said immune cell, the at least two genes being selected from the group consisting of 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, CD7, and combinations thereof. In some embodiments, three, four, five, or six or more genes are disrupted.

[0014] In certain embodiments, the immune cells are T cells, NK cells, B cells or stem cells. In some embodiments, the immune cells are engineered to express a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR). The immune cells can be virus-specific, such as virus-specific T cells. The T cells can be regulatory T cells. The B cells can be regulatory B cells. In some embodiments, the stem cells are mesenchymal stem cells (MSCs) or induced pluripotent stem (iPS) cells. In certain embodiments, the T cells are CD8 + T cells, CD4 + The immune cells are T cells or gamma-delta T cells. The immune cells can be isolated from peripheral blood, umbilical cord blood or bone marrow. In some embodiments, the umbilical cord blood is pooled from two or more individual umbilical cord blood units.

[0015] In certain embodiments, the method includes disrupting 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 ADORA2; or ADAM17, TGFβR2 NKG2A and SHP1).

[0016] In some embodiments, the disruption leads to enhanced anti-tumor cytotoxicity, in vivo proliferation, in vivo persistence and / or improved function of the immune cells. In certain embodiments, the immune cells increase secretion of IFN-γ, CD107 and / or TNFα. In some embodiments, the immune cells increase production of perforin and / or granzyme B.

[0017] In some embodiments, the cell is engineered to express CAR and / or TCR.CAR can be inserted into the endogenous inhibitory gene locus of the cell, for example, the inhibitory gene locus is selected from the group consisting of 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, CD7 and combinations thereof.In some embodiments, CAR is under the control of the endogenous promoter of inhibitory gene.In certain embodiments, CAR is inserted into the inhibitory gene locus by CRISPR-mediated gene editing.

[0018] In some embodiments, the CAR comprises an antigen-binding domain selected from the group consisting of F(ab')2, Fab', Fab, Fv and scFv. In certain embodiments, the CAR targets one or more tumor associated antigens selected from the group consisting of CD19, 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-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, and combinations thereof. In certain embodiments, the CAR comprises at least one signaling domain selected from the group consisting of CD3ξ, CD28, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, ILRB / CD122, IL-2RG / CD132, DAP12, CD70, CD40, and combinations thereof. In some embodiments, the immune cells comprise one or more heterologous cytokines (e.g., one or more of IL-7, IL-2, IL-15, IL-12, IL-18, and IL-21). In certain embodiments, the CAR further comprises a suicide gene, such as a membrane-bound non-secreted TNF-alpha mutant or inducible caspase 9.

[0019] Further provided herein is an expression vector that encodes at least one CAR and / or TCR, at least one inhibitory gene sequence and at least one gRNA.In some embodiments, the inhibitory gene sequence is derived from an inhibitory gene selected from the group consisting of 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 specific to the inhibitory gene.In some embodiments, the vector is a viral vector, such as an AAV vector.The CAR may be flanked by homology arms to the inhibitory gene. Also provided herein is a host cell (e.g., a cell of the embodiments) engineered to express the vector of the above embodiments. In aspects, the cell is a T cell, a NK cell, a B cell, or a stem cell.

[0020] Also provided herein is a pharmaceutical composition comprising the population of immune cells of the disclosed embodiments. Another embodiment provides a composition comprising the population of cells of the disclosed embodiments for treating immune-related disorders, infectious diseases and / or cancer.

[0021] In further embodiments, a method of treating a disease or disorder in a subject is provided, comprising administering to the subject an effective amount of an immune cell of the disclosed embodiments. In some aspects, the disease or disorder is an infectious disease, cancer (e.g., a solid cancer or a hematological malignancy), or an immune-related disorder. The immune-related disorder can be, for example, an autoimmune disorder, graft-versus-host disease, allograft rejection, or an inflammatory condition. In some aspects, the immune-related disorder is an inflammatory condition, and the immune cells are essentially free of glucocorticoid receptor expression. In certain aspects, the immune cells are autologous or allogeneic to the recipient individual.

[0022] In additional embodiments, the method further comprises administering at least a second therapeutic agent to the individual receiving the immune cells. In some embodiments, the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiation therapy, hormone 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, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.

[0023] Another embodiment provides a method for engineering immune cells to express a CAR, comprising inserting the CAR into the locus of an inhibitory gene of the immune cell using CRISPR gRNA.In some embodiments, the CAR is encoded by an expression vector (e.g., a retroviral vector, a plasmid, a lentiviral vector, an adenoviral vector, an adenovirus-associated virus vector, etc.).In certain embodiments, the viral vector is an adenovirus-associated vector, such as AAV6.

[0024] In some embodiments, the vector further comprises an inhibitory gene sequence, such as an inhibitory gene sequence selected from the group consisting of 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, CD7, and combinations thereof. 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 inserted into the locus of the inhibitory gene at any part of the inhibitory gene (e.g., an exon of the inhibitory gene). The CAR can be under the control of the endogenous promoter of the inhibitory gene. In a specific embodiment, the CAR disrupts the expression of an inhibitory gene.

[0025] In some embodiments, the CAR targets one or more tumor associated antigens selected from the group consisting of CD19, 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-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, and combinations thereof. In certain embodiments, the CAR comprises at least one signaling domain selected from the group consisting of CD3ξ, CD28, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, ILRB / CD122, IL-2RG / CD132, DAP12, CD70 and CD40. In some embodiments, the vector encoding the CAR also encodes a cytokine (e.g., IL-7, IL-2, IL-15, IL-12, IL-18, IL-21 or a combination thereof). In alternative cases, the cytokine is present on a vector separate from the vector encoding the CAR. In certain embodiments, the expression construct encoding the CAR further comprises a suicide gene (e.g., inducible caspase 9 or a membrane-bound non-secreted TNF-alpha mutant).

[0026] Further provided herein is an immune cell having at least one CAR inserted into an inhibitory gene of the immune cell, such as an immune cell produced by the method. Also provided herein is a composition comprising a population of immune cells of the present disclosure (e.g., a population of T cells, B cells, NK cells, NKT cells, macrophages, stem cell populations, mixtures thereof, etc.).

[0027] Another embodiment provides a composition comprising a population of cells of the above embodiments, which in certain embodiments is utilized for the treatment of any type of medical condition, such as at least for the treatment of immune-related disorders, infectious diseases and / or cancer.

[0028] Further embodiments provide a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the immune cells of the above embodiments. In some aspects, the disease or disorder is an infectious disease; cancer (e.g., solid cancer or hematological malignancy); and / or an immune-related disorder. The immune-related disorder may in some cases be an autoimmune disorder, graft-versus-host disease, allograft rejection, and / or an inflammatory condition. In some aspects, the immune-related disorder is an inflammatory condition, and the immune cells essentially do not have expression of glucocorticoid receptors. In certain aspects, the immune cells are autologous or allogeneic to the recipient individual.

[0029] In additional embodiments, the method further comprises administering at least a second therapeutic agent to the individual. In some embodiments, the at least a second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiation therapy, hormone 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, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. The immune cells and at least a second therapeutic agent may be administered simultaneously or at different times, and when administered at different times or simultaneously but not in the same formulation, they 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, but it should be understood that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [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 novel approach to simultaneously knock down (or knock out) two or more genes (e.g., genes (e.g., genes listed in Table 1, including adenosine 2a receptor, TGFβR2, NKG2A, TIGIT, and / or CISH)) in human immune cells (e.g., T cells, NK cells, CAR-transduced T cells, or CAR-transduced NK cells) using CRISPR-Cas9 technology. The immune cells can be derived from peripheral blood or umbilical cord blood or a combination thereof.

[0052] This study demonstrated that low expression of these proteins correlates with improved T and NK cell function, in vivo proliferation and persistence, and cytotoxicity. This strategy also protects T cells, NK cells, NKT cells, and iNKT cells from the immunosuppressive tumor microenvironment, which is mainly driven by TGFβ and adenosine. Thus, this method can be used to improve the efficacy of various adoptive cell therapy products (e.g., NK cells, T cells (e.g., virus-specific T cells and regulatory T cells), B cells (e.g., regulatory B cells), CAR-transduced NK cells, CAR-T cells, and TCR-engineered T and NK cells, iNKT cells, NKT cells). The adoptive cell therapy products can be used to treat various diseases, ranging from cancer (e.g., hematological or solid malignancies) to infectious diseases and immune disorders.

[0053] In certain embodiments, the immune cells express at least one CAR. CAR technology has made several advances in the past few years. Indeed, CAR-CD19 has shown impressive clinical results in patients with B-cell leukemia and lymphoma, and two CAR T products were approved by the FDA last year. CAR-transduced T cells have taken the lead in the past few years, but many preclinical studies, as well as the applicant's Phase I / II CAR NK trial, have also shown the efficacy of CAR-NK cells against cancer. Despite the advances in CAR technology, CARs are still mostly transduced into T cells or NK cells using viral vectors, which only randomly integrate into the DNA of cells, which may lead to clonal proliferation, cancerization, altered expression of transgenes, or transcriptional silencing. Therefore, it would be beneficial to find a way to target the insertion of CAR into specific DNA loci.

[0054] Thus, in one embodiment, the present disclosure provides a method for inserting a CAR into the locus of a particular gene (e.g., the locus of an inhibitory gene or checkpoint protein) using CRISPR / Cas9. Insertion of a CAR at a gene locus can also be used to disrupt expression of a gene while simultaneously placing the CAR under the control of the promoter of the gene, if desired. Specifically, the present method can use AAV6 vectors and CRISPR / Cas9 technology to guide insertion of a CAR at the locus of an inhibitory gene (e.g., genes listed in Table 1, including but not limited to NKG2A, CISH, PD-1, TIGIT, TIM3, SHP1 or TGFβR2). Insertion of a CAR at the locus of an inhibitory gene can disrupt the inhibitory effect of a checkpoint molecule (e.g., ) while also allowing expression of the CAR to be under the control of the promoter of the checkpoint and upregulated in the tumor microenvironment. This is useful when applying CAR therapy to solid tumors, where upregulation of checkpoint molecules can negatively impact the success of CAR therapy. Thus, additional methods are provided for generating adoptive cell therapy (e.g., T cells, B cells, NK, NKT or iNKT cells) using CAR insertion methods that may have a high safety profile. I. Definition

[0055] As used herein, "essentially free" of a particular component is used herein to mean that the particular component is not intentionally formulated in the composition and / or is not present even as a contaminant or in trace amounts.Thus, the total amount of the particular component resulting from any unintentional contamination of a composition is less than 0.05%, preferably less than 0.01%.Most preferred is a composition in which the amount of the particular component cannot be detected by standard analytical methods.

[0056] As used herein, "a" or "an" can mean one or more. When used in the claims, the words "a" or "an" when used with the word "comprising" can mean one or more than one. As used herein, "another" can mean at least a second or more. Still further, the terms "having," "including," "containing," and "comprising" are interchangeable, and those of skill in the art will appreciate that these terms are open-ended terms. In specific embodiments, aspects of the disclosure can "consist essentially of" or "consist of," for example, one or more sequences of the disclosure. Some embodiments of the invention can consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. The scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described herein. As used herein, the terms "or" and "and / or" are used to describe multiple elements in combination or exclusive of each other. For example, "x, y and / or z" can refer to "x" only, "y" only, "z" only, "x, y and z", "(x and y) or z", "x or (y and z)", or "x or y or z". It is expressly contemplated that x, y or z can be expressly excluded from an embodiment.

[0057] The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports the definition to refer to alternatives only and "and / or." As used herein, "another" can mean at least a second or more. The terms "about," "substantially," and "approximately" generally mean the stated value plus or minus 5%.

[0058] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0059] "Immune disorder," "immune-related disorder," or "immune-mediated disorder" refers to a disorder in which the immune response plays a significant role in the development or progression of the disease. Immune-mediated disorders include autoimmune disorders, allograft rejection, graft-versus-host disease, and inflammatory and allergic conditions.

[0060] An "immune response" is a response of a cell of the immune system, such as a B cell or T cell or an innate immune cell, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response").

[0061] As used herein, the term "inhibitory gene" refers to a gene whose product is directly or indirectly detrimental to the activity, proliferation and / or persistence of one or more types of immune cells.

[0062] "Autoimmune disease" refers to a disease in which the immune system mounts an immune response (e.g., a B cell response or a T cell response) against antigens that are part of the normal host (i.e., self-antigens), resulting in tissue damage. Self-antigens can be derived from host cells or from commensal organisms (e.g., microorganisms that normally colonize mucosal surfaces, known as commensals).

[0063] As used herein, the term "engineered" refers to entities made by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, engineered entities are synthetic and contain elements that do not occur in nature or that have been constructed for use in the present disclosure.

[0064] "Treating" a disease or condition or treatment thereof refers to carrying out a protocol that may include administering one or more drugs to a patient with the aim of alleviating the signs or symptoms of the disease. The desired effects of treatment include slowing the progression of the disease, reversing or alleviating the disease state, and remission or improvement of prognosis. Alleviation can occur before as well as after the signs or symptoms of the disease or condition appear. Thus, "treating" or "treatment" can include "preventing" or the "prevention" of a disease or undesirable condition. Furthermore, "treating" or "treatment" does not require complete alleviation of signs or symptoms, does not require a cure, and in particular includes protocols that have only marginal effects on the patient.

[0065] The term "therapeutic effect" or "therapeutically effective" as used throughout this application refers to anything that enhances or improves the well-being of a subject with respect to the medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of a sign or symptom of a disease. For example, treating cancer can include, for example, reducing tumor size, reducing the invasiveness of a tumor, reducing the rate of growth of a cancer, or preventing metastasis. Treating cancer can also refer to extending the survival time of a subject with cancer.

[0066] "Subject" and "patient" refer to humans or non-humans, e.g., primates, mammals, and vertebrates. 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 mammalian class of higher vertebrates, including humans, and may be characterized by live birth, body hair, and mammary glands in females that secrete milk to feed their young. Additionally, mammals are characterized by the ability to maintain a constant body temperature despite fluctuating climatic conditions. Examples of mammals are humans, cats, dogs, cows, mice, rats, horses, goats, sheep, and chimpanzees. A mammal may be referred to as a "patient" or a "subject" or an "individual."

[0068] The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not cause adverse, allergic or other untoward reactions when appropriately administered to animals, such as humans. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients is known to those skilled in the art in light of the present disclosure. Furthermore, it is understood that for administration to animals (e.g., humans), preparations should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0069] As used herein, "pharmaceutical acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents and inert gases), isotonicity agents, absorption retardants, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, flow and nutritional supplements, such similar materials and combinations thereof, as known to those skilled in the art. The pH and exact concentration of various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0070] As used herein, the "disruption" of a gene refers to the absence or reduction of expression of one or more gene products encoded by a gene of interest in a cell, compared to the expression level of the gene product in the absence of the disruption. Exemplary gene products include the mRNA and protein products encoded by the gene. The disruption is in some cases transient or reversible, and in other cases permanent. Despite the fact that truncated or non-functional products may be generated, the disruption is in some cases the disruption of functional or full-length protein or mRNA. In some embodiments herein, the activity or function of a gene is disrupted quite differently than its expression. Gene disruption is generally induced by artificial methods, i.e., the addition or introduction of a compound, molecule, complex or composition, and / or the disruption of the nucleic acid of the gene or the nucleic acid associated with the gene, such as at the DNA level. Exemplary methods for gene disruption include gene disruption techniques such as gene silencing, knockdown, knockout, and / or gene editing. Examples include antisense techniques (e.g., RNAi, siRNA, shRNA and / or ribozymes), which generally result in a transient reduction in expression, and gene editing techniques that result in targeted gene inactivation or disruption, for example by cleavage and / or induction of homologous recombination. Examples include insertion, mutation and deletion. Disruption usually prevents expression of the normal or "wild type" product encoded by the gene, and / or completely eliminates its expression. Illustrative of such gene disruption are insertion, frameshift and missense mutation, deletion, knock-in and knock-out (including deletion of the entire gene) of a gene or part of a gene. Such disruption can occur in a coding region, for example, in one or more exons, such that a full-length product, a functional product or any product cannot be produced, such as by inserting a stop codon. Such disruption can also occur by disruption in a promoter or enhancer or other region that affects transcription activation, so as to prevent transcription of the gene.Gene disruption includes gene targeting, which includes targeted gene inactivation by homologous recombination. II. Multiplex gene editing

[0071] In certain embodiments, the present disclosure relates to multiplex gene editing of any type of immune cell.CRISPR is one example that can be used to disrupt the expression of two or more genes (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more genes) in immune cells. These genes include those listed in Table 1, such as 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 protein (CISH, CIS-1, SOCS), forkhead box O1 (FOXO1), transforming growth factor beta receptor 2 (TGFβR2), T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96, adenosine receptor 2A (ADORA2), nuclear receptor subfamily 3 group C member 1 (NR3C1), protease inhibitor 1 (PT1), and IFN-γ receptor 1 (IFN-γ). The inhibitory genes may be selected from gram cell death 1 (PD1), programmed cell death 1 ligand 1 (PDL-1), programmed cell death 1 ligand 2 (PDL-2), CD47, signal regulatory protein alpha (SIRPA), SH2 domain-containing inositol 5-phosphatase 1 (SHIP1), ADAM metallopeptidase domain 17 (ADAM17), ribosomal protein S6 (RPS6), eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), CD25, CD40, interleukin 21 receptor (IL21R), intercellular adhesion molecule 1 (ICAM1), CD95, CD80, CD86, interleukin 21 receptor (IL10R), CD5, CD7, or other inhibitory genes may be present. Gene editing allows the expression of multiple genes to be disrupted simultaneously.

[0072] In some embodiments, gene disruption is performed by creating a disruption in the gene (e.g., knockout, insertion, missense mutation or frameshift mutation, e.g., biallelic frameshift mutation, deletion of all or part of the gene, e.g., deletion of one or more exons or therefore parts, and / or knock-in).For example, disruption can be created by sequence-specific or targeted nucleases, including DNA-binding targeted nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), specifically designed to target the sequence of a gene or a part thereof, and RNA-guided nucleases such as CRISPR-associated nucleases (Cas).

[0073] In some embodiments, the disruption is transient or reversible, and expression of the gene is restored at a later time, in other embodiments, the disruption is not reversible or transient, e.g., permanent.

[0074] In some embodiments, gene disruption is carried out by inducing one or more double-strand breaks and / or one or more single-strand breaks in the gene, usually in a targeted manner.In some embodiments, double-strand or single-strand breaks are carried out by nucleases, for example endonucleases such as gene-targeting nucleases.In some embodiments, the breaks are induced in the coding region of gene, for example, in exons.For example, in some embodiments, the induction occurs near the N-terminal part of the coding region, for example, the first exon, the second exon or subsequent exons.

[0075] Said immune cell can be introduced with guide RNA and CRISPR enzyme or mRNA encoding CRISPR enzyme.In some embodiments, one, two, three, four, five or more guide RNAs are introduced into the cell at the same time.For example, one, two or three guide RNAs can be introduced into the cell during the first electroporation, and then one, two or three additional guide RNAs can be further introduced into the cell during the second electroporation, etc.

[0076] In some embodiments, gene disruption is achieved using antisense techniques (e.g., RNA interference (RNAi), small interfering RNA (siRNA), short hairpin (shRNA) and / or ribozymes), which selectively suppress or block the expression of genes. siRNA technology is RNAi, which uses double-stranded RNA molecules with a sequence that is homologous to and complementary to the nucleotide sequence of the mRNA transcribed from a gene. siRNAs can generally be siRNAs that are homologous / complementary to one region of the mRNA transcribed from a gene, or that contain multiple RNA molecules that are homologous / complementary to different regions. In some embodiments, siRNAs are included in polycistronic constructs.

[0077] In some embodiments, the disruption is achieved using a DNA targeting molecule (e.g., a DNA binding protein or a DNA binding nucleic acid) or a complex, compound or composition comprising the same that specifically binds or hybridizes to the gene. In some embodiments, the DNA targeting molecule comprises a DNA binding domain, such as a DNA binding domain of a zinc finger protein (ZFP), a DNA binding domain of a transcription activator-like protein (TAL) or a DNA binding domain of a TAL effector (TALE), a DNA binding domain of a clustered regularly interspaced short palindromic repeats (CRISPR), or a DNA binding domain of a meganuclease. The binding domain of zinc finger, TALE and CRISPR systems can be engineered to bind to a predetermined nucleotide sequence, for example, through the manipulation (changing one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein. The engineered DNA binding protein (zinc finger or TALE) is a protein that does not occur in nature. Rational criteria for design include the application of substitution rules and computer algorithms to process information in databases that store information on existing ZFP and / or TALE design and binding data.

[0078] In the case of CRISPR-mediated destruction, guide RNA and endonuclease can be introduced into immune cells by any means known in the art that allows delivery inside cells or intracellular compartments, and the agents / chemicals and / or molecules (proteins and nucleic acids) that can be used include, but are not limited to, liposomal delivery means, polymeric carriers, chemical carriers, lipoplexes, polyplexes, dendrimers, nanoparticles, emulsions, natural endocytosis or phagocytosis pathways, and physical methods such as electroporation.In a specific embodiment, electroporation is used to introduce guide RNA and endonuclease or the nucleic acid that codes for endonuclease.

[0079] In one exemplary specific method, the method for CRISPR knockout of multiple genes may include the isolation of immune cells, such as NK cells, from umbilical cord blood or peripheral blood. The NK cells may be isolated and seeded on a culture plate with irradiated feeder cells, such as in a ratio of 1:2. The cells may then be electroporated with gRNA and Cas9 in the presence of IL-2, such as at a concentration of 200 IU / mL. The medium may be changed, for example, every other day. After 1-3 days, the NK cells may be isolated to remove the feeder cells, and then transduced with a CAR construct. The NK cells may then be subjected to a second CRISPR Cas9 knockout for additional genes. After electroporation, the NK cells may be seeded with the feeder cells, such as for 5-9 days.

[0080] [Table 1]

[0081] [Table 2]

[0082] In some embodiments, the immune cells of the present disclosure are modified to change the expression of two or more genes. In some embodiments, the change in gene expression is achieved by creating a disruption in the gene (e.g., knockout, insertion, missense mutation or frameshift mutation, e.g., biallelic frameshift mutation, deletion of all or part of the gene, e.g., deletion of one or more exons or therefore parts, and / or knock-in). In a specific embodiment, the change in gene expression can be achieved by sequence-specific nuclease or targeted nuclease, including DNA-binding targeted nuclease specifically designed to target the sequence of a gene or part thereof, e.g., RNA-guided nuclease such as CRISPR-associated nuclease (Cas).

[0083] In some embodiments, the expression, activity and / or function of a gene is altered by disrupting the gene. In some embodiments, the gene is modified so that its expression is reduced by at least or about 10, 20, 30 or 40%, usually at least or about 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, compared to the expression in the absence of gene modification or the expression in the absence of the introduction of the component that causes the modification.

[0084] In some embodiments, the alteration is transient or reversible, and expression of the gene is restored at a later time, if desired. In other embodiments, the alteration is not reversible or transient, e.g., permanent.

[0085] In some embodiments, gene modification is performed by inducing one or more double-strand breaks and / or one or more single-strand breaks in the gene, usually in a targeted manner.In some embodiments, double-strand or single-strand breaks are performed by nucleases, for example endonucleases such as gene-targeting nucleases.In some embodiments, the breaks are induced in the coding region of gene, for example, in exons.For example, in some embodiments, the induction occurs near the N-terminal part of the coding region, for example, the first exon, the second exon or subsequent exons.

[0086] In some embodiments, the double-stranded or single-stranded break undergoes repair via cellular repair processes, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some embodiments, this repair process leads to gene disruption, e.g., frameshift mutations, e.g., biallelic frameshift mutations, which are error-prone and can result in complete knockout of the gene. For example, in some embodiments, the disruption includes inducing deletions, mutations, and / or insertions. In some embodiments, the disruption results in the premature presence of a stop codon. In some embodiments, the insertion, deletion, translocation, frameshift mutation, and / or the presence of a premature stop codon disrupts the expression, activity, and / or function of the gene.

[0087] In some embodiments, the modification is carried out using one or more DNA-binding nucleic acids, such as modification via RNA-guided endonuclease (RGEN).For example, the modification can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins.Generally, "CRISPR system" refers collectively to CRISPR-associated ("Cas") genes (including sequences encoding Cas genes), tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeat sequences" in the context of endogenous CRISPR systems, and partial direct repeat sequences processed by tracrRNA), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or transcripts and other elements involved in or directing the expression of other sequences and transcripts from CRISPR locus.

[0088] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner, and a Cas protein (e.g., Cas9) that has a nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system, for example, from a particular organism that contains an endogenous CRISPR system (e.g., Streptococcus pyogenes).

[0089] In some embodiments, Cas nuclease and gRNA (comprising a fusion of a target sequence-specific crRNA and a given tracrRNA) are introduced into cells. In general, a target site at the 5' end of gRNA targets Cas nuclease to its target site, e.g., a gene, by complementary base pairing. Target sites can be selected based on the location immediately 5' of a protospacer adjacent motif (PAM) sequence (e.g., typically NGG or NAG). In this regard, gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems feature elements that promote the formation of a CRISPR complex at the site of the target sequence. In general, "target sequence" generally refers to a sequence that the guide sequence is designed to have complementarity with, and hybridization of the target sequence with the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not necessary, as long as there is sufficient complementarity to cause hybridization and promote formation of the CRISPR complex.

[0090] The CRISPR system can induce a double-strand break (DSB) at the target site followed by a disruption or modification as discussed herein. In other embodiments, a Cas9 variant considered a "nickase" is used to nick a single strand at the target site. For example, to improve specificity, a pair of nickases can be used, each of which is guided by a different pair of gRNAs targeting sequences, and a 5' overhang is introduced when a nick is introduced simultaneously. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or transcriptional activator, to affect gene expression.

[0091] The target sequence may comprise 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 in a cell organelle. In general, the sequence or template that can be used for recombination into the targeted locus that comprises the target sequence is referred to as "editing template" or "editing polynucleotide" or "editing sequence". In some embodiments, an exogenous template polynucleotide may be referred to as an editing template. In some embodiments, the recombination is a homologous recombination.

[0092] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) results in cleavage of one or both strands at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more of the target sequence). A tracr sequence that may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about 20 nucleotides, about 26 nucleotides, about 32 nucleotides, about 45 nucleotides, about 48 nucleotides, about 54 nucleotides, about 63 nucleotides, about 67 nucleotides, about 85 nucleotides or more, or more than about 20 nucleotides, about 26 nucleotides, about 32 nucleotides, about 45 nucleotides, about 48 nucleotides, about 54 nucleotides, about 63 nucleotides, about 67 nucleotides, about 85 nucleotides or more of the wild-type tracr sequence) may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence (e.g., at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned) to hybridize and participate in the formation of a CRISPR complex.

[0093] One or more vectors can be introduced into a cell to drive the expression of one or more elements of the CRISPR system, so that the expression of those elements directs the formation of a CRISPR complex at one or more target sites. Also, components can be delivered to a cell as protein and / or RNA. For example, Cas enzyme, guide sequence linked to tracr mate sequence, and tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector, where one or more additional vectors provide any components of the CRISPR system that are not included in the first vector. The vector can include one or more insertion sites (also referred to as "cloning sites"), such as restriction endonuclease recognition sequences. In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in a cell.

[0094] The vector can include regulatory elements operably linked to 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, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof or modified versions thereof. These enzymes are known; for example, the amino acid sequence of the Cas9 protein of S. pyogenes can be found in the SwissProt database under accession number Q99ZW2.

[0095] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct the cleavage of one or both strands at the location of the target sequence (e.g., within the target sequence and / or within the complementary strand of the target sequence). The vector can encode a CRISPR enzyme mutated relative to the corresponding wild-type enzyme, which lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands into a nickase (cleaves a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence, e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination can nick both strands and is used to induce NHEJ or HDR.

[0096] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell can be a cell of a particular organism (e.g., a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate) or a cell derived from those organisms. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the natural sequence with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the natural amino acid sequence. Different species show a certain bias for certain codons of a particular amino acid. Codon bias (the difference in codon usage between organisms) often correlates with the translation efficiency of messenger RNA (mRNA), which is believed to depend, among other things, on the properties of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The tRNA that is predominantly selected in a cell usually reflects the codon that is most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be tailored for optimal gene expression in a given organism.

[0097] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and a corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0098] Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0099] CRISPR enzyme can be part of a fusion protein that contains one or more heterologous protein domains. CRISPR enzyme fusion protein can contain any additional protein sequence, and optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription blocking activity, transcription termination factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of fusion proteins containing CRISPR enzymes are 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 relates to the insertion of a CAR and / or a TCR at a specific genetic locus of an immune cell. The CAR and / or the TCR may be inserted into an inhibitory gene locus (e.g., a gene selected from the group consisting of NKG2A, Siglec7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, adenosine receptor 2A, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPa, SHIP1, ADAM17, pS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and combinations thereof).

[0101] The insertion of one or more CARs and / or TCRs in any of the methods disclosed herein can be site-specific. For example, one or more CARs and / or TCRs can be inserted adjacent to or near a promoter. In another example, one or more transgenes can be inserted adjacent to, near, or within an exon of a gene (e.g., an inhibitory gene). Such an insertion can be used to knock in a CAR and / or TCR while simultaneously disrupting the expression of the gene. In another example, one or more CARs and / or TCRs can be inserted adjacent to, near, or within an intron of a gene. A CAR and / or TCR can be introduced by an adeno-associated virus (AAV) viral vector and integrated into a targeted genomic location. In some cases, a rAAV vector can be used to direct the insertion of a transgene into a specific location. For example, in some cases, the CAR and / or TCR can be integrated into at least a portion of the NKG2A, Siglec7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, adenosine receptor 2A, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPa, SHIP1, ADAM17, pS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, or CD7 genes by rAAV or AAV vectors.

[0102] The modification of the targeted locus of a cell can be carried out by introducing DNA into the cell, where the DNA has homology to the target locus. The DNA can contain a marker gene, which allows the selection of cells containing the integrated construct. The complementary DNA in the target vector can recombine with the chromosomal DNA at the target locus. The marker gene can be flanked by a complementary DNA sequence, a 3' recombination arm and a 5' recombination arm. Multiple loci in a cell can be targeted. For example, transgenes with recombination arms specific to one or more target loci can be introduced simultaneously, so that multiple genome modifications are carried out in one step. The homology arms can be from about 0.2 kb to about 5 kb in length (e.g., from about 0.2 kb, 0.4 kb 0.6 kb, 0.8 kb, 1.0 kb, 1.2 kb, 1.4 kb, 1.6 kb, 1.8 kb, 2.0 kb, 2.2 kb, 2.4 kb, 2.6 kb, 2.8 kb, 3.0 kb, 3.2 kb, 3.4 kb, 3.6 kb, 3.8 kb, 4.0 kb, 4.2 kb, 4.4 kb, 4.6 kb, 4.8 kb to about 5.0 kb in length, etc.).

[0103] In one method, the guide RNA can be designed to target a region of the locus of the inhibitory gene (e.g., a region adjacent to the promoter, exon, or intron of the gene). The guide RNA can target the 5' end of an exon (e.g., the first, second, or third exon) of the inhibitory gene. The guide RNA can be included in an AAV vector repair matrix. The AAV vector can encode a self-cleaving 2A peptide, such as a P2A peptide, followed by a CAR cDNA. The CAR cassette and guide RNA sequence can be adjacent to homology arms to the inhibitory gene. The immune cell can then be introduced (e.g., electroporated) with the AAV vector and Cas9 (e.g., Cas9 mRNA). IV. Immune cells

[0104] Certain embodiments of the present disclosure relate to immune cells engineered to have knockouts of multiple genes and / or knock-ins of CARs at the locus of inhibitory genes. These immune cells include T cells (e.g., regulatory T cells, CD4 + T cells, CD8 + The immune cells may be 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 (iPSC) cells). The immune cells may be virus-specific, express a CAR, and / or express a TCR. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. Also provided herein are methods of making and manipulating immune cells, as well as using and administering the cells for adoptive cell therapy, where the cells may be autologous or allogeneic. Thus, the immune cells may be used as immunotherapy, such as to target cancer cells.

[0105] The immune cells can be isolated from a subject, particularly a human subject. The immune cells can be obtained from a subject of interest (e.g., a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, or a subject undergoing treatment for a particular disease or condition). The immune cells can be collected from any location where they are present in a subject, including, but not limited to, blood, umbilical cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells can be used directly or can 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 reside, including, but not limited to, blood (including blood collected by blood bank or umbilical cord blood bank), spleen, bone marrow, tissue removed and / or exposed during surgical procedures, and tissue obtained via biopsy procedures. The tissue / organ from which immune cells are enriched, isolated and / or purified may be isolated from both living and non-living subjects, where the non-living subject is an organ donor. In certain embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood or a mixture thereof. In some embodiments, immune cells isolated from umbilical cord blood have high immunoregulatory capacity, as measured by suppression of CD4+ or CD8+ T cells. In a specific embodiment, immune cells are isolated from pooled blood, particularly pooled umbilical cord blood, for high immunoregulatory capacity. Pooled blood may be blood from two or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

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

[0108] When the immune cell population is obtained from a donor different from the subject, the donor is preferably allogeneic, provided that the cells obtained are matched with the subject in that the cells can be introduced into the subject. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) matched. AT cells

[0109] In some embodiments, the immune cell is a T cell. Several basic approaches for the induction, activation and expansion of functional anti-tumor effector cells have been reported in the past 20 years. These include autologous cells such as tumor-infiltrating lymphocytes (TILs); T cells activated ex vivo using autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T cell ligands and activating antibodies, or cells isolated by target cell membrane capture; allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs); and non-tumor specific autologous or allogeneic cells that are genetically reprogrammed or "re-instructed" to express tumor-reactive or chimeric TCR molecules that exhibit antibody-like tumor recognition capabilities known as "T-bodies". These approaches have led to numerous protocols for preparing and immunizing T cells that can be used in the methods described herein.

[0110] In some embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some embodiments, the cells are human cells. The cells are usually primary cells, e.g., cells isolated directly from a subject, and / or cells isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types (e.g., the entire T cell population, CD4 + cells, CD8 +The present invention includes cells and subpopulations thereof, such as subpopulations defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization and / or survival, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. The cells can be allogeneic and / or autologous cells with respect to the subject to be treated. In some embodiments, such as in the case of existing technologies, the cells are pluripotent and / or multipotent (e.g., stem cells such as induced pluripotent stem cells (iPSCs)). In some embodiments, the method includes isolating cells from the subject, preparing, treating, culturing and / or manipulating them as described herein, and reintroducing them into the same patient before or after cryopreservation.

[0111] T cells (e.g., CD4 + and / or CD8 + T cell) subtypes and subpopulations include 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 Memory T (TC M ), Effector Memory T (T EM ) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, intrinsic and adaptive regulatory T (Treg) cells, helper T cells (e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells), alpha / beta T cells and delta / gamma T cells.

[0112] In some embodiments, one or more of the T cell populations are enriched or depleted in cells that are positive for a specific marker, such as a surface marker, or that are negative for a specific marker. In some cases, such markers are markers that are absent or expressed at relatively low levels on certain T cell populations (e.g., non-memory cells) but present or expressed at relatively high levels on certain other T cell populations (e.g., memory cells).

[0113] In some embodiments, T cells are separated from the PBMC sample by negative selection of a marker (e.g., CD14) expressed on non-T cells (e.g., B cells, monocytes or other leukocytes). + or CD8 + By using a selection process, CD4 + Helper T cells and CD8 + Cytotoxic T cells are isolated. Such CD4 + and CD8 + The population can be further sorted into subpopulations by positive or negative selection of markers that are expressed or expressed to a relatively high degree on one or more subpopulations of naive, memory and / or effector T cells.

[0114] In some embodiments, CD8 + The T cells are further enriched or depleted for naive, central memory, effector memory and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CM ) Enrichment of cells is performed to increase efficacy, such as improving long-term survival, expansion and / or engraftment following administration, which in some embodiments is particularly robust in such subpopulations.

[0115] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a suspension of single cells is obtained. The suspension of single cells is then purified in any suitable manner, for example mechanically (e.g., by gentleMACS TM Tumors can be disaggregated using a ELISA kit (Microbial Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Single cell suspensions of the enzymatic digest of tumors are cultured in interleukin-2 (IL-2).

[0116] The cultured T cells can be pooled and rapidly expanded. Rapid expansion over about 10 to about 14 days 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, rapid expansion over about 10 to about 14 days increases the number of antigen-specific T cells by at least about 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900, or more).

[0117] Expansion can be accomplished by any of several methods known in the art. For example, T cells can be readily expanded using non-specific T cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. The non-specific T cell receptor stimulation can include approximately 30 ng / ml of OKT3 (available from Ortho-McNeil®, Raritan, NJ), a mouse monoclonal anti-CD3 antibody. Alternatively, T cells can be readily expanded by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with one or more cancer antigens (including antigenic portions thereof, such as epitopes, or cells) in the presence of a T cell growth factor (e.g., 300 IU / ml IL-2 or IL-15 (IL-2 is preferred)), which may be expressed from a vector, optionally, such as a human leukocyte antigen A2 (HLA-A2) binding peptide. The in vitro induced T cells are rapidly expanded by restimulation with the same cancer antigen pulsed against antigen presenting cells expressing HLA-A2. Alternatively, the T cells can be rapidly expanded by restimulation with, for example, irradiated autologous lymphocytes or irradiated HLA-A2 antigen-presenting cells. + They can be restimulated with allogeneic lymphocytes and IL-2.

[0118] The autologous T cells can be modified to express T cell growth factors that promote the proliferation and activation of the autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable modification methods are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rded., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain embodiments, the modified autologous T cells express high levels of T cell growth factors. T cell growth factor coding sequences (e.g., coding sequences for IL-12) are readily available in the art, as are promoters whose operably linked to the T cell growth factor coding sequences promote high level expression. B.NK cells

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

[0120] In certain embodiments, NK cells are obtained from human peripheral blood mononuclear cells (PBMCs), unstimulated leukapheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow or umbilical cord blood by methods well known in the art. In particular, umbilical cord blood is used to obtain NK cells. In certain aspects, NK cells are isolated and expanded by a previously reported method of ex vivo expansion of NK cells (Spanholtz et al., 2011; Shah et al., 2013). In this method, CB mononuclear cells are isolated by Ficoll density gradient centrifugation and cultured in a bioreactor with IL-2 and artificial antigen presenting cells (aAPCs). After 7 days, the cell culture is depleted of any cells expressing CD3 and re-cultured for another 7 days. Again, cells are depleted for CD3 and CD56 + / CD3 - Another method uses umbilical cord blood and characterizes it to measure the percentage of CD34 cells or NK cells. + Isolation of CD56 cells and culturing them in medium containing SCF, IL-7, IL-15 and IL-2 + / 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 a specific case, the expansion of the NK cells includes stimulating mononuclear cells (MNCs) from umbilical cord blood in the presence of antigen-presenting cells (APCs) and IL-2; and restimulating the cells with APCs to generate expanded NK cells, at least in some cases, the method is performed in a bioreactor. The stimulation step can direct the MNCs to NK cells. The restimulation step may or may not include the presence of IL-2. In certain embodiments, the method does not include the removal or addition of any medium components during the stimulation step. In certain embodiments, the method is performed within a certain time frame (e.g., less than 15 days, e.g., 14 days).

[0122] In certain embodiments, the NK cells are expanded by an ex vivo method for expansion, which includes the steps of (a) obtaining a starting population of mononuclear cells (MNCs) from umbilical cord blood; (b) stimulating the MNCs 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 being performed in a bioreactor and in compliance with good manufacturing practice (GMP). The stimulation in step (b) can direct the MNCs towards NK cells. Step (c) may or may not include the presence of IL-2. In certain embodiments, the method does not include the removal or addition of any media components during step (b). In certain embodiments, the method is performed in less than 15 days (e.g., 14 days).

[0123] In some embodiments, the method further comprises a step of depleting cells positive for one or more specific markers, such as CD3. In certain embodiments, the depletion step is performed between steps (b) and (c). In some embodiments, the cells are removed from the bioreactor for CD3 depletion and placed in the bioreactor for step (c).

[0124] In certain embodiments, obtaining a starting population of MNCs from cord blood comprises thawing cord blood in the presence of dextran, human serum albumin (HSA), DNAse and / or magnesium chloride. In certain embodiments, obtaining a starting population of MNCs from cord blood comprises thawing cord blood in the presence of dextran and / or DNase. In specific embodiments, cord blood is washed in the presence of 5-20% (e.g., 10%) dextran. In certain embodiments, cord blood is suspended in the presence of magnesium chloride at a concentration of 100-300 mM, particularly 200 mM. In some embodiments, obtaining comprises Ficoll density gradient centrifugation to obtain mononuclear cells (MNCs).

[0125] In certain embodiments, the bioreactor is a gas permeable bioreactor. In certain embodiments, the gas permeable bioreactor is a G-Rex100M or a G-Rex100. In some embodiments, the stimulation of step (b) is performed in 3-5 L of medium (e.g., 3, 3.5, 4, 4.5, or 5 L).

[0126] In some embodiments, the APCs are gamma irradiated. In certain embodiments, the APCs are engineered to express membrane-bound IL-21 (mbIL-21). In certain embodiments, the APCs are engineered to express IL-21, IL-15 and / or IL-2. In some embodiments, the MNCs and APCs are cultured at a ratio of 1:2. In some embodiments, the IL-2 is present at a concentration of 50-200 IU / mL (e.g., 100 IU / mL). In certain embodiments, the IL-2 is replenished every 2-3 days.

[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 some embodiments, step (c) does not include splitting the cells. In certain embodiments, the cells are fed with IL-2 twice during step (c), and in certain cases, no other medium components are added or removed during step (c).

[0128] In some embodiments, the method comprises the use of 3, 4, 5, or 6 bioreactors. In certain embodiments, the method comprises the use of less than 10 bioreactors.

[0129] In specific embodiments, the NK cells are expanded at least 500-fold, 800-fold, 1000-fold, 1200-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, or 5000-fold. In certain embodiments, culturing the NK cells in a bioreactor generates 1000-fold more NK cells compared to static liquid culture.

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

[0131] In some embodiments, the expanded NK cells have higher anti-tumor activity than NK cells expanded from peripheral blood. In certain embodiments, the expanded NK cells have higher expression of one or more cell cycle genes, one or more cell division genes, and / or one or more DNA replication genes than NK cells expanded from peripheral blood. In some embodiments, the expanded NK cells have higher proliferation capacity than NK cells expanded from peripheral blood. In some embodiments, the expanded NK cells do not show exhaustion. In certain embodiments, exhaustion is detected by measuring the expression of perforin, granzymes, CD57, KLRG1, and / or PD1. In some embodiments, the expanded NK cells have high expression of perforin and / or granzymes. In certain embodiments, the expanded NK cells have low or no expression of CD57, 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 has been tested for 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 pooled cord blood, such as from 3, 4, 5, 6, 7, or 8 individual cord blood units.

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

[0134] In some embodiments, the APC is a universal antigen presenting cell (uAPC). In certain embodiments, the uAPC is engineered to express (1) CD48 and / or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 41BB ligand (41BBL). In some embodiments, the uAPC expresses CD48. In certain embodiments, the uAPC expresses CS1. In certain embodiments, the uAPC expresses CD48 and CS1. In some embodiments, the uAPC has essentially no endogenous expression of HLA class I, II, and / or CD1d molecules. In certain embodiments, the uAPC expresses ICAM-1 (CD54) and / or LFA-3 (CD58). In certain embodiments, the uAPC is further defined as an aAPC derived from a leukemia cell, such as a K562 cell. C. Stem cells

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

[0136] Pluripotent stem cells used herein can be induced pluripotent stem (iPS) cells, usually abbreviated as iPS cells or iPSCs. Any cell can be used as the starting point of iPSCs, except germ cells. For example, the cell type can be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells or stomach cells. There is no limit to the degree of cell differentiation or the age of the animal from which the cells are collected. Undifferentiated progenitor cells (including somatic stem cells) and even terminally differentiated mature cells can be used as the source of somatic cells in the methods disclosed herein.

[0137] Somatic cells can be reprogrammed to generate iPS cells using methods known to those skilled in the art.Generally, nuclear reprogramming factors are used to generate pluripotent stem cells from somatic cells.In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog and Lin28 are used.In other embodiments, Oct3 / 4, Sox2, c-Myc and Klf4 are used, or Oct3 / 4, Sox2, Nanog and Lin28 are used.

[0138] Once induced, iPSCs can be cultured in a medium sufficient to maintain pluripotency. In certain embodiments, indefinite conditions can be used. For example, pluripotent cells can be cultured on fibroblast feeder cells or on medium exposed to fibroblast feeder cells to maintain the stem cells in an undifferentiated state. In some embodiments, the cells are cultured with mouse fetal fibroblasts as feeder cells that have been irradiated or treated with antibiotics to terminate cell division. Alternatively, pluripotent cells can be cultured in TESR. TM Medium or E8 TM / Essential 8 TM The cells can be cultured and maintained in an essentially undifferentiated state using a feeder-independent defined culture system such as culture medium. V. Genetically Engineered Antigen Receptors

[0139] The immune cells of the present disclosure can be genetically engineered to express antigen receptors (e.g., engineered TCRs, CARs, chimeric cytokine receptors, chemokine receptors, combinations thereof, etc.). For example, immune cells are modified to express CARs and / or TCRs with antigen specificity for cancer antigens. Multiple CARs and / or TCRs (e.g., for different antigens) can be added to immune cells. In some embodiments, immune cells are engineered to express CARs or TCRs by knocking in the CARs or TCRs at the locus of inhibitory genes using CRISPR.

[0140] Suitable modification methods are known in the art. For example, see Sambrook and Ausubel, supra. For example, the above cells can be transduced to express TCR with antigen specificity for cancer antigen using the transduction method described in Heemskerk et al., 2008 and Johnson et al., 2009.

[0141] As an option to overcome the long-term problems of autoreactivity caused by pairing of retrovirally transduced TCR chains with endogenous TCR chains, electroporation of RNA encoding full-length TCR α and β (or γ and δ) chains can be used. Even if such alternative pairing occurs in a transient transfection strategy, the introduced TCR α and β chains are only expressed transiently, so that any autoreactive T cells that may be generated lose this autoreactivity after a while. When the expression of the introduced TCR α and β chains decreases, only normal autologous T cells remain. This is not the case when the full-length TCR chains are introduced by stable retroviral transduction, the introduced TCR chains are not lost and autoreactivity is constantly present in the patient.

[0142] In some embodiments, the cells contain one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors, and the genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not normally present in the cell or sample obtained from the cell (e.g., obtained from another organism or cells that are not normally found, e.g., in the cell being engineered and / or the organism from which such cell is derived). In some embodiments, the nucleic acids are not naturally occurring, such as nucleic acids not found in nature (e.g., chimeras).

[0143] In some embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the cell surface. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen (e.g., a peptide antigen of an intracellular protein) that is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule, similar to a TCR.

[0144] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for engineering and introducing those receptors into cells are described in, for example, International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748 ... Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, and European Patent Application No. EP 2537416, and / or Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some embodiments, the genetically engineered antigen receptor includes a CAR, such as that described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO / 2014055668A1. A. Chimeric antigen receptor

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

[0146] In some embodiments, engineered antigen receptors include CARs, including activating or stimulatory CARs, costimulatory CARs (see WO2014 / 055668) and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). Such CARs generally comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some embodiments via a linker and / or transmembrane domain. Such molecules typically mimic or mimic the signaling through a natural antigen receptor, through such receptors in conjunction with costimulatory receptors, and / or through costimulatory receptors alone.

[0147] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding antigen-specific CAR polypeptides (including CARs humanized to reduce immunogenicity (hCARs)) that include an intracellular signaling domain, a transmembrane domain, and an extracellular domain that includes one or more signaling motifs. In certain embodiments, the CAR may recognize an epitope that includes a space shared between one or more antigens. In certain embodiments, the binding region may include a complementarity determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide that binds to a receptor (e.g., a cytokine).

[0148] It is contemplated that the human CAR nucleic acid may be a human gene used to enhance cellular immunotherapy for human patients. In a specific embodiment, the present invention comprises a full-length cDNA or coding region of a CAR. The antigen-binding region or domain is the V of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody.H Chain and V L The fragments may include fragments of the chains (e.g., those described in U.S. Patent No. 7,109,304, incorporated herein by reference). The fragments may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragments are antigen-specific scFvs encoded by sequences optimized for human codon usage for expression in human cells.

[0149] The arrangement can be a multimer (e.g., a diabody or multimer). The multimer is most likely formed by cross-pairing the variable parts of the light and heavy chains into a diabody. The hinge part of the construct can have multiple options ranging from complete deletion, maintaining the first cysteine, proline substitution instead of serine substitution, truncation up to the first cysteine. The Fc part can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. Only one of the Fc domains can be used, for example the CH2 or CH3 domain of a human immunoglobulin. The hinge, CH2 and CH3 regions of a human immunoglobulin modified to improve dimerization can also be used. Only the hinge part of an immunoglobulin can be used. A part of CD8 alpha can also be used.

[0150] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Other costimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12 and 4-1BB (CD137). In addition to the primary signal elicited by CD3ζ, the additional signal provided by the human costimulatory receptor inserted into the human CAR is important for the complete activation of NK cells and can help improve in vivo persistence and therapeutic success of adoptive immunotherapy.

[0151] In some embodiments, the CAR is constructed to have specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type targeted by adoptive therapy, e.g., a cancer marker and / or an antigen intended to induce an attenuated response (e.g., an antigen expressed on a normal or non-diseased cell type). Thus, the CAR typically comprises, in its extracellular portion, one or more antigen-binding molecules (e.g., one or more antigen-binding fragments, antigen-binding domains or antigen-binding portions) or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR comprises an antigen-binding portion of an antibody molecule (e.g., a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb)).

[0152] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as the extracellular domain containing the antigen-binding region) comprises a tumor-associated antigen or a pathogen-specific antigen-binding domain. Antigens include carbohydrate antigens recognized by pattern recognition receptors such as Dectin-1. The tumor-associated antigen may be of any type, so long as it is expressed on the cell surface of the tumor cell. Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, and the like. In certain embodiments, the CAR may be co-expressed with a cytokine to improve persistence when the amount of tumor-associated antigen is low. For example, the CAR may be co-expressed with one or more cytokines, such as IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, or a combination thereof.

[0153] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or can be synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns have been found to stabilize mRNA. It may also be further beneficial to use endogenous or foreign non-coding regions to stabilize mRNA.

[0154] It is contemplated that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector.Methods for stably transfecting cells by electroporation with naked DNA are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA that codes for the chimeric receptor that is contained in a plasmid expression vector in the proper orientation for expression.

[0155] Alternatively, viral vectors (such as retroviral vectors, adenoviral vectors, adeno-associated viral vectors or lentiviral vectors) can be used to introduce chimeric constructs into immune cells.The vectors suitable for use according to the method of the present disclosure are non-replicative vectors in immune cells.There are many known virus-based vectors (such as HIV, SV40, EBV, HSV or BPV-based vectors), and the copy number of the virus that is maintained in cells is low enough to maintain the viability of the cells.

[0156] In some embodiments, the antigen-specific binding component or antigen-specific recognition component is linked to one or more transmembrane domains and intracellular signaling domains.In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR.In one embodiment, the transmembrane domain that naturally associates with one of the domains in the CAR is used.In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid such domains binding to the transmembrane domain of the same or different surface membrane protein to minimize interaction with other members of the receptor complex.

[0157] The transmembrane domain, in some embodiments, is derived from natural or synthetic origin. If the origin is natural, the domain, in some embodiments, is derived from any membrane-bound or transmembrane protein. The transmembrane region includes the transmembrane region derived from (i.e., at least includes) the transmembrane region of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D and DAP molecules. Alternatively, the transmembrane domain, in some embodiments, is a synthetic transmembrane domain. In some embodiments, the synthetic transmembrane domain mainly comprises hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine may be found at each end of the synthetic transmembrane domain.

[0158] In certain embodiments, platform technologies disclosed herein for genetically modifying immune cells, such as NK cells, include (i) non-viral gene transfer using an electroporation device (e.g., nucleofector), (ii) CARs that signal through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations), (iii) CARs with an extracellular domain of variable length that connects the antigen recognition domain to the cell surface, and in some cases, (iv) CARs + and artificial antigen-presenting cells (aAPCs) derived from K562, which can robustly and numerically expand immune cells ( Singh et al., 2008 ; Singh et al., 2011 ). BT cell receptor (TCR)

[0159] In some embodiments, genetically engineered antigen receptors include recombinant TCRs and / or TCRs cloned from naturally occurring T cells. "T cell receptor" or "TCR" refers to a molecule that contains a variable a chain and a variable β chain (also known as TCRα and TCRβ, respectively) or a variable γ chain and a variable δ chain (also known as TCRγ and TCRδ, respectively) and that can specifically bind to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is of the αβ type.

[0160] TCRs, which usually exist as αβ and γδ types, are generally similar in structure, but the T cells expressing them may differ in anatomical location or function. TCRs may be found on the cell surface or in soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes) and are usually involved in the recognition of antigens bound to major histocompatibility complex (MHC) molecules on the surface. In some embodiments, TCRs may also include a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al, 1997). For example, in some embodiments, each chain of the TCR may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs of the αβ or γδ types.

[0161] Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment (e.g., an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e., an MHC-peptide complex). An "antigen-binding portion" or antigen-binding fragment of a TCR, which may be used interchangeably, refers to a molecule that contains only a portion of the structural domain of the TCR but binds to the antigen (e.g., an MHC-peptide complex) that the complete TCR binds. In some cases, the antigen-binding portion includes sufficient variable domains of the TCR (e.g., the variable a and variable β chains of the TCR) to form a binding site for binding to a specific MHC-peptide complex, e.g., each chain typically includes three complementarity determining regions.

[0162] In some embodiments, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) similar to immunoglobulins, which provide antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule. Usually, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, for example, Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR involved in the recognition of processed antigens, but CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of the peptide. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the beta chain may contain an additional hypervariable (HV4) region.

[0163] In some embodiments, the TCR chain comprises a constant domain. For example, similar to an immunoglobulin, the extracellular portion of a TCR chain (e.g., a chain, β chain) comprises two immunoglobulin domains, a variable domain (e.g., V a or Vp; usually Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.), and one constant domain adjacent to the cell membrane (e.g., the a chain constant domain or C aThe TCR may comprise a α-chain constant domain or Cp, typically amino acids 117-259 based on Kabat, and a β-chain constant domain or Cp, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by the two chains comprises two membrane proximal constant domains, and two membrane distal variable domains that include the CDRs. The constant domain of the TCR domain comprises a short connecting sequence in which cysteine ​​residues form disulfide bonds, thereby forming a link between the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α-chain and the β-chain such that the TCR comprises two disulfide bonds in the constant domain.

[0164] In some embodiments, the TCR chain may include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic tail. In some cases, due to its structure, the TCR can associate with other molecules such as CD3. For example, a TCR that includes a constant domain together with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0165] In general, CD3 is a multiprotein complex that may have three different chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, the complex may include CD3γ, CD3δ, two CD3ε, and a homodimeric CD3ζ chain. CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of CD3γ, CD3δ, and CD3ε chains are negatively charged, a property that allows these chains to associate with the positively charged T cell receptor chain. Each intracellular tail of CD3γ, CD3δ, and CD3ε chains contains a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3ζ chain contains three. In general, ITAMs are involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in the propagation of signals from the TCR to the cell. The CD3 chain and the ζ chain associate with the TCR to form a complex known as the T cell receptor complex.

[0166] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α and β or γ and δ) linked by a disulfide bond or the like. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, a nucleic acid encoding the TCR may be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, e.g., a cell, e.g., a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, the T cell may be obtained from an in vivo isolated cell. In some embodiments, a high affinity T cell clone may be isolated from a patient and the TCR may be isolated. In some embodiments, the T cell may be a cultured T cell hybridoma or clone. In some embodiments, the TCR clone against the target antigen is a clone produced in a transgenic mouse engineered with human immune system genes (e.g., human leukocyte antigen system or HLA). See, for example, tumor antigens (see, for example, Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate a TCR against the target antigen (see, for example, Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR or antigen-binding portion thereof can be synthetically produced with knowledge of the sequence of the TCR. C. Antigen-presenting cells

[0167] Antigen-presenting cells, including macrophages, B lymphocytes, and dendritic cells, are identified by the expression of specific MHC molecules. APCs internalize antigens and re-express a portion of the antigen along with MHC molecules on the outer membrane of their cell membrane. The MHC is a large genetic complex that contains multiple loci. The MHC loci code for two major classes of MHC membrane molecules, termed class I and class II MHC. T helper lymphocytes generally recognize antigens associated with MHC class II molecules, while T cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. The MHC is referred to as the HLA complex in humans and the H-2 complex in mice.

[0168] In some cases, aAPC is useful when preparing the therapeutic composition and cell therapy products of the above embodiment.For general guidance on the preparation and use of antigen-presenting system, see, for example, U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S. Patent Application Publication Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. WO2007 / 103009.

[0169] The aAPC system may include at least one exogenous auxiliary molecule. Any suitable number and combination of auxiliary molecules may be used. The auxiliary molecules may be selected from auxiliary molecules such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include CD86, CD64 (FcγRI), 41BB ligand and IL-21. Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane-binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins such as intercellular adhesion molecules (ICAMs), which promote, for example, cell-cell or cell-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs such as ICAM-1. Techniques, methods and reagents useful for the selection, cloning, preparation and expression of exemplary auxiliary molecules, including costimulatory molecules and adhesion molecules, are illustrated, for example, in U.S. Pat. Nos. 6,225,042, 6,355,479 and 6,362,001. D. Antigen

[0170] Antigens targeted by genetically engineered antigen receptors include antigens expressed in the context of the disease, condition or cell type targeted via adoptive cell therapy. These diseases and conditions include proliferative, neoplastic and malignant diseases and disorders, including cancers and tumors, including blood cancers, cancers of the immune system (e.g., lymphomas, leukemias and / or myelomas, e.g., B, T and myeloid leukemias, lymphomas and multiple myelomas). In some embodiments, the antigen is selectively expressed or overexpressed on the cells of the disease or condition, e.g., on tumor cells or pathogenic cells, compared to normal cells or tissues or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0171] Any suitable antigen can be targeted in this method. The antigen may be associated with a certain cancer cell, but in some cases may not be associated with non-cancerous cells. Exemplary antigens include, but are not limited to, infectious agents, auto / self antigens, tumor-associated / cancer-associated antigens, and antigenic molecules derived from tumor neo-antigens (Linnemann et al., 2015). In certain embodiments, the antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and CEA. In certain embodiments, antigens for two or more antigen receptors include, but are not limited to, CD19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and / or CEA. Sequences for these antigens are known in the art, for example, the following in the GenBank® database: CD19 (Accession No. NG_007275.1), EBNA (Accession No. NG_002392.2), WT1 (Accession No. NG_009272.1), CD123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_007726.4), HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL / DR4, and / or CEA. 29383.1), HER2 (accession number NG_007503.1), CA-125 (accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession number NC_000003.12), and / or CEA (accession number NC_000019.10).

[0172] Tumor-associated antigens can be derived from, for example, prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelioma, ovarian cancer, liver cancer, brain cancer, bone cancer, stomach cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer or melanoma.Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3 and MAGE4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO1); SAGE; and HAGE or GAGE.These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma and bladder cancer.See, for example, U.S. Patent No. 6,544,518. Tumor-associated antigens of prostate cancer include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphate, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).

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

[0174] Tumor antigens include tumor antigens derived from cancers characterized by the expression of tumor-associated antigens, such as expression of HER-2 / neu. Tumor-associated antigens of interest include lineage-specific tumor antigens, such as melanocyte-melanoma lineage antigen MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase-related proteins. Illustrative tumor-associated antigens include p53, Ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-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 antigen (WT1), AFP, -catenin / m, caspase-8 / m, CEA, CDK-4 / m, ELF2M, GnT-V, G250 , HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling transduction 1 (TACSTD1), TACSTD2, receptor tyrosine kinase (TKI) For example, epidermal growth factor receptor (EGFR) (particularly EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinase (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-kappa B (NF-B),Notch receptors (e.g., Notch1-4), c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAG Tumor antigens derived from or including any one or more of E1, 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 idiotypes, but are not limited to these.

[0175] Antigens can include epitopic regions or epitopic peptides derived from genes mutated in tumor cells or from genes that are transcribed at different levels in tumor cells compared to normal cells (e.g., aberrantly expressed intronic sequences such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and N-acetylglucosaminyltransferase-V); clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myelomas and B-cell lymphomas; tumor antigens that contain epitopic regions or epitopic peptides derived from oncoviral processes (e.g., human papillomavirus proteins E6 and E7); Epstein-Barr virus protein LMP2; and non-mutated oncofetal proteins with tumor-selective expression (e.g., carcinoembryonic antigen and alpha-fetoprotein).

[0176] In other embodiments, antigens are obtained or derived from pathogenic or opportunistic pathogenic microorganisms (also referred to herein as infectious disease microorganisms) (e.g., viruses, fungi, parasites, and bacteria). In certain embodiments, antigens derived from such microorganisms include full-length proteins.

[0177] Illustrative pathogenic organisms having antigens contemplated for use in the methods described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B and C, vesicular stomatitis virus (VSV), polyomaviruses (e.g., BK virus and JC virus), adenovirus, Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species, including Streptococcus pneumoniae. As one of skill in the art will appreciate, proteins from these and other pathogenic microorganisms for use as antigens as described herein, as well as the nucleotide sequences encoding those proteins, may be identified in publications and public databases, such as GENBANK®, SWISS-PROT®, and TREMBL®.

[0178] Antigens derived from Human Immunodeficiency Virus (HIV) include HIV virion structural proteins (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or any of the HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0179] Antigens derived from herpes simplex viruses (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed from HSV late genes. The late group of genes primarily encode proteins that form virion particles. Such proteins include the five proteins that form the viral capsid (UL): UL6, UL18, UL35, UL38, and the major capsid proteins UL19, UL45, and UL27, each of which can be used as an antigen as described herein. Other illustrative HSV proteins contemplated for use as antigens herein include ICP27 (H1, H2), glycoprotein B (gB), and glycoprotein D (gD) proteins. 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 expressed during immediate early and early viral replication, glycoproteins I and III, capsid proteins, coat proteins, lower matrix proteins pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products of the UL128-UL150 gene cluster (Rykman, et al., 2006), envelope glycoproteins B (gB), gH, gN, and pp150. As one of skill in the art will appreciate, CMV proteins for use as antigens described herein may be identified in public databases such as GENBANK®, SWISS-PROT®, and TREMBL® (see, e.g., Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

[0181] Antigens derived from Epstein-Van virus (EBV) contemplated for use in certain embodiments include EBV proteins produced during the latent infection cycle, including EBV lytic proteins gp350 and gp110, Epstein-Van nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), and latent membrane protein (LMP)-1, LMP-2A, and LMP-2B (see, e.g., Lockey et al., 2008).

[0182] Antigens derived from respiratory syncytial virus (RSV) contemplated for use herein include any of 11 proteins or antigenic fragments thereof 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 control), RNA polymerase, and phosphoprotein P.

[0183] Antigens derived from vesicular stomatitis virus (VSV) contemplated for use include any one of the five major proteins and their antigenic fragments encoded by the VSV genome: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P) and matrix protein (M) (see, e.g., Rieder et al., 1999).

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

[0185] Exemplary viral antigens include adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigens), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (including hepatitis B core or surface antigens, hepatitis C virus E1 or E2 glycoproteins, core or nonstructural proteins), herpesvirus polypeptides (including herpes simplex virus or varicella zoster virus glycoproteins), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, and the like. Also included are, but are not limited to, polypeptides of viruses, orthomyxoviruses, papillomaviruses, polypeptides of parainfluenza viruses (e.g., hemagglutinin and neuraminidase polypeptides), polypeptides of paramyxoviruses, polypeptides of parvoviruses, polypeptides of pestiviruses, polypeptides of picornaviruses (e.g., capsid polypeptides of polioviruses), polypeptides of poxviruses (e.g., polypeptides of vaccinia viruses), polypeptides of rabies viruses (e.g., glycoprotein G of rabies virus), polypeptides of reoviruses, polypeptides of retroviruses, and polypeptides of rotaviruses.

[0186] In certain embodiments, the antigen may be a bacterial antigen. In certain embodiments, the bacterial antigen of interest may be a secreted polypeptide. In certain other embodiments, the bacterial antigen includes an antigen that has a portion of the polypeptide exposed on the extracellular surface of the bacteria.

[0187] Antigens from Staphylococcus species, including Methicillin-resistant Staphylococcus aureus (MRSA), that are intended to be used include virulence regulators, such as Agr system, Sar and Sae, Arl system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), Srr system and TRAP.Other Staphylococcus proteins that can serve as antigens include Clp protein, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, for example, Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes of two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example, at PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As one of skill in the art will appreciate, Staphylococcus proteins for use as antigens may also be identified in other public databases, such as GenBank®, Swiss-Prot®, and TrEMBL®.

[0188] Antigens from Streptococcus pneumoniae contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and may be used as antigens in some embodiments (see, for example, Zysk et al., 2000). The complete genome sequence of a virulent strain of Streptococcus pneumoniae has been sequenced, and as one of skill in the art will appreciate, S. pneumoniae proteins for use herein may also be identified in other public databases, such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins of particular interest as antigens according to the present disclosure include virulence factors and proteins predicted to be exposed on the surface of S. pneumoniae (see, e.g., Frolet et al., 2010).

[0189] Examples of bacterial antigens that can be used as antigens include Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae b-type outer membrane proteins), Helicobacter polypeptides, Klebsiella polypeptides, L-type bacteria polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacterium polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Streptococcus pneumoniae polypeptides (i.e., S. pn eumoniae polypeptides) (see description herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Roshalimea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, Group A Streptococcus polypeptides (e.g., the M protein of S. pyogenes), Group B Streptococcus (S. agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., the F1 and V antigens of Y pestis).

[0190] Examples of fungal antigens include Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucous membrane polypeptides, Pecorinolytic enzymes, and the like. These include, but are not limited to, polypeptides of the genus Silomyces, Polypeptides of the genus Penicillium, Polypeptides of the genus Phialemonium, Polypeptides of the genus Phialophora, Polypeptides of the genus Prototheca, Polypeptides of the genus Pseudoalescheria, Polypeptides of the genus Pseudomicrodochium, Polypeptides of the genus Phythium, Polypeptides of the genus Rhinosporidium, Polypeptides of the genus Rhizopus, Polypeptides of the genus Scolecobasidium, Polypeptides of the genus Sporothrix, Polypeptides of the genus Stemphylium, Polypeptides of the genus Trichosporon, Polypeptides of the genus Xylohypha.

[0191] Examples of protozoan parasite antigens include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, and Plasmodium polypeptides. Examples of helminth parasite antigens include Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Cabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Dipridium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Ancylostoma polypeptides, Nematogyrus polypeptides, Heterodera polypeptides, Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagia polypeptides, Parafilaria polypeptides, Paragonimus polypeptides, Parascaris polypeptides, Physaloptera polypeptides,Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides, Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichuris polypeptides, Trichuria polypeptides, Uncinaria polypeptides and Uchleria polypeptides (e.g., P. falciparum circumsporozoite polypeptide (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSA1 c-terminus), and export protein 1 (PfExp-1), Pneumocystis polypeptide, Sarcocystis polypeptide, Schistosoma polypeptide, Theileria polypeptide, Toxoplasma polypeptide, and Trypanosoma polypeptide.

[0192] Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens and allergens) from fleas; ticks, including hard mites and ulcerative mites; flies, such as midges, mosquitoes, sand flies, black flies, bot flies, horn flies, deer flies, tsetse flies, stable flies, flies that cause myiasis and small biting gnats; ants; spiders, lice; mites; and hemipteran insects, such as bedbugs and assassin bugs. E. Suicide gene

[0193] In some cases, any cell of the present disclosure is modified to produce one or more agents other than heterologous cytokines, engineered receptors, etc. In specific embodiments, cells such as NK cells are engineered to have one or more suicide genes, the term "suicide gene" as used herein being defined as a gene whose gene product changes into a compound that kills the host cell when a prodrug is administered. In some cases, NK cell therapy may be subject to the utilization of one or more suicide genes of any kind when an individual undergoing NK cell therapy and / or an individual undergoing NK cell therapy exhibits one or more symptoms of one or more adverse events (e.g., cytokine release syndrome, neurotoxicity, anaphylaxis / allergy and / or on-target / off-tumor toxicity (as examples)) or is considered at risk of having one or more symptoms (including imminent cases). The use of a suicide gene may be part of a planned protocol for treatment or may be used only when the need for its use is recognized. In some cases, cell therapy is terminated by using an agent that targets the suicide gene or its gene product because it is no longer needed.

[0194] Examples of suicide genes include engineered non-secreted (including membrane-bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see PCT / US19 / 62009, incorporated herein by reference in its entirety), which can be targeted by delivery of antibodies that bind to the TNF-alpha mutants. Examples of suicide gene / prodrug combinations that can be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. The so-called suicide gene, purine nucleoside phosphorylase from E. coli, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, can be used. Other examples of suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP). F. Delivery method

[0195] Those of skill in the art would be well equipped to construct vectors by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference) to express the antigen receptors of the present disclosure. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses and plant viruses) and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors (including replication competent, replication defective and gutless forms thereof), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, Maraba virus vectors, These include, but are not limited to, group B adenovirus (A virus) vectors and group B adenovirus enadenotucirev vectors.

[0196] In a specific embodiment, the vector is a multicistronic vector as described in PCT / US19 / 62014, which is incorporated herein by reference in its entirety. In such cases, a single vector may encode a CAR or TCR (whose expression constructs may be configured in a modular fashion to allow for interchangeability of portions of the CAR or TCR), a suicide gene, and one or more cytokines. A. Viral vectors

[0197] A viral vector encoding an antigen receptor can be provided in certain embodiments of the present disclosure. When making a recombinant viral vector, non-essential genes are usually replaced with genes or coding sequences of heterologous (or non-native) proteins. A viral vector is a type of expression construct that utilizes viral sequences to introduce nucleic acid and possibly proteins into cells. The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into the genome of host cells and stably and efficiently express viral genes makes them attractive candidates for transferring foreign nucleic acid into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver the nucleic acid of certain embodiments of the present invention are described below.

[0198] Lentiviruses are complex retroviruses, and in addition to the common retroviral genes gag, pol and env, they also contain other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, for example, U.S. Patent Nos. 6,013,516 and 5,994,136).

[0199] Recombinant lentivirus vector can infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression both in vivo and ex vivo.For example, recombinant lentivirus can infect non-dividing cells, where suitable host cells are transfected with two or more vectors with packaging function, namely gag, pol and env, and rev and tat, as described in US Patent No. 5,994,136, which is incorporated herein by reference. B. Regulatory elements

[0200] The expression cassette contained in the vector useful in the present disclosure includes, in particular, a eukaryotic transcription promoter operably linked to a protein coding sequence, a splice signal including an intervening sequence, and a transcription termination / polyadenylation sequence (5' to 3' direction). The promoters and enhancers that control the transcription of protein-coding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery can collect and integrate the regulatory information carried by each element, allowing different genes to develop different patterns of transcriptional control, which are often complex patterns. Promoters used in the context of the present disclosure include constitutive promoters, inducible promoters and tissue-specific promoters. (i) Promoter / enhancer

[0201] The expression constructs provided herein include a promoter that drives the expression of the antigen receptor. Promoters generally contain sequences that function to locate the start site for RNA synthesis. The best known example of this is the TATA box, but some promoters, such as the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter, lack the TATA box and have discrete elements that overlap the start site itself to help fix the location of start. Additional promoter elements control the frequency of transcription initiation. Usually, these are located in the region 30-110 bp upstream of the start site, but some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control of" a promoter, the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA and promotes expression of the encoded RNA.

[0202] Spacing between promoter elements is often variable, and promoter function is preserved even when elements are inverted or moved relative to one another. In the tk promoter, spacing between promoter elements can be increased by up to 50 bp before activity begins to decrease. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0203] A promoter may be a promoter naturally associated with a nucleic acid sequence, such as may be obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as an "endogenous" promoter. Similarly, an enhancer may be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of the sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not naturally associated with a nucleic acid sequence in its natural environment. Also, a recombinant or heterologous enhancer refers to an enhancer that is not naturally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., that contain various elements of the various transcriptional control regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp-) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, recombinant cloning, and / or PCR TMUsing nucleic acid amplification techniques such as those described herein, sequences can be generated in connection with the compositions disclosed herein. Additionally, it is contemplated that regulatory sequences that direct transcription and / or expression of sequences in organelles other than the nucleus (e.g., mitochondria, chloroplasts, etc.) can be used as well.

[0204] Naturally, it becomes important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of a combination of promoter, enhancer and cell type for protein expression (see, for example, Sambrook et al. 1989, which is incorporated herein by reference). The promoter used can be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a promoter that directs high-level expression of the introduced DNA segment and is useful under appropriate conditions (e.g., a promoter that is useful in large-scale production of recombinant proteins and / or recombinant peptides). The promoter can be heterologous or endogenous.

[0205] Additionally, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Data Base EPDB via the World Wide Web at epd.isb-sib.ch / ) can be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another viable embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if a suitable bacterial polymerase is provided as part of the delivery complex or as an additional genetic expression construct.

[0206] Non-limiting examples of promoters include early or late viral promoters (e.g., SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter); eukaryotic promoters (e.g., beta actin promoter, GADPH promoter, metallothionein promoter); and chained response element promoters (e.g., cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA) and minimal TATA box proximal response element promoter (tre)). It is also possible to use a human growth hormone promoter sequence (e.g., human growth hormone minimal promoter described in Genbank®, accession number X05244, nucleotides 283-341) or a mouse mammary tumor promoter (available from ATCC, Cat. No. ATCC45007). In certain embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, however, any other promoter useful for driving expression of therapeutic genes is applicable to the practice of the present disclosure.

[0207] In certain embodiments, the method of the present disclosure also relates to enhancer sequences, i.e., nucleic acid sequences that increase the activity of promoters, and have the ability to act in cis and in any orientation over relatively long distances (up to several kilobases away from the target promoter).However, enhancer function is not necessarily limited to such long distances, since enhancers can also function proximally to a given promoter. (ii) Initiation signal and ligated expression

[0208] Specific initiation signals may also be used in the expression constructs provided in this disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals (including the ATG initiation codon) may need to be provided. Those skilled in the art will be able to easily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. The exogenous translational control signals and initiation codons may be natural or synthetic. Expression efficiency may be increased by including appropriate transcriptional enhancer elements.

[0209] In certain embodiments, the use of an internal ribosome entry site (IRES) element is used to generate multigene messages, i.e., polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) and IRES from mammalian messages have been reported. IRES elements can link heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together, generating polycistronic messages. Thanks to the IRES element, each open reading frame becomes accessible to ribosomes for efficient translation. Multiple genes can also be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0210] In addition, certain 2A sequence elements can be used to provide for linked or simultaneous expression of genes in the constructs provided in the present disclosure. For example, a truncation sequence can be used to link open reading frames to form a single cistron to simultaneously express genes. An exemplary truncation sequence is F2A (foot and mouth disease virus 2A) or a "2A-like" sequence (e.g., Thosea asigna virus 2A; T2A). (iii) Replication origin point To propagate a vector in a host cell, the vector may contain one or more origins of replication (often referred to as "ori"), such as a specific nucleic acid sequence at which replication is initiated, such as the oriP of EBV as described above, or a genetically engineered oriP with similar or enhanced function in programming. Alternatively, origins of replication of other viruses that replicate extrachromosomally, as described above, or autonomously replicating sequences (ARS) can be used. c. Selectable and screenable markers

[0211] In some embodiments, cells containing the constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker brings about an identifiable change in cells that allows cells containing the expression vector to be easily identified. In general, a selection marker is a marker that confers a property that allows selection. A positive selection marker is a marker whose presence allows its selection, and a negative selection marker is a marker whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0212] Typically, the inclusion of a drug selection marker aids in cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows the differentiation of transformants based on the implementation of conditions, other types of markers are contemplated, including colorimetrically based screenable markers such as GFP. Alternatively, screenable enzymes can be utilized as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art will also know how to use immunological markers, possibly in conjunction with FACS analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection markers and screenable markers are well known to those skilled in the art. d. Other nucleic acid delivery methods

[0213] In addition to viral delivery of nucleic acids encoding antigen receptors, the following methods are contemplated in the present disclosure as being additional methods of recombinant gene delivery into a given host cell.

[0214] The introduction of nucleic acid such as DNA or RNA into immune cells of the present disclosure can use any method suitable for nucleic acid delivery to transform cells, as described herein or known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA (e.g., ex vivo transfection, injection (including microinjection)); electroporation; calcium phosphate precipitation; use of DEAE-dextran followed by polyethylene glycol; direct sonication; liposome-mediated transfection and receptor-mediated transfection; microprojectile bombardment; agitation with silicon carbide fiber; Agrobacterium-mediated transformation; desiccation / inhibition-mediated DNA uptake, and any combination of such methods. By applying such methods, organelles, cells, tissues or organisms can be transformed stably or transiently. VI. Treatment Method

[0215] The embodiments of the present disclosure include a method of treating an individual for cancer, any kind of infectious disease, and any immune disorder.The individual may use the treatment method of the present disclosure as a first treatment, or after or during another treatment.The immunotherapy method can be tailored to the needs of the individual with cancer based on the type or stage of cancer, and at least in some cases, the immunotherapy can be modified for the individual during the treatment period.

[0216] In specific cases, the treatment methods are as follows: 1) adoptive cell therapy using T or NK cells (ex vivo expanded T or NK cells, or T or NK cells expressing CAR or TCR) to treat cancer patients with any type of hematological malignancy, (2) adoptive cell therapy using T or NK cells (ex vivo expanded T or NK cells, or T or NK cells expressing CAR or TCR) to treat cancer patients with any type of solid cancer, (3) adoptive cell therapy using Treg and regulatory B cells (ex vivo expanded or expressing CAR or TCR) to treat patients with immune disorders, (4) adoptive cell therapy using T or NK cells (ex vivo expanded T or NK cells, or T or NK cells expressing CAR or TCR) to treat patients with infectious diseases. The present disclosure is the first to show that knocking down / knockout multiple genes in human NK cells contributes to improved cell function and resistance to the tumor microenvironment. In specific embodiments, this has direct implications for patient care using novel immunotherapy approaches that enhance the function of the patient's own or adoptively transferred immune cells. The embodiments provide novel approaches to generate highly functional T, NK and B cells (ex vivo expanded and CAR or TCR engineered cells) for immunotherapy. These include targeting cancer (both hematological and solid tumors) (NK and T cells, as well as CAR T and CAR NK cells), autoimmune and alloimmune disorders (B cells, regulatory B and regulatory T cells), and treating infectious diseases (pathogen-specific T cells).

[0217] In some embodiments, the present disclosure provides a method for immunotherapy, comprising administering an effective amount of the immune cells of the present disclosure.In one embodiment, medical disease or disorder is treated by transferring immune NK cell populations that induce immune response.In certain embodiments of the present disclosure, cancer or infectious disease is treated by transferring immune cell populations that induce immune response.A method for treating cancer or delaying the progression of cancer in an individual is provided herein, comprising administering an effective amount of antigen-specific cell therapy to the individual.The method can be applied to the treatment of immune disorders, solid cancers, blood cancers and viral infections.

[0218] The tumors that this treatment method is useful for include any malignant cell type, such as the cell type found in solid tumors or blood tumors.Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate and breast.Exemplary blood tumors include bone marrow tumors, T or B cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric or stomach cancer (including digestive and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0219] The cancer may specifically be cancer of the following histological types, but is not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma-cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumor; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cellular carcinoma. follicular carcinoma;follicular adenocarcinoma;papillary-follicular adenocarcinoma;nonencapsulated sclerosing carcinoma;adrenal cortical carcinoma;endometrioid carcinoma;adnexal carcinoma;apocrine gland carcinoma;sebaceous gland carcinoma;earwax gland carcinoma;mucoepidermoid carcinoma;cystadenocarcinoma;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, breast;acinic cell carcinoma;adenosquamous 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 tumor, malignant;lipid cell tumor, malignant;paraganglioma tumor, malignant;extramammary paraganglioma, malignant;pheochromocytoma;glomus angiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;lentigo maligna melanoma;acral lentigo melanoma;nodular melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymoma, malignant;Brenner tumor, malignant;phyllodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysgerminoma;fetal carcinoma;teratoma, malignant;ovarial goiter, malignant;choriocarcinoma;mesonephroma, malignant;angiosarcoma;hemangioendothelioma, malignant;Kaposi's sarcoma;hemangiopericytoma, malignant;lymphangiosarcoma;osteosarcoma;paracortical osteosarcoma;chondrosarcoma;chondroblastoma, malignant;mesenchymal chondrosarcoma;giant cell tumor of bone;Ewing's sarcoma;odontogenic tumor, malignant;ameloblastoma, malignant;ameloblastoma, malignant;ameloblastoma fibrosarcoma;pinealoma, malignant;chordoma;glioma, malignant;ependymoma;astrocytoma;protoplasmic astrocytoma;fibrillary astrocytoma;astroblastoma;glioblastoma;oligodendroglioma;primitive neuroectodermal;Cerebellar sarcoma;Ganglioneuroblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma, malignant;Neurofibrosarcoma;Neurilemoma, malignant;Granular cell tumor, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin;Side granuloma;Malignant lymphoma, small lymphocytic;Malignant lymphoma, large cell, diffuse;Malignant 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 NHL;Intermediate-grade diffuse NHL;High-grade immunoblastic NHL;High-grade lymphoblastic NHL;High-grade small noncleaved cell NHL;Bulky disease disease)NHL;mantle cell lymphoma;AIDS-related lymphoma;Waldenstrom's macroglobulinemia;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphoblastic cell leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;hairy cell leukemia;chronic lymphocytic leukemia (CLL);acute lymphoblastic leukemia (ALL);acute myeloid leukemia (AML);and chronic myeloblastic leukemia. ;

[0220] Certain embodiments relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by abnormal proliferation (production by division) of blood cells, usually white blood cells (leukocytes). Leukemia is part of a broad group of diseases called hematological neoplasms. Leukemia is a broad term that covers a wide variety of diseases. Leukemia is clinically and pathologically divided into acute and chronic forms.

[0221] In certain embodiments of the present disclosure, immune cells are delivered to an individual in need thereof (e.g., an individual with cancer or infectious disease). These cells then boost the individual's immune system to attack the respective cancer cells or pathogenic cells. In some cases, the individual is provided with immune cells one or more times. When the individual is provided with immune cells two or more times, the time between administrations should be sufficient for the immune cells to spread in the individual, and in specific embodiments, the time between administrations is 1, 2, 3, 4, 5, 6, 7 days or more.

[0222] Certain embodiments of the present disclosure provide a method for treating or preventing immune-mediated disorders. In one embodiment, the subject has an autoimmune disease. Non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac polydermatitis, and the like. spate-dermatitis), chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (e.g., microvascular fibrosis, pulmonary ... These include: inflammatory bowel disease (e.g., pulmonary edema, focal glomerulosclerosis or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff man syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (e.g., polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis or dermatitis herpetiformis vasculitis), vitiligo and Wegener's granulomatosis. Thus, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis or psoriasis.Subjects can also have allergic disorders, such as asthma.

[0223] In yet another embodiment, the subject is a recipient of a transplanted organ or stem cells, and the immune cells are used to prevent and / or treat rejection. In certain embodiments, the subject has or is at risk of developing graft-versus-host disease. GVHD is a possible complication of any transplant that uses or includes stem cells from related or unrelated donors. There are two types of GVHD: acute and chronic. Acute GVHD appears within the first three months after transplant. Signs of acute GVHD include a reddish rash on the hands and feet, which may spread with peeling or blistering of the skin and may become more severe. Acute GVHD may also affect the stomach and intestines, where muscle cramps, nausea, and diarrhea are observed. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD is affecting the liver. Chronic GVHD is graded based on its severity: stage / grade 1 is mild; stage / grade 4 is severe. Chronic GVHD develops 3 months or later after transplantation. Symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD can affect the mucous glands of the eye, the salivary glands of the mouth, and the glands that lubricate the stomach wall and intestine. Any immune cell population disclosed herein can be used. Examples of transplanted organs include organ grafts, such as kidney, liver, skin, pancreas, lung, and / or heart, or cell grafts, such as islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The graft can be a composite graft, such as facial tissue. Immune cells can be administered before, at the same time, or after transplantation. In some embodiments, the immune cells are administered prior to transplantation, e.g., at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month prior to transplantation. In one specific, non-limiting example, administration of a therapeutically effective amount of immune cells occurs 3-5 days prior to transplantation.

[0224] In some embodiments, the subject may be administered non-myeloablative lymphodepleting chemotherapy prior to immune cell therapy. The non-myeloablative lymphodepleting chemotherapy may be any suitable such treatment that may be administered by any suitable route. The non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, particularly when the cancer is melanoma that may be metastatic. An exemplary administration route for cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In certain embodiments, approximately 60 mg / kg of cyclophosphamide is administered for two days, followed by approximately 25 mg / m 2 of fludarabine is administered for 5 days.

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

[0226] A therapeutically effective amount of the immune cells can be administered by several routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal or intraarticular injection or infusion.

[0227] The therapeutically effective amount of immune cells for use in adoptive cell therapy is the amount that achieves the desired effect in the subject being treated.For example, it can be the amount of immune cells required to inhibit the progression or the amount of immune cells required to reverse autoimmune disease or alloimmune disease, or the amount that can relieve symptoms caused by autoimmune disease, such as pain and inflammation.It can be the amount required to relieve symptoms associated with inflammation, such as pain, edema and elevated body temperature.It can also be the amount required to reduce or prevent rejection of transplanted organs.

[0228] The immune cell population may be administered in a treatment regimen consistent with the disease, for example, once or several times over one to several days, to ameliorate the disease state, or in regular administration over an extended period of time to inhibit disease progression and prevent disease recurrence. The exact dose employed in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the physician and each patient's circumstances. 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, the dose that may be used in the treatment of a human subject is at least 3.8×10 4 , 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 or at least 3.8×10 10 immune cells / m 2 In certain embodiments, the dose used in treating a human subject is about 3.8×10 9 ~Approx. 3.8×10 10 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 Cells ~ approx. 5 x 108 Cells / kg body weight or approximately 5 x 10 7 cells ~ approx. 2 x 10 8 The amount of immune cells may vary by cell / kg body weight.The exact amount of immune cells can be readily determined by those skilled in the art based on the age, weight, sex and physiological condition of the subject.Effective amounts can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0229] The immune cells can be administered in combination with one or more other therapeutic agents for treating an immune-mediated disorder. Combination therapy may include, but is not limited to, one or more antibacterial agents (e.g., antibiotics, antiviral agents, and antifungal agents), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunodepleting agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine or glucocorticoids, such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids (e.g., hydrocortisone, dexamethasone, or prednisone) or nonsteroidal anti-inflammatory agents (e.g., acetylsalicylic acid, ibuprofen, or naproxen sodium)), cytokines (e.g., interleukin-10 or transforming growth factor-beta), hormones (e.g., estrogen), or vaccines. In addition, immunosuppressants or immune tolerance inducers may be administered, including but not limited to calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., antibodies that recognize CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); irradiation; or chemokines, interleukins, or inhibitors thereof (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional pharmaceutical agents may be administered before, during, or after administration of the immune cells, depending on the desired effect. This administration of the cells and agents may be by the same route or different routes, and at the same site or different sites. A. Pharmaceutical Compositions

[0230] Also provided herein are pharmaceutical compositions and formulations comprising immune cells (eg, T cells, B cells, or NK cells) and a pharma- ceutically acceptable carrier.

[0231] Pharmaceutical compositions and formulations as described herein comprise the active ingredient (e.g., an antibody or polypeptide) having a desired degree of purity in one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 22 ndPharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include buffers (e.g., phosphoric acid, citric acid, and other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol alcohol, butyl alcohol, or benzyl alcohol; alkyl parabens (e.g., methylparaben or propylparaben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues). polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., polyethylene glycol (PEG)). Exemplary pharmacologic acceptable carriers herein further include interstitial drug dispersion agents, such as neutral active soluble hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is used in combination with one or more additional glycosaminoglycanases, such as chondroitinases. B. Combination Therapy

[0232] In certain embodiments, the compositions and methods of the present embodiment comprise immune cell populations that are combined with at least one additional treatment.The additional treatment can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy or the combination of the above.The additional treatment can be in the form of adjuvant therapy or neoadjuvant therapy.

[0233] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent aimed at reducing the incidence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents known in the art.

[0234] Immune cell therapy may be administered before, during, after, or in various combinations with additional cancer therapy, such as immune checkpoint therapy. Their administration may occur at intervals ranging from simultaneously to minutes, days, or weeks. In embodiments in which immune cell therapy is provided to a patient separately from an additional therapeutic agent, it is common to ensure that no significant period of time passes between each delivery time point so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is contemplated that the antibody therapy and the anti-cancer therapy may be provided to the patient within about 12-24 or 72 hours of each other, more particularly within about 6-12 hours of each other. In some situations, when several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between the respective administrations, it may be desirable to extend the treatment period considerably.

[0235] Various combinations can be used. In the following example, the immune cell therapy is "A" and the 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 / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

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

[0237] A wide variety of chemotherapeutic agents can be used according to this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity in cells, for example, whether they affect the cell cycle and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[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, meturedopa, and uredopa); ethylenimines and methylamelamines (altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylol melamine); trimethylolomelamine); acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatins; kallistatins; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleuthecins. eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenestrine, rine, prednimustine, 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 1I and calicheamicin omega 11)); dynemicins (including dynemicin A); bisphosphonates (e.g., clodronate); esperamicin;and the neocarzinostatin chromophore and related chromoproteins, the enediyne antibiotic chromophores, aclacinomycin, actinomycin, autarubicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L -norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g. mitomycin C), mycophenolic acid, nogalarnicin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folate analogs (e.g., denopterin, pteropterin, and trimetrexate); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprin e) and thioguanine); pyrimidine analogues (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine); androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane and testolactone); anti-adrenals (e.g., mitotane and trilostane); folic acid supplements (e.g., frolinic acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;Bisantrene; edatraxate; defofamine; demecolcine; diazicon; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids (e.g., maytansine and ansamitocins); mitoguazone; mitoxantrone; mopidanmol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid);2-ethylhydrazide;procarbazine;PSK polysaccharide complex;razoxane;rhizoxin;schizophyllan;spirogermanium;tenuazonic acid;triaziquone;2,2',2"-trichlorotriethylamine;trichothecines (especially T-2 toxin, veracrine A, roridin A and anguidine);urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacytosine;arabinosides ("Ara-C");cyclophosphamide;taxoids, e.g. paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes (e.g., cisplatin, oxaliplatin, and carboplatin); vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids (e.g., retinoic acid); capecitabine;Carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, as well as pharmaceutically 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 what are commonly known as gamma radiation, X-rays and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam irradiation and UV irradiation are also contemplated. All of these agents most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells. 3. Immunotherapy

[0240] Those skilled in the art will understand that additional immunotherapy may be used in conjunction or in conjunction with the methods of the above embodiments. In the context of cancer treatment, immunotherapeutics usually rely on the use of immune effector cells and immune effector molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody may function alone as an effector of therapy or may recruit other cells to actually affect cell killing. The antibody may also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte with a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0241] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) covalently linked to cytotoxic drugs and can be used in combination therapy. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver payloads (drugs) to tumor cells that have abundant levels of antigen. Targeted delivery of drugs also minimizes exposure to normal tissues, resulting in reduced toxicity and improved therapeutic index. Exemplary ADC drugs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).

[0242] In one aspect of immunotherapy, the tumor cells must have some marker that is amenable to targeting, i.e., some marker that is not present on most other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (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 effects with immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0243] Examples of immunotherapies include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapy, such as interferon alpha, beta, and gamma, IL-1, GM-CSF, and TNF; gene therapy, such as TNF, IL-1, IL-2, and p53; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185. It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0244] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either strengthen or weaken signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), 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 (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0245] The immune checkpoint inhibitor can be a drug, such as a small molecule, a recombinant ligand or receptor, or an antibody, such as a human antibody. A known inhibitor of immune checkpoint protein or its analogue can be used, and in particular, a chimeric, humanized or human antibody can be used. As those skilled in the art will recognize, alternative and / or equivalent names can be used for certain antibodies described in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known as MK-3475 and pembrolizumab, which are alternative and equivalent names.

[0246] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand binding partner. In a specific embodiment, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits PDL1 from binding to its binding partner. In a specific embodiment, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits PDL2 from binding to its binding partner. In a specific embodiment, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, 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., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-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 (e.g., an immunoadhesin that includes an extracellular portion or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody that may be used. Pembrolizumab, also known as MK-3475, Merck3475, Lambrolizumab, KEYTRUDA® and SCH-900475, is an exemplary anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.

[0248] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 resembles CD28, a T cell costimulatory protein, and both molecules bind to CD80 and CD86 (also called B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA4 is also found on regulatory T cells and may be important for the function of those cells. Activation of T cells via the T cell receptor and CD28 leads to high expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0249] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0250] Anti-human CTLA-4 antibodies (or VH and / or VL domains therefrom) suitable for use in the present method can be made using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101 and Yervoy®) or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or heavy and light chain VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to the same epitope on CTLA-4 as the above-mentioned antibodies 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 amino acid sequence identity to the above-mentioned antibody (eg, at least about 90%, 95% or 99% variable region identity to ipilimumab). 4.Surgery

[0251] Approximately 60% of people with cancer undergo some type of surgery, including preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, which physically removes, excises, and / or destroys all or part of the cancerous tissue, and may be used in conjunction with other therapies (e.g., the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies). Lumpectomy refers to the physical removal of at least a portion of the tumor. Surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery) in addition to lumpectomy.

[0252] When removing part or all of cancerous cells, tissues or tumors, a cavity may be formed in the body. Treatment may be achieved by perfusion, direct injection or local application of the area with additional anticancer therapy. Such treatment may be repeated, 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, 10, 11 or 12 months. These treatments may also be of various dosages. 5. Other agents

[0253] It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing signaling between cells by increasing the number of gap junctions may enhance the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that enhance the sensitivity of hyperproliferative cells to apoptosis (e.g., antibody c225) may be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment. VII. Products or Kits

[0254] Also provided herein is an article of manufacture or kit that includes the immune cells. The article of manufacture or kit may further include a package insert that includes instructions for using the immune cells to treat or delay the progression of cancer in an individual or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein may be included in the article of manufacture or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin) or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation and a label that is attached to or associated with the container, and the container may indicate how to use. The article of manufacture or kit may further include other materials that are desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts that include instructions for use. In some embodiments, the article of manufacture further includes one or more additional agents (e.g., chemotherapeutic and antineoplastic agents). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes. EXAMPLES

[0255] VIII. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be recognized by those skilled in the art that the procedures disclosed in the following examples are procedures that the inventors have discovered to work well in the practice of the invention and therefore can be considered to be preferred modes for its practice. However, those skilled in the art should recognize in light of this disclosure that many changes can be made in the specific embodiments disclosed that still yield the same or similar results without departing from the spirit and scope of the invention. Example 1 – Multiplex gene editing

[0256] To test the efficacy of simultaneously disrupting the expression of multiple genes in immune cells such as T cells and NK cells, several studies were performed to test the disruption of different gene combinations using CRISPR. In the first study, CRISPR / Cas9 was used to disrupt the expression of NKG2A, CD47, TGFBR2 and CISH in NK cells. In this gene set, NKG2A and CD47 were knocked out in the first electroporation, and CISH and TGFBR2 were targeted in the second electroporation. Both electroporation rounds were successfully validated for knockout efficiency using PCR and flow cytometry (Figure 1).

[0257] Multiple gene disruption methods were validated in an additional set of genes including TIGIT, CD96, CISH, adenosine (Figure 2), and NKG2A, CD47, TGFBR2, and CISH (Figure 3). Multiple gene disruption was found to enhance function against target tumor cells. 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. After stimulation with the target cell lines, there was increased secretion of IFN-γ, TNFα, and CD107 (Figure 3).

[0258] This enhancement of function was confirmed by disruption of NKG2A, CD47, TGFBR2 and CISH in NK cells. 51The ADAM17-positive NK cells showed increased antitumor cytotoxicity against K562 cells as measured by Cr release assay (Figure 4A). Furthermore, pSMAD activity was measured by flow cytometry 30 min after treatment with recombinant TGF-B (50 ng / ml) (Figure 4B). We also observed that NK cells lost expression of CD16 and CD62L upon cytokine stimulation or target recognition (Figure 5), and that knockout of ADAM17 in NK cells prevented CD16 and CD62L shedding (Figure 6) and improved ADCC and cytotoxicity against K562 targets (Figure 7).

[0259] Further studies showed that disrupting SHP1 in NK cells enhanced the antitumor effect (Figures 9 and 10). NK cells were co-cultured with K562 / Raji cells at a 1:1 ratio for 4 hours. After incubation, the cells were stained with Annexin V to analyze live and dead cells. K562 cells are sensitive to NK cell killing, and Raji cells are resistant to NK cell killing. Furthermore, disrupting NKG2A in NK-CAR cells enhanced the antitumor effect against Raji targets (Figure 12).

[0260] This approach was further validated with additional gene sets, namely TIGIT, CD96, CISH and adenosine, and NKG2A, CISH, TGFBRII and adenosine. NK cell function was assessed by flow cytometry measurements, and an increase in TNFα, IFNγ and CD107a was observed in the cells upon stimulation with the target cell line (Figures 13-14).

[0261] Thus, the method can be used to simultaneously disrupt the expression of multiple genes in an immune cell to enhance the function of that immune cell. Example 2 - Method

[0262] sgRNA-Cas9 pre-complexing and electroporation: One or two sgRNAs spanning adjacent regions were designed and used for each gene. Reactions of 1ug cas9 (PNA Bio) and 500ng sgRNA (total of all sgRNAs) were made for each gene and incubated on ice for 20 minutes. After 20 minutes, 250,000 NK cells were incubated in T-buffer for 20 minutes. * (Attached to the Neon Electroporation Kit, Invitrogen; the total volume including RNP complexes and cells should be 14 ul) and electroporated with a 10 ul electroporation tip using the Neon Transfection System. The electroporation conditions for NK cells were 1600V, 10 ms, and 3 pulses. * The cells were then added to culture plates containing APC (1NK:2APC), SCGM medium (preferentially without antibiotics), 200 IU / ml IL2, and allowed to recover in a 37° C. incubator.

[0263] Precomplexation and electroporation of crRNA: The crRNA and tracrRNA duplexes were mixed using a pipette and centrifuge. The mixture was incubated at 95°C for 5 min in a thermocycler and then cooled to room temperature on the benchtop.

[0264] [Table 3]

[0265] [Table 4]

[0266] [Table 5]

[0267] The crRNA:tracrRNA duplex was combined with the Cas9 nuclease mix using a pipette and incubated at room temperature for 15 minutes. The mixture was then combined with the crRNA.

[0268] [Table 6]

[0269] Electroporation was performed by first preparing a culture plate containing APC (1NK:2APC), SCGM medium (preferably without antibiotics) and 200 IU / mL IL-2. The preparation of 250,000 cells per well was resuspended in 7.5 ul of T buffer immediately before use. The electroporation conditions were 1600V, 10 ms and 3 pulses. * The cells were then added to culture plates and allowed to recover in a 37° C. incubator.

[0270] NK cell expansion: Isolate NK cells from umbilical cord blood or peripheral blood using Miltenyi's NK cell isolation kit (130-092-657). Place NK cells in SCGM medium with feeder cells in a 1:2 ratio (1 NK cell 2 feeder cells) in the presence of IL2 (200 IU / ml). Change medium with IL2 every other day. On day 4, select NK cells again using the NK cell isolation kit and remove feeder cells or wait until day 7 until all feeder cells are dead. Transduce chimeric antigen receptors or electroporate for CRISPR-Cas9.

[0271] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and steps or steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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Claims

1. 1. An in vitro method for disrupting two or three genes in a NK cell, comprising: the NK cells express a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR); and (aa) the two genes are (a) NKG2A and CISH, (b) NKG2A and TGFBRII, (c) CISH and TGFBRII, (j) CISH and TIGIT, (q) CD47 and CISH, (r) CD47 and TGFBRII, (u) CD47 and TIGIT, (y) ADAM17 and CISH, (z) TGFBRII and ADAM17, (b1) SHP1 and CISH, (c1) CISH and TGFBRII, (d1) SHP1 and TGFBRII, and (e1) SHP1 and TIGIT; or (bb) the three genes are selected from the group consisting of NKG2A, CISH, TGFBRII, TIGIT, CD96, CD47, SHP1, and ADAM17; method.

2. 2. The method of claim 1, wherein the disruption comprises introducing a guide RNA (gRNA) for each gene into the NK cell.

3. 2. The method of claim 1, wherein the three genes are selected from the group consisting of: (1) NKG2A, CISH and TGFBRII, (3) TGFBRII, CD96 and TIGIT, (4) TGFBR2, CISH and TIGIT, (9) CD47, CISH and TGFBRII, (11) TGFBRII, CD47 and TIGIT, (14) TGFBRII, CISH and ADAM17, (17) SHP1, CISH and TGFBRII, (18) TGFBRII, CISH and SHP1, and (19) TGFBRII, SHP1 and TIGIT.

4. The method of claim 1, further comprising the step of introducing an RNA-guided endonuclease.

5. 5. The method of claim 4, wherein the RNA-guided endonuclease is Cas9.

6. The method of claim 4, wherein the step of introducing the RNA-guided endonuclease comprises a step of introducing a nucleic acid encoding the RNA-guided endonuclease into the NK cell.

7. The method of claim 6 , wherein the nucleic acid is mRNA.

8. An in vitro method for disrupting a gene in a NK cell, comprising: the NK cells express a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR); and The method, wherein the genes comprise two of the following subgroups: (a) NKG2A and CISH, (b) NKG2A and TGFBRII, (c) CISH and TGFBRII, (e) TIGIT and TGFBRII, (g) CD96 and TGFBRII, (i) CD96 and TIGIT, (j) CISH and TIGIT, (q) CD47 and CISH, (r) CD47 and TGFBRII, (u) CD47 and TIGIT, (y) ADAM17 and CISH, (z) TGFBRII and ADAM17, (b1) SHP1 and CISH, (c1) CISH and TGFBRII, (d1) SHP1 and TGFBRII, and (e1) SHP1 and TIGIT.

9. An in vitro method for disrupting a gene in a NK cell, comprising: the NK cells express a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR); and The method, wherein the genes comprise two of the following subgroups: The gene is one of the following subgroups: (a) NKG2A and CISH, (b) NKG2A and TGFBRII, (c) CISH and TGFBRII, (e) TIGIT and TGFBRII, (g) CD96 and TGFBRII, (i) CD96 and TIGIT, (j) CISH and TIGIT, (q) CD47 and CISH, (r) CD47 and TGFBRII, (u) CD47 and TIGIT, (y) ADAM17 and CISH, (z) TGFBRII and ADAM17, (bl) SHP1 and CISH, (cl) CISH and TGFBRII, (dl) SHP1 and TGFBRII, and (el) SHP1 and TIGIT. and One of the following subgroups: (1) NKG2A, CISH, and TGFBRII, (3) TGFBRII, CD96, and TIGIT, (4) TGFBR2, CISH, and TIGIT, (9) CD47, CISH, and TGFBRII, (11) TGFBRII, CD47, and TIGIT, (14) TGFBRII, CISH, and ADAM17, (17) SHP1, CISH, and TGFBRII, (18) TGFBRII, CISH, and SHP1, and (19) TGFBRII, SHP1, and TIGIT.

10. An in vitro method for disrupting a gene in a NK cell, comprising: the NK cells express a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR); and The method, wherein the genes comprise two of the following subgroups: (1) NKG2A, CISH, and TGFBRII, (3) TGFBRII, CD96, and TIGIT, (4) TGFBR2, CISH, and TIGIT, (5) TIM3, CISH, and TGFBRII, (9) CD47, CISH, and TGFBRII, (11) TGFBRII, CD47, and TIGIT, (14) TGFBRII, CISH, and ADAM17, (17) SHP1, CISH, and TGFBRII, (18) TGFBRII, CISH, and SHP1, and (19) TGFBRII, SHP1, and TIGIT.

11. The method of claim 1 , wherein the disruption is simultaneous.

12. The method of claim 2, wherein the NK cells are virus-specific.

13. The method of claim 1 , wherein the NK cells are derived from peripheral blood, umbilical cord blood, bone marrow or a mixture thereof.

14. 14. The method of claim 13, wherein the cord blood is pooled from two or more individual cord blood units.

15. The method of claim 2 , wherein the introducing comprises transfection or transduction.

16. The method of claim 2 , wherein the introducing comprises electroporation.

17. 17. The method of claim 16, wherein electroporation is performed more than once.

18. The method of claim 16, wherein two electroporations are performed.

19. 20. The method of claim 18, wherein a first group of CRISPR gRNAs is introduced in a first electroporation and a second group of CRISPR gRNAs is introduced in a second electroporation.

20. 20. The method of claim 19, wherein the first and / or second group of CRISPR gRNAs comprises one, two, three or four CRISPR gRNAs.

21. 20. The method of claim 18, wherein two CRISPR gRNAs are introduced in a first electroporation and two CRISPR gRNAs are introduced in a second electroporation.

22. An in vitro method for disrupting a NK cell gene, comprising: the NK cells express a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR); and The method includes disrupting NKG2A, CD47, TGFBR2 and CISH; NKG2A, TGFBRII and CISH; or ADAM17, TGFBRII NKG2A and SHP1. A method comprising:

23. The method of claim 1, wherein said disruption results in enhanced anti-tumor cytotoxicity, improved in vivo expansion, in vivo persistence and / or function of said NK cells.

24. The method of claim 23, wherein the NK cells have increased secretion of IFN-γ, CD107 and / or TNFα.

25. 24. The method of claim 23, wherein the NK cells have increased production of perforin and / or granzyme B.

26. NK cells in which expression of two or three genes in the NK cell is disrupted, wherein the NK cell expresses a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), and is either (aa) the two genes are (a) NKG2A and CISH, (b) NKG2A and TGFBRII, (c) CISH and TGFBRII, (j) CISH and TIGIT, (q) CD47 and CISH, (r) CD47 and TGFBRII, (u) CD47 and TIGIT, (y) ADAM17 and CISH, (z) TGFBRII and ADAM17, (b1) SHP1 and CISH, (c1) CISH and TGFBRII, (d1) SHP1 and TGFBRII, and (e1) SHP1 and TIGIT; or (bb) the three genes are selected from the group consisting of NKG2A, CISH, TGFBR2, TIGIT, CD96, CD47, SHP1, and ADAM17. NK cells.

27. The cell of claim 26, wherein the NK cell is virus-specific.

28. 27. The cell of claim 26, wherein the NK cells are isolated from peripheral blood, umbilical cord blood, bone marrow or a mixture thereof.

29. 30. The cells of claim 28, wherein the cord blood is pooled from two or more individual cord blood units.

30. A NK cell in which expression of a NK cell gene is disrupted, wherein the NK cell expresses a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR); A cell, wherein NKG2A, CD47, TGFβR2 and CISH, or; NKG2A, TGFBR2 and CISH, of said NK cell; or ADAM17, TGFBR2 NKG2A and SHP1, of said NK cell, are disrupted.

31. The cell of claim 26, wherein the NK cell has enhanced anti-tumor cytotoxicity, improved in vivo proliferation, in vivo persistence and / or function.

32. The cell of claim 26, which increases the secretion of IFN-γ, CD107 and / or TNFα by the NK cells.

33. The cell of claim 26, wherein the NK cell has increased production of perforin and / or granzyme B.

34. 27. The cell of claim 26, wherein the CAR is inserted into an endogenous inhibitory gene locus of the cell, The locus of said inhibitory gene is selected from the group consisting of NKG2A, CISH, TGFBR2, TIGIT, CD96, CD47, SHIP1, ADAM17.

35. The cell of claim 34, wherein the CAR is under the control of an endogenous promoter of the inhibitory gene.

36. 35. The cell of claim 34, wherein the CAR is inserted into the locus of the inhibitory gene by CRISPR-mediated gene editing.

37. The cell of claim 26, wherein the CAR comprises an antigen-binding domain selected from the group consisting of F(ab')2, Fab', Fab, Fv and scFv.

38. The CAR is selected from the group consisting of CD19, 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, and CD23. , CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, and CD99.

39. The cell of claim 26, wherein the CAR comprises at least one signaling domain selected from the group consisting of CD3ξ, CD28, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, ILRB / CD122, IL-2RG / CD132, DAP12, CD70, and CD40.

40. 27. The cell of claim 26, wherein the cell is engineered to express a heterologous cytokine selected from the group consisting of IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, and combinations thereof.

41. The cell of claim 26 , wherein the cell further comprises a suicide gene.

42. The cell of claim 41, wherein the suicide gene is a membrane-bound tumor necrosis factor (TNF)-alpha mutant gene.

43. An expression vector encoding a gRNA for a CAR, and / or TCR, inhibitory gene sequence, The inhibitory gene sequence is (aa) two genes, (a) NKG2A and CISH, (b) NKG2A and TGFBRII, (c) CISH and TGFBRII, (j) CISH and TIGIT, (q) CD47 and CISH, (r) CD47 and TGFBRII, (u) CD47 and TIGIT, (y) ADAM17 and CISH, (z) TGFBRII and ADAM17, (b1) SHP1 and CISH, (c1) CISH and TGFBRII, (d1) SHP1 and TGFBRII, (e1) SHP1 and TIGIT, and (f1) SHP1 and TIM3; or (bb) three genes selected from the group consisting of NKG2A, CISH, TGFBR2, TIGIT, CD47, SHP1, ADAM17, and CD96; Inhibitory genes are derived from vectors.

44. 44. The vector of claim 43, wherein the gRNA is specific for the inhibitory gene.

45. The vector of claim 43 , wherein the vector is a retroviral vector.

46. The vector of claim 43, wherein the vector is an AAV vector.

47. 44. The vector of claim 43, wherein the CAR is flanked by homology arms to the inhibitory gene.

48. A host cell engineered to express the vector of claim 43.

49. 49. The cell of claim 48, wherein the cell is a T cell, a NK cell, a B cell or a stem cell.

50. 50. The cell of claim 49, wherein the cell is a cell of claim 26.

51. A pharmaceutical composition comprising a population of NK cells according to any one of claims 26 to 42.

52. A composition comprising the population of cells according to any one of claims 26 to 42 for treating an immune-related disorder, an infectious disease or cancer in a subject.

53. 53. The composition of claim 52, wherein the immune-related disorder is an autoimmune disorder, graft-versus-host disease, allograft rejection or an inflammatory condition.

54. 53. The composition of claim 52, wherein the immune-related disorder is an inflammatory condition and the NK cells have essentially no expression of glucocorticoid receptors.

55. 53. The composition of claim 52, wherein the NK cells are autologous or allogeneic to the subject.

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