Methods for engineering immune cells for enhanced efficacy and durability, and the use of engineered cells in immunotherapy

Abstract

Some embodiments of the methods and compositions disclosed herein relate to immune cells engineered to express chimeric antigen receptors and gene-edited or otherwise engineered to alter the effectiveness with which other immune cells can target and induce cytotoxic effects against the engineered cells. In some embodiments, the methods and compositions disclosed herein reduce fratricide within a therapeutic cell population, in some instances containing a mixture of immune cell types. Additionally, the methods and compositions disclosed herein enhance one or more aspects of the efficacy of immune cells in cellular immunotherapy, including cytotoxicity and / or persistence, and reduction of graft-versus-host or host-versus-graft effects.

Description

Technical field

[0001] Related applications This application is filed in U.S. Provisional Patent Application No. 63 / 121,206, filed on December 3, 2020, U.S. Provisional Patent Application No. 63 / 201,159, filed on April 15, 2021, and August 23, 2021. U.S. Provisional Patent Application No. 63 / 236,167 filed on November 17, 2021, and U.S. Provisional Patent Application No. 63 / 264,234 filed on November 17, 2021. No. 63 / 264,223, each of which is incorporated by reference in its entirety.

[0002] field Some embodiments disclosed herein relate to methods and compositions comprising genetically engineered cells, particularly combinations of genetically engineered immune cell types, for cancer immunotherapy. In some embodiments, the present disclosure relates to cells engineered to express chimeric antigen receptors. In some embodiments, further manipulation is performed to increase efficacy and / or reduce potential side effects when the cells are used in cancer immunotherapy. [Background technology]

[0003] As more knowledge is gained about different cancers and what properties cancerous cells have that can be used to specifically distinguish cancerous cells from healthy cells, Therapeutic drugs that exploit salient features of cells are under development. Immunotherapy using engineered immune cells is one approach to treating cancer.

[0004] Incorporating information by reference in an ASCII text file This application incorporates by reference the Sequence Listing contained in the following ASCII text file, filed at the same time: Filename: NKT068WO_ST25.txt; Created on December 2, 2021 and is 2,456,860 bytes in size. [Summary of the invention] [Means to solve the problem]

[0005] Immunotherapy offers new technological advances in the treatment of disease, in which immune cells are engineered to express specific targeting and / or effector molecules that specifically identify and respond to diseased or damaged cells. There is. This is a promising advance due, at least in part, to the possibility of specifically targeting diseased or damaged cells, as opposed to more traditional approaches such as chemotherapy, where all cells are affected. and the desired outcome is that enough healthy cells survive for the patient to survive. One immunotherapeutic approach is the recombinant expression of chimeric receptors in immune cells and, in addition, to achieve efficient and sustained targeted recognition and destruction of the desired aberrant cells, harmful immune responses against the treated cells can be avoided. The idea is to manipulate or gene edit cells to avoid this.

[0006] In some embodiments, genetically engineered and gene-edited immune cell populations are provided that express cytotoxic receptors, including those that target tumor markers expressed by target tumor cells. Immune cells, including immune cells, are gene edited at one or more targeted positions in the CISH gene encoding a CIS protein, and the editing is compared to immune cells that are not edited at one or more positions in the CISH gene. resulting in reduced expression and / or function of the CIS, the immune cell is gene edited at one or more targeted locations in a further gene encoding a further protein; resulting in reduced expression and / or function of additional proteins compared to unedited immune cells at multiple positions, and genetically engineered and edited immune cells do not contain the above gene edits at CISH and additional genes Exhibits one or more of increased expansion capacity, increased cytotoxicity to target cells, increased persistence, or other beneficial characteristics compared to immune cells.

[0007] In some embodiments, the cell has been edited with an additional target site in the Cbl proto-oncogene B protein (CBLB) gene. In some embodiments, the cell is edited with an additional target site in the transforming growth factor receptor beta2 (TGFBR2) gene. In some embodiments, the cell has been edited with additional target sites in the TIGIT gene. Similarly, editing combinations are provided, such as cells being edited with CISH / CBLB / TGFBR2, CISH / CBLB / TIGIT, CISH / TGFBR2 / TIGIT, etc. In some embodiments, a guide sequence of either SEQ ID NOs: 153-157 or 562-565 is used to target the CISH gene. In some embodiments, a guide sequence of either SEQ ID NOs: 164-166 or 552-555 is used to target the CBLB gene. In some embodiments, a guide sequence of either SEQ ID NOs: 147-152 or 544-547 is used to target the TGFBR2 gene. In some embodiments, a guide sequence of any of SEQ ID NOs: 507-510 is used to target the TIGIT gene. In some embodiments, the cell has one or more additional targets in genes encoding ADARA2, SMAD3, MAPKAPK3, CEACAM1, DDIT4, NKG2A, SOCS2, B2M, PD-1, TIM-3, CD38, or TCR alpha. The parts have been further edited.

[0008] For example, in some embodiments, the cell contains (i) a gene encoding NKG2A, in which a guide sequence of any of SEQ ID NOS: 548-551 is used to target the NKG2A gene; (ii) SEQ ID NOS: (iii) the guide sequence of SEQ ID NOs: 199 to 208 is used to target the B2M gene; (iv) a gene encoding PD-1, in which a guide sequence of any of SEQ ID NOs: 511 to 514 is used to target the PD-1 gene, (v) ) a gene encoding TIM-3, in which the guide sequence of any of SEQ ID NOs: 515 to 518 is used to target the TIM-3 gene, and (vi) a guide sequence of any of SEQ ID NOs: 566 to 568 is further edited in one or more of the genes encoding TCR alpha, which is used to target the TCR alpha gene. In some embodiments, the cell comprises (i) a gene encoding an adenosine A2 receptor, wherein a guide sequence of any of SEQ ID NOs: 503-506 is used to target the adenosine A2 receptor gene; ii) a gene encoding SMAD3, in which a guide sequence of SEQ ID NOs: 491 to 493 is used to target the SMAD3 gene; (iii) a guide sequence of any of SEQ ID NOs: 494 to 496 targets the MAPKAPK3 gene; (iv) a gene encoding MAPKAPK3, which is used to target the CEACAM1 gene, (iv) a guide sequence of any of SEQ ID NOs: 497 to 499 is used to target the CEACAM1 gene, (v) ) a gene encoding DDIT4, in which any guide sequence of SEQ ID NOs: 500 to 502 is used to target the DDIT4 gene, and (vi) a guide sequence of any of SEQ ID NOs: 519 to 522 is used to target the CD38 gene. Further edits are made in one or more of the genes encoding CD38, which are used for targeting. In some embodiments, other guide sequences targeting these genes are also used. In some embodiments, the cells are not edited at the CD70 gene target site.

[0009] In some embodiments, the cytotoxic receptor is one of the following ligands expressed by the target tumor cells: NKG2D, CD19, CD70, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, KREMEN2. Target one or more, or any combination thereof. However, in some embodiments, the cells have not been engineered to express a cytotoxic receptor complex that targets CD70. In some embodiments, the cells have not been engineered to express a cytotoxic receptor complex that targets CD19. In further embodiments, the cell has not been engineered to express a cytotoxic receptor complex that targets a ligand for the NKG2D receptor.

[0010] In some embodiments, at least some of the genetically engineered immune cells are engineered to express membrane-bound IL-15. In some embodiments, a fusion of mbIL15 and a natural receptor (eg, an IL-15 receptor unit or subunit) is provided. In some embodiments, the genetically engineered and edited immune cells reduce the risk of graft-versus-host disease and / or have greater cytotoxicity directed against tumor cells (thereby Suitable for use in allogeneic cancer cell therapy (resulting in more effective treatment).

[0011] Methods of treating cancer in a subject are provided herein, and in some embodiments, methods of treating cancer in a subject include at least a portion of a genetically engineered and gene edited population of immune cells as provided herein. including administering to Also provided is the use of genetically engineered and edited immune cells, as provided herein, to treat cancer and / or to prepare a medicament for treating cancer.

[0012] Also provided herein is a method of producing a gene-edited immune cell population for cancer immunotherapy, comprising: contacting the immune cell population with a first gene-editing complex; Editing at one or more target sites in the CISH gene of immune cells such that the body results in expression of reduced levels of the CIS protein encoded by the CISH gene compared to immune cells that have not been edited with the CISH gene. and contacting the population of immune cells with a second gene editing complex, wherein the second gene editing complex increases the amount of protein encoded by the additional gene compared to immune cells not edited with the additional gene. editing at one or more target sites in a further gene of the immune cell to result in a reduced level of expression, the gene-edited immune cell comprising editing the gene-edited one or more target sites in a further gene of the immune cell. exhibits one or more of increased expansion capacity, increased cytotoxicity to target cells, and increased persistence compared to immune cells without.

[0013] In some embodiments, the second gene editing complex results in the expression of a reduced level of the CBLB protein encoded by the CBLB gene compared to immune cells that have not been edited with the CBLB gene. Edit at one or more target sites in the cell's CBLB gene. In some embodiments, the second gene editing complex results in the expression of a reduced level of TGFBR2 protein encoded by the TGFBR2 gene compared to immune cells that have not been edited with the TGFBR2 gene. Edit at one or more target sites in the cell's TGFBR2 gene. In some embodiments, the second gene editing complex results in the expression of reduced levels of the TIGIT protein encoded by the TIGIT gene compared to immune cells that have not been edited with the TIGIT gene. Edit at one or more target sites in the cell's TIGIT gene. As discussed herein, combinations of edits are also provided. In some embodiments, in one or more genes encoding one of ADARA2, SMAD3, MAPKAPK3, CEACAM1, DDIT4, NKG2A, SOCS2, B2M, PD-1, TIM-3, CD38, or TCR alpha Further editing.

[0014] In some embodiments, the method comprises targeting at least a portion of the population of immune cells to a polynucleotide encoding a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. further comprising contacting the vector with a vector containing the vector. In some embodiments, the cytotoxic receptor is one of the following ligands expressed by the target tumor cells: NKG2D, CD19, CD70, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, KREMEN2. Target one or more, or any combination thereof. However, in some embodiments, the cytotoxic receptor does not target CD70, does not target CD19, and / or does not target NKG2D ligand. Furthermore, in some embodiments, the method does not include editing the gene encoding CD70.

[0015] In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject a population of genetically engineered immune cells, wherein the immune cells have an extracellular ligand binding domain, a transmembrane domain, and a transmembrane domain. Immune cells that express cytotoxic receptors containing cytotoxic signaling complexes are gene edited at one or more targeted locations in the CISH gene encoding CIS proteins; Immune cells are edited at one or more of the CBLB gene, TGFBR2 gene, and / or TIGIT gene, resulting in reduced expression and / or function of CIS compared to immune cells that are not edited at multiple positions. genetically engineered and edited resulting in reduced levels of expression or activity of CBLB protein, TGFBR2 protein, and / or TIGIT protein compared to immune cells that have not been edited with the CBLB gene, TGFBR2 gene, or TIGIT gene. The edited immune cells exhibit one of the following: increased expansion capacity, increased cytotoxicity to target cells, and increased persistence compared to immune cells that do not contain the gene-edited target site or sites described above. Indicates one or more.

[0016] In some embodiments, the method further comprises editing the immune cell with at least one additional target gene. In some embodiments, the at least one additional target gene is not a gene encoding CD70. In some embodiments, the additional target gene encodes ADAR2A, SMAD3, MAPKAPK3, CEACAM1, DDIT4, NKG2A, SOCS2, B2M, PD-1, TIM-3, CD38, or TCR alpha. In some embodiments, the cytotoxic receptor expressed by the immune cell is an epitope of one or more of CD19, CD70, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, KREMEN2; or combine them into combinations. However, in some embodiments, the cytotoxic receptor expressed by the immune cell does not target any of the CD19, CD70, or NKG2D ligands.

[0017] In some embodiments, the immune cells include natural killer (NK) cells, T cells, induced pluripotent stem cells (iPSCs), iPSC-derived NK cells, NK-92 cells, or combinations thereof. In some embodiments, the immune cells include a mixture of NK cells and T cells, or a mixture of iPSC-derived NK cells and T cells. In some embodiments, the immune cells include a purified or substantially purified NK cell population. In some embodiments, the immune cells administered are allogeneic to the subject. In some embodiments, the method further comprises administering IL2.

[0018] Some embodiments include genetically engineered immune cells that express cytotoxic receptors that include extracellular ligand binding domains, transmembrane domains, and cytotoxic signaling complexes. a population of immune cells is provided, the extracellular ligand-binding domain is targeted to a tumor marker expressed by the target tumor cells, and at least a portion of the immune cells are genetically engineered to express membrane-bound IL-15. one in the immune cell's genome such that the engineered immune cell results in the expression of reduced levels of the protein encoded by the gene containing the edited target site compared to the non-edited immune cell. The genetically engineered and edited immune cells that are gene-edited with one or more of the target sites have an increased ability to expand, an increased ability to target the target cells, compared to immune cells that do not contain one or more of the gene-edited target sites. exhibiting one or more of increased cytotoxicity, and increased persistence. In some embodiments, the immune cells are gene edited at a targeted site in the gene encoding the adenosine A2 receptor so as to result in reduced levels of adenosine receptor A2 expression compared to unedited immune cells. and a guide sequence of SEQ ID NOs: 503 to 506 is used to target the adenosine receptor A2 gene. In some embodiments, the cell is one of CISH, SMAD3, MAPKAPK3, CEACAM1, DDIT4, TGFBR2R, NKG2A, SOCS2, CBLB, B2M, TIGIT, PD-1, TIM-3, CD38, and TCR alpha It is further edited with genes encoding the above. For example, in some embodiments, the cell contains (i) a gene encoding CIS, in which a guide sequence of any of SEQ ID NOs: 562-565 is used to target the gene encoding CIS; ) the gene encoding SMAD3, in which a guide sequence of SEQ ID NOs: 491 to 493 is used to target the SMAD3 gene; (iii) a guide sequence of SEQ ID NOs: 494 to 496 targets the MAPKAPK3 gene; A gene encoding MAPKAPK3, (iv) a gene encoding CEACAM1 and a guide sequence of any one of SEQ ID NOs: 497 to 499, (v) a guide sequence of any one of SEQ ID NOs: 500 to 502, used for (vi) a guide sequence of any of SEQ ID NOs: 544 to 547 is used to target the TGFBR2 gene, encoding TGFBR2; (vii) a gene encoding NKG2A, in which the guide sequence of SEQ ID NOs: 548-551 is used to target the NKG2A gene, (viii) the guide sequence of SEQ ID NOs: 556-561 is used to target the NKG2A gene; (ix) a gene encoding CBLB, in which a guide sequence of any of SEQ ID NOs: 552 to 555 is used to target the CBLB gene; , (x) a gene encoding B2M, where any guide sequence of SEQ ID NOs: 290 to 299 is used to target the B2M gene, (xi) a guide sequence of any of SEQ ID NOs: 507 to 510 is TIGIT (xii) the gene encoding TIGIT, which is used to target the PD-1 gene; (xiii) a gene encoding TIM-3, in which the guide sequence of any of SEQ ID NOs: 515 to 518 is used to target the TIM-3 gene, (xiv) any of SEQ ID NOs: 519 to 522; any of the guide sequences are used to target the CD38 gene, the gene encoding CD38; and / or (xv) any of the guide sequences of SEQ ID NOs: 566-569 are used to target the TCR alpha gene. It has been further edited with the gene encoding TCR alpha, which is used for

[0019] In some embodiments, the extracellular ligand binding domain comprises a receptor with tropism for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. For example, in some embodiments, the cytotoxic receptor expressed by NK cells comprises (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z co-stimulatory domain. Contains a signaling complex that includes a stimulatory subdomain.

[0020] In some embodiments, the cytotoxic receptor is one of the NKG2D receptor ligands, CD19, CD70, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, or KREMEN2 expressed by the target tumor cells. Target one or more of the following: In some embodiments, the cytotoxic signaling complex comprises an OX40 subdomain or a 4-1BB domain and a CD3 zeta subdomain.

[0021] In some embodiments, the immune cell population comprises natural killer (NK) cells, T cells, induced pluripotent stem cells (iPSCs), iPSC-derived NK cells, NK-92 cells, or combinations thereof. In some embodiments, the immune cells are suitable for use in allogeneic cancer cell therapy to reduce the risk of graft-versus-host disease and / or to increase cytotoxic activity between genetically engineered immune cells. Suitable for use in allogeneic cancer cell therapy to reduce risk. Methods of treating cancer by administering such cells are described herein, as are the use of such cells to treat cancer or to prepare a medicament for treating cancer. Provided in

[0022] Further provided herein is a method of producing a genetically engineered immune cell population for cancer immunotherapy, wherein the immune cell population has an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. contacting a population of immune cells with a vector comprising a polynucleotide encoding a cytotoxic receptor comprising a cytotoxic receptor; comprising contacting a population of immune cells with a gene editing complex, wherein the gene editing complex Genetically engineered and edited immune cells that edit at a target site result in reduced levels of expression of the protein encoded by the gene containing the edited target site compared to non-edited immune cells. exhibiting one or more of increased expansion capacity, increased cytotoxicity to target cells, and increased persistence compared to immune cells that do not include the gene-edited one or more target sites. In some embodiments, the edit (or edits) includes CISH, ADARA2, SMAD3, MAPKAPK3, CEACAM1, DDIT4, TGFBR2R, NKG2A, SOCS2, CBLB, B2M, TIGIT, PD-1, TIM-3, CD38, and in genes encoding one or more of TCR alpha.

[0023] Also provided are methods of treating cancer in a subject, comprising administering to the subject a population of genetically engineered immune cells, wherein the immune cells have an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling domain. the cell is engineered to express membrane-bound IL-15; the cytotoxic signaling complex includes an OX-40 subdomain and a CD3 zeta subdomain; one or more target sites in the immune cell's genome such that the immune cell results in expression of reduced levels of the protein encoded by the gene containing the edited target site compared to an unedited immune cell The genetically engineered and edited immune cells have increased expansion capacity, increased cytotoxicity against target cells, compared to immune cells that do not contain the gene-edited target site or sites described above. , and exhibiting one or more of increased persistence. In some embodiments, the cells encode CISH, ADAR2A, SMAD3, MAPKAPK3, CEACAM1, DDIT4, TGFBR2R, NKG2A, SOCS2, CBLB, B2M, TIGIT, PD-1, TIM-3, CD38, and TCR alpha Edited in at least two genes selected from genes. In some embodiments, the cytotoxic receptor is (i) an NKG2D ligand binding domain expressed by the target tumor cell, or CD19, CD70, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, or a binding domain that binds to one or more epitopes of KREMEN2, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 costimulatory subdomain and a CD3z costimulatory subdomain. In some embodiments, the immune cells include natural killer (NK) cells, T cells, induced pluripotent stem cells (iPSCs), iPSC-derived NK cells, NK-92 cells, or combinations thereof. Furthermore, in some embodiments, the immune cells administered are allogeneic to the subject.

[0024] In some embodiments, a genetically engineered immune cell population for cancer immunotherapy is provided and includes a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. wherein at least a portion of the genetically engineered immune cells are engineered to express membrane-bound IL-15, and the genetically engineered immune cells express at least one immunosuppressive engineered to express effectors, the at least one immunosuppressive effector exerts a suppressive effect against the undesired cytotoxic activity of the suppressor cells, and the genetically engineered immune cells can be combined with cells that do not contain said immunosuppressive effector. In comparison, it exhibits one or more of increased expansion capacity, increased cytotoxicity to target cells, and increased persistence. In some embodiments, the population includes one or more of genetically engineered NK cells and genetically engineered T cells. In some embodiments, the suppressor cell comprises a host cell, or one or more of a non-engineered natural killer cell, a non-engineered T cell, or an engineered suppressor cell. In some embodiments, the immunosuppressive effector-free cells are either non-engineered cells or engineered cells.

[0025] In some embodiments, the genetically engineered immune cells are also gene edited to reduce expression of beta-2 microglobulin (B2M), and the reduced expression of B2M expresses endogenous levels of B2M. Enables the use of immune cells in allogeneic cancer immunotherapy to reduce host versus graft rejection compared to immune cells.

[0026] In some embodiments, the at least one immunosuppressive effector comprises a virus-derived peptide that may be derived from a retrovirus and may be derived from an envelope protein of a retrovirus. In some embodiments, the at least one immunosuppressive effector is bound to the extracellular membrane of the immune cell, the at least one immunosuppressive effector comprises a transmembrane protein, and the transmembrane protein is CD8α, CD4, CD3ε, CD3γ, CD3δ , CD3ζ, CD28, CD137, glycophorin A, glycophorin D, nicotinic acetylcholine receptor, GABA receptor, FcεRIγ, and T cell receptor.

[0027] In some embodiments, the immunosuppressive effector is at least one of SEQ ID NOs: 199-215, 219, 220, 223, 225, 228, 230, 235, 238, 240, 243, 250, or 253. Contains one or more peptides with 90% sequence identity. In some embodiments, the at least one immunosuppressive effector comprises at least a portion of a human protein and / or at least a portion of a human protein complex. In some embodiments, the immunosuppressive effector has at least 90% sequence identity with one or more of SEQ ID NOs: 248, 245, 273, 276, 278, 279, 286, 287, 288, and 289. Contains peptides. In some embodiments, the at least one immunosuppressive effector comprises a chimeric construct comprising at least one virus-derived peptide and at least a portion of a human protein and / or a human protein complex. In some embodiments, the chimeric immunosuppressive effector construct comprises (i) two or more of truncated human CD47, p15E peptide, HIV peptide, and HTLV peptide; (ii) truncated human CD47 domain and at least one p15E peptide, HIV peptide, and HTLV peptide, (iii) truncated human CD47 domain and at least one HIV peptide, (iv) truncated human CD47 domain and at least one HTLV peptide, (v) SEQ ID NO: 256, 259, 262 , 265, 268, 271; (vi) a viral UL18 protein; In some embodiments, the chimeric immunosuppressive effector construct comprises a viral UL18 protein and has a sequence that has at least 95% sequence identity with SEQ ID NO: 280, or the chimeric immunosuppressive effector construct comprises a viral UL18 protein and A sequence comprising a human B2M domain and having at least 95% sequence identity with SEQ ID NO: 283 or 285. In some embodiments, the immunosuppressive effector construct comprises an scFv between the transmembrane domain and the extracellular ligand binding domain, or within the extracellular ligand binding domain, and the chimeric immunosuppressive effector construct comprises an scFv. If incorporated into the linker region of the scFv, either within the linker region of the scFv, within the N-terminal region of the cytotoxic receptor located distal to the extracellular ligand binding domain, or within the cytotoxic receptor's cell It is incorporated into cytotoxic receptors at multiple locations within the external region. In some embodiments, at least one chimeric immunosuppressive effector construct is bound to the extracellular membrane of an immune cell. In some embodiments, the cytotoxic receptor comprises at least one immunosuppressive effector and the genetically engineered immune cell expresses at least one membrane-bound immunosuppressive effector.

[0028] In some embodiments, genetically engineered immune cells are suitable for use in allogeneic cancer cell therapy to reduce the risk of graft versus host disease. In a further embodiment, the genetically engineered immune cells are suitable for use in allogeneic cancer cell therapy to reduce the risk of cytotoxic activity among the genetically engineered immune cells.

[0029] Also provided herein is a method of producing genetically engineered immune cell populations for cancer immunotherapy, wherein the immune cell population has an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling domain. contacting the immune cell population with an additional polynucleotide encoding at least one immunosuppressive effector, the at least one immunosuppressive effector comprising: contacting the immune cell population with a polynucleotide encoding a cytotoxic receptor comprising a complex; Exerting a suppressive effect on the cytotoxic activity of suppressor cells, genetically engineered immune cells have an increased ability to expand, an increased ability to target target cells, compared to cells that do not contain the at least one immunosuppressive effector mentioned above. exhibiting one or more of the following: toxicity, and increased persistence.

[0030] Also provided are methods of engineering genetically engineered immune cell populations for cancer immunotherapy, which transform immune cell populations into cells containing extracellular ligand-binding domains, transmembrane domains, and cytotoxic signaling complexes. contacting a polynucleotide encoding a toxin receptor and encoding at least one immunosuppressive effector, the at least one immunosuppressive effector exerting an inhibitory effect on the cytotoxic activity of the suppressor cell, and the genetically engineered The treated immune cells exhibit one or more of increased expansion capacity, increased cytotoxicity to target cells, and increased persistence compared to cells that do not contain the at least one immunosuppressive effector. In some embodiments, the polynucleotide further encodes membrane-bound IL15. In some embodiments, the method further comprises contacting the population of immune cells with a polynucleotide encoding a membrane-bound immunosuppressive effector.

[0031] In some embodiments, a method of reducing fratricide in a mixed population of genetically engineered immune cells for cancer immunotherapy is provided, the first fratricide of immune cells from the mixed immune cell population being reduced. contacting a subpopulation of the immune cells with a polynucleotide encoding at least one immunosuppressive effector, wherein expression of the at least one immunosuppressive effector by the first subpopulation of immune cells is directed toward the first subpopulation of immune cells. and contacting the second subpopulation from the mixed immune cell population with a polynucleotide encoding at least an additional immunosuppressive effector to reduce the suppressive activity of a second subpopulation of immune cells having a Expression of at least a further immunosuppressive effector by the subpopulation of the second subpopulation comprises reducing the suppressive activity of the first subpopulation of immune cells having a tropism for the second subpopulation of immune cells; and expression of an immunosuppressive effector by the second subpopulation reduces fratricide among cells within the genetically engineered immune cell population.

[0032] Also provided are methods of engineering genetically engineered immune cell populations for enhancement of allogeneic cancer immunotherapy, and in some embodiments, expression of human leukocyte antigens (HLA) on the surface of the immune cells. Gene editing a mixed population of immune cells, including NK cells and T cells, such that reduced expression of HLA on the surface of immune cells increases T cell-mediated cytotoxicity against the edited immune cell population. and reduced expression of HLA on the surface of cells renders the edited immune cell population susceptible to NK-mediated cytotoxicity against edited immune cells; as well as NK-mediated cytotoxicity against mixed populations of immune cells. The edited cells are genetically engineered to express one or more immunosuppressive effectors that reduce sexual cytotoxicity, the one or more immunosuppressive effectors being viral immunosuppressive peptides, viral proteins that are HLA homologs, one or more of HLA-E, HLA-G, a human protein or fragment thereof that reduces cellular phagocytosis, a viral immunosuppressive peptide, and a human protein or fragment thereof that reduces cellular phagocytosis. , or a combination thereof, reduced T cell-mediated cytotoxicity decreases engineered T cell-mediated fratricide-type cytotoxicity to engineered NK cells, and upon administration, reduced T cell-mediated cytotoxicity to engineered NK cells. Reduced host T cell-mediated cytotoxicity reduces engineered NK cell-mediated fratricide-type cytotoxicity to engineered T cells, and upon administration, reduced NK cell-mediated cytotoxicity reduced host NK cell-mediated cytotoxicity towards engineered NK and engineered T cells, thereby allowing for increased persistence of engineered mixed populations of immune cells upon administration to allogeneic subjects and Enables the enhancement of cancer-based cancer immunotherapy. In some embodiments, the method alters the expression of one or more of the following: CISH gene, B2M gene, CD70 gene, adenosine receptor gene, NKG2A gene, CIITA gene, TGFBR gene, or any combination thereof. The method further includes genetically editing the DNA of the genetically engineered immune cells so as to do so.

[0033] As described herein, some embodiments include genetically engineered immune cells that express cytotoxic receptors that include an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. A genetically engineered and gene-edited population of immune cells is provided in which the extracellular ligand-binding domain targets a tumor marker expressed by the target tumor cell, and the immune cells have one or more of the CISH genes encoding CIS proteins. gene edited at the targeted location, the editing results in decreased expression and / or function of the CIS compared to immune cells that are not edited at one or more locations in the CISH gene; edited with one or more additional target sites in the genome, resulting in reduced levels of expression of the protein encoded by the gene containing the edited target site compared to non-edited immune cells, resulting in the CISH gene and Editing of one or more additional target sites is performed using the Crispr / Cas9 system, Crispr / CasX system, Crispr / CasY system, or another RNA-guided endonuclease, and the genetically engineered and edited immune cells are exhibiting one or more of increased expansion capacity, increased cytotoxicity to target cells, and increased persistence compared to immune cells that do not include the gene-edited target site or sites. In some embodiments, a guide sequence of either SEQ ID NOs: 153-157 or 562-565 is used to target the CISH gene.

[0034] In some embodiments, the cell is edited with additional target sites in the CBLB gene, resulting in a cell containing edits in at least CISH and CBLB. In some embodiments, a guide sequence of either SEQ ID NOs: 164-166 or 552-555 is used to target the CBLB gene.

[0035] In some embodiments, the cell is edited at an additional target site of the TGFBR2 gene, resulting in a cell containing an edit in at least CISH and TGFBR2. In some embodiments, a guide sequence of either SEQ ID NOs: 147-152 or 544-547 is used to target the TGFBR2 gene.

[0036] In some embodiments, the cell is edited at an additional target site of the TIGIT gene, resulting in a cell containing an edit in at least CISH and TIGIT. In some embodiments, a guide sequence of any of SEQ ID NOs: 507-510 is used to target the TIGIT gene.

[0037] In some embodiments, the genetically engineered and gene edited immune populations are not edited at the CD70 gene target site. However, in some embodiments they are edited with CD70 and also edited with one or more additional genes, such as CD70 / CISH / CBLB, CD70 / CISH / TGFBR2, CD70 / CISH / TIGIT, etc. Any combination of target genes can be edited according to embodiments disclosed herein.

[0038] In some embodiments, at least some of the genetically engineered immune cells are engineered to express membrane-bound IL-15.

[0039] In some embodiments, the genetically engineered and gene edited immune cell populations have not been engineered to express cytotoxic receptor complexes that target CD70. In some embodiments, the cells have not been engineered to express a cytotoxic receptor complex that targets CD19. In some embodiments, the cell has not been engineered to express a cytotoxic receptor complex that targets a ligand for the NKG2D receptor. However, in some embodiments, the cell is engineered to express an anti-CD70 CAR encoded by a polynucleotide at least 95% identical to one or more of SEQ ID NOs: 300-382. In some embodiments, the polynucleotide encodes an amino acid sequence that is at least 95% identical to one or more of SEQ ID NOs: 383-465. In some embodiments, the cell is engineered to express an anti-CD70 CAR that is at least 95% identical to one or more of SEQ ID NOs: 912-985. In some embodiments, the cell has a polynucleotide at least 95% identical to one or more of SEQ ID NO: 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, or 197. engineered to express an anti-CD19 CAR encoded by In some embodiments, the cell is engineered to express an anti-CD19 CAR that is at least 95% identical to one or more of SEQ ID NOs: 900-911. In some embodiments, the cell is engineered to express a chimeric receptor that targets the NKG2D ligand encoded by a polynucleotide at least 95% identical to SEQ ID NO: 475. In some embodiments, the cell is engineered to express a chimeric receptor targeting an NKG2D ligand that is at least 95% identical to SEQ ID NO:899.

[0040] In some embodiments, the cells encode adenosine A2 receptor, SMAD3, MAPKAPK3, CEACAM1, DDIT4, TGFBR2R, NKG2A, SOCS2, B2M, TIGIT, PD-1, TIM-3, CD38, or TCR alpha 1 further edited in more than one gene.

[0041] In some embodiments, the cell is further edited with a gene encoding an adenosine A2 receptor, and the nuclease is guided to an adenosine A2 receptor targeting sequence of any of SEQ ID NOs: 503-506. In some embodiments, the cell is further edited with the gene encoding SMAD3 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 491-493. In some embodiments, the cell is further edited with a gene encoding MAPKAPK3 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 494-496. In some embodiments, the cell is further edited with the gene encoding CEACAM1 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 497-499. In some embodiments, the cell is further edited with the gene encoding DDIT4 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 500-502. In some embodiments, the cells are further edited with a gene encoding NKG2A and the nuclease is guided to a target sequence of any of SEQ ID NOs: 548-551. In some embodiments, the cell is further edited with the gene encoding SOCS2 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 556-561. In some embodiments, the cell is further edited with a gene encoding B2M and the nuclease is guided to a target sequence of any of SEQ ID NOs: 290-299. In some embodiments, the cell is further edited with the gene encoding PD-1 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 511-514. In some embodiments, the cell is further edited with the gene encoding TIM-3 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 515-518. In some embodiments, the cells are further edited with the gene encoding CD38 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 519-522. In some embodiments, the cell is further edited with a gene encoding TCR alpha and the nuclease is guided to a target sequence of any of SEQ ID NOs: 566-569.

[0042] Depending on the embodiment, the genetically engineered and gene-edited immune cell population includes natural killer (NK) cells, T cells, induced pluripotent stem cells (iPSCs), iPSC-derived NK cells, NK-92 cells, or the like. may include a combination of. In some embodiments, genetically engineered and edited immune cells are suitable for use in allogeneic cancer cell therapy to reduce the risk of graft versus host disease.

[0043] Also provided herein are methods of treating cancer in a subject, comprising administering to the subject at least a portion of an immune cell population, according to embodiments discussed herein. Also provided is the use of the genetically engineered and edited immune cells provided herein to treat cancer and / or prepare a medicament for treating cancer.

[0044] Also provided herein is a method of producing a gene-edited immune cell population for cancer immunotherapy, comprising contacting the immune cell population with a first Cas-gRNA ribonucleoprotein complex (RNP). , at one or more target sites in the CISH gene of immune cells such that the RNP results in the expression of reduced levels of the CIS protein encoded by the CISH gene compared to immune cells that have not been edited with the CISH gene. editing, the Cas of the RNP comprises Cas9, CasX, CasY, or a combination thereof, and contacting the immune cell population with a second RNP complex, the second RNP being edited with the CBLB gene. RNP Cas, Cas9, edits at one or more target sites in the CBLB gene of immune cells, resulting in the expression of reduced levels of the CBLB protein encoded by the CBLB gene compared to immune cells that do not. Gene-edited immune cells containing CasX, CasY, or a combination thereof have increased expansion capacity, increased targeting of target cells, compared to immune cells that do not contain the gene-edited target site or sites described above. exhibiting one or more of increased cytotoxicity, and increased persistence.

[0045] Also provided herein is a method of producing a gene-edited immune cell population for cancer immunotherapy, comprising contacting the immune cell population with a first Cas-gRNA ribonucleoprotein complex (RNP). , at one or more target sites in the CISH gene of immune cells such that the RNP results in the expression of reduced levels of the CIS protein encoded by the CISH gene compared to immune cells that have not been edited with the CISH gene. editing, the Cas of the RNP comprises Cas9, CasX, CasY, or a combination thereof, and contacting the immune cell population with a second RNP complex, the second RNP being edited with the TGFBR2 gene. RNP Cas, Cas9, etc. edit at one or more target sites in the TGFBR2 gene of immune cells, resulting in the expression of reduced levels of the TGFBR2 protein encoded by the TGFBR2 gene compared to immune cells that do not. Gene-edited immune cells containing CasX, CasY, or a combination thereof have increased expansion capacity, increased targeting of target cells, compared to immune cells that do not contain the gene-edited target site or sites described above. exhibiting one or more of increased cytotoxicity, and increased persistence.

[0046] Also provided herein is a method of producing a gene-edited immune cell population for cancer immunotherapy, comprising contacting the immune cell population with a first Cas-gRNA ribonucleoprotein complex (RNP). , at one or more target sites in the CISH gene of immune cells such that the RNP results in the expression of reduced levels of the CIS protein encoded by the CISH gene compared to immune cells that have not been edited with the CISH gene. editing, the Cas of the RNP comprises Cas9, CasX, CasY, or a combination thereof, and contacting the immune cell population with a second RNP complex, the second RNP being edited with the TIGIT gene. The RNP Cas, Cas9, edits at one or more target sites in the TIGIT gene of immune cells, resulting in the expression of reduced levels of the TIGIT protein encoded by the TIGIT gene compared to immune cells that do not. Gene-edited immune cells containing CasX, CasY, or a combination thereof have increased expansion capacity, increased targeting of target cells, compared to immune cells that do not contain the gene-edited target site or sites described above. exhibiting one or more of increased cytotoxicity, and increased persistence.

[0047] Also provided herein is a method of producing a gene-edited immune cell population for cancer immunotherapy, comprising contacting the immune cell population with a first Cas-gRNA ribonucleoprotein complex (RNP). , at one or more target sites in the CISH gene of immune cells such that the RNP results in the expression of reduced levels of the CIS protein encoded by the CISH gene compared to immune cells that have not been edited with the CISH gene. editing, the Cas of the RNP comprises Cas9, CasX, CasY, or a combination thereof, and contacting the immune cell population with a second RNP complex, the second RNP being edited with an additional gene. RNP Cas, Cas9, CasX edits at one or more target sites in additional genes in immune cells, resulting in reduced levels of expression of proteins encoded by additional genes compared to immune cells that do not. , CasY, or a combination thereof, the gene-edited immune cells have an increased ability to expand, an increased ability to target cells, compared to immune cells that do not contain the gene-edited target site or sites described above. exhibiting one or more of cytotoxicity, and increased persistence.

[0048] In some embodiments, the method comprises targeting a population of immune cells with a vector comprising a polynucleotide encoding a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. Further comprising contacting. In some embodiments, the cytotoxic receptor does not target CD70. In other embodiments, the cytotoxic receptor targets CD70. In some embodiments, the method does not include editing the gene encoding CD70. In some embodiments, at least CD70 is edited. In some embodiments, the cytotoxic receptor does not target CD19. In some embodiments, the cytotoxic receptor targets CD19. In some embodiments, the cytotoxic receptor does not target NKG2D ligand. In some embodiments, the cytotoxic receptor targets NKG2D ligand.

[0049] In some embodiments, the method encodes one of ADARA2, SMAD3, MAPKAPK3, CEACAM1, DDIT4, TGFBR2R, NKG2A, SOCS2, B2M, PD-1, TIM-3, CD38, or TCR alpha Further comprising making further edits in one or more genes.

[0050] In some embodiments, the cytotoxic receptor is one of the following ligands expressed by the target tumor cells: NKG2D, CD19, CD70, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, KREMEN2. Target one or more, or any combination thereof. Depending on the embodiment, the immune cells can include natural killer (NK) cells, T cells, induced pluripotent stem cells (iPSCs), iPSC-derived NK cells, NK-92 cells, or combinations thereof.

[0051] In some embodiments, a method of treating cancer in a subject is provided that includes administering to the subject a population of genetically engineered immune cells, wherein the immune cells have an extracellular ligand binding domain, a transmembrane domain, and a transmembrane domain. Immune cells that express cytotoxic receptors, including cytotoxic signaling complexes, are gene edited at one or more targeted locations in the CISH gene encoding the CIS protein, and the editing occurs at one or more of the CISH genes. resulting in reduced levels of expression and / or function of CIS compared to immune cells not edited in multiple positions, and immune cells compared to immune cells not edited in the CBLB gene, TGFBR2 gene or TIGIT gene. and the editing is RNA guided Immune cells that have been genetically engineered and edited using endonucleases have an increased ability to expand, an increased ability to target cells, compared to immune cells that do not contain the gene-edited target site or sites described above. exhibiting one or more of cytotoxicity, and increased persistence.

[0052] In some embodiments, the RNA-guided endonuclease is a Crispr / CasX, Crispr / CasY, or Crispr / Cas9 system. In some embodiments, the method further comprises editing the immune cell with at least one additional target gene. In some embodiments, the at least one additional target gene is not a gene encoding CD70. In some embodiments, the cells are edited with additional target genes encoding ADAR2A, SMAD3, MAPKAPK3, CEACAM1, DDIT4, NKG2A, SOCS2, B2M, PD-1, TIM-3, CD38, or TCR alpha . In some embodiments, the cytotoxic receptor expressed by the immune cell does not target CD70, does not target CD19, or does not target NKG2D ligand. However, in some embodiments, the cytotoxic receptor expressed by the immune cell is one of CD19, CD70, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, KREMEN2, or a combination thereof. binds to one or more epitopes of

[0053] In some embodiments, the immune cells include natural killer (NK) cells, T cells, induced pluripotent stem cells (iPSCs), iPSC-derived NK cells, NK-92 cells, or combinations thereof. In some embodiments, the immune cells include a mixture of NK cells and T cells, or a mixture of iPSC-derived NK cells and T cells. In some embodiments, the immune cells administered are allogeneic to the subject. In some embodiments, the method further comprises administering IL2.

[0054] Accordingly, in some embodiments, genetically engineered immune cell populations for cancer immunotherapy are provided and include genetically engineered immune cells that express cytotoxic receptors, the immune cells being edited Gene-edited at one or more target sites in the immune cell's genome to result in the expression of reduced levels of the protein encoded by the genetically engineered gene, the genetically engineered immune cell may also be a natural killer cell and / or The resulting genetically engineered and edited immune cells are engineered to express at least one immunosuppressive effector that exerts a suppressive effect on the cytotoxic activity of T cells, and the resulting genetically engineered and edited immune cells are Exhibits one or more of increased expansion capacity, increased cytotoxicity to target cells, and increased persistence compared to cells without the gene.

[0055] Also provided herein are genetically engineered immune cell populations for cancer immunotherapy, including one or more of genetically engineered NK cells and genetically engineered T cells. The immune cells are engineered to express cytotoxic receptors, herein the immune cells are engineered to express reduced levels of the protein encoded by the edited gene (or genes). Gene-edited at one or more target sites in the immune cell's genome, genetically engineered immune cells are known as non-engineered natural killer cells, non-engineered T cells, or unwanted engineered cells. The genetically engineered and edited immune cells are further genetically engineered to express at least one immunosuppressive effector that exerts a suppressive effect on the cytotoxic activity of the undesired cells of the cell, and the genetically engineered and edited immune cells contain the immunosuppressive effector and the edited gene. exhibiting one or more of increased expansion capacity, increased cytotoxicity to target cells, and increased persistence compared to cells without.

[0056] Additionally, provided herein are genetically engineered immune cell populations for cancer immunotherapy, including one or more of genetically engineered NK cells and genetically engineered T cells, wherein multiple genetically engineered immune cell populations are provided. Engineered immune cells are engineered to express cytotoxic receptors, and immune cells are also encoded by edited genes that contain edited target sites compared to non-edited immune cells. gene edited at one or more target sites in the genome to result in the expression of reduced levels of a protein that causes immune cells to be configured to produce a suppressive effect against the unwanted cytotoxic activity of suppressor cells. The engineered immune cells are further genetically engineered to express at least one immunosuppressive effector, and the genetically engineered immune cells have increased expansion capacity, increased cytotoxicity toward target cells, compared to cells that do not contain said immunosuppressive effector. , and exhibiting one or more of increased persistence.

[0057] In some embodiments, suppressor cells include host cells. In some embodiments, the suppressor cells include one or more of non-engineered natural killer cells, non-engineered T cells, or engineered suppressor cells. In some embodiments, engineered suppressor cells include genetically engineered immune cells (eg, other cells within the cell population used for therapy). In some embodiments, the immunosuppressive effector-free cells are non-engineered cells (e.g., host cells) or engineered cells (other engineered cells that have not been edited or engineered with immunosuppressive effectors). ).

[0058] In some embodiments, the cytotoxic receptor expressed by the cell is a chimeric antigen receptor or other chimeric receptor that includes an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. It is. In some embodiments, the cytotoxic signaling complex comprises an OX40 subdomain or a 4-1BB domain and a CD3 zeta subdomain. In some embodiments, the cytotoxic receptor targets one or more of NKG2D, CD19, BCMA, CD70, and CD38 expressed by the target tumor cells.

[0059] In some embodiments, editing is performed using a targeted endonuclease. In some embodiments, the endonuclease is an RNA guided endonuclease. In some embodiments, editing is performed using the Crispr / Cas system.

[0060] According to some embodiments, the genetically engineered immune cells have also been gene edited to reduce expression of beta-2 microglobulin (B2M). In some embodiments, reduced expression of B2M reduces host versus graft rejection compared to immune cells expressing endogenous levels of B2M using immune cells in allogeneic cancer immunotherapy. make it possible to

[0061] In some embodiments, at least one immunosuppressive effector comprises a virus-derived peptide. In some embodiments, the immunosuppressive effector comprises a peptide derived from a retrovirus, such as a peptide derived from a retroviral envelope protein. In further embodiments, the at least one immunosuppressive effector comprises at least a portion of a human protein and / or at least a portion of a human protein complex. In some embodiments, the at least one immunosuppressive effector comprises at least a portion of a human protein. In yet another embodiment, the at least one immunosuppressive effector comprises a chimeric construct comprising at least one virus-derived peptide and at least a portion of a human protein and / or at least a portion of a human protein complex.

[0062] In some embodiments, at least one immunosuppressive effector is incorporated into the cytotoxic receptor at one or more positions within the receptor. For example, in some embodiments, at least one immunosuppressive effector is incorporated into the cytotoxic receptor between the transmembrane domain and the extracellular ligand binding domain. In other embodiments, at least one immunosuppressive effector is incorporated into the cytotoxic receptor within the extracellular ligand binding domain. In yet another embodiment, when the extracellular ligand binding domain comprises an scFv, at least one immunosuppressive effector is incorporated into the linker region of the scFv. In some embodiments, at least one immunosuppressive effector is incorporated into the cytotoxic receptor within the N-terminal region of the cytotoxic receptor located distal from the extracellular ligand binding domain. In some embodiments, the at least one immunosuppressive effector is incorporated into the cytotoxic receptor at multiple locations within the extracellular region of the cytotoxic receptor, e.g., multiple copies of the effector are incorporated within the receptor. placed at various locations in the body to induce enhanced immunosuppressive effects.

[0063] In addition to or in place of effectors incorporated into receptor complexes, in some embodiments at least one immunosuppressive effector is bound to the extracellular membrane of the immune cell. In some such embodiments, the at least one immunosuppressive effector comprises a transmembrane protein. Non-limiting examples of transmembrane proteins that can be used in accordance with embodiments disclosed herein include CD8α, CD4, CD3ε, CD3γ, CD3δ, CD3ζ, CD28, CD137, glycophorin A, glycophorin D, nicotinic These are acetylcholine receptors, GABA receptors, FcεRIγ, and T cell receptors. According to some embodiments, combinations of transmembrane domains are used. In some embodiments, the transmembrane protein comprises CD8α transmembrane protein. In a further embodiment, the at least one immunosuppressive effector is expressed on the immune cell by a disulfide trap single chain trimer (dtSCT).

[0064] In some embodiments, the immunosuppressive effector comprises a peptide encoded by a nucleic acid or having at least 85% sequence identity with one or more of the nucleotide or amino acid sequences of SEQ ID NOs: 683-894. In some embodiments, the immunosuppressive effector comprises a peptide that has at least 95% sequence identity to SEQ ID NO: 689. In some embodiments, the immunosuppressive effector is encoded by a nucleic acid that has at least 95% sequence identity to SEQ ID NO: 690.

[0065] In some embodiments, the genetically engineered immune cells include genetically engineered natural killer (NK) cells, genetically engineered T cells, or a combination thereof. In some embodiments, genetically engineered immune cells are suitable for use in allogeneic cancer cell therapy to reduce the risk of graft versus host disease. Additionally, in some embodiments, the genetically engineered immune cells are suitable for use in allogeneic cancer cell therapy, which reduces the risk of cytotoxic activity among the genetically engineered immune cells. In some embodiments, at least a portion (e.g., at least 50%, at least 60%, at least 75%, or more) of the genetically engineered immune cells are engineered to express membrane-bound IL-15. Ru. In some embodiments, at least about 60% of the NK cells are positive for NKG2A. In some embodiments, at least about 80% of the NK cells are positive for NKG2A. In some embodiments, elevated expression levels of NKG2A enhance the efficacy of engineered and edited cells in cancer immunotherapy due to decreased cytotoxicity within the cells of the population administered to the subject. .

[0066] In some embodiments, methods of treating cancer in a subject are also provided that include administering to the subject genetically engineered and edited immune cells according to embodiments disclosed herein. Some embodiments also include the use of genetically engineered and edited immune cells in accordance with embodiments described herein to treat cancer or to prepare a medicament for treating cancer. Also provided.

[0067] In some embodiments, a method of producing a genetically engineered immune cell population for cancer immunotherapy is provided, wherein the population encoded by a gene containing an edited target site compared to non-edited immune cells is provided. contacting the population of immune cells with an RNA-guided endonuclease to genetically edit one or more target sites in the genome of the immune cells so as to result in expression of a reduced level of a protein to be treated; contacting the immune cell population with a polynucleotide encoding a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex; the immunosuppressive effector is a peptide encoded by the nucleic acid or having at least 85% sequence identity with one or more of the nucleotide or amino acid sequences of SEQ ID NOs: 683-894. including; the at least one immunosuppressive effector exerts a suppressive effect on the cytotoxic activity of the suppressor cells, and the genetically engineered immune cells have an increased exhibiting one or more of increased expansion capacity, increased cytotoxicity toward target cells, and increased persistence. In some embodiments, the gene editing target is one or more of the following: CISH gene, B2M gene, CD70 gene, adenosine receptor gene, NKG2A gene, CIITA gene, TGFBR gene, or any combination thereof .

[0068] Also provided herein are methods for engineering genetically engineered immune cell populations for the enhancement of allogeneic cancer immunotherapy, disrupting beta-2 microglobulin (B2M) expression and Gene editing a mixed population of immune cells, including NK cells and T cells, to coordinately reduce the expression of human leukocyte antigen (HLA) on the surface of the immune cells, resulting in reduced expression of HLA on the surface of the immune cells. Reduced expression of HLA on the surface of the cells reduces T cell-mediated cytotoxicity against the edited immune cell population, and reduces T cell-mediated cytotoxicity against the edited immune cell population. genetically engineered cells to express one or more immunosuppressive effectors that reduce NK-mediated cytotoxicity to mixed populations of immune cells; , viral immunosuppressive peptides, viral proteins that are HLA homologs, HLA-E, HLA-G, human proteins or fragments thereof that reduce cellular phagocytosis, viral immunosuppressive peptides and human proteins that reduce cellular phagocytosis. or a fragment thereof, or a combination thereof, the reduced T cell-mediated cytotoxicity is determined by the engineered T cell-mediated fratricide-type cytotoxicity against the engineered NK cells. and upon administration, decreased host T cell-mediated cytotoxicity against engineered NK cells, and decreased NK cell-mediated cytotoxicity was associated with engineered NK cell-mediated fratricide against engineered T cells. upon administration, decreases host NK cell-mediated cytotoxicity to engineered NK and engineered T cells, thereby reducing immune cell manipulation upon administration to allogeneic subjects. Enables increased persistence in mixed populations and enables enhancement of allogeneic cancer immunotherapy. In some embodiments, at least about 60% of the NK cells in the mixed population are positive for NKG2A expression.

[0069] In some embodiments, the method comprises contacting a mixed population of immune cells with a polynucleotide encoding a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. It further includes causing. Depending on the embodiment, the polynucleotide encodes a cytotoxic receptor that targets one or more of NKG2D, CD19, CD70, BCMA, and CD38 expressed by the target tumor cells.

[0070] In some embodiments, the method alters the expression of one or more of the CISH gene, the CD70 gene, the adenosine receptor gene, the NKG2A gene, the CIITA gene, the TGFBR gene, or any combination thereof. , further comprising gene editing the DNA of the genetically engineered immune cells. In some embodiments, gene editing to decrease expression or gene editing to induce expression is performed using the CRISPR-Cas system. In some embodiments, the Cas is Cas9, Cas12, Cas13, CasX or CasY.

[0071] Genetically engineered immune cell populations, including one or more of genetically engineered NK cells and genetically engineered T cells, are also provided based on the generation methods disclosed herein, including genetically engineered NK cells and genetically engineered T cells. At least approximately 70% of NK cells express NKG2A, and several genetically engineered immune cells express cytotoxic receptors that contain extracellular ligand-binding domains, transmembrane domains, and cytotoxic signaling complexes. The engineered immune cells are genetically engineered to express at least one immunosuppressive effector derived from a human leukocyte antigen (HLA), and the at least one immunosuppressive effector is a desired component of the suppressor cell. The genetically engineered immune cells, if appropriate, have been incorporated within the extracellular domain of one or more of the following: IL-15, mbIL-15, cytotoxic receptors. at least one viral immunosuppressive peptide expressed on the cell membrane of a genetically engineered immune cell; incorporated within one or more extracellular regions of a cytotoxic receptor; at least one portion of a human immunosuppressive protein or protein complex, at least one portion of a human immunosuppressive protein or protein complex expressed on the cell membrane of the genetically engineered immune cell, and / or the genetically engineered immune cell further engineered to express one or more of at least one chimeric virus-human immunosuppressive construct expressed on the cell membrane of and / or incorporated into one or more extracellular regions of a cytotoxic receptor. has been done. In some embodiments, the HLA-derived immunosuppressive effector comprises at least one HLA-E peptide.

[0072] In some embodiments, provided herein are genetically engineered and gene edited immune cell populations, wherein the cells are genetically engineered in one or more respects, and the cells are also genetically engineered in one or more respects. Edited. In some embodiments, the cells are genetically engineered to express one or more cytotoxic receptors that target tumor markers expressed by the target tumor cells. In some embodiments, the gene-edited immune cells result in reduced levels of expression of a protein encoded by a target gene that includes the edited target site compared to non-edited immune cells. , gene editing at one or more target sites in the immune cell genome of the immune cell, thereby altering one or more of the cell's genotype, phenotype or function. In some embodiments, gene editing is performed using guided nucleases.

[0073] In some embodiments, herein includes genetically engineered immune cells that express cytotoxic receptors that include an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. , genetically engineered and gene-edited immune cell populations are provided in which extracellular ligand binding domains target tumor markers expressed by target tumor cells. In some embodiments, the immune cell's genome is modified such that the immune cell's genome results in expression of a reduced level of a protein encoded by a gene that includes the edited target site compared to an unedited immune cell. The gene has been edited at one or more target sites in the gene. In some embodiments, gene editing is performed using the CRISPR / CasX system. In some embodiments, gene editing is performed using the CRISPR / CasY system. In some embodiments, gene editing is performed using the CRISPR / Cas9 system. In some embodiments, the CRISPR / Cas systems used herein are configured to reduce potentially deleterious off-target effects. In some embodiments, gene editing is performed using a non-Cas guided nuclease.

[0074] In some embodiments, at least some of the genetically engineered immune cells are engineered to express membrane-bound IL-15.

[0075] In some embodiments, the genetically engineered and gene-edited immune cells have increased expansion capacity, increased cell-to-target cells, as compared to immune cells that do not contain the gene-edited target site or sites. exhibiting one or more of the following: toxicity, and increased persistence.

[0076] In some embodiments, within the immune cell population, the cells have reduced levels of expression of the adenosine A2 receptors CISH, SMAD3, MAPKAPK3, CEACAM1, DDIT4 compared to non-edited immune cells. , TGFBR2, NKG2A, SOCS2, CBLB, B2M, TIGIT, PD-1, TIM-3, CD38, and TCR alpha. In some embodiments, within the immune cell population, the nuclease is guided by an adenosine A2 receptor targeting sequence of any of SEQ ID NOs: 503-506. In some embodiments, within the immune cell population, cells are edited with a gene encoding CIS and the nuclease is guided to a target sequence of any of SEQ ID NOs: 562-565. In some embodiments, within the immune cell population, cells are edited with the gene encoding SMAD3 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 491-493. In some embodiments, within the immune cell population, cells are edited with the gene encoding MAPKAPK3 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 494-496. In some embodiments, within the immune cell population, cells are edited with the gene encoding CEACAM1 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 497-499. In some embodiments, within the immune cell population, cells are edited with the gene encoding DDIT4 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 500-502. In some embodiments, within the immune cell population, cells are edited with the gene encoding TGFBR2 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 544-547. In some embodiments, within the immune cell population, cells are edited with the gene encoding NKG2A and the nuclease is guided to a target sequence of any of SEQ ID NOs: 548-551. In some embodiments, within the immune cell population, cells are edited with the gene encoding SOCS2 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 556-561. In some embodiments, within the immune cell population, cells are edited with a gene encoding CBLB and the nuclease is guided to a target sequence of any of SEQ ID NOs: 552-555. In some embodiments, within the immune cell population, cells are edited with the gene encoding B2M and the nuclease is guided to a target sequence of any of SEQ ID NOs: 290-299. In some embodiments, within the immune cell population, cells are edited with a gene encoding TIGIT and the nuclease is guided to a target sequence of any of SEQ ID NOs: 507-510. In some embodiments, within the immune cell population, cells are edited with the gene encoding PD-1 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 511-514. In some embodiments, within the immune cell population, cells are edited with the gene encoding TIM-3 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 515-518. In some embodiments, within the immune cell population, cells are edited with the gene encoding CD38 and the nuclease is guided to a target sequence of any of SEQ ID NOs: 519-522. In some embodiments, within the immune cell population, cells are edited with a gene encoding TCR alpha and the nuclease is guided to a target sequence of any of SEQ ID NOs: 566-569.

[0077] In some embodiments, the extracellular ligand binding domain comprises a receptor with tropism for at least one tumor marker selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the cytotoxic receptor expressed by the NK cell comprises (i) an NKG2D ligand binding domain, (ii) a CD8 transmembrane domain, and (iii) a signaling complex. In some embodiments, the cytotoxic signaling complex comprises an OX40 subdomain and / or a 4-1BB domain, and a CD3 zeta subdomain.

[0078] In some embodiments, the cytotoxic receptor is NKG2D, CD19, CD70, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, ALPPL2, CLDN4, CLDN6, or Target one or more of KREMEN2.

[0079] In some embodiments, the immune cell population can include natural killer (NK) cells, T cells, induced pluripotent stem cells (iPSCs), iPSC-derived NK cells, NK-92 cells, or combinations thereof. . In some embodiments, the immune cell population comprises engineered and edited immune cells suitable for use in allogeneic cancer cell therapy to reduce the risk of graft versus host disease. In some embodiments, the immune cell population comprises genetically engineered and edited immune cells suitable for use in allogeneic cancer cell therapy to reduce the risk of cytotoxic activity among genetically engineered immune cells. including. In some embodiments, the immune cell population comprises engineered and edited immune cells to treat cancer or to prepare a medicament for treating cancer.

[0080] In some embodiments, methods of treating cancer in a subject are provided that include administering to the subject a population of genetically engineered and edited immune cells.

[0081] In some embodiments, provided herein are methods of producing genetically engineered and gene-edited populations of immune cells, wherein the cells are genetically engineered in one or more respects, and the cells are also genetically engineered in one or more ways. Gene editing is performed in the above points. In some embodiments, the vector includes a polynucleotide encoding a cytotoxic receptor. In some embodiments, the cytotoxic receptor includes an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. In some embodiments, the method further comprises contacting the immune cell population with a Cas-gRNA ribonucleoprotein complex (RNP). In some embodiments, the RNP is isolated in the genome of the immune cell such that it results in reduced levels of expression of the protein encoded by the gene containing the edited target site compared to the non-edited immune cell. Edit at one or more target sites.

[0082] Also provided herein is a method of producing genetically engineered immune cell populations for cancer immunotherapy, wherein the immune cell population has an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling domain. contacting a population of immune cells with a vector containing a polynucleotide encoding a cytotoxic receptor containing complex, and contacting the immune cell population with a Cas-gRNA ribonucleoprotein complex (RNP), wherein the RNP is unedited. Cas edits and RNPs at one or more target sites in the immune cell's genome result in reduced levels of expression of the protein encoded by the gene containing the edited target site compared to the immune cell. , Cas9, CasX, CasY, or a combination thereof, genetically engineered and edited immune cells exhibit increased expansion capacity, increased ability to target cells, compared to immune cells that do not contain the gene-edited target site. exhibiting one or more of cytotoxicity, and increased persistence.

[0083] In some embodiments, the method comprises detecting the following among CISH, ADARA2, SMAD3, MAPKAPK3, CEACAM1, DDIT4, TGFBR2, NKG2A, SOCS2, CBLB, B2M, TIGIT, PD-1, TIM-3, CD38, and TCR alpha. further comprising gene editing performed in a gene encoding one or more of the following: In some embodiments, the immune cell population is contacted with a vector comprising a polynucleotide encoding a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex, and the immune cell population is The outer ligand binding domain comprises a receptor with tropism for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the immune cell population is contacted with a vector comprising a polynucleotide encoding a cytotoxic receptor, wherein the cytotoxic receptor expressed by the NK cell comprises (i) an NKG2D ligand binding domain; (ii) a CD8 transmembrane domain, and (iii) a signaling complex that includes an OX40 costimulatory subdomain and a CD3z costimulatory subdomain. In some embodiments, the immune cell population is contacted with a vector comprising a polynucleotide encoding a cytotoxic receptor, wherein the cytotoxic receptor is NKG2D, CD19, CD70, BCMA expressed by the target tumor cells. , CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, ALPPL2, CLDN4, CLDN6, or KREMEN2.

[0084] In some embodiments, the method comprises contacting an immune cell population with a vector comprising a polynucleotide encoding a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. The cytotoxic signaling complex comprises an OX40 subdomain and / or a 4-1BB domain, and a CD3 zeta subdomain.

[0085] In some embodiments, the method further comprises contacting the immune cell population with a vector comprising a polynucleotide, the polynucleotide dicistronically encoding membrane-bound IL15. In some embodiments, the vector is a retroviral vector.

[0086] In some embodiments, the method further comprises contacting a population of immune cells, where the immune cells include natural killer (NK) cells, T cells, induced pluripotent stem cells (iPSCs), iPSC-derived NK cells, Contains NK-92 cells, or a combination thereof.

[0087] Also provided herein are methods of treating cancer in a subject that include administering to the subject a population of genetically engineered and / or edited immune cells. In some embodiments, the immune cell expresses a cytotoxic receptor that includes an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. In some embodiments, the cytotoxic signaling complex comprises an OX-40 subdomain and a CD3 zeta subdomain. In some embodiments, the method further comprises where the cell is engineered to express membrane-bound IL-15. In some embodiments, the immune cells are gene edited. In some embodiments, the immune cell's genome is modified such that the immune cell produces a reduced level of expression of a protein encoded by a gene that includes the edited target site compared to an unedited immune cell. The gene has been edited at one or more target sites in the gene. In some embodiments, editing is performed using Crispr / CasX, Crispr / CasY, or Crispr / Cas9 systems, or combinations thereof. In some embodiments, the genetically engineered and edited immune cells have increased expansion capacity, increased cytotoxicity toward the target cells, as compared to immune cells with or without the gene-edited target site. , and exhibiting one or more of increased persistence.

[0088] Some embodiments provide that the immune cells contain CISH, ADAR2A, SMAD3, MAPKAPK3, CEACAM1, DDIT4, TGFBR2, NKG2A, SOCS2, CBLB, B2M, TIGIT, PD-1, TIM-3, CD38, and TCR alpha. relating to how to be edited with a gene encoding one or more of the following:

[0089] In some embodiments, the cytotoxic receptor expressed by the immune cell comprises (i) an NKG2D ligand binding domain, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z costimulatory subdomain. Contains signaling complexes containing domains. In some embodiments, the cytotoxic receptor expressed by the immune cell is (i) CD19, CD70, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, expressed by the target tumor cell; a signaling complex that includes a binding domain that binds to one or more epitopes of ALPPL2, CLDN4, CLDN6, or KREMEN2, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z costimulatory subdomain Including the body.

[0090] In some embodiments, methods of treating cancer in a subject are provided that include administering to the subject a population of genetically engineered immune cells, where the immune cells include natural killer (NK) cells, T cells, induced Contains pluripotent stem cells (iPSCs), iPSC-derived NK cells, NK-92 cells, or a combination thereof. In some embodiments, the genetically engineered immune cell population comprises a mixture of NK cells and T cells, or a mixture of iPSC-derived NK cells and T cells.

[0091] In some embodiments, the method further provides that the NK cells and / or iPSC-derived NK cells are edited to at least reduce CISH expression, and / or the T cells target CD19 or NKG2D. . In some embodiments, the method includes administering an immune cell, the immune cell being allogeneic to the subject. In some embodiments, the method further comprises administering IL2.

[0092] As disclosed herein, the host immune response against the administered cells (eg, allogeneic engineered cells) can limit the efficacy and persistence of the administered cells. In some embodiments, mixed populations of immune cells are used for immunotherapy as disclosed herein. In such cases, the host immune response not only presents a challenge, but the administered cells act on each other as well as on host tissues (e.g., off-target effects) in a manner that suppresses the efficacy of the immunotherapy. It also presents possibilities. In some embodiments, mixed cell immunotherapy approaches therefore require further manipulation and treatment to reduce graft versus host effects, host versus graft rejection, and maintenance of compatibility between mixed immune cell types in treatment / or require gene editing. In the context of allogeneic immunotherapy, engineered T cells expressing CARs can cause graft-versus-host rejection as T cells recognize host cells as non-self cells. This recognition occurs through the graft T cell receptor, which interacts with host cell HLA. Thus, in some embodiments, gene editing to disrupt T cell receptors can eliminate T cell-based graft-versus-host rejection. In the opposite situation, i.e. host versus graft rejection, host T cells recognize graft immune cells (e.g. NK cells and / or T cells) as non-self and, by destroying the administered cells, therefore Reduces the persistence and effectiveness of immunotherapy. Since it is impractical to edit T cell receptors from host cells, gene editing of the administered immune cells can be performed as described herein. In some embodiments, gene editing to reduce / eliminate HLA expression on engineered immune cells allows them to avoid being identified as non-self by host cells. Beta-2 microglobulin (B2M) gene editing / knockout is used to accomplish this according to some embodiments disclosed herein. However, this renders the edited cells susceptible to NK cell cytotoxicity not only from host NK cells, but also from NK cells within the administered immune cells (host vs. grafts and fratricide). This is because editing to remove B2M expression causes the loss of all HLA molecules, including normally expressed signals that function to inhibit NK cell activity (KIR molecules). Thus, as disclosed in more detail below, in some embodiments, further modifications include allogeneic mixed immunity from both the host immune system and fratricide from other immune cells in the mixed immune cell product. It is done to mask cell products. In some embodiments, various proteins can be expressed on the surface of allogeneic immune cells that inhibit them from being targeted by NK cells (both host cells and allogeneic cells). For example, the "don't eat me" signal, which normally allows cells to evade phagocytosis by macrophages, can be inhibited by NK and / or T cells expressing allogeneic CAR to reduce targeting by NK cells. can be expressed on cells. Other approaches that provide a number of advantages, such as reducing NK cell-mediated suppression while also enhancing persistence of engineered NK cells, are also disclosed herein. As discussed in more detail below, in some embodiments, immune cells (e.g., CAR-expressing T cells) are engineered to express HLA-E, giving these cells the ability to express HLA-E present on NK cells. Inhibition confers the ability to interact with the NKG2A receptor (host or engineered allogeneic). This interaction induces a temporary inhibition of NK cell activity, whether host or engineered allogeneic NK cells. Regarding host NK cells, this suppression reduces host NK cell activity against T cells, allowing them to act against target tumor cells. Temporary suppression of engineered allogeneic NK cells helps prevent NK cell exhaustion, as engineered allogeneic NK cells have periods of reduced cytotoxic activity (eg, resting periods). When the temporary suppression is relieved, the engineered allogeneic NK cells return to exerting cytotoxic effects against target tumor cells. Thus, in some embodiments, the suppressive effect actually serves to enhance the persistence of allogeneic NK cells in mixed immune cell populations for immunotherapy. In a further embodiment, the virus-derived peptide or protein is expressed by engineered allogeneic cells to exhibit transient suppressive activity against NK cells (both host and engineered NK cells in a mixed population). and thus increase the overall persistence of the allogeneic mixed immune cell population. In some embodiments, the combination of acute activity (even with periods of suppression) of engineered NK cells and longer sustained activity of engineered T cells may result in cytotoxic lysis of tumor cells. bring about. The resulting tumor pieces are processed by host immune cells (eg, dendritic cells) and tumor antigens are therefore presented to T cells, thereby recruiting host immune cells to fight the tumor. In some embodiments, engineered cells are engineered to undergo temporary suppression (eg, a period of reduced cytotoxic activity or a period of quiescence). This suppression may be a reduction in cellular or other activity by at least 10-90% (e.g., 10-30%, 30-50%, 50-70%, 70-90% and overlapping ranges therein). . In one embodiment, greater than 90% inhibition is achieved. Temporal can include, for example, minutes, hours, or days. Cells may be permanently suppressed in one embodiment.

[0093] Included herein, in some embodiments, are genetically engineered immune cells that express cytotoxic receptors that include an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. , a genetically engineered immune cell population for cancer immunotherapy is provided, wherein the genetically engineered immune cells are engineered to express at least one immunosuppressive effector, and the at least one immunosuppressive effector is naturally occurring. Exerting a suppressive effect on the cytotoxic activity of killer cells and / or T cells, genetically engineered immune cells have an increased ability to expand and target cells compared to cells that do not contain the above-mentioned immunosuppressive effectors. exhibiting one or more of increased cytotoxicity, and increased persistence.

[0094] Also provided herein are genetically engineered immune cell populations for cancer immunotherapy, including one or more of genetically engineered NK cells and genetically engineered T cells. The genetically engineered immune cells are engineered to express cytotoxic receptors, the genetically engineered immune cells are genetically engineered to express at least one immunosuppressive effector, and the at least one immunosuppressive effector is desirably exerts an inhibitory effect on the cytotoxic activity of non-desired cells, including one or more of non-engineered natural killer cells, non-engineered T cells, or undesired engineered cells. , the genetically engineered immune cells exhibit one or more of increased expansion capacity, increased cytotoxicity to target cells, and increased persistence compared to cells that do not contain the immunosuppressive effector.

[0095] Additionally, herein, in some embodiments, genetically engineered immune cells for cancer immunotherapy include one or more of genetically engineered NK cells and genetically engineered T cells. a population is provided, a plurality of genetically engineered immune cells are engineered to express a cytotoxic receptor, and the genetically engineered immune cells are genetically engineered to express at least one immunosuppressive effector. , the at least one immunosuppressive effector exerts a suppressive effect on the undesirable cytotoxic activity of the suppressor cells, and the genetically engineered immune cells exhibit increased expansion compared to cells without said immunosuppressive effector. exhibiting one or more of the following: potency, increased cytotoxicity toward target cells, and increased persistence.

[0096] Also provided herein, in some embodiments, is a genetically engineered immune cell population comprising one or more of a genetically engineered NK cell and a genetically engineered T cell; The genetically engineered immune cell is engineered to express a cytotoxic receptor that includes an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex, and the genetically engineered immune cell has at least genetically engineered to express one immunosuppressive effector, the at least one immunosuppressive effector exerts a suppressive effect against the undesired cytotoxic activity of the suppressor cells, and where appropriate, the genetically engineered immune cells are: :IL-15, mbIL-15, at least one viral immunosuppressive peptide incorporated within the extracellular region of one or more cytotoxic receptors, at least one viral immunosuppressive peptide expressed on the cell membrane of genetically engineered immune cells. one viral immunosuppressive peptide, at least one portion of a human immunosuppressive protein or protein complex incorporated within the extracellular region or regions of a cytotoxic receptor, on the cell membrane of a genetically engineered immune cell; at least one portion of an expressed human immunosuppressive protein or protein complex, and / or expressed on the cell membrane of a genetically engineered immune cell, and / or one or more extracellular regions of a cytotoxic receptor. further engineered to express one or more of the integrated at least one chimeric virus-human immunosuppressive construct.

[0097] In some embodiments, suppressor cells include host cells (eg, NK cells and / or T cells). In some embodiments, the suppressor cells include one or more of non-engineered natural killer cells, non-engineered T cells, or engineered suppressor cells. In some embodiments, engineered suppressor cells include genetically engineered immune cells (eg, those administered to a patient for cancer immunotherapy). In some embodiments, the immunosuppressive effector-free cells are either non-engineered cells or engineered cells. In some embodiments, the immunosuppressive effector exerts a transient immunosuppressive effect. In some embodiments, a transient immunosuppressive effect is beneficial to reduce potential exhaustion of one or more genetically engineered immune cells. In some embodiments, one immunosuppressive effector (or effectors) is not engineered to be expressed in a consistent manner across different cell types within the genetically engineered immune cells. For example, in some embodiments, T cells may be engineered to express one specific immunosuppressive effector (or multiple effectors), whereas NK cells are not so engineered and have a different profile. Expressing an immunosuppressive effector (or multiple effectors).

[0098] In some embodiments, the cytotoxic receptor includes an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. In some embodiments, the cytotoxic receptor targets one or more of NKG2D, CD19, and CD70 expressed by the target tumor cells. In some embodiments, the cytotoxic signaling complex comprises an OX40 subdomain or a 4-1BB domain, and a CD3 zeta subdomain, or any combination thereof (including duplications of one or more subdomains). include.

[0099] Furthermore, in some embodiments, at least some of the genetically engineered immune cells are engineered to express membrane-bound IL15. In some embodiments, some of the cells are not engineered to express membrane-bound IL15. In some embodiments, NK cells are engineered to express mbIL15 while T cells are not. In some embodiments, both NK cells and T cells are engineered to express mbIL15. In some embodiments, T cells but not NK cells are engineered to express mbIL15, but NK cells are not engineered. In some embodiments, the NK cells and / or T cells are engineered to express soluble IL15 in addition to or in place of mbIL15. In some embodiments, mbIL15 is the nucleic acid sequence of SEQ ID NO: 489, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identical to SEQ ID NO: 489. encoded by a sequence with a specific character. In some embodiments, mbIL15 has the amino acid sequence of SEQ ID NO: 490, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 490. It has a sequence with a characteristic.

[0100] In some embodiments, the genetically engineered immune cells have also been gene edited to reduce expression of beta-2 microglobulin (B2M). In some embodiments, reduced expression of B2M reduces host versus graft rejection compared to immune cells expressing endogenous levels of B2M using the immune cells in allogeneic cancer immunotherapy. make it possible.

[0101] In some embodiments, at least one immunosuppressive effector comprises a virus-derived peptide. In some embodiments, the at least one immunosuppressive effector comprises a retrovirus-derived peptide. In some embodiments, the at least one immunosuppressive effector comprises a peptide derived from a retroviral envelope protein. In some embodiments, the at least one immunosuppressive effector comprises at least a portion of a human protein and / or at least a portion of a human protein complex. In some embodiments, the at least one immunosuppressive effector comprises at least a portion of a human protein. In some embodiments, the at least one immunosuppressive effector comprises a chimeric construct comprising at least one virus-derived peptide and at least a portion of a human protein and / or a human protein complex. In some embodiments, the chimeric immunosuppressive effector construct comprises two or more of truncated human CD47, p15E peptide, HIV peptide, and HTLV peptide. In some embodiments, the chimeric immunosuppressive effector construct comprises a truncated human CD47 domain and at least one of a p15E peptide, an HIV peptide, and an HTLV peptide. In some embodiments, the chimeric immunosuppressive effector construct comprises a truncated human CD47 domain and at least one HIV peptide. In some embodiments, the chimeric immunosuppressive effector construct comprises a truncated human CD47 domain and at least an HTLV peptide. In some embodiments, the chimeric immunosuppressive effector construct comprises a viral UL18 protein. In some embodiments, the chimeric immunosuppressive construct comprises a human B2M domain.

[0102] In some embodiments, at least one immunosuppressive effector is incorporated into a cytotoxic receptor. In some embodiments, at least one immunosuppressive effector is incorporated into the cytotoxic receptor between the transmembrane domain and the extracellular ligand binding domain. In some embodiments, at least one immunosuppressive effector is incorporated into the cytotoxic receptor within the extracellular ligand binding domain. In some embodiments, the extracellular ligand binding domain comprises an scFv and at least one immunosuppressive effector is incorporated into the linker region of the scFv. In some embodiments, at least one immunosuppressive effector is incorporated into the cytotoxic receptor within the N-terminal region of the cytotoxic receptor located distal from the extracellular ligand binding domain. Depending on the embodiment, the immunosuppressive effector is incorporated into the cytotoxic receptor at multiple locations within the extracellular region of the cytotoxic receptor. In some embodiments, the immunosuppressive effector at the first position is different from the immunosuppressive effector at the different position(s). In some embodiments, the same immunosuppressive effector is incorporated at multiple positions. In some embodiments, different immunosuppressive effectors are incorporated at various positions.

[0103] In some embodiments, at least one immunosuppressive effector is bound to the extracellular membrane of the immune cell. In some embodiments, the at least one immunosuppressive effector comprises a transmembrane protein. In some embodiments, the transmembrane proteins include CD8α, CD4, CD3ε, CD3γ, CD3δ, CD3ζ, CD28, CD137, glycophorin A, glycophorin D, nicotinic acetylcholine receptor, GABA receptor, FcεRIγ, and T cell receptor. Selected from the body. In some embodiments, the transmembrane protein comprises CD8α transmembrane protein.

[0104] In some embodiments, the genetically engineered immune cell comprises at least one immunosuppressive effector and a cytotoxic receptor comprising at least one membrane-bound immunosuppressive effector.

[0105] In some embodiments, a spacer sequence (also called a hinge) is used to separate the immunosuppressive effector or CAR from the transmembrane domain, whether the membrane binds or incorporates the CAR. In some embodiments, the spacer is selected from or derived from CD8a, IgG1, IgG2, IgG3, IgG4, or CD28 spacers, or is a completely synthetic sequence. It can be. In some embodiments, the hinge region comprises one or more of SEQ ID NOs: 479-487. In some embodiments, the hinge region is at least about 80%, at least about 85%, at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about any of SEQ ID NOs: 479-486. Amino acid sequences having about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence.

[0106] In some embodiments, the immunosuppressive effector has at least about 80%, at least about 85%, at least about 90%, at least about 94%, or at least about 95% with one or more of SEQ ID NOs: 199-215. Contains one or more peptides with sequence identity. In some embodiments, the immunosuppressive effector comprises a peptide having at least about 80%, at least about 85%, at least about 90%, at least about 94%, or at least about 95% sequence identity to SEQ ID NO: 199. . In some embodiments, the immunosuppressive effector comprises an amino acid that has at least about 80%, at least about 85%, at least about 90%, at least about 94%, or at least about 95% sequence identity with SEQ ID NO: 219. . In some embodiments, the immunosuppressive effector is at least about 80%, at least about 85%, at least about 90%, at least about 94% with one or more of SEQ ID NOs: 220, 225, 230, 235, and 250. , or peptides having at least about 95% sequence identity. In some embodiments, the immunosuppressive effector is at least about 80%, at least about 85%, at least about 90%, at least about 94% with one or more of SEQ ID NOs: 223, 228, 233, 238, and 253. , or amino acids with at least about 95% sequence identity. In some embodiments, the immunosuppressive effector comprises a peptide having at least about 80%, at least about 85%, at least about 90%, at least about 94%, or at least about 95% sequence identity to SEQ ID NO: 240. . In some embodiments, the immunosuppressive effector comprises an amino acid that has at least about 80%, at least about 85%, at least about 90%, at least about 94%, or at least about 95% sequence identity with SEQ ID NO: 243. .

[0107] In some embodiments, the immunosuppressive effector has at least about 80%, at least about 85%, at least Includes peptides having about 90%, at least about 94%, or at least about 95% sequence identity. In some embodiments, the immunosuppressive effector comprises an amino acid that has at least about 80%, at least about 85%, at least about 90%, at least about 94%, or at least about 95% sequence identity with SEQ ID NO: 248. .

[0108] In some embodiments, the immunosuppressive effector is membrane-bound and at least about 80%, at least about 85%, at least Chimeric immunosuppressive effector constructs comprising sequences having about 90%, at least about 94%, or at least about 95% sequence identity.

[0109] In some embodiments, the chimeric immunosuppressive effector construct comprises a viral UL18 protein and has at least about 80%, at least about 85%, at least about 90%, at least about 94%, or at least about 95% of SEQ ID NO: 280. Contains sequences with sequence identity. In some embodiments, the chimeric immunosuppressive effector construct comprises a viral UL18 protein and a human B2M domain, and has at least about 80%, at least about 85%, at least about 90%, at least about 94%, SEQ ID NO: 283 or 285; or comprising sequences having at least about 95% sequence identity.

[0110] In some embodiments, the genetically engineered immune cells include genetically engineered natural killer (NK) cells, genetically engineered T cells, or a combination thereof. In some embodiments, other immune cells may be included as well. In some embodiments, genetically engineered immune cells are suitable for use in allogeneic cancer cell therapy to reduce the risk of graft versus host disease. Advantageously, in some embodiments, the genetically engineered immune cells are suitable for use in allogeneic cancer cell therapy, which reduces the risk of cytotoxic activity among the genetically engineered immune cells.

[0111] Also provided herein are methods of treating cancer in a subject comprising administering to the subject genetically engineered immune cells according to embodiments of the present disclosure. Also provided is the use of genetically engineered immune cells according to embodiments of the present disclosure to treat cancer, as well as their use in the preparation of a medicament to treat cancer.

[0112] Further, in some embodiments, a method of producing a genetically engineered immune cell population for cancer immunotherapy is provided, comprising: contacting the immune cell population with a polynucleotide encoding a cytotoxic receptor; contacting the population of immune cells with an additional polynucleotide encoding at least one immunosuppressive effector. In some embodiments, a method of engineering a genetically engineered immune cell population for cancer immunotherapy is provided, the immune cell population encoding a cytotoxic receptor and at least one immunosuppressive effector. contacting the encoding polynucleotide. In some embodiments, the at least one immunosuppressive effector exerts a suppressive effect on the cytotoxic activity of the suppressor cell, and in some embodiments, the genetically engineered immune cell is Exhibits one or more of increased expansion capacity, increased cytotoxicity to target cells, and increased persistence compared to cells without suppressive effectors. In some embodiments, the method alters the expression of one or more of the following: CISH gene, B2M gene, CD70 gene, adenosine receptor gene, NKG2A gene, CIITA gene, TGFBR gene, or any combination thereof. The method further includes genetically editing the DNA of the genetically engineered immune cells so as to do so. In some embodiments, the polynucleotide may further encode membrane-bound IL15. In some embodiments, the method further comprises contacting the population of immune cells with a polynucleotide encoding a membrane-bound immunosuppressive effector.

[0113] Also provided herein is a method of reducing fratricide in a mixed population of genetically engineered immune cells for cancer immunotherapy, wherein a first subpopulation of immune cells derived from the mixed immune cell population is contacting a polynucleotide encoding at least one immunosuppressive effector, and contacting a second subpopulation derived from the mixed immune cell population with a polynucleotide encoding at least one additional immunosuppressive effector; Expression of immunosuppressive effectors by one subpopulation and a second subpopulation reduces fratricide in the genetically engineered immune cell population.

[0114] In some embodiments, expression of the at least one immunosuppressive effector by the first subpopulation of immune cells comprises, at least temporarily, a second subpopulation of immune cells that has a tropism for the first subpopulation of immune cells. expression of at least an additional immunosuppressive effector by the second subpopulation of immune cells reduces the suppressive activity of the subpopulation of immune cells, at least temporarily, reduces the suppressive activity of the first subpopulation of immune cells with a tropism toward the second subpopulation of immune cells. decreases the inhibitory activity of a subpopulation of . In some embodiments, the method comprises contacting a population of immune cells with a polynucleotide encoding a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. further including. In some embodiments, the method alters the expression of one or more of the following: CISH gene, B2M gene, CD70 gene, adenosine receptor gene, NKG2A gene, CIITA gene, TGFBR gene, or any combination thereof. The method may further include gene editing the DNA of the genetically engineered immune cells to do so.

[0115] In some embodiments, methods of engineering genetically engineered immune cell populations for enhancement of allogeneic cancer immunotherapy are provided, including genetically editing mixed populations of immune cells including NK cells and T cells. one or more immunization regimens that reduce human leukocyte antigen (HLA) expression on the surface of immune cells and genetically engineer the edited cells to reduce NK-mediated cytotoxicity against a mixed population of immune cells. including expressing inhibitory effectors. In some embodiments, reduced expression of HLA on the surface of immune cells reduces T cell-mediated cytotoxicity against the edited immune cell population. In some embodiments, reduced expression of HLA on the surface of the cell makes the edited immune cell population susceptible to NK-mediated cytotoxicity against the edited immune cells. In some embodiments, the one or more immunosuppressive effectors are viral immunosuppressive peptides, viral proteins that are HLA homologs, HLA-E, HLA-G, human proteins or fragments thereof that reduce cellular phagocytosis; One or more of a chimeric construct comprising a viral immunosuppressive peptide and a human protein or fragment thereof that reduces cellular phagocytosis, or a combination thereof. In some embodiments, the reduced T cell-mediated cytotoxicity at least temporarily reduces engineered T cell-mediated fratricide-type cytotoxicity toward engineered NK cells, and upon administration, decreases host T cell-mediated cytotoxicity against NK cells. In some embodiments, the reduced NK cell-mediated cytotoxicity at least temporarily reduces engineered NK cell-mediated fratricide-type cytotoxicity toward engineered T cells, and upon administration, reduced host NK cell-mediated cytotoxicity towards engineered NK and engineered T cells, thereby allowing for increased persistence of engineered mixed populations of immune cells upon administration to allogeneic subjects and Enabling the enhancement of allogeneic cancer immunotherapy.

[0116] In some embodiments, the method comprises contacting a mixed population of immune cells with a polynucleotide encoding a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex. It further includes causing. In some embodiments, the polynucleotide encodes a cytotoxic receptor that targets one or more of NKG2D, CD19, and CD70 expressed by the target tumor cell. In some embodiments, the cytotoxic signaling complex comprises an OX40 subdomain or a 4-1BB domain, and a CD3 zeta subdomain, or any combination thereof. In some embodiments, the method alters the expression of one or more of the following: CISH gene, B2M gene, CD70 gene, adenosine receptor gene, NKG2A gene, CIITA gene, TGFBR gene, or any combination thereof. The method further includes genetically editing the DNA of the genetically engineered immune cells so as to do so. In some embodiments, gene editing to decrease expression or gene editing to induce expression is performed using the CRISPR-Cas system. In some embodiments, the CRISPR-Cas system comprises a Cas selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, CasX, CasY, and combinations thereof. In some embodiments, the Cas is Cas9.

[0117] In some embodiments, cells for immunotherapy have been genetically modified to increase one or more properties of the cells that result in more effective treatment. In some embodiments, one or more of expansion potential, cytotoxicity, and / or persistence of the genetically modified immune cells is increased. In some embodiments, the immune cells are also engineered to express cytotoxic receptors that target tumors. Provided herein, in some embodiments, are genetically engineered NK cell populations for cancer immunotherapy that include a plurality of natural killer (NK) cells, wherein the plurality of NK cells are extracellular. Engineered to express a cytotoxic receptor containing a ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex, NK cells have a higher concentration of cytotoxic receptors encoded by the CISH gene compared to non-engineered NK cells. Gene-edited to express reduced levels of cytokine-inducible SH2-containing (CIS) protein, reduced CIS expression is engineered through editing of the CISH gene, and engineered NK cells are Compared to NK cells expressing levels of CIS, they exhibit increased expansion capacity, increased cytotoxicity toward target cells, and increased persistence. In some embodiments, the cytotoxic signaling complex comprises an OX-40 subdomain and a CD3 zeta subdomain. In some embodiments, the NK cells are engineered to express membrane-bound IL-15. In some embodiments, T cells are engineered and used instead of or in addition to NK cells. In some embodiments, NKT cells are not included in the engineered immune cell population. In some embodiments, the immune cell population comprises, consists of, or consists essentially of engineered NK cells.

[0118] In some embodiments, the extracellular ligand binding domain comprises a receptor with tropism for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the cytotoxic receptor expressed by the NK cell comprises (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z costimulatory subdomain. comprises or consists essentially of a signaling complex containing domains. In some embodiments, the cytotoxic receptor is a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 145. encoded by nucleotides. In some embodiments, the cytotoxic receptor has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 174 or 899. have

[0119] In some embodiments, the cytotoxic receptor expressed by the NK cell comprises (i) a tumor binding domain comprising an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain. and a chimeric antigen receptor (CAR) comprising or consisting essentially of a signaling complex comprising a CD3z co-stimulatory subdomain. In some embodiments, the anti-CD19 antibody comprises a variable heavy chain (VH) domain of a single chain variable fragment (scFv) and a variable light chain (VL) domain of an scFv, wherein the VH domain is equal to or equal to SEQ ID NO: 120. The VL domain comprising and encoding the amino acid sequence comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, the CAR expressed by the T cell has at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 178 or 901. In some embodiments, anti-CD19 antibody fragments reduce the potential antigenicity of the encoded protein and / or improve one or more properties of the encoded protein (e.g., target recognition and / or binding). characteristics) are designed (e.g., manipulated) to increase Thus, according to some embodiments, the anti-CD19 antibody fragment does not include specific sequences. For example, according to some embodiments, the anti-CD19 antibody fragment is not encoded by SEQ ID NO: 116, does not include the VL region of SEQ ID NO: 105 or 107, or the VH region of SEQ ID NO: 104 or 106. In some embodiments, the anti-CD19 antibody fragment does not include one or more CDRs selected from SEQ ID NOs: 108-115.

[0120] In some embodiments, expression of CIS is substantially reduced compared to non-engineered NK cells. According to certain embodiments provided herein, gene editing increases the expression of a target protein, such as CIS (or others disclosed herein) by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99%, or less or more (including any amount between those listed). In some embodiments, the gene is completely knocked out such that expression of the target protein is undetectable. Thus, in some embodiments, the immune cells (eg, NK cells) do not express detectable levels of CIS protein.

[0121] In some embodiments, the NK cells are further genetically engineered to express reduced levels of transforming growth factor beta receptor (TGFBR) compared to unengineered NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of TGFBR. In some embodiments, the NK cells have been further gene edited to express reduced levels of beta-2 microglobulin (B2M) compared to unengineered NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of B2M surface protein. In some embodiments, the NK cells are further gene edited to express reduced levels of CIITA (class II major histocompatibility complex transactivator) compared to unengineered NK cells. There is. In some embodiments, at least 50% of the NK cell population does not express detectable levels of CIITA. In some embodiments, the NK cells have been further genetically edited to express reduced levels of natural killer group 2, member A (NKG2A) receptors compared to unengineered NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of NKG2A. In some embodiments, the NK cells have been further gene edited to express reduced levels of the Cbl proto-oncogene B protein, encoded by the CBLB gene, compared to unengineered NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of Cbl proto-oncogene B protein. In some embodiments, the NK cells are further gene edited to express reduced levels of the tricomponent motif-containing protein 29 protein, encoded by the TRIM29 gene, compared to unengineered NK cells. There is. In some embodiments, at least 50% of the NK cell population does not express detectable levels of TRIM29 protein. In some embodiments, the NK cells are further gene edited to express reduced levels of suppressor of cytokine signaling 2 protein, encoded by the SOCS2 gene, compared to non-engineered NK cells. ing. In some embodiments, at least 50% of the NK cell population does not express detectable levels of SOCS2 protein. Depending on the embodiment, editing any combination of the above target proteins / genes to desired levels, including in combination with CIS, such that the proteins are not expressed at detectable levels. I can do it. In some embodiments, some amount of detectable protein may remain, but the function of the protein is destroyed, substantially destroyed, eliminated, or substantially eliminated. Ru. In some embodiments, even if some functionality remains, the positive effects imparted to the engineered immune cells (e.g., NK cells or T cells) remain and the cell's one or more anti-inflammatory Helps increase cancer aspects.

[0122] In some embodiments, the NK cells are further genetically edited to disrupt expression of at least one immune checkpoint protein by the NK cells. In some embodiments, the at least one immune checkpoint protein is CTLA4, PD-1, lymphocyte activation gene (LAG-3), NKG2A receptor, KIR2DL-1, KIR2DL-2, KIR2DL-3, KIR2DS -1 and / or KIR2DA-2, and combinations thereof.

[0123] In some embodiments, gene editing is used to "knock in" or alternatively increase expression of a target protein. In some embodiments, the expression of the target protein is about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about It may be increased by 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). For example, in some embodiments, the NK cells have been further gene edited to express CD47. In some embodiments, the NK cells are further genetically engineered to express HLA-E. A knocked-in gene can be knocked-in in combination with either a knocked-out or a disrupted gene.

[0124] In some embodiments, the genetically engineered NK cell population further comprises a genetically engineered T cell population. In some embodiments, the T cell population is at least partially, if not substantially, non-alloreactive. In some embodiments, the non-alloreactive T cell has at least one T cell receptor (TCR) such that the non-alloreactive T cell does not exhibit alloreactive effects on cells of the recipient subject. Contains two gene-edited subunits. In some embodiments, the T cell population is engineered to express a chimeric antigen receptor (CAR) with tropism for a tumor marker, the tumor marker being CD19, CD123, CD70, Her2, mesothelin, claudin 6. , BCMA, PD-L1, EGFR. In some embodiments, combinations of two or more of these tumor markers can be targeted. In some embodiments, the CAR expressed by the T cell has tropism for CD19. In some embodiments, the CAR expressed by the T cell has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or Has 99% sequence identity. In some embodiments, the CAR targets CD19. In some embodiments, the CAR decreases the antigenic potential of the encoded protein and / or increases one or more properties of the encoded protein (e.g., target recognition and / or binding properties). designed (e.g., operated) to Thus, according to some embodiments, the anti-CD19 CAR does not include specific sequences. For example, according to some embodiments, the anti-CD19 CAR is not constituted by SEQ ID NO: 116, SEQ ID NO: 105, 107, 104 or 106. In some embodiments, the anti-CD19 antibody fragment does not include one or more CDRs selected from SEQ ID NOs: 108-115.

[0125] In some embodiments, the TCR subunit of the modified T cell is TCRα. In some embodiments, the modification to the TCR of a T cell causes at least 80%, 85%, or 90% of the T cell population to no longer express detectable levels of the TCR. Similar to the edited NK cells disclosed herein, in some embodiments the T cells have increased levels of CIS, TGFBR, B2M, CIITA, TRIM29 and SOCS2 compared to unengineered T cells. have been further gene edited to reduce the expression of one or more of the following: or to express CD47 or HLA-E. In some embodiments, the T cell is further gene edited to disrupt expression of at least one immune checkpoint protein by the T cell, and the at least one immune checkpoint protein is CTLA4, PD-1, and lymphoma. selected from the globular activation gene (LAG-3).

[0126] Depending on the embodiment, gene editing of NK cells and / or T cells to reduce expression and / or gene editing to induce expression is performed using the CRISPR-Cas system. In some embodiments, the CRISPR-Cas system comprises a Cas selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, and combinations thereof. In some embodiments, the Cas is Cas9. In some embodiments, the CRISPR-Cas system includes Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1 , and combinations thereof. In some embodiments, gene editing of NK cells and / or T cells to reduce expression and / or gene editing to induce expression is performed using zinc finger nucleases (ZFNs). In some embodiments, gene editing of NK cells and / or T cells to reduce expression and / or gene editing to induce expression is performed using transcription activator-like effector nucleases (TALENs). be exposed.

[0127] In some embodiments, the genetically engineered NK cell and / or genetically engineered T cell is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 5. It has an OX40 subdomain. In some embodiments, the genetically engineered NK cell and / or genetically engineered T cell is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 7. CD3 zeta subdomain. In some embodiments, the genetically engineered NK cell and / or genetically engineered T cell has a sequence that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 11 or 489. It has mbIL15 encoded by.

[0128] The present invention also includes methods for treating cancer in a subject, including administering to the subject a genetically engineered NK cell population (and / or a genetically engineered T cell population), as disclosed herein. A method is provided for treating. Also provided herein is the use of genetically engineered NK cell populations (and / or genetically engineered T cell populations) as disclosed herein in the treatment of cancer. Also disclosed herein is the use of genetically engineered NK cell populations (and / or genetically engineered T cell populations) as disclosed herein in the manufacture of medicaments for the treatment of cancer. is provided.

[0129] Also provided herein are methods of treating cancer. In some embodiments, a method of treating cancer in a subject is provided that includes administering to the subject a population of genetically engineered immune cells, comprising: (i) a plurality of NK cells, the plurality of NK cells; but engineered to express cytotoxic receptors containing an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex, NK cells have significantly lower NK cells compared to non-engineered NK cells. cells are gene-edited to express reduced levels of the cytokine-inducible SH2-containing (CIS) protein, encoded by the CISH gene; reduced CIS expression is engineered through gene editing of the CISH gene and Engineered NK cells exhibit multiple factors that exhibit one or more of increased expansion capacity, increased cytotoxicity toward target cells, and increased persistence compared to NK cells expressing natural levels of CIS. NK cells; and optionally (ii) multiple T cells.

[0130] In some embodiments, the cytotoxic signaling complex comprises an OX-40 subdomain and a CD3 zeta subdomain. In some embodiments, NK cells are also engineered to express membrane-bound IL-15.

[0131] In some embodiments, if included, the plurality of T cells are substantially non-alloreactive. Advantageously, in some embodiments, the non-alloreactive T cell has a T cell receptor (TCR ) contains at least one modification to the subunit. In some embodiments, the T cells also have chimeric antigens with tropism for tumor markers that may be selected from CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, EGFR, and combinations thereof. engineered to express a receptor (CAR).

[0132] In some embodiments, the cytotoxic receptor expressed by the NK cell comprises (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z costimulatory subdomain. Contains signaling complexes containing domains. In some embodiments, the cytotoxic receptor is encoded by a polynucleotide that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 145. In some embodiments, the cytotoxic receptor has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 174 or 899. In some embodiments, the cytotoxic receptor expressed by the NK cell has tropism for CD19. In some embodiments, the cytotoxic receptor expressed by the NK cell has at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 178 or 901. . In some embodiments, the CAR expressed by the T cell has tropism for CD19. In some embodiments, CARs expressed by T cells (and NK cells) include (i) a tumor-binding domain that includes an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) an OX40 co-stimulatory sub-domain. contains a signaling complex that includes a CD3z co-stimulatory subdomain. In some embodiments, the polynucleotide encoding the CAR also encodes membrane-bound IL15. In some embodiments, the anti-CD19 antibody fragment comprises a single chain variable fragment (scFv) variable heavy chain (VH) domain and an scFv variable light chain (VL) domain. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 120 and the VL domain comprises the amino acid sequence of SEQ ID NO: 118.

[0133] In some embodiments, the NK cells and / or T cells have decreased expression of one or more of CIS, TGFBR, B2M, CIITA, TRIM29 and SOCS2 compared to unengineered T cells. , or further gene edited to express CD47 or HLA-E.

[0134] In some embodiments, the NK cells and / or T cells are further gene edited to disrupt expression of at least one immune checkpoint protein by the cells, and the at least one immune checkpoint protein is CTLA4, PD- 1, and lymphocyte activation gene (LAG-3), NKG2A receptor, KIR2DL-1, KIR2DL-2, KIR2DL-3, KIR2DS-1 and / or KIR2DA-2.

[0135] In some embodiments, the OX40 subdomain is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:5. In some embodiments, the CD3 zeta subdomain is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:7. In some embodiments, mbIL15 is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:11.

[0136] Depending on the embodiment of the methods disclosed herein applied, gene editing of NK cells and / or T cells to reduce expression, and / or gene editing to induce expression of CRISPR- This is done using the Cas system. In some embodiments, the CRISPR-Cas system comprises a Cas selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, and combinations thereof. In some embodiments, the Cas is Cas9. In some embodiments, the CRISPR-Cas system includes Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1 , and combinations thereof. In some embodiments, gene editing of NK cells and / or T cells to reduce expression and / or gene editing to induce expression is performed using zinc finger nucleases (ZFNs). In some embodiments, gene editing of NK cells and / or T cells to reduce expression and / or gene editing to induce expression is performed using transcription activator-like effector nucleases (TALENs). be exposed.

[0137] Additionally, provided herein is a mixed population of engineered cells for cancer immunotherapy that includes a plurality of NK cells, wherein the plurality of NK cells have an extracellular ligand binding domain, a transmembrane domain, and a transmembrane domain. Engineered to express cytotoxic receptors, including cytotoxic signaling complexes, NK cells exhibit reduced levels of cytotoxicity, encoded by the CISH gene, when compared to non-engineered NK cells. The reduced CIS expression was engineered through gene editing of the CISH gene, and the engineered NK cells expressed natural levels of CIS. T cells exhibit increased expansion capacity, increased cytotoxicity toward target cells, and increased persistence compared to NK cells, and multiple T cells have an increased ability to target at least one subunit of the T cell receptor (TCR). T cell populations are selected from one or more of CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, and EGFR. It has been engineered to express a chimeric antigen receptor (CAR) with tropism for specific tumor markers. In some embodiments, the cytotoxic receptor and / or CAR cytotoxic signaling complex comprises an OX-40 subdomain and a CD3 zeta subdomain. In some embodiments, the NK cells and / or T cells are engineered to express membrane-bound IL-15. In some embodiments, the cytotoxic receptor expressed by the NK cell has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 174 or 899. In some embodiments, the cytotoxic receptor expressed by the NK cell has at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 178 or 901. . In some embodiments, the CAR expressed by the T cell has at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 178 or 901.

[0138] Provided herein, in some embodiments, are genetically engineered immune cell populations for cancer immunotherapy that reduce the expression of cytokine-induced SH2-containing proteins encoded by the CISH gene by immune cells. Genetically modified to reduce the expression of transforming growth factor beta receptor by immune cells, Genetically modified to reduce the expression of natural killer group 2, member A (NKG2A) by immune cells and genetically modified to decrease the expression of the Cbl proto-oncogene B protein encoded by the CBLB gene by immune cells and to decrease the expression of the tricomponent motif-containing protein 29 protein encoded by the TRIM29 gene by immune cells. Genetically modified and / or genetically modified to reduce expression of the Suppressor of Cytokine Signaling 2 protein encoded by the SOCS2 gene by immune cells, as well as having tropism for tumor markers present on target tumor cells Contains a population of immune cells that have been genetically engineered to express chimeric antigen receptors (CARs). In some embodiments, the population comprises, consists of, or consists essentially of natural killer cells. In some embodiments, the population further comprises T cells. In some embodiments, the CAR is directed against CD19. In some embodiments, the CAR includes one or more humanized CDR sequences. In some embodiments, the CAR is directed against an NKG2D ligand. In some embodiments, genetic modifications to cells are performed using the CRISPR-Cas system. In some embodiments, the CRISPR-Cas system comprises a Cas selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, and combinations thereof. In some embodiments, the Cas is Cas9. In some embodiments, the modification is to CISH and the CRISPR-Cas system is guided by one or more guide RNAs selected from those comprising the sequence SEQ ID NO: 153, 154, 155, 156, or 157. the modification is to TGFBR2 and the CRISPR-Cas system is guided by one or more guide RNAs selected from those comprising the sequence SEQ ID NO: 147, 148, 149, 150, 151, or 152; is for NKG2A and the CRISPR-Cas system is guided by one or more guide RNAs selected from those containing the sequence SEQ ID NO: 158, 159, or 160; the modification is for CBLB and the CRISPR-Cas system is for NKG2A; The Cas system is guided by one or more guide RNAs selected from those containing the sequences SEQ ID NO: 164, 165, or 166; the modification is to TRIM29; or 169; and / or the modification is to SOCS2 and the CRISPR-Cas system has a sequence of SEQ ID NO: 171, 172, or guided by one or more guide RNAs selected from:

[0139] In some embodiments, genetic modification(s) are performed using zinc finger nucleases (ZFNs). In some embodiments, genetic modification(s) are performed using transcription activator-like effector nucleases (TALENs).

[0140] In some embodiments, the genetically modified immune cells exhibit increased cytotoxicity, increased survival, and / or increased anti-tumor cytokine release profile compared to unmodified immune cells. In some embodiments, the genetically modified immune cells are further genetically modified to reduce alloreactivity towards the cells when administered to a subject who is not the donor of the cells.

[0141] Also provided herein is a mixed population for cancer immunotherapy, through at least one modification to a subunit of the T cell receptor (TCR) selected from TCRα, TCRβ, TCRγ, and TCRδ. contains a T cell population that is substantially non-alloreactive, such that the TCR does not recognize major histocompatibility complex differences among the T cells of a recipient subject to whom a mixed population of immune cells has been administered. First, the T cell population is engineered to express chimeric antigen receptors (CARs) with tropism for tumor markers, including CD19, CD123, CD70, mesothelin, claudin 6, BCMA, PD-L1, A natural killer (NK) cell population selected from the group consisting of EGFR, and combinations thereof engineered to express a chimeric receptor containing an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex. and the extracellular ligand binding domain has tropism for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the modified TCR subunit is TCRα.

[0142] In some embodiments, the T cells and / or NK cells express reduced levels of MHC I and / or MHC II molecules such that the NK cells and T cells are allogeneic in the recipient. Modified to induce a reduced immune response from the subject's immune system. In some embodiments, the MHC I and / or MHC II molecules are beta-microglobulin and / or CIITA (class II major histocompatibility complex transactivator). In some embodiments, the T cell and / or NK cell further comprises a modification that disrupts expression of at least one immune checkpoint protein by the T cell and / or NK cell. Depending on the embodiment, the at least one immune checkpoint protein is CTLA4, PD-1, lymphocyte activation gene (LAG-3), NKG2A receptor, KIR2DL-1, KIR2DL-2, KIR2DL-3, KIR2DS -1 and / or KIR2DA-2, and combinations thereof.

[0143] In some embodiments, the NK cells and / or T cells are further modified to reduce or substantially eliminate CIS expression and / or function. In some embodiments, the NK cells are further engineered to express membrane-bound IL-15.

[0144] In some embodiments, the CAR expressed by the T cell comprises (i) a tumor binding domain comprising an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z costimulatory domain. Contains signaling complexes containing subdomains. In some embodiments, the T cells also express membrane-bound IL15. In some embodiments, mbIL15 is encoded by the same polynucleotide that encodes the CAR. In some embodiments, the anti-CD19 antibody comprises a single chain variable fragment (scFv) variable heavy chain (VH) domain and an scFv variable light chain (VL) domain. In some such embodiments, the VH domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 120. In some embodiments, the encoded VL domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 118. In some embodiments, the OX40 subdomain is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:5. In some embodiments, the CD3 zeta subdomain is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:7. In some embodiments, mbIL15 is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:11. In some embodiments, the CAR expressed by the T cell has at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 178 or 901. In some embodiments, the chimeric receptor expressed by the NK cell comprises (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z costimulatory subdomain. including signaling complexes. In some embodiments, the NK cell is further engineered to express membrane-bound IL15 (which may be encoded by the same polynucleotide encoding the chimeric receptor). In some embodiments, the chimeric receptor is encoded by a polynucleotide that has at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 145. In some embodiments, the chimeric receptor has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 174 or 899.

[0145] In some embodiments, the modification to the TCR causes at least 80% of the T cell population to no longer express detectable levels of TCR, but at least 70% of the T cell population expresses detectable levels of CAR. . In some embodiments, the T cells and / or NK cells contain B2M surface protein, cytokine-inducible SH2-containing protein (CIS) encoded by the CISH gene, transforming growth factor beta receptor, natural killer group 2, member A (NKG2A) receptor, Cbl proto-oncogene B protein encoded by the CBLB gene, ternary motif-containing protein 29 protein encoded by the TRIM29 gene, cytokine signaling encoded by the SOCS2 gene by T cells and / or NK cells 2 is further modified to reduce expression of one or more of the protein suppressors. In some embodiments, gene editing reduces the expression of any of these target proteins by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, It can be reduced by about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99%, or more (including any amounts between those listed). In some embodiments, the gene is completely knocked out such that expression of the target protein is undetectable. In some embodiments, target protein expression is about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% %, about 97%, about 98%, about 99%, or more (including any amounts between those listed). For example, in some embodiments, the T cells and / or NK cells have been further gene edited to express CD47. In some embodiments, the NK cells are further genetically engineered to express HLA-E. Any gene that is knocked in can be knocked in in combination with either a knocked out or alternatively disrupted gene.

[0146] In some embodiments, modification(s) to the TCR or further modification of NK cells or T cells is performed using the CRISPR-Cas system. In some embodiments, the CRISPR-Cas system comprises a Cas selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, and combinations thereof. In some embodiments, the Cas is Cas9. In some embodiments, the CRISPR-Cas system includes Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1 , and combinations thereof. In some embodiments, modification(s) to the TCR or further modification of NK cells or T cells is performed using zinc finger nucleases (ZFNs). In some embodiments, modification(s) to the TCR or further modification of NK cells or T cells is performed using transcription activator-like effector nucleases (TALENs).

[0147] Also provided herein is a mixed population of immune cells for cancer immunotherapy that provides a substantially non-allogeneic response due to at least one modification to a subunit of the T cell receptor (TCR). a T-cell population that is non-alloreactive, such that non-alloreactive T cells do not exhibit alloreactive effects on cells of the recipient subject; A population of T cells that has been engineered to express chimeric antigen receptors (CARs) with tropism for tumor markers selected from claudin-6, BCMA, PD-L1, EGFR, and combinations thereof, and natural killer ( NK) cell population engineered to express a chimeric receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex, the extracellular ligand binding domain comprising: and a NK cell population with tropism for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0148] Also provided herein are methods of treating cancer in a subject without inducing graft-versus-host disease, comprising administering to the subject a mixed population of immune cells according to the present disclosure. Provided herein is the use of mixed populations of immune cells according to the present disclosure in the treatment of cancer. Provided herein is the use of a mixed population of immune cells according to the present disclosure in the manufacture of a medicament for treating cancer.

[0149] In some embodiments, a method for treating cancer in a subject is provided comprising administering to the subject at least a first dose of a mixed population of immune cells, the mixed population of cells comprising CD19, CD123, , CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, EGFR, and combinations thereof. a non-alloreactive T cell population, a natural killer (NK) cell population engineered to express a chimeric receptor containing an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex. , the extracellular ligand binding domain has tropism for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0150] In some embodiments, the non-alloreactive T cell is a subunit of the T-cell receptor (TCR) such that the non-alloreactive T cell does not exhibit alloreactive effects on cells of the recipient subject. Contains at least one qualification to In some embodiments, the CAR expressed by the T cell has tropism for CD19. In some embodiments, the CAR expressed by the T cell comprises (i) a tumor binding domain comprising an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z costimulatory domain. Contains signaling complexes containing subdomains. In some embodiments, the polynucleotide encoding the CAR also encodes membrane-bound IL15. In some embodiments, the anti-CD19 antibody comprises a single chain variable fragment (scFv) variable heavy chain (VH) domain and an scFv variable light chain (VL) domain. In some embodiments, the VH domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 120, and the VL domain comprises, consists of, or consists of the amino acid sequence of SEQ ID NO: 118, or essentially consists of it. In some embodiments, the CAR expressed by the T cell has at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 178 or 901. In some embodiments, the chimeric receptor expressed by the NK cell comprises (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z costimulatory subdomain. including signaling complexes. In some embodiments, the polynucleotide encoding the chimeric receptor also encodes membrane-bound IL15. In some embodiments, the chimeric receptor is encoded by a polynucleotide that has at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 145. In some embodiments, the chimeric receptor has at least 95%80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 174 or 899. In some embodiments, the OX40 subdomain of the CAR and / or chimeric receptor is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:5. In some embodiments, the CD3 zeta subdomain of the CAR and / or chimeric receptor is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO:7. In some embodiments, mbIL15 expressed by T cells and / or NK cells is encoded by a sequence that has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 11.

[0151] In some embodiments, the cytotoxic receptor expressed by the immune cell targets CD19 and has SEQ ID NO: 179, 181, 183, 185, 187, 189, 191, 193, 195, and / or 197. Having at least 80%, 85%, 90%, or 95% sequence identity with the indicated nucleic acid sequence. In some embodiments, the CAR expressed by the immune cell targets CD19 and comprises SEQ ID NOs: 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, and / or 900-911. have at least 80%, 85%, 90%, or 95% sequence identity with any described amino acid sequence.

[0152] In some embodiments, a mixed population of immune cells for cancer immunotherapy is provided, the mixed population being a T cell population expressing a CAR with tropism for a tumor antigen, wherein the T cells are substantially a population of T cells that have been genetically modified to be non-alloreactive to the same tumor antigen, and a population of NK cells that express a CAR with tropism for the same tumor antigen. In some embodiments, a mixed population of immune cells for cancer immunotherapy is provided, the mixed population being a T cell population expressing a CAR with tropism for a tumor antigen, wherein the T cells are substantially and a population of NK cells that express a CAR with tropism for additional tumor antigens. In some embodiments, a mixed population of immune cells for cancer immunotherapy is provided, the mixed population comprising a substantially non-alloreactive T cell population and a chimeric receptor targeting tumor ligand. NK cell population expressing .

[0153] In some embodiments, the non-alloreactive T cell comprises at least one modification to a subunit of a T cell receptor (TCR), whereby the TCR is administered to a subject to whom the mixed population of immune cells is administered. recognize antigens without recognizing major histocompatibility complex differences between T cells. In some embodiments, the non-alloreactive T cell population is engineered to express a chimeric antigen receptor (CAR) with tropism for a tumor marker (eg, a tumor-associated antigen or tumor antigen). Depending on the embodiment, the CAR targets one or more of CD19, CD123, CD70, Her2, mesothelin, claudin 6 (but not other claudins), BCMA, PD-L1, EGFR. can be operated on.

[0154] In some embodiments, the NK cell population is engineered to express a chimeric receptor that includes an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex, and the extracellular ligand binding domain is MICA , MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, NK cells can also be engineered to express CARs, including CD19, CD123, CD70, Her2, mesothelin, claudin 6 (but not other claudins), BCMA, PD -L1, EGFR (or any other antigen such that both T cells and NK cells target the same antigen of interest).

[0155] In some embodiments, the T cell comprises a mutation that disrupts expression of at least one immune checkpoint protein by the T cell. For example, T cells can be mutated for immune checkpoint proteins selected from CTLA4, PD-1 and combinations thereof. In some embodiments, B7-1 / B7-2 blockade of CTLA4 is also used to reduce T cells that are maintained in an inactive state. Thus, in some embodiments, T cells are engineered such that they express mismatched or mutated CTLA4, while in some embodiments T cells are modified using exogenous agents, e.g. The ability of B7-1 / B7-2 to bind to presenting cells and / or alternatively interact with CTLA4 on antigen presenting cells can be inhibited. Similarly, in some embodiments, NK cells can be modified to disrupt expression of at least one checkpoint inhibitor. In some embodiments, eg, CDTLA4 or PD-1 is modified, eg, mutated, to reduce the ability of such checkpoint inhibitors to reduce NK cytotoxic responses. In some embodiments, lymphocyte activation gene 3 (LAG-3, CD223) is disrupted in NK cells (and / or T cells). In some embodiments, the inhibiting NKG2A receptor is mutated, knocked out, or inhibited, eg, by an antibody. Monalizumab, by way of non-limiting example, is used in some embodiments to disrupt inhibitory signaling by NKG2A receptors. In some embodiments, one or more killer inhibitory receptors (KIRs) on NK cells are disrupted and / or blocked (eg, via genetic modification). For example, in some embodiments, one or more of KIR2DL-1, KIR2DL-2, KIR2DL-3, KIR2DS-1 and / or KIR2DA-2 is disrupted or blocked, thereby causing HLA- C prevents their binding to MHC molecules. Additionally, in some embodiments, TIM3 is modified, mutated (e.g., via gene editing), or alternatively functionally disrupted (e.g., blocked by an antibody), thereby The immune cell's normal ability to suppress its response upon binding of the ligand is disrupted. In some such embodiments, disruption of TIM3 expression or function (e.g., by CRISP or other methods disclosed herein), optionally in combination with disruption of one or more immune checkpoint modulators, The administered T cells and / or NK cells have increased antitumor activity. Tim-3 is involved in galectin-9 secretion, the latter of which functions to impair the anticancer activity of cytotoxic lymphoid cells, including natural killer (NK) cells. TIM3 is also expressed in soluble form and prevents interleukin-2 (IL-2) secretion. Thus, in some embodiments, disruption of TIM3, expression, secretion, or pathway functionality provides for increased T cell and / or NK cell activity.

[0156] In some embodiments, TIGIT (also referred to as VSTM3) is modified, mutated (e.g., through gene editing), or alternatively functionally disrupted (e.g., blocked by an antibody), so that it disrupts its normal ability to suppress immune cell responses upon ligand binding. CD155 is a ligand for TIGIT. In some embodiments, TIGIT expression is reduced or knocked out. In some embodiments, TIGIT is blocked by a non-activating ligand or its activity is reduced through a competitive inhibitor of CD155 (the inhibitor does not activate TIGIT). TIGIT contains an inhibitory ITIM motif and, in some embodiments, is excised via gene editing, eg, using CRISP, or other methods disclosed herein. In such embodiments, TIGIT function is reduced, allowing for increased T cell and / or NK cell activity.

[0157] In some embodiments, adenosine receptor A1 is modified, mutated (e.g., via gene editing), or alternatively functionally disrupted (e.g., blocked by an antibody), thereby The immune cell's normal ability to suppress its response upon binding of the ligand is disrupted. Adenosine signaling is involved in tumor immunity as a result of its function as an immunosuppressive metabolite. Thus, in some embodiments, adenosine receptor A1 expression is reduced or knocked out. In some embodiments, adenosine receptor A1 is blocked by a non-activating ligand or its activity is reduced through a competitive inhibitor of adenosine, which inhibits the adenosine signaling pathway. not activated). In some embodiments, the adenosine receptor is modified, e.g., by gene editing using CRISpr, or other methods disclosed herein, to reduce its function or expression and to reduce its function or expression in T cells and / or NK. Allows for increased cellular activity.

[0158] In some embodiments, the modified TCR subunit is selected from TCRα, TCRβ, TCRγ, and TCRδ. In some embodiments, the modified TCR subunit is TCRα.

[0159] In some embodiments, modifications to the TCR are made using the CRISPR-Cas system. In some embodiments, disruption of expression of at least one immune checkpoint protein by T cells or NK cells is performed using a CRISPR-Cas system. For example, the Cas may be selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, and combinations thereof. In some embodiments, the Cas is Cas9. In some embodiments, the CRISPR-Cas system includes Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1 , and combinations thereof.

[0160] In some embodiments, modifications to the TCR are performed using zinc finger nucleases (ZFNs). In some embodiments, disruption of expression of at least one immune checkpoint protein by T cells or NK cells is performed using zinc finger nucleases (ZFNs). In some embodiments, modifications to the TCR are performed using transcription activator-like effector nucleases (TALENs). In some embodiments, disruption of expression of at least one immune checkpoint protein by T cells or NK cells is performed using transcription activator-like effector nucleases (TALENs). Combinations of ZFNs and TALENs (and optionally CRISPR-Cas) are used in some embodiments to modify either or both NK cells and T cells.

[0161] According to some embodiments, either the NK cells, the non-alloreactive T cells, or both have been further engineered to express membrane-bound IL-15.

[0162] Advantageously, mixed cell populations are useful in the methods provided herein and cancer in a subject can be treated without inducing graft-versus-host disease. In some embodiments, the method comprises administering to the subject a mixed population of non-alloreactive T cells expressing a CAR and engineered NK cells expressing a chimeric receptor. Also, the use of mixed populations of non-alloreactive T cells expressing CARs and engineered NK cells expressing chimeric receptors in the treatment of cancer and / or in the manufacture of drugs for the treatment of cancer. is provided. In still further embodiments, the NK cells and T cells are allogeneic with respect to the subject receiving them. In some embodiments, such combinations involve NK cells and T cells with tropism for the same target antigen. For example, in some embodiments, both the NK cells and the T cells (e.g., non-alloreactive T cells) are allogeneic with respect to the subject receiving them and target the same antigen, e.g., CAR It has been manipulated to express. In some embodiments, NK cells and T cells have another marker, such as CD123, CD70, Her2, mesothelin, claudin 6 (but not other claudins), BCMA, PD-L1, EGFR (or T Both NK cells and NK cells are comprised of target cells that express the same antigen of interest (such as any other antigen).

[0163] In some embodiments, the modification to the TCR causes at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the T cell population to no longer express detectable levels of the TCR, while , at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of the T cell population expresses a detectable level of the CAR. These cells are therefore primarily non-alloreactive and activated with anti-tumor-directed CARs. Additionally, in some embodiments, to help limit the immune response from allogeneic T cells, at least 50% of the engineered T cells express a detectable level of CAR, and at least 50% of the engineered T cells express a detectable level of CAR. does not express TCR surface proteins or B2M surface proteins.

[0164] In some embodiments, the NK cells are genetically modified to reduce the immune response that an allogeneic host may generate against non-autologous NK cells. In some embodiments, NK cells have been engineered such that they exhibit reduced expression of one or more MHC class I and / or one or more MHC class II molecules. In some embodiments, the expression of beta-microglobulin is expressed by expressing (or causing to express) a CAR with tropism for a tumor antigen, such as CD19 (or any other antigen disclosed herein). modified) NK cells are substantially, significantly, or completely reduced in at least a portion of the NK cells. In some embodiments, the expression of CIITA (class II major histocompatibility complex transactivator) has a tropism for a tumor antigen, such as CD19 (or any other antigen disclosed herein). At least a portion of NK cells that express (or are modified to express) CAR are substantially, significantly, or completely reduced. In some embodiments, such genetically modified NK cells are generated using CRIS Pr-Cas systems, TALENs, zinc fingers, RNAi or other gene editing techniques. As discussed herein, in some embodiments, alloreduced NK cells are used in combination with non-alloreactive T cells. In some embodiments, the NK cells are modified to express CD47, which helps the modified NK cells evade detection by the recipient's endogenous innate immune cells. In some embodiments, the T cells are modified in a similar manner. In some embodiments, both NK cells and T cells are modified to express CD47, which aids in the persistence of NK and / or T cells in the recipient, thus increasing anti-tumor efficacy. In some embodiments, the NK cells are modified to express HLA-G, which helps the modified NK cells evade detection by the recipient's endogenous innate immune cells. In some embodiments, the T cells are modified in a similar manner. In some embodiments, both NK cells and T cells are modified to express HLA-G, which aids in NK and / or T cell persistence in the recipient and thus increases anti-tumor efficacy. There is. In some embodiments, T cells and NK cells engineered to have reduced alloreactivity and express CARs against the same antigen are used to treat cancer in allogeneic patients. .

[0165] In some embodiments, genetically modified immune cell populations for cancer immunotherapy are provided that are genetically modified to reduce expression of transforming growth factor beta receptors by immune cells and that are genetically modified to reduce expression of transforming growth factor beta receptors by immune cells. contains a population of immune cells genetically engineered to express chimeric antigen receptors (CARs) with tropism for tumor markers present in the human body. In a further embodiment, a genetically modified immune cell population for cancer immunotherapy is provided, wherein the population is genetically modified to reduce expression of natural killer group 2, member A (NKG2A) receptors by immune cells, and which target It includes a population of immune cells that have been genetically engineered to express chimeric antigen receptors (CARs) with tropism for tumor markers present on tumor cells. In further embodiments, genetically modified immune cell populations for cancer immunotherapy are provided, genetically modified to reduce expression of cytokine-inducible SH2-containing protein encoded by the CISH gene by immune cells, and targeted It includes a population of immune cells that have been genetically engineered to express chimeric antigen receptors (CARs) with tropism for tumor markers present on tumor cells. CISH is an inhibitory checkpoint in NK cell-mediated cytotoxicity. In a further embodiment, a genetically modified immune cell population for cancer immunotherapy is provided, which is genetically modified to reduce expression of the Cbl proto-oncogene B protein encoded by the CBLB gene by immune cells and targeted tumor. It includes a population of immune cells that have been genetically engineered to express chimeric antigen receptors (CARs) with tropism for tumor markers present on the cells. CBLB is an E3 ubiquitin ligase and a negative regulator of NK cell activation. In a further embodiment, a genetically modified immune cell population for cancer immunotherapy is provided, wherein the population is genetically modified to reduce expression of the tricomponent motif-containing protein 29 protein encoded by the TRIM29 gene by the immune cells; It comprises a population of immune cells genetically engineered to express chimeric antigen receptors (CARs) with tropism for tumor markers present on target tumor cells. TRIM29 is an E3 ubiquitin ligase and a negative regulator of NK cell function after activation. In a further embodiment, a genetically modified immune cell population for cancer immunotherapy is provided, wherein the population is genetically modified to reduce expression of a suppressor of cytokine signaling 2 protein encoded by the SOCS2 gene by immune cells. , comprising a population of immune cells genetically engineered to express chimeric antigen receptors (CARs) with tropism for tumor markers present on target tumor cells. SOCS2 is a negative regulator of NK cell function. In some embodiments, the genetically modified immune cell population comprises NK cells, T cells, or a combination thereof. In some embodiments, additional immune cells such as gamma delta T cells, NK T cells, etc. are also included. In some embodiments, the CAR is directed against CD19. In some such embodiments, the CAR includes one or more humanized CDR sequences. In further embodiments, the CAR is directed against CD123. In some embodiments, the genetically modified cell is engineered to express more than one CAR with tropism for more than one target. Optionally, mixed populations of T cells and NK cells are used, each of which may or may not have tropism for the same cancer marker, depending on the embodiment. can express at least one CAR. In some embodiments, the cell expresses a CAR with tropism for an NKG2D ligand.

[0166] As discussed above, in some embodiments, cells are edited using a CRISpr-based approach. In some embodiments, the modification is to TGFBR2 and the CRISPR-Cas system is directed to the sequence SEQ ID NO: 147, 148, 149, 150, 151, or 152; 151 or 152 sequences and at least 80%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Guided by one or more guide RNAs selected from those comprising sequences with 96%, 97%, 98%, or 99% homology. In some embodiments, the modification is to NKG2A and the CRISPR-Cas system has at least 80 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology guided by one or more guide RNAs selected from those comprising a sequence having the following: In some embodiments, the modification is to CISH and the CRISPR-Cas system is directed to the sequence SEQ ID NO: 153, 154, 155, 156, or 157; and at least 80%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% homology. In some embodiments, the modification is to CBLB and the CRISPR-Cas system is at least 80% compatible with the sequence of SEQ ID NO: 164, 165, or 166, or a sequence comprising the sequence of SEQ ID NO: 164, 165, or 166. , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology. guided by one or more guide RNAs selected from those comprising sequences with In some embodiments, the modification is to TRIM29 and the CRISPR-Cas system is at least 80% compatible with the sequence of SEQ ID NO: 167, 168, or 169, or a sequence comprising the sequence of SEQ ID NO: 167, 168, or 169. , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology. guided by one or more guide RNAs selected from those comprising sequences with In some embodiments, the modification is to SOCS2 and the CRISPR-Cas system is at least 80% compatible with the sequence of, or comprising the sequence of SEQ ID NO: 171, 172, or 173. , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology. guided by one or more guide RNAs selected from those comprising sequences with In some embodiments, the guide RNA has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides.

[0167] In some embodiments, methods are provided for generating engineered T cells suitable for allogeneic transplantation, the methods comprising providing the T cells with an RNA-guided nuclease, a gRNA that targets a T cell receptor gene, and a CAR. The CAR comprises a vector containing a donor template containing a nucleic acid encoding (i) a tumor-binding domain containing an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and CD3z costimulatory a signaling complex containing subdomains, and (iv) a nucleic acid containing membrane-bound IL15 and encoding a CAR, flanked by left and right homologous arms at the T cell receptor locus, and (b) an engineered T cell in culture. Expand cells.

[0168] Further methods are also provided for engineered T cells suitable for allogeneic transplantation, methods targeting RNA-guided nucleases, and T cell receptor genes to disrupt expression of at least one subunit of the TCR. A nucleic acid vector containing a nucleic acid encoding a CAR is delivered to a T cell, and the CAR contains (i) a tumor-binding domain containing an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) a signaling complex containing an OX40 co-stimulatory subdomain and a CD3z costimulatory subdomain, and (iv) containing membrane-bound IL15 to expand engineered T cells in culture.

[0169] Additionally, methods are provided, e.g., methods of generating engineered T cells suitable for allogeneic transplantation, comprising: disrupting expression of at least one subunit of a TCR in a targeted region of a T cell receptor gene. delivering a nuclease capable of inducing a targeted double-stranded DNA break to a T cell, delivering a vector containing a nucleic acid encoding a CAR to the T cell, the CAR comprising: (i) CD19, CD123, CD70; , a tumor-binding domain comprising an antibody fragment that recognizes one or more of the following: , Her2, mesothelin, claudin-6, BCMA, PD-L1, and EGFR; (ii) a CD8 transmembrane domain; and (iii) an OX40 co-stimulatory subdomain. and (iv) membrane-bound IL15 to expand the engineered T cells in culture. In some embodiments, the method further comprises modifying the T cell by inactivating at least a first gene encoding an immune checkpoint protein. In some embodiments, the immune checkpoint gene is selected from the group consisting of PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, and 2B4.

[0170] A method of treating cancer is provided, the method generating T cells suitable for allogeneic transplantation according to embodiments disclosed herein, the T cells being donor-derived and expanded from the same donor. activated chimera containing an extracellular ligand binding domain with tropism for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. engineered NK cells that express receptors, generate engineered NK cell populations, and may further expand T cells and / or engineered NK cell populations, making T cells suitable for allogeneic transplantation. combining the populations and administering the combined NK cell and T cell populations to a subject that is allogeneic with respect to the donor.

[0171] A method of treating cancer is provided, the method generating T cells suitable for allogeneic transplantation according to embodiments disclosed herein, the T cells being derived from a donor and containing CD19, CD123, CD70. , modified to express a CAR with tropism for Her2, mesothelin, claudin 6 (but not other claudins), BCMA, PD-L1, or EGFR and transfected into a population of NK cells expanded from the same donor. Introducing and engineered NK cell populations to express CARs with tropism for CD19, CD123, CD70, Her2, mesothelin, claudin 6 (but not other claudins), BCMA, PD-L1, or EGFR. The T cells and / or engineered NK cell population may be further expanded by generating T cells suitable for allogeneic transplantation, combining the engineered NK cell population with the combined NK cell and T cell population. to a subject that is allogeneic with respect to the donor.

[0172] Further methods for treating a subject with cancer are also provided, the methods generating T cells suitable for allogeneic transplantation according to embodiments disclosed herein, the T cells being derived from a first donor. transducing a population of NK cells derived from a second donor and expanded from a second donor to confer tropism for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. Expressing activated chimeric receptors containing extracellular ligand-binding domains with and generating engineered NK cell populations, T cells and / or engineered NK cell populations may be further expanded by allogeneic transplantation. combining suitable T cells with an engineered NK cell population and administering the combined NK cell and T cell population to a subject that is allogeneic with respect to the first and second donors.

[0173] In some embodiments, provided herein are immune cells, and even immune cell populations, that express a CD19-directed chimeric receptor, the chimeric receptor comprising an extracellular anti-CD19 binding moiety, a hinge, and / or a chimeric receptor. or a transmembrane domain, and an intracellular signaling domain. Also provided herein are polynucleotides encoding CD19-directed chimeric antigen receptors, chimeric antigen receptors that include an extracellular anti-CD19 binding portion, a hinge and / or transmembrane domain, and an intracellular signaling domain (and , vectors for transfecting cells with the same) are provided.

[0174] Also provided herein, in some embodiments, is a polynucleotide encoding a CD19-directed chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular anti-CD19 binding moiety; The scFv hinge is a CD8 alpha hinge, contains a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain includes a CD3 zeta ITAM.

[0175] Provided herein, in some embodiments, are polynucleotides encoding CD19-directed chimeric antigen receptors, wherein the chimeric antigen receptor is an extracellular anti-CD19 binding portion, and the anti-CD19 binding portion is a variable of an scFv. Contains heavy chain or variable light chain of scFv, hinge is CD8 alpha hinge, transmembrane domain, transmembrane domain contains CD8 alpha transmembrane domain, intracellular signaling domain, intracellular signaling domain is CD3 zeta ITAM including, containing.

[0176] In some embodiments, the transmembrane domain comprises the CD8 alpha transmembrane domain. In some embodiments, the transmembrane domain comprises an NKG2D transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0177] In some embodiments, the intracellular signaling domain comprises or further comprises a CD28 signaling domain. In some embodiments, the intracellular signaling domain comprises or further comprises a 4-1BB signaling domain. In some embodiments, the intracellular signaling domain comprises or further comprises an OX40 domain. In some embodiments, the intracellular signaling domain comprises or further comprises a 4-1BB signaling domain. In some embodiments, the intracellular signaling domain comprises or further comprises a domain selected from ICOS, CD70, CD161, CD40L, CD44, and combinations thereof.

[0178] In some embodiments, the polynucleotide also encodes a truncated epidermal growth factor receptor (EGFRt). In some embodiments, EGFRt is expressed intracellularly as a soluble factor. In some embodiments, EGFRt is expressed in a membrane-bound form. In some embodiments, the polynucleotide also encodes membrane-bound interleukin-15 (mbIL15). Also provided herein are engineered immune cells (e.g., NK or T cells, or mixtures thereof) that express a CD19-directed chimeric antigen receptor encoded by the polynucleotides disclosed herein. provided. Further provided are methods of treating cancer in a subject that includes administering to the subject an engineered immune cell expressing a chimeric antigen receptor disclosed herein. In some embodiments, there is provided use of a polynucleotide disclosed herein in the treatment of cancer and / or in the manufacture of a medicament for the treatment of cancer.

[0179] In some embodiments, the anti-CD19 binding portion comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the VH domain has at least 95% identity to the VH domain amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the VL domain has at least 95% identity to the VL domain amino acid sequence set forth in SEQ ID NO:32. In some embodiments, the anti-CD19 binding portion is derived from the VH and / or VL sequences of SEQ ID NO: 33 or 32. For example, in some embodiments, the VH and VL sequences of SEQ ID NO: 33 and / or 32 are the subject of a humanization campaign and are therefore more easily expressed and / or more immune when administered to human subjects. Low origin. In some embodiments, the anti-CD19 binding moiety comprises an scFv that targets CD19, and the scFv comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 35 or a sequence at least 95% identical to SEQ ID NO: 35. In some embodiments, the anti-CD19 binding moiety comprises an scFv that targets CD19, and the scFv comprises a light chain variable region comprising the sequence of SEQ ID NO: 36 or a sequence at least 95% identical to SEQ ID NO: 36. In some embodiments, the anti-CD19 binding moiety comprises a light chain CDR comprising a first, second, and third complementarity determining region (LC CDR1, LC CDR2, and LC CDR3, respectively), and / or a first , the heavy chain CDRs containing the second and third complementarity determining regions (HC CDR1, HC CDR2, and HC CDR3, respectively). Depending on the embodiment, various combinations of LC CDRs and HC CDRs are used. For example, in one embodiment, the anti-CD19 binding moiety includes LC CDR1, LC CDR3, HC CD2, and HC, CDR3. In some embodiments other combinations are used. In some embodiments, the LC CDR1 comprises the sequence of SEQ ID NO: 37, or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 37. In some embodiments, the LC CDR2 comprises the sequence of SEQ ID NO: 38, or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 38. In some embodiments, the LC CDR3 comprises the sequence of SEQ ID NO: 39, or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 39. In some embodiments, the HC CDR1 comprises the sequence SEQ ID NO: 40, or a sequence at least about 95% homologous to the sequence SEQ ID NO: 40. In some embodiments, the HC CDR2 comprises a sequence of SEQ ID NO: 41, 42, or 43, or a sequence that is at least about 95% homologous to a sequence of SEQ ID NO: 41, 42, or 43. In some embodiments, the HC CDR3 comprises the sequence of SEQ ID NO: 44, or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 44.

[0180] In some embodiments, an anti-CD19 binding portion is also provided that includes a light chain variable region (VL) and a heavy chain variable region (HL), where the VL region comprises a first, second, and third complementarity determining region ( VL CDR1, VL CDR2, and VL CDR3, respectively), and the VH region includes first, second, and third complementarity determining regions (VH CDR1, VH CDR2, and VH CDR3, respectively). In some embodiments, the VL region comprises a sequence of SEQ ID NO: 45, 46, 47, or 48, or a sequence that is at least about 95% homologous to a sequence of SEQ ID NO: 45, 46, 47, or 48. In some embodiments, the VH region comprises a sequence of SEQ ID NO: 49, 50, 51 or 52, or a sequence that is at least about 95% homologous to a sequence of SEQ ID NO: 49, 50, 51 or 52.

[0181] In some embodiments, an anti-CD19 binding moiety is also provided that includes a light chain CDR that includes a first, second, and third complementarity determining region (LC CDR1, LC CDR2, and LC CDR3, respectively). In some embodiments, the anti-CD19 binding portion further comprises a heavy chain CDR that includes a first, second, and third complementarity determining region (HC CDR1, HC CDR2, and HC CDR3, respectively). In some embodiments, the LC CDR1 comprises the sequence SEQ ID NO: 53, or a sequence at least about 95% homologous to the sequence SEQ ID NO: 53. In some embodiments, the LC CDR2 comprises the sequence SEQ ID NO: 54, or a sequence at least about 95% homologous to the sequence SEQ ID NO: 54. In some embodiments, the LC CDR3 comprises the sequence SEQ ID NO: 55, or a sequence at least about 95% homologous to the sequence SEQ ID NO: 55. In some embodiments, the HC CDR1 comprises the sequence SEQ ID NO: 56, or a sequence at least about 95% homologous to the sequence SEQ ID NO: 56. In some embodiments, the HC CDR2 comprises the sequence SEQ ID NO: 57, or a sequence at least about 95% homologous to the sequence SEQ ID NO: 57. In some embodiments, the HC CDR3 comprises the sequence SEQ ID NO: 58, or a sequence at least about 95% homologous to the sequence SEQ ID NO: 58.

[0182] In some embodiments, the intracellular signaling domain of the chimeric receptor comprises an OX40 subdomain. In some embodiments, the intracellular signaling domain further comprises a CD3 zeta subdomain. In some embodiments, the OX40 subdomain comprises the amino acid sequence of SEQ ID NO: 6 (or a sequence at least about 95% homologous to the sequence of SEQ ID NO: 6) and the CD3 zeta subdomain comprises the amino acid sequence of SEQ ID NO: 8 ( or a sequence at least about 95% homologous to the sequence of SEQ ID NO: 8).

[0183] In some embodiments, the hinge domain comprises a CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises the amino acid sequence of SEQ ID NO: 2 (or a sequence at least about 95% homologous to the sequence of SEQ ID NO: 2).

[0184] In some embodiments, the immune cells also express membrane-bound interleukin-15 (mbIL15). In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 12, or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 12.

[0185] In some embodiments, the chimeric receptor further comprises the extracellular domain of an NKG2D receptor. In some embodiments, the immune cell expresses a second chimeric receptor comprising the extracellular domain of the NKG2D receptor, the transmembrane domain, the cytotoxic signaling complex, and optionally mbIL15. In some embodiments, the extracellular domain of the NKG2D receptor comprises the amino acid sequence of SEQ ID NO: 26, or a functional fragment of NKG2D comprising a sequence at least about 95% homologous to the sequence of SEQ ID NO: 26. In various embodiments, the chimeric antigen receptors disclosed herein and / or the immune cells engineered to express the chimeric receptors are NK cells. In some embodiments, T cells are used. In some embodiments, a combination of NK and T cells (and / or other immune cells) is used.

[0186] In some embodiments, provided herein are methods of treating cancer in a subject comprising administering to the subject an engineered immune cell that targets CD19 as disclosed herein. Ru. Also provided herein is the use of the CD19-targeting immune cells disclosed herein for the treatment of cancer. Also provided herein is the use of the CD19-targeting immune cells disclosed herein in the preparation of a medicament for the treatment of cancer. In some embodiments, the cancer treated is acute lymphocytic leukemia.

[0187] Some embodiments of the methods and compositions described herein relate to immune cells. In some embodiments, the immune cell expresses a CD19-directed chimeric receptor that includes an extracellular anti-CD19 moiety, a hinge and / or transmembrane domain, and / or an intracellular signaling domain. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cell is a T cell.

[0188] In some embodiments, the hinge domain comprises a CD8a hinge domain. In some embodiments, the hinge domain comprises an Ig4 SH domain.

[0189] In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD3 transmembrane domain.

[0190] In some embodiments, the signaling domain comprises an OX40 signaling domain. In some embodiments, the signaling domain comprises a 4-1BB signaling domain. In some embodiments, the signaling domain comprises a CD28 signaling domain. In some embodiments, the signaling domain comprises an NKp80 signaling domain. In some embodiments, the signaling domain comprises a CD16 IC signaling domain. In some embodiments, the signaling domain comprises a CD3zeta or CD3ζ ITAM signaling domain. In some embodiments, the signaling domain comprises an mbIL-15 signaling domain. In some embodiments, the signal transduction domain comprises a 2A cleavage domain. In some embodiments, the mIL-15 signaling domain is separated from the rest or another portion of the CD19-directed chimeric receptor by a 2A cleavage domain.

[0191] Some embodiments relate to methods comprising administering an immune cell described herein to a subject in need thereof. In some embodiments, the subject has cancer. In some embodiments, the administration treats, inhibits, or prevents the progression of cancer. [Brief explanation of drawings]

[0192]

Figure 1

[0193]

Figure 2

[0194] [Figure 3] Figure 3 shows further non-limiting examples of tumor-tropic chimeric antigen receptors.

[0195] [Figure 4] Figure 4 shows further non-limiting examples of tumor-tropic chimeric antigen receptors.

[0196] [Figure 5] Figure 5 shows further non-limiting examples of tumor-tropic chimeric antigen receptors.

[0197] [Figure 6-1] Figure 6 shows non-limiting examples of tumor-tropic chimeric antigen receptors with tropism for non-limiting examples of tumor markers. [Figure 6-2] Same as above.

[0198] [Figure 7] FIG. 7 shows further non-limiting examples of tumor-tropic chimeric antigen receptors with tropism for non-limiting examples of tumor markers.

[0199] [Figure 8-1] Figures 8A-8I schematically depict various pathways that are modified via the gene editing techniques disclosed herein. Figure 8A shows a schematic diagram of the inhibitory effect of TGF-beta release by tumor cells in the tumor microenvironment. Figure 8B shows a schematic diagram of the CIS / CISH negative regulatory pathway on IL-15 function. FIG. 8C shows a non-limiting sample for generating engineered non-alloreactive T cells and engineered NK cells for use in combination therapy, according to some embodiments disclosed herein. A schematic process flow is shown. Figure 8D shows a schematic diagram of signaling pathways that can lead to graft-versus-host disease. FIG. 8E shows a schematic diagram of how some embodiments disclosed herein reduce and / or eliminate graft-versus-host disease. Figure 8F shows a schematic diagram of the signaling pathways that can lead to host vs. graft rejection. FIG. 8G shows a schematic diagram of several embodiments disclosed herein that can reduce and / or eliminate host versus graft rejection. Figure 8H shows a schematic diagram of how edited immune cells can act on other edited immune cells in a mixed cell product. FIG. 8I shows a schematic diagram of how some embodiments disclosed herein can reduce and / or eliminate host immune effects on edited immune cells. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 8-5] Same as above. [Figure 8-6] Same as above. [Figure 8-7] Same as above. [Figure 8-8] Same as above. [Figure 8-9] Same as above.

[0200] [Figure 9-1] Figures 9A-9G show flow cytometry data regarding the use of various guide RNAs to reduce the expression of TGFBR2 by NK cells. Figure 9A shows control data. Figure 9B shows the data obtained from the use of guide RNA1; Figure 9C shows the data obtained from the use of guide RNA2; Figure 9D shows the data obtained from the use of guide RNA3; and Figure 9E shows the data obtained from the use of guide RNA1. Figure 9F shows the data obtained from the use of guide RNA1 and guide RNA2; Figure 9G shows the data obtained from the use of guide RNA2 and guide RNA3. Expression was assessed 7 days after electroporation with the indicated guide RNA. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above. [Figure 9-6] Same as above. [Figure 9-7] Same as above.

[0201] [Figure 10-1] Figures 10A-10G show next generation sequencing data on decreased expression of TGFBR2 by NK cells in response to electroporation with various guide RNAs. Figure 10A shows control data. Figure 10B shows the data obtained from the use of guide RNA1; Figure 10C shows the data obtained from the use of guide RNA2; Figure 10D shows the data obtained from the use of guide RNA3; Figure 10E shows the data obtained from the use of guide RNA1. Figure 10F shows the data obtained from the use of guide RNA1 and guide RNA2; Figure 10G shows the data obtained from the use of guide RNA2 and guide RNA3. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 10-6] Same as above. [Figure 10-7] Same as above.

[0202] [Figure 11-1] Figures 11A-11D show data comparing the cytotoxicity of NK cells against tumor cells in the presence or absence of TGF beta after knockdown of TGFBR2 expression by CRIS Pr / Cas9. Figure 11A shows changes in cytotoxicity after TGFBR2 knockdown using guide RNAs 1 and 2. Figure 11B shows changes in cytotoxicity after TGFBR2 knockdown using guide RNAs 1 and 3. Figure 11C shows changes in cytotoxicity after TGFBR2 knockdown using guide RNAs 2 and 3. FIG. 11D shows data for mock TGFBR2 knockdown. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above.

[0203] [Figure 12-1] Figures 12A-12F show flow cytometry data for reduced expression of TGFBR2 with additional guide RNAs. Figure 12A shows an unstained control of the same cells expressing TGFBR2. Figure 12B shows positive control data for NK cells expressing TGFBR2 in the absence of electroporation with CRIS Pr / Cas9 gene editing elements. Figure 12C shows knockdown of TGFBR2 expression using guide RNA4. Figure 12D shows knockdown of TGFBR2 expression using guide RNA5. Figure 12E shows knockdown of TGFBR2 expression using guide RNA6. Figure 12F shows knockdown of TGFBR2 expression using a 1:1 ratio of guide RNAs 2 and 3. Data were collected 4 days after electroporation with CRISPr / Cas9 gene editing elements. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above. [Figure 12-5] Same as above. [Figure 12-6] Same as above.

[0204] [Figure 13-1] Figures 13A-13F show flow cytometry data regarding the expression of a chimeric antigen receptor (here, anti-CD19 CAR, NK19-1) by NK cells when subjected to CRISPr / Cas9-mediated knockdown of TGFBR2. Figure 13A shows a negative control for NK cells not engineered to express NK19-1. Figure 13B shows positive control data for NK cells engineered to express NK19-1 but not electroporated with CRIS Pr / Cas9 gene editing elements. Figure 13C shows data on NK19-1 expression in NK cells subjected to electroporation with guide RNA4 to knock down TGFBR2 expression. Figure 13D shows data regarding NK19-1 expression in NK cells subjected to electroporation with guide RNA5 to knock down TGFBR2 expression. Figure 13E shows data regarding NK19-1 expression in NK cells subjected to electroporation with guide RNA6 to knock down TGFBR2 expression. Figure 13F shows data regarding NK19-1 expression in NK cells subjected to electroporation with guide RNAs 2 and 3 to knock down TGFBR2 expression. Data were collected 4 days after transduction using a vector encoding NK19-1. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above. [Figure 13-5] Same as above. [Figure 13-6] Same as above.

[0205] [Figure 14-1] Figures 14A-14D show the resistance of NK cells expressing a non-limiting example of a CAR (here, an anti-CD19 CAR, NK19-1) to TGFbeta inhibition as a result of single guide RNA knockdown of TGFBR2 expression. Indicates data regarding gender. Figure 14A shows the cytotoxicity of NK cells against Nalm6 tumor cells where NK cells were cultured with Nalm6 cells in TGF beta to recreate the tumor microenvironment. Figures 14B and 14C show control data (14C) in which the TGFB2 receptor was not knocked out, and Figure 14C shows selected data curves extracted from 14A to more clearly show the selected curves. . Figure 14D shows a schematic diagram of NK cell processing. NK cells were subjected to electroporation with CRISPr / Cas9 and a single guide RNA on day 0, cultured in high IL-2 medium for 1 day, and subsequently incubated with feeder cells (e.g., 4-1BBL and / or or modified K562 cells expressing mbIL15). On day 7, NK cells were transduced with virus encoding the NK19-1 CAR construct. On day 14, the cytotoxicity of the obtained NK cells was evaluated. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above.

[0206] [Figure 15-1] Figures 15A-15D show data regarding increased cytokine secretion by primary and NK19-1 expressing NK cells. Figure 15A shows data regarding secretion of IFNgamma. Figure 15B shows data on secretion of GM-CSF. Figure 15C shows data regarding granzyme B secretion. Figure 15D shows data regarding the secretion of TNF-alpha. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above.

[0207] [Figure 16-1] Figures 16A-16D show data regarding the knockout of NKG2A expression by NK cells using CRISPr / Cas9. Figure 16A shows the expression of NKG2A by NK cells subjected to a simulated gene editing protocol. Figure 16B shows NKG2A expression by NK cells after editing with CRIS Pr / Cas9 and guide RNA1. Figure 16C shows NKG2A expression by NK cells after editing with CRIS Pr / Cas9 and guide RNA2. Figure 16D shows NKG2A expression by NK cells after editing with CRIS Pr / Cas9 and guide RNA3. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above.

[0208] [Figure 17-1] Figures 17A-17B show data regarding the cytotoxicity of NK cells with knockout NKG2A expression (compared to mock cells). Figure 17A shows the cytotoxicity of NKG2A edited NK cells against REH cells 7 days after electroporation with CRIS Pr / Cas9 gene editing elements. Figure 17B shows flow cytometry data regarding the extent of HLA-E expression in REH cells. [Figure 17-2] Same as above.

[0209] [Figure 18] Figure 18 shows data regarding the cytotoxicity of NK cells in which cytokine-inducible SH2-containing protein (CIS) expression has been knocked out by mock NK cells or by gene editing of the CISH gene encoding CIS in humans. CIS is an inhibitory checkpoint in NK cell-mediated cytotoxicity. NK cell cytotoxicity against REH tumor cells was measured 7 days after electroporation with CRISPr / Cas9 gene editing elements.

[0210] [Figure 19-1] Figures 19A-19E present data regarding the effect of CISH knockout on the expression of a non-limiting example of a chimeric antigen receptor construct (here, anti-CD19 CAR, NK19-1) by NK cells. Figure 19A shows CD19 CAR expression (measured by FLAG expression included in this construct for detection purposes, whereas a further embodiment of the CAR does not include a tag) in control (untreated) NK cells. Figure 19B shows anti-CD19 CAR expression in NK cells subjected to CISH knockdown using CRIS Pr / Cas9 and guide RNA1. Figure 19C shows anti-CD19 CAR expression in NK cells subjected to CISH knockdown using CRIS Pr / Cas9 and guide RNA2. Figure 19D shows anti-CD19 CAR expression in NK cells subjected to simulated gene editing conditions (electroporation only). Figure 19E shows a Western blot showing the disappearance of the CIS protein band at 35 kDa, indicating knockout of the CISH gene. [Figure 19-2] Same as above. [Figure 19-3] Same as above. [Figure 19-4] Same as above. [Figure 19-5] Same as above.

[0211] [Figure 20-1] Figures 20A-20B show data from cytotoxicity assays using donor NK cells modified and / or engineered via gene editing to express CAR against Nalm6 tumor cells. Figure 20A shows data from a single challenge assay at a 1:2 effector:target ratio with data collected 7 days after transduction with the indicated CAR constructs. Figure 20B shows data from a dual challenge model in which control, edited, and / or edited / engineered NK cells were challenged with Nalm6 tumor cells at two time points. [Figure 20-2] Same as above.

[0212] [Figure 21-1] Figures 21A-21B show data related to long-term CISH knockout NK cell survival and cytotoxicity in culture. Figure 21A shows NK cell survival data over time when NK cells were treated as indicated. Figure 21B shows NK cell cytotoxicity data against tumor cells after 100 days of culture. [Figure 21-2] Same as above.

[0213] [Figure 22-1] Figures 22A-22E show cytokine release data by NK cells treated with indicated control, gene editing, or gene editing + manipulation to express CAR status. Figure 22A shows data on interferon gamma release. Figure 22B shows data on tumor necrosis factor alpha release. Figure 22C shows data on GM-CSF release. Figure 22D shows data on granzyme B release. Figure 22E shows data on perforin release. [Figure 22-2] Same as above. [Figure 22-3] Same as above.

[0214] [Figure 23-1] Figures 23A-23C are from cytotoxicity assays of mock NK cells or NK cells in which either Cbl proto-oncogene B (CBLB) or tripartite motif-containing protein 29 (TRIM29) expression was knocked out by CRISPR / Cas9 gene editing. The following data is shown. Figure 23A shows cytotoxicity data for NK cells knocked out with three different CBLB gRNAs, CISH gRNA5, or mock NK cells. Figure 23B shows cytotoxicity data for NK cells knocked out with three different TRIM19 gRNAs, CISH gRNA5, or mock NK cells. Figure 23C shows the timeline of electroporation and cytotoxicity assay. [Figure 23-2] Same as above. [Figure 23-3] Same as above.

[0215] [Figure 24-1] Figures 24A-24C show data from time course cytotoxicity assays of mock NK cells or NK cells in which either suppressor of cytokine signaling 2 (SOCS2) or CISH expression was knocked out by CRISPR / Cas9 gene editing. shows. Figure 24A shows cytotoxicity data over time of NK cells knocked out with three different SOCS2 gRNA, CISH gRNA2, or CD45 gRNA using the MaxCyte electroporation system. Figure 24B shows cytotoxicity data over time for NK cells knocked out with three different SOCs2 gRNA, CISH gRNA2 or CD45 gRNA using the Lonza electroporation system. Figure 24C shows the timeline of electroporation and cytotoxicity assay. [Figure 24-2] Same as above. [Figure 24-3] Same as above.

[0216] [Figure 25-1]Figures 25A-25D show schematic diagrams of non-limiting modifications made to CARs according to embodiments disclosed herein. Figure 25A shows a non-limiting embodiment in which a single immunosuppressive effector is incorporated into the hinge domain of the CAR. Figure 25B shows a non-limiting embodiment in which multiple domains are incorporated into a CAR and the first immunosuppressive effector is of a different type than the second immunosuppressive effector. In some embodiments, multiple immunosuppressive effectors are included (e.g., 2, 3, 4, 5, or more), and the immunosuppressive effector is the same as any other immunosuppressive effector in the CAR. may be different or different. Figure 25C shows a non-limiting embodiment in which the immunosuppressive effector is located in the target binding region (eg, in the linker between the heavy and light chains of the scFv). Figure 25D shows a non-limiting embodiment in which the immunosuppressive effector is located at the N-terminus of the chimeric antigen receptor. [Figure 25-2] Same as above. [Figure 25-3] Same as above. [Figure 25-4] Same as above.

[0217] [Figure 26-1] Figures 26A-26J show schematic diagrams of non-limiting modifications made to immune cells, which may be allogeneic immune cells. Figure 26A shows immune cells engineered to express immunosuppressive effectors in a membrane-bound format based on being tethered to transmembrane proteins (or fragments thereof). Figure 26B shows immune cells engineered to express multiple immunosuppressive effectors in a membrane-bound format. Figure 26C shows immune cells engineered to express multiple immunosuppressive effectors in a membrane-bound format, each two effectors being different from each other. Figure 26D shows immune cells engineered to express multiple immunosuppressive effectors tethered to a single transmembrane protein. Figure 26E shows immune cells engineered to express multiple immunosuppressive effectors tethered to a single transmembrane protein, with domains distinct from each other. Figure 26F shows an immune cell engineered to express multiple immunosuppressive effectors in a membrane-bound format, with each of the two effectors tethered to one of the transmembrane proteins being different from each other. FIG. 26G shows membrane-bound immunosuppressive effectors engineered to express membrane-bound immunosuppressive effectors in conjunction with a CAR containing one or more immunosuppressive effectors (although in some embodiments the CAR does not contain an immunosuppressive effector domain). Showing immune cells. Figure 26H shows immune cells engineered to express two immunosuppressive effectors in a membrane-bound format in conjunction with a CAR containing one or more immunosuppressive effectors. Figure 26I expresses a membrane-bound immunosuppressive effector in conjunction with a CAR containing one or more immunosuppressive effectors and also expresses one or more of HLA-E and / or HLA-G. Shown are engineered immune cells. In some embodiments, as schematically depicted, the engineered cells are NK cells that have been gene edited to disrupt B2M expression. Figure 26J shows CARs containing multiple immunosuppressive effectors, at least one immunosuppressive effector, as well as HLA-E and / or HLA-G, CD47, PD-L1, and / or poliovirus receptor (PVR). Immune cells engineered to express Although all are shown in a single cell, any combination or only one of the immunosuppressive effectors shown in the figures or disclosed herein may be used. [Figure 26-2] Same as above. [Figure 26-3] Same as above.

[0218] [Figure 27-1] Figures 27A-27JJ show schematic diagrams of non-limiting embodiments of immunosuppressive effector constructs provided herein. [Figure 27-2] Same as above. [Figure 27-3] Same as above. [Figure 27-4] Same as above. [Figure 27-5] Same as above. [Figure 27-6] Same as above. [Figure 27-7] Same as above. [Figure 27-8] Same as above. [Figure 27-9] Same as above. [Figure 27-10] Same as above.

[0219] [Figure 28] Figure 28 shows a non-limiting schematic of immune cells engineered to express chimeric UL18-B2M constructs.

[0220] [Figure 29-1] Figures 29A-29C show schematic diagrams related to the constructs and experimental designs disclosed herein. Figure 29A shows a schematic diagram of various scenarios in which immunosuppressive editing is performed or immunosuppressive constructs are created and the self versus non-self results obtained. Figure 29B shows a schematic diagram of a disulfide trap single chain trimer (dtSCT) used to express various immune evasion peptides (modified from Hansen et al. Current Protocols in Immunology, 2009). FIG. 29C shows a schematic of the experimental design to evaluate the efficacy of the hypoimmune constructs provided herein. [Figure 29-2] Same as above.

[0221] [Figure 30-1] Figures 30A-30C show data on the survival of B2M knockout T cells (as a percentage of viable T cells) after co-culture with NK cells engineered to express a non-limiting anti-CD19 CAR. Figure 30A shows the survival level at a 1:4 NK cell:T cell ratio. Figure 30B shows survival at a NK:T ratio of 1:2. Figure 30C shows survival at a 1:1 NK:T ratio. [Figure 30-2] Same as above.

[0222] [Figure 31-1] Figures 31A-31C show further data regarding HLA-E expression and the efficacy of using additional donor-derived T cells to reduce elimination of B2M-deficient T cells. Figure 31A shows T cell survival at a 1:4 NK cell:T cell ratio. Figure 31B shows T cell survival at a 1:2 NK cell:T cell ratio. Figure 31C shows T cell survival at a 1:1 NK cell:T cell ratio. [Figure 31-2] Same as above.

[0223] [Figure 32] Figure 32 shows a schematic diagram of the pathway by which HLA-E acts on NK cells.

[0224] [Figure 33-1] Figures 33A-33D show data on NKG2A expression in NK cells and B2M expression in T cells for two donors after co-culturing NK cells and T cells at various ratios. [Figure 33-2] Same as above.

[0225] [Figure 34] Figure 34 provides phenotypic characteristics for the characteristics of NK cells tested for suitability in mixed cell products.

[0226] [Figure 35] Figure 35 provides phenotypic characteristics for the characteristics of T cells tested for compatibility in mixed cell products.

[0227] [Figure 36-1] 36A-36F, T cells expressing anti-CD19 CAR and engineered for immune evasion were cultured with NK cells at various NK cell:T cell ratios according to embodiments disclosed herein. Figure 3 shows data on the percentage of B2M negative cells among live T cells in case of a. Figure 36A shows data for a NK:T ratio of 0:1, Figure 36B shows a NK:T ratio of 1:4, and Figure 36C shows a NK:T ratio of 1:2. , FIG. 36D shows a NK:T ratio of 1:1, FIG. 36E shows a NK:T ratio of 2:1, and FIG. 36F shows a NK:T ratio of 4:1. [Figure 36-2] Same as above.

[0228] [Figure 36-3] FIGS. 36G-36L show T cells expressing anti-CD19 CARs and engineered for immune evasion at various NK cell:T cell ratios, also with anti-CD19 cells, according to embodiments disclosed herein. Data are shown regarding the percentage of B2M negative cells among live T cells when cultured with CAR expressing NK cells. Figure 36G shows data for a NK:T ratio of 0:1, Figure 36H shows a NK:T ratio of 1:4, and Figure 36I shows a NK:T ratio of 1:2. , FIG. 36J shows a NK:T ratio of 1:1, FIG. 36K shows a NK:T ratio of 2:1, and FIG. 36L shows a NK:T ratio of 4:1. [Figure 36-4] Same as above.

[0229] [Figure 37-1]37A-37F show T cells expressing anti-CD19 CAR and engineered for immune evasion (harvested from a first donor) to various NK cells: T Data are shown regarding the percentage of B2M negative cells among live T cells when cultured with NK cells also expressing anti-CD19 CAR. Figure 37A shows data for a NK:T ratio of 0:1, Figure 37B shows a NK:T ratio of 1:4, and Figure 37C shows a NK:T ratio of 1:2. , FIG. 37D shows a NK:T ratio of 1:1, FIG. 37E shows a NK:T ratio of 2:1, and FIG. 37F shows a NK:T ratio of 4:1. [Figure 37-2] Same as above.

[0230] [Figure 38-1] FIGS. 38A-38F show T cells expressing anti-CD19 CAR and engineered for immune evasion (harvested from a second donor) to various NK cells: T Data are shown regarding the percentage of B2M negative cells among live T cells when cultured with NK cells also expressing anti-CD19 CAR. Figure 38A shows data for a NK:T ratio of 0:1, Figure 38B shows a NK:T ratio of 1:4, and Figure 38C shows a NK:T ratio of 1:2. , FIG. 38D shows a NK:T ratio of 1:1, FIG. 38E shows a NK:T ratio of 2:1, and FIG. 38F shows a NK:T ratio of 4:1. [Figure 38-2] Same as above.

[0231] [Figure 39-1] FIGS. 39A-39F show T cells expressing anti-CD19 CAR and engineered for immune evasion (collected from a third donor) to various NK cells: T Data are shown regarding the percentage of B2M negative cells among live T cells when cultured with NK cells also expressing anti-CD19 CAR. Figure 39A shows data for a NK:T ratio of 0:1, Figure 39B shows a NK:T ratio of 1:4, and Figure 39C shows a NK:T ratio of 1:2. , FIG. 39D shows a NK:T ratio of 1:1, FIG. 39E shows a NK:T ratio of 2:1, and FIG. 39F shows a NK:T ratio of 4:1. [Figure 39-2] Same as above.

[0232] [Figure 40-1] Figures 40A-40F show T cells expressing anti-CD19 CARs and engineered for immune evasion (either by TCR knockout, TCR knockout and B2M knockout, or B2M knockout and TCR knockout with HLA reexpression). Data on the percentage of B2M negative cells among live T cells (collected from the first donor) when cultured at various NK cell:T cell ratios with NK cells also expressing an anti-CD19 CAR. shows. Figure 40A shows data for a 0:1 NK:T ratio, Figure 40B shows a 1:4 NK:T ratio, and Figure 40C shows a 1:2 NK:T ratio. , FIG. 40D shows the NK:T ratio of 1:1, FIG. 40E shows the NK:T ratio of 2:1, and FIG. 40F shows the NK:T ratio of 4:1. [Figure 40-2] Same as above.

[0233] [Figure 41-1] Figures 41A-41F show T cells expressing anti-CD19 CARs and engineered for immune evasion (either by TCR knockout, TCR knockout and B2M knockout, or B2M knockout and TCR knockout with HLA reexpression). Data on the percentage of B2M negative cells among live T cells (collected from the first donor) when cultured at various NK cell:T cell ratios with NK cells also expressing an anti-CD19 CAR. shows. Figure 41A shows data for a 0:1 NK:T ratio, Figure 41B shows a 1:4 NK:T ratio, and Figure 41C shows a 1:2 NK:T ratio. , FIG. 41D shows the NK:T ratio of 1:1, FIG. 41E shows the NK:T ratio of 2:1, and FIG. 41F shows the NK:T ratio of 4:1. [Figure 41-2] Same as above.

[0234] [Figure 42] Figures 42A-42B show schematics of various approaches to reducing immune responses to engineered therapeutic cells. Figure 42A shows a schematic of a CAR containing a hypoimmune domain (HYPO) and various peptides or HLA sequences used to reduce the immune response to engineered therapeutic cells. Figure 42B shows various peptides and peptide combinations used to reduce immune responses to engineered therapeutic cells.

[0235] [Figure 43] FIG. 43 shows a schematic diagram of an experimental design that allows evaluation of various hypoimmunity-inducing embodiments disclosed herein.

[0236] [Figure 44] FIG. 44 shows cytotoxicity data for evaluating low immunity induction constructs according to embodiments provided herein.

[0237] [Figure 45-1] Figures 45A-45C depict additional cytotoxicity data collected while evaluating hypoimmune induction constructs in accordance with embodiments provided herein. Figure 45A shows cytotoxicity data using NK cells derived from the first donor against K562 cells expressing various hypoimmune constructs. Figure 45B shows cytotoxicity data using NK cells derived from additional donors against K562 cells expressing various hypoimmune constructs. Figure 45C shows cytotoxicity data using immortalized NK92 cells against K562 cells expressing various hypoimmune constructs. [Figure 45-2] Same as above. [Figure 45-3] Same as above.

[0238] [Figure 46] Figure 46 shows analysis of NKG2A expression by donor NK cells.

[0239] [Figure 47-1] Figures 47A-47R depict experimental setup and data regarding the cytotoxicity of various immune constructs provided in embodiments disclosed herein. Figure 47A shows a non-limiting schematic diagram of an assay procedure for evaluating the immune evasion effects of various constructs provided in embodiments herein. Figure 47B shows the cytotoxicity curve after co-culturing hypoimmune modified K562 cells with activated NK cells derived from the first donor. Figure 47C shows the detection of GFP expressed by modified K562 cells and shows a histogram representing the extent of expression of the indicated hypoimmune constructs. Figure 47D shows the cytotoxicity curve after co-culturing hypoimmune modified K562 cells with activated NK cells derived from a second donor. Figure 47E shows detection of GFP expressed by modified K562 cells and shows a histogram representing the extent of expression of the indicated hypoimmune constructs. Figure 47F shows the cytotoxicity curve after co-culturing hypoimmune modified K562 cells with activated NK cells derived from a third donor. Figure 47G shows detection of GFP expressed by modified K562 cells and shows a histogram representing the extent of expression of the indicated hypoimmune constructs. Figure 47H shows the cytotoxicity curve after co-culturing hypoimmune modified K562 cells with donor-derived activated NK cells. Figure 47I shows the detection of GFP expressed by modified K562 cells and shows a histogram representing the extent of expression of the indicated hypoimmune constructs. Figure 47J shows the cytotoxicity curve after co-culturing hypoimmune modified K562 cells with donor-derived activated NK cells. Figure 47K shows the detection of GFP expressed by modified K562 cells and shows a histogram representing the extent of expression of the indicated hypoimmune constructs. Figure 47L shows the cytotoxicity curve after co-culturing hypoimmune modified K562 cells with donor-derived activated NK cells. Figure 47M shows detection of GFP expressed by modified K562 cells and shows a histogram representing the extent of expression of the indicated hypoimmune constructs. Figure 47N shows the cytotoxicity curve after co-culturing hypoimmune modified K562 cells with donor-derived activated NK cells. Figure 47O shows detection of GFP expressed by modified K562 cells and shows a histogram representing the extent of expression of the indicated hypoimmune constructs. Figure 47P shows a tabulated summary of the indicated low-immunity construct screening. Figure 47Q shows the cytotoxicity curve after co-culturing hypoimmune modified K562 cells with donor-derived activated NK cells. Figure 47R shows the detection of GFP expressed by modified K562 cells and shows a histogram representing the extent of expression of the indicated hypoimmune constructs. [Figure 47-2] Same as above. [Figure 47-3] Same as above. [Figure 47-4] Same as above. [Figure 47-5] Same as above. [Figure 47-6] Same as above. [Figure 47-7] Same as above. [Figure 47-8] Same as above. [Figure 47-9] Same as above. [Figure 47-10] Same as above. [Figure 47-11] Same as above. [Figure 47-12] Same as above. [Figure 47-13] Same as above. [Figure 47-14] Same as above. [Figure 47-15] Same as above. [Figure 47-16] Same as above. [Figure 47-17] Same as above. [Figure 47-18] Same as above.

[0240] [Figure 48-1] Figures 48A-48B show cytotoxicity data from various hypoimmune constructs provided herein. Figure 48A shows cytotoxicity curves for K562 cells modified to express the indicated immune evasion constructs co-cultured with activated donor NK cells. Figure 48B shows the detection of GFP expressed by modified K562 cells and shows a histogram representing the extent of expression of the indicated hypoimmune constructs. [Figure 48-2] Same as above.

[0241] [Figure 49-1] Figures 49A-49C show data correlating the degree of expression of selected hypoimmune constructs provided herein with immunoprotection. Figure 49A is a summary table reflecting the cytotoxicity curves for K562NR cells highly expressing HLA-E and K562NR cells expressing HLA-E lowly and the relative expression of HLA-E constructs by both sets of cells. shows. Figure 49B shows cytotoxicity curves for K562NR cells that highly express CD47 and K562NR cells that express CD47 low as well as a summary table reflecting the relative expression of the CD47 construct by both sets of cells. Figure 49C shows cytotoxicity curves for K562NR cells highly expressing CD43_TM and K562NR cells expressing low CD47_TM as well as a summary table reflecting the relative expression of the CD47_TM construct by both sets of cells. [Figure 49-2] Same as above. [Figure 49-3] Same as above.

[0242] [Figure 50-1] Figures 50A-50D show data and resulting cytotoxicity from a non-limiting embodiment of gene editing combinations performed on NK cells. Figure 50A shows data regarding directed editing performed in NK cells and evaluation of cytotoxicity against target 786-O cells 14 days after editing was performed. FIG. 50B shows data corresponding to that shown in FIG. 50A, but not in the presence of TGFb. Figure 50C shows data regarding directed editing performed in NK cells and evaluation of cytotoxicity against target 786-O cells 21 days after editing was performed. FIG. 50D shows data corresponding to that shown in FIG. 9C, but not in the presence of TGFb. [Figure 50-2] Same as above. [Figure 50-3] Same as above. [Figure 50-4] Same as above.

[0243] [Figure 51-1] Figures 51A-51D show data and resulting cytotoxicity from a non-limiting embodiment of gene editing combinations performed on NK cells. Figure 51A shows data regarding directed editing performed in NK cells and evaluation of cytotoxicity against target 786-O cells 14 days after editing was performed. Figure 51B shows data corresponding to that shown in Figure 51A, but not in the presence of TGFb. Figure 51C shows data regarding directed editing performed in NK cells and evaluation of cytotoxicity against target ACHN cells 21 days after editing was performed. Figure 51D shows data corresponding to that shown in Figure 51C, but not in the presence of TGFb. [Figure 51-2] Same as above. [Figure 51-3] Same as above. [Figure 51-4] Same as above.

[0244] [Figure 52-1] Figures 52A-52B show data regarding the cytotoxicity of NK cells edited in genes directed against target cells. Figure 52A shows data regarding directed editing performed in NK cells and evaluation of cytotoxicity against target 786-O cells 21 days after editing was performed. Figure 52B shows data corresponding to that shown in Figure 52A, but not in the presence of adenosine. [Figure 52-2] Same as above.

[0245] [Figure 53-1] Figures 53A-53B show data regarding the cytotoxicity of NK cells edited at multiple gene targets. Figure 53A shows data for NK cells edited in the indicated manner and the cytotoxicity of the edited NK cells towards target 786-O cells after 14 days of editing. Figure 53B shows summary data of the trace in Figure 53A. Figure 53C shows data for NK cells edited in the indicated manner and the cytotoxicity of the edited NK cells against target 786-O cells after 14 days of editing in the presence of TGFb. Figure 53D shows summary data for the trace in Figure 53C. [Figure 53-2] Same as above. [Figure 53-3] Same as above. [Figure 53-4] Same as above.

[0246] [Figure 54-1] Figures 54A-54B show data regarding the cytotoxicity of NK cells edited at multiple gene targets. Figure 53A shows data for NK cells edited in the indicated manner and the cytotoxicity of the edited NK cells towards target ACHN cells after 14 days of editing. Figure 54B shows data for NK cells edited in the indicated manner and the cytotoxicity of the edited NK cells towards target ACHN cells after 14 days of editing in the presence of TGFb. [Figure 54-2] Same as above.

[0247] [Figure 55-1] Figures 55A-55B show data regarding the cytotoxicity of NK cells edited at multiple gene targets. Figure 55A shows data for NK cells edited in the indicated manner and the cytotoxicity of the edited NK cells towards target 786-O cells after 28 days of editing. Figure 55B shows summary data for the trace in Figure 55A. Figure 55C shows data on NK cells edited in the indicated manner and their cytotoxicity against target 786-O cells after 28 days of editing in the presence of TGFb. Figure 55D shows summary data for the trace in Figure 55C. [Figure 55-2] Same as above. [Figure 55-3] Same as above. [Figure 55-4] Same as above.

[0248] [Figure 56-1] Figures 56A-56D show data regarding the cytotoxicity of NK cells edited at multiple gene targets. Figure 56A shows data for NK cells edited in the indicated manner and the cytotoxicity of the edited NK cells towards target ACHN cells after 28 days of editing. Figure 56B shows summary data for the trace in Figure 56A. Figure 56C shows data for NK cells edited in the indicated manner and the cytotoxicity of the edited NK cells towards target ACHN cells after 28 days of editing in the presence of TGFb. Figure 56D shows summary data for the trace in Figure 56C. [Figure 56-2] Same as above. [Figure 56-3] Same as above. [Figure 56-4] Same as above. [Form for carrying out the invention]

[0249] Some embodiments of the methods and compositions provided herein relate to engineered immune cells and combinations thereof for use in immunotherapy. In some embodiments, engineered cells are engineered in multiple ways, eg, to express cytotoxicity-inducing receptor complexes. As used herein, the term "cytotoxic receptor complex" shall be given its ordinary meaning and includes (unless otherwise specified) a chimeric antigen receptor (CAR), a chimeric receptor ( In the case of NKG2D chimeric receptors, reference shall be made to NKG2D chimeric receptors (also called activated chimeric receptors). In some embodiments, the cells are further engineered to achieve modification of the cells' reactivity toward non-tumor tissue. Some embodiments relate to the modification of T cells through various genetic engineering methods such that the resulting T cells have reduced and / or eliminated alloreactivity. Such non-alloreactive T cells can also be engineered to express chimeric antigen receptors (CARs) that allow the non-alloreactive T cells to confer cytotoxic effects on tumor cells. I can do it. In some embodiments, natural killer (NK) cells are also engineered to express an urban-inducible receptor complex (eg, a chimeric antigen receptor or a chimeric receptor). In some embodiments, combinations of these engineered immune cell types are used in immunotherapy to provide both rapid (NK cell-based) and long-lasting (T cell-based) antitumor effects; It also advantageously has little or no graft-versus-host disease. Some embodiments include methods of using the compositions or cells in immunotherapy.

[0250] The term "anticancer effect" includes, but is not limited to, a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in cancer cell proliferation, a decrease in cancer cell survival, and / or the alleviation of various physiological symptoms associated with cancerous conditions, which may be expressed by a variety of means.

[0251] cell type Some embodiments of the methods and compositions provided herein refer to cells such as immune cells, e.g., immune cells such as T cells that include a chimeric receptor, such as a CD19-directed chimeric receptor. or as described herein, to include a nucleic acid encoding the chimeric receptor. Further embodiments relate to engineering the second set of cells to express another cytotoxic receptor complex, such as the NKG2D chimeric receptor complex disclosed herein. Still additional embodiments provide T cells (e.g., , regarding further genetic manipulation of donor T cells).

[0252] Traditional anticancer therapy relied on surgical approaches, radiation therapy, chemotherapy, or a combination of these methods. As research has improved our understanding of some of the mechanisms of certain cancers, this knowledge has been used to develop targeted cancer treatments. Targeted therapy uses specific drugs that target specific genes or proteins found in cancer cells or cells that support cancer growth, such as blood vessel cells, to suppress or stop the growth of cancer cells. It is a cancer treatment. More recently, genetic engineering has made it possible to develop approaches that harness specific aspects of the immune system to fight cancer. In some cases, a patient's own immune cells are modified to specifically eradicate that patient's cancer type. Various types of immune cells can be used, such as T cells, natural killer (NK cells), or combinations thereof, as described in more detail below.

[0253] to facilitate cancer immunotherapy, including target binding moieties (e.g., extracellular binders of ligands, or tumor marker-directed chimeric receptors expressed by cancer cells) and cytotoxic signaling complexes. Provided herein are polynucleotides, polypeptides, and vectors encoding chimeric antigen receptors (CARs). For example, some embodiments use tumor markers, e.g., CD19, CD123, CD70, Her2, among others, to facilitate targeting of immune cells to cancer and exert cytotoxic effects on cancer cells. , mesothelin, claudin 6, BCMA, EGFR. Also provided are engineered immune cells (eg, T cells or NK cells) that express such CARs. Also provided herein are polynucleotides, polypeptides, and vectors encoding constructs that, in some embodiments, include an extracellular domain that includes two or more subdomains, e.g., a first CD19 The targeting subdomain includes a CD19 binding moiety disclosed herein, and the second subdomain includes a C-type lectin-like receptor and a cytotoxic signaling complex. Also provided are engineered immune cells (eg, T cells or NK cells) that express such bispecific constructs. Also provided herein are methods of treating cancer and other uses of such cells for cancer immunotherapy.

[0254] Additionally, to facilitate cancer immunotherapy, polypeptides encoding chimeric receptors containing target binding moieties (e.g., extracellular binders of ligands expressed by cancer cells) and cytotoxic signaling complexes have been developed. Nucleotides, polypeptides, and vectors are provided herein. For example, some embodiments target tumor markers, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, among others, to facilitate targeting of immune cells to cancer and exert a cytotoxic effect on cancer cells. , ULBP4, ULBP5, and ULBP6. Also provided are engineered immune cells (eg, T cells or NK cells) that express such chimeric receptors. Also provided herein are constructs that, in some embodiments, include two or more subdomains, e.g., an extracellular domain that includes a first and second ligand-binding receptor and a cytotoxic signaling complex. Polynucleotides, polypeptides, and vectors encoding are provided. Additionally, engineered immune cells (e.g., T cells or NK cells) expressing such bispecific constructs (in some embodiments, the first and second ligand binding domains target the same ligand) ) will be provided. Also provided herein are methods of treating cancer and other uses of such cells for cancer immunotherapy.

[0255] Engineered cells for immunotherapy In some embodiments, cells of the immune system are engineered to have increased cytotoxic effects on target cells, such as tumor cells. For example, cells of the immune system can be engineered to contain tumor-tropic chimeric receptors and / or tumor-tropic CARs, as described herein. In some embodiments, white blood cells or leukocytes are used because their natural function is to protect the body against abnormal cell growth and infection. There are various types of white blood cells that play specific roles in the human immune system and are therefore a preferred starting point for the cell manipulations disclosed herein. White blood cells include granulocytes and agranulocytes (the presence or absence of granules in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils, and mast cells. Agranulocytes include lymphocytes and monocytes. Cells such as the following or other cells described herein encode a chimeric receptor, e.g., an NKG2D chimeric receptor, and / or a CAR, e.g., a CD19-directed CAR, or a chimeric receptor or CAR. can be engineered to contain nucleic acids that In some embodiments, cells may be engineered to co-express a membrane-bound interleukin 15 (mbIL15) costimulatory domain. As discussed in more detail below, in some embodiments, cells, particularly T cells, are further genetically modified to reduce and / or eliminate cell alloreactivity.

[0256] Monocytes for immunotherapy Monocytes are a subtype of white blood cells. Monocytes can differentiate into macrophages and myeloid dendritic cells. Monocytes are associated with the adaptive immune system and are responsible for the major functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of uptake of cellular material, or whole cells, followed by digestion and destruction of the engulfed cellular material. In some embodiments, monocytes are used in conjunction with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions described herein relate to monocytes comprising a tumor-tropic CAR, or a nucleic acid encoding a tumor-tropic CAR. Some embodiments of the methods and compositions disclosed herein provide tumor markers such as CD19, CD123, CD70, Her2, among others disclosed herein. For monocytes engineered to express CARs that target mesothelin, claudin 6, BCMA, EGFR, and membrane-bound interleukin 15 (mbIL15) costimulatory domains. Some embodiments of the methods and compositions disclosed herein are directed to ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally membrane Pertains to monocytes engineered to express activated chimeric receptors that target the binding interleukin 15 (mbIL15) co-stimulatory domain.

[0257] Lymphocytes for immunotherapy Lymphocytes are the other primary subtypes of white blood cells, including T cells (cell-mediated, cytotoxic adaptive immunity), natural killer cells (cell-mediated, cytotoxic innate immunity), and B cells (humoral , antibody-driven adaptive immunity). Although B cells are engineered according to some embodiments disclosed herein, some embodiments also include engineered T cells or engineered NK cells (a mixture of T cells and NK cells). , in some embodiments used either from the same donor or from different donors). Some embodiments of the methods and compositions disclosed herein provide tumor markers such as CD19, CD123, CD70, Her2, among others disclosed herein. Regarding lymphocytes engineered to express CARs that target mesothelin, claudin 6, BCMA, EGFR, and membrane-bound interleukin 15 (mbIL15) costimulatory domains. Some embodiments of the methods and compositions disclosed herein are directed to ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally membrane Relates to lymphocytes engineered to express activated chimeric receptors that target the binding interleukin 15 (mbIL15) co-stimulatory domain.

[0258] T cells for immunotherapy T cells can be distinguished from other lymphocyte subtypes (eg, B cells or NK cells) based on the presence of T cell receptors on the cell surface. T cells are divided into various subtypes including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosa-associated invariant T cells, and gamma delta T cells. It will be done. In some embodiments, specific subtypes of T cells are engineered. In some embodiments, mixed pools of T cell subtypes are manipulated. In some embodiments, there is no specific selection of T cell types engineered to express the cytotoxic receptor complexes disclosed herein. In some embodiments, specific techniques, such as the use of cytokine stimulation, are used to increase the expansion / collection of T cells with specific marker profiles. For example, in some embodiments, activation of certain human T cells, eg, CD4+ T cells, CD8+ T cells, is achieved through the use of CD3 and / or CD28 as stimulatory molecules. In some embodiments, the method comprises administering a therapeutically effective amount of T cells expressing a cytotoxic receptor complex and / or homing moiety, as described herein. A method of treating or preventing a disease is provided. In some embodiments, the engineered T cells are autologous cells, while in some embodiments the T cells are allogeneic cells. Some embodiments of the methods and compositions disclosed herein provide tumor markers such as CD19, CD123, CD70, Her2, among others disclosed herein. Concerning T cells engineered to express CARs that target mesothelin, claudin 6, BCMA, EGFR, and membrane-bound interleukin 15 (mbIL15) costimulatory domains. Some embodiments of the methods and compositions disclosed herein are directed to activated chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally relates to T cells engineered to express the membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0259] NK cells for immunotherapy Some embodiments include administering a therapeutically effective amount of natural killer (NK) cells expressing a cytotoxic receptor complex and / or homing moiety, as described herein. Methods of treating or preventing cancer or infectious diseases are provided. In some embodiments, the engineered NK cells are autologous cells, while in some embodiments the NK cells are allogeneic cells. In some embodiments, NK cells are preferred due to their relatively high natural cytotoxic potential. In some embodiments, the engineered cells disclosed herein can further upregulate NK cell cytotoxic activity against target cells (e.g., tumors or other disease cells). It would be unexpectedly beneficial to provide even more effective activity. Some embodiments of the methods and compositions described herein provide tumor markers such as CD19, CD123, CD70, Her2, among others disclosed herein. Concerning NK cells engineered to express CARs that target mesothelin, claudin 6, BCMA, EGFR, and optionally membrane-bound interleukin 15 (mbIL15) costimulatory domains. Some embodiments of the methods and compositions disclosed herein target activated chimeric receptors with ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and regarding NK cells engineered to express ULBP6 (among others), and optionally the membrane-bound interleukin 15 (mbIL15) costimulatory domain. In some embodiments, the NK cells are derived from the cell line NK-92. NK-92 cells are derived from NK cells but lack the major inhibitory receptors presented by normal NK cells, while retaining the majority of activating receptors. Some embodiments of the NK-92 cells described herein are engineered to silence certain additional inhibitory receptors, such as SMAD3, interferon-γ (IFNγ), granzyme B, and / or or regarding NK-92 cells that allow upregulation of perforin products. Further information regarding the NK-92 cell line is disclosed in WO1998 / 49268 and US Patent Application Publication No. 2002-0068044, which are incorporated herein by reference in their entirety. NK-92 cells, in some embodiments, are used in combination with one or more of the other cell types disclosed herein. For example, in one embodiment, NK-92 cells are used in combination with the NK cells disclosed herein. In further embodiments, NK-92 cells are used in combination with T cells disclosed herein.

[0260] Hematopoietic stem cells for cancer immunotherapy In some embodiments, hematopoietic stem cells (HSCs) are used in the immunotherapy methods disclosed herein. In some embodiments, the cell is engineered to express a homing moiety and / or a cytotoxic receptor complex. HSCs are used, in some embodiments, to take advantage of their ability to engraft for long-term blood cell production, e.g., to fight cancer remission, to provide targeted anti-cancer therapy. It can provide a continuous source of effector cells. In some embodiments, this continued production helps offset anergy or depletion of other cell types by, for example, the tumor microenvironment. In some embodiments, allogeneic HSCs are used, while in some embodiments autologous HSCs are used. In some embodiments, HSCs are used in combination with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions described herein provide tumor markers such as CD19, CD123, CD70, Her2, among others disclosed herein. Relating to stem cells such as hematopoietic stem cells engineered to express CARs that target mesothelin, claudin 6, BCMA, EGFR, and optionally the membrane-bound interleukin 15 (mbIL15) costimulatory domain. Some embodiments of the methods and compositions disclosed herein are directed to ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally membrane Pertains to hematopoietic stem cells engineered to express activated chimeric receptors that target the binding interleukin 15 (mbIL15) costimulatory domain.

[0261] induced pluripotent stem cells In some embodiments, induced pluripotent stem cells (iPSCs) are used in the immunotherapy methods disclosed herein. Using iPSCs, some embodiments include one or more genetic modifications at selected sites through iPSC differentiation, including but not limited to CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem and progenitor cells). ), hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, non-pluripotent cells such as T cells, NKT cells, NK cells, and B cells, or the same genetic modification at the same selected site. Take advantage of the ability of iPSCs to differentiate and induce into poorly differentiated cells. In some embodiments, iPSCs are used to generate iPSC-derived NK or T cells. In some embodiments, the cell is engineered to express a homing moiety and / or a cytotoxic receptor complex. In some embodiments, iPSCs are used in combination with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions described herein include tumor markers, e.g., CD19, CD123, CD70, Her2, mesothelin, among any other disclosed herein. Concerning stem cells such as induced pluripotent stem cells engineered to express CARs that target claudin-6, BCMA, EGFR, and optionally membrane-bound interleukin-15 (mbIL15) costimulatory domains. Some embodiments of the methods and compositions disclosed herein target activated chimeric receptors with ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and to induced pluripotent stem cells engineered to express ULBP6 (among others), and optionally the membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0262] Genetic manipulation of immune cells As discussed above, various cell types can be utilized for cellular immunotherapy. Additionally, as explained in more detail below and shown in the Examples, genetic modifications may affect these cells by increasing their efficacy (e.g., cytotoxicity) and / or persistence (e.g., active lifespan). ) can be done to increase one or more aspects of

[0263] gene editing targets Additional cell engineering strategies are provided herein that help further enhance the persistence of allogeneic cell therapy products, such as allogeneic CAR-T cells and / or allogeneic CAR-NK cells. Provided herein, in some embodiments, are genetically engineered immune cell populations for cancer immunotherapy, wherein the genetically engineered immune cells include cells such as NK cells and / or T cells. Genetically modified (eg, gene edited) at one, two, three or more genetic loci to enhance cytotoxic activity, persistence, or other characteristics.

[0264] As discussed herein, in some embodiments, NK cells are used for immunotherapy. In some embodiments provided herein, gene editing of NK cells advantageously causes the edited NK cells to resist and / or overcome various inhibitory signals generated in the tumor microenvironment. ability can be given. Tumors are known to produce a variety of signaling molecules that are intended to reduce the antitumor effects of immune cells. As discussed in more detail below, in some embodiments, gene editing of NK cells is performed on NK cells, T cells, a combination of NK and T cells, or any edited / manipulated cells provided herein. This tumor microenvironment suppresses the effect on immune cells. As discussed below, in some embodiments, gene editing is employed to reduce or knock out expression of a target protein, eg, by disrupting the underlying gene encoding the protein. In some embodiments, gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90% %, about 95%, about 97%, about 98%, about 99%, about 99%, or more (including any amounts between those listed). In some embodiments, the gene is completely knocked out such that expression of the target protein is undetectable. In some embodiments, gene editing is used to "knock in" or alternatively increase expression of a target protein. In some embodiments, the expression of the target protein is about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about It may be increased by 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). Unless otherwise indicated, the sequences provided in the guide RNA listed with deoxyribonucleotides refer to the target DNA and must also be taken as a reference to the actual guide used (e.g. When using ribonucleotides, the ribonucleotide uracil is used in place of the deoxyribonucleotide thymine, or vice versa, when thymine is used in place of uracil, and when enumerating either RNA or DNA sequences, Both form a complementary base pair to adenine). For example, a gRNA with the sequence ATGCTCAATGCGTC (SEQ ID NO: 995) also refers to the sequence AUGCUCAAUGCGUC (SEQ ID NO: 996), or a gRNA with the sequence AUGCUCAAUGCGUC (SEQ ID NO: 996) also refers to the sequence ATGCTCAATGCGTC (SEQ ID NO: 995). ).

[0265] As a non-limiting example, TGF-beta is one such cytokine released by tumor cells that results in immunosuppression within the tumor microenvironment. Immunosuppression reduces the ability of immune cells, even engineered CAR-immune cells, to destroy tumor cells, thereby allowing tumor progression. In some embodiments, immune checkpoint inhibitors are destroyed via gene editing, as discussed in detail below. In some embodiments, blockers of immunosuppressive cytokines in the tumor microenvironment are used, including blockers of their release or competitive agents that reduce the ability of signaling molecules to bind to and inhibit immune cells. Contains inhibitors. Such signaling molecules include, but are not limited to, TGF-beta, IL10, arginase, inducible NOS, reactive NOS, Arg1, indoleamine 2,3-dioxygenase (IDO), and PGE. 2 is included. However, in further embodiments, immune cells, such as NK cells, are provided in which the ability of NK cells (or other cells) to respond to certain immunosuppressive signaling molecules is disrupted and / or eliminated. For example, in some embodiments, NK cells or T cells have been gene edited to have decreased sensitivity to TGF-beta. TGF-beta is an inhibitor of NK cell function, at least to the level of proliferation and cytotoxicity. See, for example, FIG. 8A, which schematically shows some of the inhibitory pathways by which TGF-beta reduces NK cell activity and / or proliferation. Thus, according to some embodiments, TGF-beta receptor expression is knocked through gene editing such that the edited NK is resistant to the immunosuppressive effects of TGF-beta in the tumor microenvironment. be knocked down or knocked out. In some embodiments, the TGFB2 receptor is knocked down or knocked out via gene editing, for example, by using CRISPR-Cas editing. In other embodiments, small interfering RNAs, antisense RNAs, TALENs or zinc fingers are used. Other isoforms of the TGF-beta receptor (eg, TGF-beta 1 and / or TGF-beta 3) are edited in some embodiments. In some embodiments, TGF-beta receptors on T cells are knocked down via gene editing.

[0266] According to further embodiments, other regulators of one or more aspects of NK cell (or T cell) function are modulated via gene editing. Various cytokines provide negative or positive signals (similar to TGF-beta above) to immune cells. As a non-limiting example, IL15, as disclosed herein, is a positive regulator of NK cells, and is associated with NK cell homing, NK cell migration, NK cell expansion / proliferation, NK cell One or more of cytotoxicity and / or NK cell persistence can be increased. To suppress NK cells under normal physiological circumstances, the cytokine-inducible SH2-containing protein (CIS, encoded by the CISH gene) acts as an important negative regulator of IL-15 signaling in NK cells. do. As discussed herein, this suggests that IL15 biology is involved in multiple functions of NK cell functionality including, but not limited to, proliferation / expansion, activation, cytotoxicity, persistence, homing, migration, etc., among others. This is because it affects the side. Thus, according to some embodiments, editing CISH increases NK cell functionality across multiple functions, resulting in more effective and long-lasting NK cell therapy. In some embodiments, inhibitors of CIS are used in conjunction with engineered NK cell administration. In some embodiments, CIS expression is knocked down or knocked out through gene editing of the CISH gene, eg, by use of CRISPR-Cas editing. In other embodiments, small interfering RNAs, antisense RNAs, TALENs or zinc fingers are used. In some embodiments, CIS expression in T cells is knocked down via gene editing.

[0267] In some embodiments, CISH gene editing confers on NK cells an increased ability to home to target sites. In some embodiments, CISH gene editing confers on NK cells an increased ability to migrate, eg, within a tissue, eg, in response to a chemoattractant or away from a repellent. In some embodiments, CISH gene editing confers on NK cells an increased ability to be activated and thus exert an anti-tumor effect, for example. In some embodiments, CISH gene editing confers increased proliferative capacity on NK cells and, in some embodiments, allows generation of robust NK cell numbers from donor blood samples. Furthermore, in such embodiments, NK cells edited for CISH and engineered to express a CAR are more easily, robustly, and consistently expanded in culture. In some embodiments, CISH gene editing confers increased cytotoxicity on NK cells. In some embodiments, editing CISH synergistically increases the cytotoxic effect of engineered NK cells and / or engineered T cells expressing a CAR.

[0268] In some embodiments, CISH gene editing activates or inhibits a broad range of pathways. CIS proteins are negative regulators of IL15 signaling, for example by inhibiting the JAK-STAT signaling pathway. These pathways typically result in transcription of IL15-responsive genes (including CISH). In some embodiments, knockdown of CISH disinhibits JAK-STAT (eg, JAK1-STAT5) signaling and increases transcription of IL15-responsive genes. In some embodiments, knockout of CISH results in increased signaling through the mammalian target of rapamycin (mTOR), with a corresponding increase in expression of genes related to cellular metabolism and respiration. In some embodiments, knockout of CISH increases IL15-induced expression of IL-2Rα (CD25), but not IL-15Rα or IL-2 / 15Rβ, NK of IL15 and / or IL2. leading to increased cell membrane binding, increased phosphorylation of STAT-3 and / or STAT-5, and increased expression of anti-apoptotic proteins such as Bcl-2. In some embodiments, CISH knockout results in IL15-induced regulation of selected genes related to mitochondrial function (eg, electron transport chain and cellular respiration) and cell cycle. Thus, in some embodiments, knocking out CISH by gene editing increases NK cell cytotoxicity and / or persistence, at least in part through metabolic reprogramming. In some embodiments, a negative regulator of cellular metabolism, such as TXNIP, is downregulated in response to CISH knockout. In some embodiments, promoters for cell survival and proliferation, including BIRC5 (survivin), TOP2A, CKS2, and RACGAP1, are upregulated after CISH knockout, whereas promoters such as TGFB1, ATM, and PTCH1 are upregulated after CISH knockout. Proliferative or proapoptotic proteins are downregulated. In some embodiments, the CISH knockout is CXCL-10, IL2, TNF, IFNg, IL13, IL4, Jnk, PRF1, STAT5, PRKCQ, IL2 receptor beta, SOCS2, MYD88, STAT3, STAT1, TBX21, LCK, altering the state of signaling (e.g. activation or inactivation do).

[0269] In some embodiments, gene editing of immune cells can also provide an unexpected increase in expansion, persistence and / or cytotoxicity of the edited immune cells. As disclosed herein, engineered cells (eg, cells expressing CAR) can also be edited, and the combination provides robust cells for immunotherapy. In some embodiments, the editing unexpectedly allows for improved NK cell expansion, persistence, and / or cytotoxicity. In some embodiments, knockout of CISH expression in NK cells removes a strong negative regulator of IL15-mediated signaling in NK cells, disinhibits NK cells, and increases NK cell homing, NK cells enabling one or more of migration, NK cell activation, expansion, cytotoxicity and / or persistence. Additionally, in some embodiments, editing can increase NK cell and / or T cell function in an otherwise suppressive tumor microenvironment. In some embodiments, CISH gene editing results in increased NK cell expansion, persistence and / or cytotoxicity without the need for exogenously provided Notch ligands.

[0270] As discussed above, T cells engineered to express a CAR or chimeric receptor are employed in some embodiments. Also, as mentioned above, T cells express T cell receptors (TCR) on their surface. As disclosed herein, in some embodiments, autoimmune cells are returned to the original donor of the cells. In such embodiments, immune cells such as NK cells or T cells are obtained from a patient, expanded, genetically modified (e.g., with a CAR or chimeric receptor), and / or further expanded. It is also often reintroduced to the patient. As disclosed herein, in some embodiments, allogeneic immune cells are transferred to a subject that is not the original donor of the cells. In such embodiments, immune cells such as NK cells or T cells are obtained from a donor, expanded, genetically modified (e.g., with a CAR or chimeric receptor), and / or further expanded. It is also often administered to a subject.

[0271] Allogeneic immunotherapy has several hurdles to overcome. In immunocompetent hosts, administered allogeneic cells are rapidly rejected, known as host versus graft rejection (HvG). This substantially limits the effectiveness of the administered cells, especially their persistence. In immunocompetent hosts, allogeneic cells are able to engraft. However, when the administered cells include T cells (some embodiments disclosed herein use mixed populations of NK cells and T cells), the endogenous T cell receptor (TCR) specificity is , recognizing host tissue as foreign, resulting in graft-versus-host disease (GvHD). GvHD can cause significant tissue damage in the host (cell recipient). Some embodiments disclosed herein address both of these hurdles, thereby enabling effective and safe allogeneic immunotherapy. In some embodiments, gene editing can advantageously serve to reduce and / or avoid graft-versus-host disease (GvHD). A non-limiting embodiment of such an approach using a mixed population of NK cells and T cells is schematically illustrated in Figure 8C, where NK cells are engineered to express a CAR and T cells are engineered to express a CAR. as well as being edited to make T cells non-alloreactive. Figure 8D schematically depicts the mechanism by which graft-versus-host disease occurs. Both allogeneic T cells and allogeneic NK cells are engineered to express tumor-targeting CARs and introduced into the host. However, T cells still have a natural T cell receptor (TCR). This TCR recognizes the HLA type of the host cell as "non-self" and can exert cytotoxicity against the host cell. FIG. 8E shows a non-limiting embodiment of how graft-versus-host disease can be reduced or alternatively avoided by gene editing of T cells. Briefly, this approach can perform gene editing to knock out the native TCR on T cells, as discussed in more detail below. When the TCR is deficient, allogeneic T cells are unable to detect the "non-self" HLA of the host cell and are therefore not induced to exert cytotoxicity against the host cell. Accordingly, in some embodiments, T cells are subjected to gene editing to reduce native T cell functionality and / or reduce or eliminate native T cell expression. In some embodiments, CRISPR is used to knock out the TCR. These and other embodiments are discussed below.

[0272] T cell receptors (TCRs) are cell surface receptors involved in T cell activation in response to antigen presentation. The TCR consists of two different protein chains, which are heterodimers. The majority of human T cells have a TCR consisting of alpha (α) and beta (β) chains (encoded by separate genes). A small proportion of T cells have a TCR consisting of gamma and delta chains (γ / δ chains) (the cells are known as gamma-delta T cells).

[0273] Rather than recognizing intact antigens (like immunoglobulins), T cells are activated by processed peptide fragments in association with MHC molecules. This is known as MHC restriction. When the TCR recognizes differences between donor and recipient MHC, that recognition stimulates T cell proliferation and potential development of GVHD. In some embodiments, genes encoding any of TCRα, TCRβ, TCRγ, and / or TCEδ reduce the tendency of donor T cells to recognize differences between donor MHC and host MHC, thereby reducing allogeneic Destroyed or alternatively modified to reduce antigen and GVHD recognition.

[0274] T cell-mediated immunity involves a balance between costimulatory and inhibitory signals that help fine-tune the immune response. Inhibitory signals, also known as immune checkpoints, allow the avoidance of autoimmunity (eg, self-tolerance) and also limit immune-mediated damage. Immune checkpoint protein expression is often altered by tumors, increasing immune resistance in tumor cells and limiting the effectiveness of immunotherapy. CTLA4 downregulates the amplitude of T cell activation. In contrast, PD1 limits T cell effector function in peripheral tissues during inflammatory responses and also limits autoimmunity. Immune checkpoint blockade, in some embodiments, helps overcome barriers to activation of functional cellular immunity. In some embodiments, it comprises cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4; also known as CD152) and programmed cell death protein 1 (PD1 or PDCD1, also known as CD279). Competitive antibodies specific for inhibitory ligands on T cells are used to augment immunotherapy.

[0275] In some embodiments, genetically modified T cells are provided that are non-alloreactive and highly active. In some embodiments, the T cells are further modified such that certain immune checkpoint genes are inactivated, and thus immune checkpoint proteins are not expressed by the T cells. In some embodiments, this is done in the absence of manipulation or disruption of the CD3z signaling domain (eg, the TCR is still able to initiate T cell signaling).

[0276] In some embodiments, genetic inactivation of TCR alpha and / or TCR beta in combination with inactivation of immune checkpoint genes in T lymphocytes from allogeneic donors significantly reduces the risk of GVHD. In some embodiments, this includes at least a portion of one or more substituted protein chains (alpha, beta, gamma, and / or delta) involved in the recognition of MHC differences between donor and recipient cells. This is done by removing. In some embodiments, this is done while still allowing T cell proliferation and activity.

[0277] In some embodiments where allogeneic cells are administered, the receiving subject may receive some other adjunctive treatment to support or alternatively augment the function of the administered immune cells. be. In some embodiments, the subject may be pretreated (eg, with radiation or chemotherapy). In some embodiments, adjuvant treatment includes administration of lymphocyte growth factors (such as IL-2).

[0278] Additionally, in some embodiments, the editing improves the persistence of the administered cells (either NK cells, T cells, or other), e.g., by masking the cells against the host immune response. be able to. In some cases, the recipient's immune cells attack donor cells, especially donor cells from allogeneic donors, and this is known as host versus graft disease (HvG). Figure 8F shows a schematic diagram of HvG in which host T cells, along with native / functional TCRs, identify HLA on donor T and / or donor NK cells as non-self. In such cases, binding of the host T cell TCR to the allogeneic cell HLA results in the removal of the allogeneic cell and reduces the persistence of the donor engineered NK / T cells. Regarding HvG, subjects receiving the cells require suppression of their immune systems to prevent rejection of the administered allogeneic T cells. In some embodiments, glucocorticoids are used and include, but are not limited to, beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, among others. Activation of glucocorticoid receptors in the recipient's own T cells changes the expression of genes involved in the immune response and leads to decreased levels of cytokine production, which leads to T cell anergy and T cell activation. interference (in the recipient). Other embodiments relate to the administration of antibodies that can deplete specific types of recipient immune cells. One such target is CD52, which is expressed at high levels on T and B lymphocytes and at lower levels on monocytes, but is absent on granulocytes and myeloid progenitors. Treatment or pretreatment of recipients with alemtuzumab, a humanized monoclonal antibody directed against CD52, has been shown to induce rapid depletion of circulating lymphocytes and monocytes, which is associated with Given the reduction in , it reduces the possibility of HvG. Immunosuppressive drugs may limit the effectiveness of administered allogeneic engineered T cells. Accordingly, as disclosed herein, some embodiments relate to genetically engineered allogeneic donor cells that are resistant to immunosuppressive therapy. In some embodiments, as discussed in more detail below, immune cells such as NK cells and / or T cells achieve their persistence by evading cytotoxic responses from host immune cells. Edited to be elongated (in addition to being engineered to express a CAR). In some embodiments, gene editing to remove one or more HLA molecules from allogeneic NK and / or T cells reduces removal by host T cells. In some embodiments, the allogeneic NK and / or T cells are edited to knock out one or more of beta-2 microglobulin (HLA class I molecule) and CIITA (HLA class II molecule). Ru. Figure 8G schematically illustrates this approach.

[0279] In some embodiments of mixed allogeneic cell therapy, engineered cell populations actually target each other, for example, when treated cells are edited to remove HLA molecules to avoid HvG. become For example, such editing of CAR T cells can render the edited allogeneic CAR T cells vulnerable to cytotoxic attack by CAR NK cells and removal by host NK cells. This is caused by the lack of "self" inhibitory signals commonly presented by KIR molecules. Figure 8H schematically depicts this process. In some embodiments, gene editing is used to knock in the expression of one or more "masking" molecules that mask allogeneic cells from the host immune system and from fratricide by other administered engineered cells. can do. Figure 8I schematically illustrates this approach. In some embodiments, the protein can be expressed on the surface of allogeneic cells to inhibit targeting by NK (both engineered and host NK); Prolongs the persistence of both CAR-T and CAR-NK. In some embodiments, gene editing is used to knock in CD47, whose expression effectively functions as a "don't eat me" signal. In some embodiments, gene editing is used to knock in expression of HLA-E. HLA-E binds to both the inhibitory and activating receptors NKG2A and NKG2C, respectively, present on the surface of NK cells. However, NKG2A is more highly expressed on most human NK cells, and therefore, in some embodiments, expression on engineered cells of HLA-E is less likely to be edited into HLA-E (or resulting in an inhibitory effect of NK cells (both host and donor) on naturally expressed) cells. Additionally, in some embodiments, one or more viral HLA homologues are knocked in such that they are expressed by engineered NK and / or T cells, thus the ability of the virus to evade the host immune system. is applied onto the cells. In some embodiments, these approaches advantageously prolong the persistence of both allogeneic CAR-T and CAR-NK.

[0280] In some embodiments, gene editing ( Either knockout or knockin) is achieved through the targeted introduction of DNA breaks and subsequent DNA repair mechanisms. In some embodiments, double-strand breaks in DNA are repaired by non-homologous end joining (NHEJ), and enzymes are used to directly join the DNA ends together to repair the break. However, in some embodiments, double-strand breaks are repaired by homology-directed repair (HDR), which is advantageously more accurate, thereby allowing sequence-specific cleavage and repair. HDR is used for the regeneration of the defective DNA sequence at the break point, such as a vector carrying the desired genetic element (e.g., an insertion element that disrupts the coding sequence of the TCR) in the sequence that is homologous to the flanking sequences of the double-strand break. using the homologous sequence as a template. This results in the desired change (eg, insertion) being inserted at the site of the DSB.

[0281] In some embodiments, gene editing is accomplished by one or more of a variety of engineered nucleases. In some embodiments, restriction enzymes are used, particularly when double-strand breaks are desired in multiple regions. In some embodiments, bioengineered nucleases are used. using one or more of the following: Znc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and / or clustered regularly spaced short palindromic repeat (CRISPR / Cas9) systems. to specifically edit genes encoding one or more TCR subunits.

[0282] Meganucleases are characterized by their ability to recognize and cleave large DNA sequences (14-40 base pairs). In some embodiments, meganucleases from the LAGLIDADG family are used and subjected to mutagenesis and screening to target specific sites such as in the TCR or CISH, or any other target gene disclosed herein. Meganuclease variants are generated that recognize unique sequence(s) of . TCR target sites can be easily identified. Further information on target sites within the region of the TCR can be found in U.S. Patent Application Publication No. 2018 / 0325955, U.S. Patent Application Publication No. 2015 / 0017136, each of which is incorporated herein by reference in its entirety. be incorporated into. In some embodiments, two or more meganucleases or functional fragments thereof are fused to create a hybrid enzyme that recognizes a desired target sequence within a target gene (eg, CISH).

[0283] In contrast to meganucleases, ZFNs and TALENs have a function based on a nonspecific DNA cleavage catalytic domain linked to a specific DNA sequence that recognizes peptides such as zinc fingers or transcription activator-like effectors (TALEs). . Advantageously, ZFNs and TALENs thus enable sequence-independent DNA cleavage and have a high degree of sequence specificity in target recognition. Zinc finger motifs naturally function in transcription factors to recognize specific DNA sequences for transcription. The C-terminal portion of each finger is responsible for specific recognition of DNA sequences. Although the sequences recognized by ZFNs are relatively short (e.g., approximately 3 base pairs), in some embodiments the recognition site has been characterized. , combinations of 10 or more zinc fingers are used, thereby allowing targeting of specific sequences such as parts of the TCR (or immune checkpoint inhibitors). The combined ZFNs are then fused with the catalytic domain(s) of an endonuclease, such as FokI (optionally FokI heterodimer), to induce targeted DNA cleavage. Additional information regarding the use of ZFNs to edit TCR and / or immune checkpoint inhibitors can be found in US Pat. No. 9,597,357, incorporated herein by reference.

[0284] Transcription activator-like effector nucleases (TALENs) are specific DNA-binding proteins characterized by an array of 33 or 34 amino acid repeats. Similar to ZFNs, TALENs fuse the DNA cleavage domain of a nuclease to a TALE domain, allowing for the introduction of sequence-independent double-stranded DNA breaks, allowing highly precise target site recognition. do. TALENs can create double-strand breaks at target sites, which are repaired by error-prone non-homologous end joining (NHEJ), resulting in gene disruption by introducing small insertions or deletions. TALENs are advantageously used in some embodiments, at least in part due to their higher specificity in DNA binding, reduced off-target effects, and ease of construction of DNA binding domains. .

[0285] CRISPR (Clustered Regularly Arranged Short Palindromic Repeats) is a genetic element that bacteria use as a defense against viruses. This repeat is a short sequence derived from the viral genome and integrated into the bacterial genome. Cas (CRISPR-associated protein) processes these sequences and cleaves matching viral DNA sequences. By introducing a plasmid containing a Cas gene into eukaryotic cells and specifically constructing CRISPR, a eukaryotic genome can be cleaved at any desired position. Additional information regarding CRISPR can be found in US Patent Publication No. 2014 / 0068797, which is incorporated herein by reference. In some embodiments, CRISPR is used to manipulate gene(s) encoding target genes to be knocked out or knocked in, e.g., CISH, TGFBR2, TCR, B2M, CIITA, CD47, HLA-E, etc. be done. In some embodiments, CRISPR is used to edit genes encoding one or more of the TCRs of a T cell and / or one or more immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor is selected from one or more of CTLA4 and PD1. In some embodiments, CRISPR is used to cleave one or more of TCRα, TCRβ, TCRγ, and TCRδ. In some embodiments, the TCR is truncated without affecting the function of the CD3z signaling domain of the TCR. Depending on the embodiment and which target gene is to be edited, class 1 or class 2 Cas are used. In some embodiments, class 1 Cas is used, and the Cas types are: I, IA, IB, IC, ID, IE, IF, IU, III, IIIA, IIIB, IIIC, IIID, IV, Selected from IVA, IVB, and combinations thereof. In some embodiments, the Cas is Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, and the like. selected from the group consisting of a combination of In some embodiments, class 2 Cas is used and the Cas type is selected from II, IIA, IIB, IIC, V, VI, and combinations thereof. In some embodiments, the Cas is from the group consisting of Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a (formerly known as C2c2), Cas13b, Cas13c, CasX, CasY, and combinations thereof. selected. In some embodiments, class 2 CasX is used, the CasX can form a complex with a guide nucleic acid, the complex can bind to target DNA, and the target DNA binds the non-target strand and the target strand. include. In some embodiments, class 2 CasY is used, and the CasY is capable of binding and modifying the target nucleic acid and / or the polypeptide associated with the target nucleic acid.

[0286] In some embodiments, as discussed above, editing of CISH advantageously confers increased expansion, cytotoxicity and / or persistence on edited cells, particularly edited NK cells. Furthermore, in some embodiments, modifications to the TCR, including modifications to TCRα, do not affect signaling through the CD3 complex and allow T cell proliferation. In one embodiment, TCRα is inactivated by expression of pre-Tα in the cell, thus restoring a functional CD3 complex in the absence of a functional alpha / beta TCR. As disclosed herein, non-alloreactive modified T cells also express CARs to direct non-alloreactive T cell specificity toward tumor markers, but appear to be independent of MHC. be operated on. Combinations of editing are used in some embodiments, such as, for example, a combined TCR and CISH knockout, or a CISH knockout and a CD47 knockin, as non-limiting examples.

[0287] In some embodiments, expression of ADORA2A (adenosine 2a receptor) increases overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. to be reduced and / or eliminated. In some embodiments, ADORA2A is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, ADORA2A is isolated using a Cas nuclease guided by another use of the following ADORA2A-specific guide RNA: Crispr as disclosed elsewhere herein. - disrupted and / or knocked out using a Cas-mediated approach (eg, Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). Loss of ADORA2A expression induces decreased sensitivity to adenosine, a well-established immunosuppressant for T cells and NK cells. Thus, according to some embodiments, the gene-edited ADORA2A increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0288] In some embodiments, TGFBR2 expression is decreased and / or to increase overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. Remove. In some embodiments, TGFBR2 is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, TGFBR2 is isolated using a Cas nuclease guided by another use of the following TGFBR2-specific guide RNA: Crispr as disclosed elsewhere herein. - disrupted and / or knocked out using a Cas-mediated approach (eg, Cas9). In some embodiments, gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). In NK cells, TGFBR2 is a powerful checkpoint in NK cell-mediated tumor immunity, whereas in T cells, knockout of TGFBR2 rescues TGF-β1-induced car T cell exhaustion. Thus, according to some embodiments, gene-edited TGFBR2 increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0289] In some embodiments, NKG2A expression is decreased and / or reduced to increase overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. or be removed. In some embodiments, NKG2A is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, NKG2A is isolated using a Cas nuclease guided by another use of the following NKG2A-specific guide RNA: Crispr as disclosed elsewhere herein. - disrupted and / or knocked out using a Cas-mediated approach (eg, Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). NKG2A binds to HLA-E and is recognized as an MHC recognition receptor. Since NKG2A is an inhibitory receptor, loss of NKG2A expression induces increased activation of constituent cells. In NK and T cells, loss of NKG2A results in increased activation and cytotoxicity towards tumor cells expressing HLA-E. Thus, according to some embodiments, gene-edited NKG2A increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0290] In some embodiments, expression of cytokine signaling 2 (SOCS2) increases overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. to be reduced and / or eliminated. In some embodiments, SOCS2 is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, SOCS2 is isolated using a Cas nuclease guided by another use of the following SOCS2-specific guide RNA: Crispr as disclosed elsewhere herein. - disrupted and / or knocked out using a Cas-mediated approach (eg, Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). SOCS proteins are negative regulators of cytokine responses, and SOCS2 specifically negatively regulates NK cell development through inhibition of JAK2 activity. Loss of SOCS2 expression in NK cells induces increased NK cell development and overall cytotoxicity. Thus, according to some embodiments, gene-edited SOCS2 increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0291] In some embodiments, expression of Casitas B-lineage lymphoma-b (Cbl-b) results in overall activity in T cells and / or NK cells, or other cell types provided herein. reduced and / or removed to increase the In some embodiments, Cbl-b is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, Cbl-b is isolated using a Cas nuclease guided by another use of the following Cbl-b-specific guide RNA: Crispr-b as disclosed elsewhere herein. Disrupted and / or knocked out using Cas-mediated approaches (eg, Cas9). In some embodiments, expression of Casitas B-lineage lymphoma-b (Cbl-b) is reduced and / or eliminated to increase overall activation in the resulting T cells and NK cells. In some embodiments, Cbl-b is isolated using a Crispr-Cas-mediated approach (e.g., Cas9) disclosed elsewhere herein, but with the following Cbl-b-specific guide RNA: SEQ ID NO. Destroyed and / or knocked out by another use of one or more uses of 552-555. In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). Cbl-b is an E3 ubiquitin ligase that negatively regulates T cell activation. Loss of Cbl-b expression on NK cells and T cells indicates increased anti-tumor immunity. Furthermore, Cbl-b-deficient T cells and NK cells are resistant to PD-L1 / PD-1-mediated suppression. Thus, according to some embodiments, the gene-edited Cbl-b increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein. , or otherwise enhance its effectiveness.

[0292] In some embodiments, expression of beta-2 microglobulin (B2-microglobulin) reduces overall activity in the resulting T cells and / or NK cells, or other cell types provided herein. reduced and / or removed to increase the In some embodiments, B2-microglobulin is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, B2-microglobulin is produced using a Cas nuclease guided by another use of the following B2-microglobulin-specific guide RNAs: SEQ ID NOS: 290-299. disrupted and / or knocked out using Crispr-Cas-mediated approaches (eg, Cas9) as disclosed in the section. In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). Loss of B2-microglobulin expression induces markedly reduced levels of MHC class I molecules, in both NK cells and T cells, and a reduction in B2-microglobulin throughout autologous and allogeneic cells. can modulate cellular recognition. Thus, according to some embodiments, gene-edited B2-microglobulin inhibits cytotoxicity, persistence, and immune evasion of engineered NK cells, T cells, or other cells disclosed herein. increase or otherwise enhance its effectiveness.

[0293] In some embodiments, Ig and ITIM domains (TIGIT) to increase overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. reduce and / or eliminate expression of T cell immune receptors with In some embodiments, TIGIT is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, TIGIT is produced using a Cas nuclease guided by another use of the following TIGIT-specific guide RNA: Crispr as disclosed elsewhere herein. - disrupted and / or knocked out using a Cas-mediated approach (eg, Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). TIGIT is a checkpoint receptor associated with T cell and NK cell exhaustion. Loss of TIGIT expression on NK cells prevents NK cell exhaustion and promotes NK cell-dependent tumor immunity. Loss of TIGIT expression in T cells likewise results in downstream activation of T cells. Thus, according to some embodiments, gene editing TIGIT increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0294] In some embodiments, the expression of programmed cell death protein-1 (PD-1) increases overall cell death in the resulting T cells and / or NK cells, or other cell types provided herein. reduced and / or removed to increase active activation. In some embodiments, PD-1 is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, PD-1 is synthesized using a Cas nuclease guided by another use of the following PD-1-specific guide RNAs: SEQ ID NOS: 511-514, as described elsewhere herein. Disrupted and / or knocked out using disclosed Crispr-Cas-mediated approaches (eg, Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). PD-1 plays an inhibitory role in immune regulation, downregulating global function by suppressing immune cell activity. Loss of PD-1 expression in NK cells increases overall cytotoxicity due to increased secretion of interferon-gamma, granzyme B, and perforin. Similarly, T cells with loss of PD-1 expression exhibit increased cytotoxicity and global caspase activation. Thus, according to some embodiments, gene-edited PD-1 increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein. or otherwise enhance its effectiveness.

[0295] In some embodiments, expression of T cell immunoglobulin and mucin domain-containing-3 (TIM-3) in the resulting T cells and / or NK cells, or other cell types provided herein. reduced and / or removed to increase overall activation. In some embodiments, TIM-3 is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, TIM-3 is synthesized using a Cas nuclease guided by another use of the following TIM-3-specific guide RNAs: SEQ ID NOS: 515-518 as described elsewhere herein. Disrupted and / or knocked out using disclosed Crispr-Cas-mediated approaches (eg, Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). TIM-3 is an inhibitory receptor involved in immune checkpoint function. Loss of TIM-3 expression increases overall cytotoxicity in engineered NK and T cells, as well as decreases NK and T cell exhaustion and increases in constitutive cells lacking TIM-3 expression. It brings you an effector function. Thus, according to some embodiments, gene-edited TIM-3 increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein. or otherwise enhance its effectiveness.

[0296] In some embodiments, CD38 expression is decreased and / or reduced to increase overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. or be removed. In some embodiments, CD38 is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, CD38 is isolated using a Cas nuclease guided by another use of the following CD38-specific guide RNA: Crispr as disclosed elsewhere herein. - disrupted and / or knocked out using a Cas-mediated approach (eg, Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). CD38 plays a role in the maturation cycle of immune cells, and hematological cancers can often present upregulated CD38. Loss of CD38 expression on constitutive NK cells allows for greater cytotoxicity due to decreased fratricide. Wild-type NK cells autoexpress CD38, which has downstream self-targeting effects on wild-type NK cells. In T cells, loss of CD38 expression on constitutive T cells results in increased cytotoxicity. Thus, according to some embodiments, gene-edited CD38 increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0297] In some embodiments, expression of T cell receptor alpha (TCRα) increases overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. reduced and / or eliminated to make In some embodiments, TCRα is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, TCRα is isolated using a Cas nuclease guided by another use of the following TCRα-specific guide RNA: Crispr as disclosed elsewhere herein. - disrupted and / or knocked out using a Cas-mediated approach (eg, Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). The T cell receptor (TCR) is a protein complex found on T cells that recognizes MHC molecules. Loss of certain TCRs and preferential expression of others can lead to increased cytotoxicity in engineered cells due to selective targeting and increased recognition by constituent cells. Thus, according to some embodiments, the gene editing TCR increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0298] In some embodiments, CISH expression is decreased and / or increased to increase overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. or remove. In some embodiments, CISH is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, CISH is performed using a Cas nuclease guided by the following CISH-specific guide RNAs: SEQ ID NOS: 562-565, or another use of other guides disclosed herein. Disrupted and / or knocked out using Crispr-Cas-mediated approaches (eg, Cas9) disclosed elsewhere herein. In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). In CD8+ T cells, CISH actively silences TCR signaling to maintain tumor resistance, and CISH has been shown to be a downstream negative regulator of IL-15 receptor signaling. ing. In NK and T cells, CISH plays a role in checkpoint maturation and proliferation. Thus, according to some embodiments, gene editing CISH increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0299] In some embodiments, CEACAM1 expression is decreased and / or reduced to increase overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. or be removed. In some embodiments, CEACAM1 is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, CEACAM1 is isolated using a Cas nuclease guided by another use of the following CEACAM1-specific guide RNA: Crispr as disclosed elsewhere herein. - disrupted and / or knocked out using a Cas-mediated approach (eg, Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). CEACAM1 is a checkpoint inhibitor for both NK cells and T cells and can inhibit the lysis of tumor cell lines that harbor CEACAM1. Loss of CEACAM1 expression can increase overall cytotoxicity to NK cells and T cells. Thus, according to some embodiments, gene-edited CEACAM1 increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0300] In some embodiments, DDIT4 expression is decreased and / or reduced to increase overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. or be removed. In some embodiments, DDIT4 is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, DDIT4 is isolated using a Cas nuclease as disclosed elsewhere herein, guided by the use of another of the following DDIT4-specific guide RNAs: SEQ ID NOS: 500-502. disrupted and / or knocked out using Crispr-Cas-mediated approaches (e.g., Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). In NK and T cells, DDIT4 is a negative regulator of mTORC1, which itself enhances IL-15-mediated NK cell survival and proliferation. Furthermore, DDIT4 is upregulated by oxidative stress conditions, as is common in the tumor microenvironment. Loss of DDIT4 function in engineered cells can increase overall glucose metabolism, promote proliferation, as well as increase overall NK or T cell cytotoxicity. Thus, according to some embodiments, gene-edited DDIT4 increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0301] In some embodiments, expression of MAPKAP kinase 3 (MAPKAPK3) increases overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. to be reduced and / or eliminated. In some embodiments, MAPKAPK3 is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, MAPKAPK3 is isolated using a Cas nuclease guided by the use of another of the following MAPKAPK3-specific guide RNAs: SEQ ID NOS: 494-496 as disclosed elsewhere herein. disrupted and / or knocked out using Crispr-Cas-mediated approaches (e.g., Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). MAPKAP kinase 3 is expressed on both NK cells and T cells. Loss of MAPKAPK3 in engineered cells is expected to increase cytotoxicity, cytokine secretion, and overall NK signaling. Thus, according to some embodiments, the gene-edited MAPKAPK3 increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0302] In some embodiments, SMAD3 expression is decreased and / or reduced to increase overall activation in the resulting T cells and / or NK cells, or other cell types provided herein. or be removed. In some embodiments, SMAD3 is disrupted and / or knocked out using one or more of the gene editing methods disclosed herein. In some embodiments, SMAD3 is detected using a Cas nuclease as disclosed elsewhere herein, guided by the use of another of the following SMAD3-specific guide RNAs: SEQ ID NOs: 491-493. disrupted and / or knocked out using Crispr-Cas-mediated approaches (e.g., Cas9). In some embodiments, the gene editing increases expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about It can be reduced by 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amounts between those listed). SMAD3 is a downstream mediator of TGF-beta and activin A signaling. Inhibition of activin A provides an effective downstream TGFBR knockout. Smad3-silenced NK cells exhibit increased proliferation and differentiation, as well as increased cytotoxicity in engineered T and NK cells. Thus, according to some embodiments, gene-edited SMAD3 increases cytotoxicity, persistence, immune evasion of engineered NK cells, T cells, or other cells disclosed herein; or otherwise enhance its effectiveness.

[0303] As discussed above, gene edited cells can be edited at multiple locations. For example, in some embodiments, the cells (eg, NK cells or T cells, or a mixture thereof) are edited at two positions. In some embodiments, one of the gene edits is performed at a target site in the CISH gene. In some embodiments, one of the gene edits is performed at a target site in the CBLB gene. In some embodiments, one of the gene edits is performed at a target site in the TGFBR2 gene. In some embodiments, one of the gene edits is performed at a target site in the TIGIT gene. Any combination of such edits is also within the embodiments provided, including, for example, dual TGFBR2 / CBLB, dual TIGIT / TGFBR2, CISH / CBLB, CISH / TGFBR2, CISH / TIGIT, and the like. Additionally, any combination of editing any of the target genes for editing (eg, with Crispr or other nucleases) can be performed according to some embodiments. Additionally, multiple edits can be made within a single target gene or multiple target genes to the extent necessary to achieve the desired amount of reduction in gene expression.

[0304] In some embodiments, gene editing is performed at a target site of the CISH gene and a target site of the CBLB gene. In some embodiments, dual editing, eg, CISH / CBLB, is performed in NK cells and / or T cells for therapeutic use. In some embodiments, the combined CISH / CBLB gene editing is performed in NK cells without editing at the CD70 gene. In some embodiments, the combined CISH / CBLB gene editing is performed in NK cells that do not include edits in any additional genes. In some embodiments, the combined CISH / CBLB gene editing is performed in NK cells that do not express any one or combination of anti-CD70 CAR, anti-CD19 CAR, or anti-NKG2D chimeric receptor. In some embodiments, triple editing, eg, CD70 / CISH / CBLB, is performed in NK cells and / or T cells for use in therapy. In some embodiments, triple editing, eg, CD70 / CISH / CBLB, is performed in NK cells and / or T cells engineered to express tumor-targeted CARs.

[0305] In some embodiments, gene editing is performed at a target site in the CISH gene and a target site in the TGFBR2 gene. In some embodiments, dual editing, eg, CISH / TGFBR2, is performed in NK cells and / or T cells for therapeutic use. In some embodiments, the combined CISH / TGFBR2 gene editing is performed in NK cells without editing at the CD70 gene. In some embodiments, the combined CISH / TGFBR2 gene editing is performed in NK cells that do not contain edits in any additional genes. In some embodiments, the combined CISH / TGFBR2 gene editing is performed in NK cells that do not express any one or combination of anti-CD70 CAR, anti-CD19 CAR, or anti-NKG2D chimeric receptor. In some embodiments, triple editing, eg, CD70 / CISH / TGFBR2, is performed in NK cells and / or T cells for use in therapy. In some embodiments, triple editing, eg, CD70 / CISH / TGFBR2, is performed in NK cells and / or T cells engineered to express a tumor-targeting CAR.

[0306] In some embodiments, gene editing is performed at a target site of the CISH gene and a target site of the TIGIT gene. In some embodiments, dual editing, eg, CISH / TIGIT, is performed in NK cells and / or T cells for therapeutic use. In some embodiments, combined CISH / TIGIT gene editing is performed in NK cells without editing at the CD70 gene. In some embodiments, combined CISH / TIGIT gene editing is performed in NK cells that do not include edits in any additional genes. In some embodiments, combined CISH / TIGIT gene editing is performed in NK cells that do not express any one or combination of anti-CD70 CAR, anti-CD19 CAR, or anti-NKG2D chimeric receptor. In some embodiments, triple editing, eg, CD70 / CISH / TIGIT, is performed in NK cells and / or T cells for therapeutic use. In some embodiments, triple editing, eg, CD70 / CISH / TIGIT, is performed in NK cells and / or T cells engineered to express a tumor-targeting CAR.

[0307] Manipulation enhanced immunosuppressive effector-mediated persistence Additional cell engineering strategies are provided herein that help further enhance the persistence of allogeneic cell therapy products, such as allogeneic CAR-T cells and / or allogeneic CAR-NK cells. As discussed herein, there are various strategies that can be used to reduce the propensity of allogeneic cell therapy products to induce host cell-mediated graft rejection. For example, in some embodiments, expression of B2M is reduced and / or eliminated to reduce host-mediated graft rejection. In some embodiments, B2M expression uses a Crispr-Cas-mediated approach (e.g., Cas9) disclosed elsewhere herein, but with the following B2M-specific guide RNA: SEQ ID NO: 290-CGCGAGCACAGCTAAGGCCA SEQ ID NO:291-GAGTAGCGCGAGCACAGCTA;SEQ ID NO:292-GCTACTCTCTCTTTCTGGCC;SEQ ID NO:293-GGCCGAGATGTCTCGCTCCG;SEQ ID NO:294-GGCCACGGAGCGAGACATCT;SEQ ID NO:295-CACAGCCCAAGATAGTTAAG;SEQ ID NO:296-AGTCACATGGTTCACACGGC;SEQ ID NO:297-AAGTCAACTTCAATGTCGGA;SEQ ID NO:298 -ACTTGTCTTTCAGCAAGGAC; and Another use of SEQ ID NO: 299-TGGGCTGTGACAAAGTCACA is disrupted and / or knocked out. Loss of expression of B2M induces complete loss of HLA expression and can reduce, and in some embodiments eliminate, host T cell-mediated graft rejection. However, this can also sensitize the administered cells to host NK cell-mediated graft rejection (and, if a mixed NK / T cell population is used, the administered engineered NK cells can also be made sensitive). This results in the loss of KIR inhibitory signals (eg, "deficit of self" signals), as discussed above. See Figures 8E-8I. In some embodiments, various proteins are administered to an allogeneic recipient to inhibit host (or administered) NK cells from targeting the administered NK and / or T cells. can be expressed on the surface of cells. In some embodiments, approaches include, for example, HLA-E / HLA-G expression, CD47, or one or more viral peptides / proteins, and combinations thereof (among those disclosed herein). ) is accompanied by the expression of

[0308] Viral immunosuppressive peptide Many viral infections occur, at least in part, due to the ability of the virus to evade the host immune system, either through camouflage or suppression of host immune reactivity. In vitro studies have shown that specific embodiments of certain viruses have such immunosuppressive effects, eg, retroviral transmembrane envelope proteins such as the p15E protein. Additionally, studies have identified conserved regions of sequence across multiple virus types, such as a 17 amino acid conserved sequence, CKS-17 (SEQ ID NO: 199), and a nucleic acid encoding the same (SEQ ID NO: 216). ), see Table 1.

table 1

[0309] In some embodiments, one or more such viral immunosuppressive peptides (also referred to as viral peptides) are used to confer resistance to inactivation of engineered immune cells by host NK cells. Depending on the embodiment and cell type used, various approaches can be taken.

[0310] According to some embodiments, one or more viral immunosuppressive peptides are incorporated into the chimeric antigen receptor and then expressed by the population of immune cells used in targeting the patient. In some embodiments, the immune cells are allogeneic to the patient. In some embodiments, the NK cell and T cell combination is engineered to express one or more CARs that include one or more viral immunosuppressive peptides. Figure 25A shows a non-limiting embodiment of a viral immunosuppressive peptide incorporated into a CAR (generally a viral immunosuppressive peptide or other immunosuppressive peptide disclosed herein, unless otherwise specified). / protein). As shown in Figure 25A, the viral immunosuppressive peptide comprises the hinge / spacer domain of the CAR (such as an scFv), which contains the target binding factor, the hinge (also referred to herein as spacer), the transmembrane domain and one or more is incorporated into the intracellular signaling domain of Depending on the embodiment, more than one (eg, 2, 3, 4, 5, or more) viral immunosuppressive peptides can be incorporated into the hinge / spacer region of the CAR. In some such embodiments, when two (or more) viral immunosuppressive peptides are used, these may be of different sequences or types, as schematically illustrated in FIG. 25B. . For example, in some embodiments, the CKS-17 peptide is incorporated into the hinge region of the CAR, eg, in conjunction with the REV-A peptide. In some embodiments, the inclusion of two or more viral immunosuppressive peptides produces a synergistic immunosuppressive effect.

[0311] Depending on the embodiment, the length of the hinge / spacer region may be modified. In some embodiments, the CD8 alpha hinge / spacer region is used, while in some embodiments longer or shorter spacers are used. The spacer can be an IgG1, IgG2, IgG3, IgG4, or CD28 spacer domain, or can be derived from IgG1, IgG2, IgG3, IgG4, CD28, or can be a completely synthetic sequence, depending on the embodiment. obtain. In some embodiments, the IgG-based spacer is edited to reduce or eliminate the spacer's ability to carry Fc receptor-bearing cells, which may cause immune cells (immunosuppressed as disclosed herein) Off-target activation of effectors (such as those bearing effectors) may be advantageously reduced. Non-limiting editing approaches include, but are not limited to, heavy chain constant 2 (CH2) domains that abrogate binding to Fc receptors or mutate specific amino acids that are essential for Fc receptor binding. Examples include deletions. In some embodiments, longer spacers are advantageous in allowing for enhanced targeting of specific membrane-proximal epitopes expressed by cancer cells and exposure of immunosuppressive effectors, thereby reducing host and / or can interact with administered immune cells and reduce undesired suppression of treated cells. In some embodiments, a single hinge region can be made longer by including multiple hinge-encoding sequences, eg, 2, 3, 4, or more hinges. In some embodiments where multiple hinge regions are used, these can be of the same type (e.g., three CD8a hinges) or of different types (e.g., one CD8a hinge, one CD28 hinge, and IgG1 hinge). In some embodiments, shorter hinges are used, which limit the ability of host phosphatases (such as CD45) to attenuate signaling of CARs expressed by engineered immune cells. In some embodiments, the hinge region comprises one or more of SEQ ID NOs: 479-487. In some embodiments, the hinge region is at least about 80%, at least about 85%, at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about any of SEQ ID NOs: 479-487. Amino acid sequences having about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence.

[0312] FIG. 25C depicts a further approach, namely engineering a viral immunosuppressive peptide into the linker between the heavy and light chains of the scFv, in accordance with some embodiments disclosed herein. In further embod...

Claims

1. A genetically engineered and gene edited immune cell population, a genetically engineered immune cell that expresses a cytotoxic receptor that includes an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex; The extracellular ligand-binding domain targets a tumor marker expressed by the target tumor cell; the immune cell is gene edited at one or more target locations in a CISH gene encoding a CIS protein, the editing resulting in reduced expression and / or function of CIS compared to an immune cell that has not been edited at said one or more locations in the CISH gene; the immune cell is gene edited at one or more target locations in a Cbl proto-oncogene B protein (CBLB) gene encoding a CBLB protein, wherein the editing results in decreased expression and / or function of the CBLB protein compared to an immune cell that has not been edited at said one or more locations in the CBLB gene; A genetically engineered and edited immune cell population, wherein the genetically engineered and edited immune cells exhibit one or more of increased expansion capability, increased cytotoxicity against target cells, and increased persistence compared to immune cells that do not include said gene edits in the CISH and CBLB genes.

2. The genetically engineered and gene-edited immune cell population of claim 1, wherein the genetically engineered and gene-edited immune cells are edited at additional target sites in genes encoding transforming growth factor receptor beta 2 (TGFBR2), TIGIT, NKG2A, SOCS2, B2M, PD-1, TIM-3, or TCR alpha.

3. 2. The genetically engineered and gene-edited immune cell population of claim 1, wherein the CISH gene is targeted using a guide sequence of any of SEQ ID NOs: 153-157 or 562-565.

4. 2. The genetically engineered and gene edited immune cell population of claim 1, wherein the CBLB gene is targeted using a guide sequence of any of SEQ ID NOs: 164-166 or 552-555.

5. 2. The genetically engineered and gene edited immune cell population of claim 1, wherein at least a portion of the genetically engineered immune cells are engineered to express membrane-bound IL-15.

6. The genetically engineered and gene-edited immune cell population of claim 1, wherein the cytotoxic signaling complex comprises the OX40 subdomain and the CD3 zeta subdomain.

7. 3. The genetically engineered and gene edited immune cell population of claim 2, wherein the TGFBR2 gene is targeted using a guide sequence of any of SEQ ID NOs: 147-152 or 544-547.

8. 3. The genetically engineered and gene edited immune cell population of claim 2, wherein the TIGIT gene is targeted using a guide sequence of any of SEQ ID NOs: 507-510.

9. The genetically engineered and gene-edited immune cell population of claim 1, wherein the genetically engineered and gene-edited immune cells comprise natural killer (NK) cells, T cells, or a combination thereof.

10. The genetically engineered and gene-edited immune cell population of claim 1, wherein the genetically engineered and gene-edited immune cells comprise natural killer (NK) cells.

11. 2. The genetically engineered and gene edited immune cell population of claim 1, wherein the cytotoxic receptor targets one or more of the following ligands expressed by the target tumor cells: CD70, NKG2D, CD19, BCMA, CD38, GPRC5D, DLL3, EGFR, PSMA, FLT3, or any combination thereof.

12. 11. The genetically engineered and gene edited immune cell population of any one of claims 1 to 10, wherein the cells have not been engineered to express a cytotoxicity receptor complex that targets CD19.

13. 12. The genetically engineered and gene edited immune cell population of any one of claims 1 to 11, wherein the cells have not been engineered to express a cytotoxic receptor complex that targets a ligand of the NKG2D receptor.

14. The genetically engineered and gene-edited immune cell population of any one of claims 1-13, wherein (i) any guide sequence of SEQ ID NOs:548-551 is used to target the NKG2A gene, (ii) any guide sequence of SEQ ID NOs:556-561 is used to target the SOCS2 gene, (iii) any guide sequence of SEQ ID NOs:199-208 is used to target the B2M gene, (iv) any guide sequence of SEQ ID NOs:511-514 is used to target the PD-1 gene, (v) any guide sequence of SEQ ID NOs:515-518 is used to target the TIM-3 gene, and (vi) any guide sequence of SEQ ID NOs:566-568 is used to target the TCR alpha gene.

15. 15. The genetically engineered and gene edited immune cell population of any one of claims 1-14, wherein (i) any guide sequence of SEQ ID NOs: 503-506 is used to target the adenosine A2 receptor gene, (ii) any guide sequence of SEQ ID NOs: 491-493 is used to target the SMAD3 gene, (iii) any guide sequence of SEQ ID NOs: 494-496 is used to target the MAPKAPK3 gene, (iv) any guide sequence of SEQ ID NOs: 497-499 is used to target the CEACAM1 gene, (v) any guide sequence of SEQ ID NOs: 500-502 is used to target the DDIT4 gene, and (vi) any guide sequence of SEQ ID NOs: 519-522 is used to target the CD38 gene.

16. 16. The genetically engineered and gene edited immune cell population of any one of claims 1 to 15, wherein the genetically engineered and edited immune cells are suitable for use in allogeneic cancer cell therapy to reduce the risk of graft versus host disease.

17. A genetically engineered and gene-edited immune cell population as described in any one of claims 1 to 16 for use in treating cancer in a subject.

18. A method for producing a gene-edited immune cell population, comprising: contacting the immune cell population with a first gene editing complex; editing at one or more target sites in the CISH gene of the immune cell, such that the first gene editing complex results in a reduced level of expression of a CIS protein encoded by the CISH gene, compared to an immune cell that has not been edited with the CISH gene; and contacting the immune cell population with a second gene editing complex; a second gene editing complex edits at one or more target sites in the CBLB gene of the immune cell to result in a reduced level of expression of a CBLB protein encoded by the CBLB gene compared to an immune cell not edited with the CBLB gene; The method, wherein the gene-edited immune cells exhibit one or more of increased expansion capability, increased cytotoxicity against target cells, and increased persistence compared to immune cells that do not contain the gene-edited one or more target sites.

19. A method for producing a gene-edited immune cell population, comprising: contacting the immune cell population with a first gene editing complex; editing at one or more target sites in the CISH gene of the immune cell, such that the first gene editing complex results in a reduced level of expression of a CIS protein encoded by the CISH gene, compared to an immune cell that has not been edited with the CISH gene; and contacting the immune cell population with a second gene editing complex; a second gene editing complex edits at one or more target sites in the TGFBR2 gene of the immune cell to result in a reduced level of expression of a TGFBR2 protein encoded by the TGFBR2 gene compared to an immune cell that has not been edited in the TGFBR2 gene; The method, wherein the gene-edited immune cells exhibit one or more of increased expansion capability, increased cytotoxicity against target cells, and increased persistence compared to immune cells that do not contain the gene-edited one or more target sites.

20. A method for producing a gene-edited immune cell population, comprising: contacting the immune cell population with a first gene editing complex; editing at one or more target sites in the CISH gene of the immune cell, such that the first gene editing complex results in a reduced level of expression of a CIS protein encoded by the CISH gene, compared to an immune cell that has not been edited with the CISH gene; and contacting the immune cell population with a second gene editing complex; the second gene editing complex edits at one or more target sites in the TIGIT gene of the immune cell to result in a reduced level of expression of a TIGIT protein encoded by the TIGIT gene, compared to an immune cell not edited with the TIGIT gene; The method, wherein the gene-edited immune cells exhibit one or more of increased expansion capability, increased cytotoxicity against target cells, and increased persistence compared to immune cells that do not contain the gene-edited one or more target sites.

21. A method for producing a gene-edited immune cell population, comprising: contacting the immune cell population with a first gene editing complex; editing at one or more target sites in the CISH gene of the immune cell, such that the first gene editing complex results in a reduced level of expression of a CIS protein encoded by the CISH gene, compared to an immune cell that has not been edited with the CISH gene; and contacting the immune cell population with a second gene editing complex; the second gene editing complex edits at one or more target sites in the additional gene in the immune cell to result in a reduced level of expression of a protein encoded by the additional gene, compared to an immune cell that has not been edited with the additional gene; The method, wherein the gene-edited immune cells exhibit one or more of increased expansion capability, increased cytotoxicity against target cells, and increased persistence compared to immune cells that do not contain the gene-edited one or more target sites.

22. 22. The method of any one of claims 18-21, further comprising contacting the immune cell population with a vector comprising a polynucleotide encoding a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex.

23. 23. The method of any one of claims 20-22, further comprising making additional edits in one or more genes encoding one of NKG2A, SOCS2, B2M, PD-1, TIM-3, or TCR alpha.

24. 23. The method of claim 22, wherein the cytotoxic receptor targets one or more of the ligands of CD70, NKG2D, CD19, BCMA, CD38, GPRC5D, DLL3, EGFR, PSMA, FLT3, or any combination thereof expressed by the target tumor cell.

25. The method of any one of claims 18 to 24, wherein the immune cells comprise natural killer (NK) cells, T cells, or a combination thereof.

26. A genetically engineered and gene edited immune cell population, a genetically engineered immune cell that expresses a cytotoxic receptor that includes an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex; the extracellular ligand binding domain targets a tumor marker selected from the group consisting of CD70, NKG2D ligand, CD19, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3 and KREME-N2; At least a portion of the genetically engineered immune cells are engineered to express membrane-bound IL-15; The immune cell is gene edited at one or more target sites in the genome of the immune cell to result in a reduced level of expression of a protein encoded by a gene that contains the edited target site, compared to a non-edited immune cell; the protein is selected from the group consisting of CISH, adenosine A2 receptor, SMAD3, MAPKAPK3, CEACAM1, DDIT4, TGFBR2R, NKG2A, SOCS2, CBLB, B2M, TIGIT, PD-1, TIM-3, CD38, TCR alpha, and any combination thereof; A genetically engineered and edited immune cell population, wherein the genetically engineered and edited immune cells exhibit one or more of increased expansion capability, increased cytotoxicity against target cells, and increased persistence compared to immune cells that do not contain the gene edited target site or sites.

27. ​​(i) any of the guide sequences of SEQ ID NOs: 562-565 is used to target a gene encoding CIS; (ii) any of the guide sequences of SEQ ID NOs: 491-493 is used to target the SMAD3 gene; (iii) any of the guide sequences of SEQ ID NOs: 494-496 is used to target the MAPKAPK3 gene; (iv) any of the guide sequences of SEQ ID NOs: 497-499 is used to target the CEACAM1 gene; (v) any of the guide sequences of SEQ ID NOs: 500-502 is used to target the DDIT4 gene; (vi) any of the guide sequences of SEQ ID NOs: 544-547 is used to target the TGFBR2 gene; (vii) any of the guide sequences of SEQ ID NOs: 548-551 is used to target the NKG2A gene; (viii) any of the guide sequences of SEQ ID NOs: 556-561 is used to target the SOC 27. The immune cell population of claim 26, wherein a guide sequence of any of SEQ ID NOs: 552-555 is used to target the CBLB gene, (x) a guide sequence of any of SEQ ID NOs: 290-299 is used to target the B2M gene, (xi) a guide sequence of any of SEQ ID NOs: 507-510 is used to target the TIGIT gene, (xii) a guide sequence of any of SEQ ID NOs: 511-514 is used to target the PD-1 gene, (xiii) a guide sequence of any of SEQ ID NOs: 515-518 is used to target the TIM-3 gene, (xiv) a guide sequence of any of SEQ ID NOs: 519-522 is used to target the CD38 gene, and / or (xv) a guide sequence of any of SEQ ID NOs: 566-569 is used to target the TCR alpha gene.

28. 28. The immune cell population of claim 26 or 27, wherein the extracellular ligand binding domain comprises a receptor directed against a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

29. 29. The immune cell population of any one of claims 26 to 28, wherein the cytotoxic receptor expressed by the NK cell comprises a signaling complex comprising: (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z costimulatory subdomain.

30. 30. The immune cell population of any one of claims 26 to 29, wherein the cytotoxic receptor targets one or more of the following expressed by the target tumor cells: NKG2D receptor ligand, CD70, CD19, BCMA, CD38, GPRC5D, CD138, DLL3, EGFR, PSMA, FLT3, or KREME-N2.

31. The immune cell population of any one of claims 26 to 30, wherein the cytotoxic signaling complex comprises an OX40 subdomain or a 4-1BB domain, and a CD3 zeta subdomain.

32. 32. The immune cell population of any one of claims 26 to 31, wherein the immune cells comprise natural killer (NK) cells, T cells, induced pluripotent stem cells (iPSCs), iPSC-derived NK cells, NK-92 cells, or a combination thereof.

33. 33. The immune cell population of any one of claims 26 to 32, wherein the genetically engineered and edited immune cells are suitable for use in allogeneic cancer cell therapy to reduce the risk of graft versus host disease and / or to reduce the risk of cytotoxic activity between the genetically engineered immune cells.

34. A genetically engineered immune cell population for cancer immunotherapy, comprising: a genetically engineered immune cell that expresses a cytotoxic receptor that includes an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex; At least a portion of the genetically engineered immune cells are engineered to express membrane-bound IL-15; The engineered immune cells are engineered to express at least one immune suppressive effector comprising: (i) two or more of a truncated human CD47, a p15E peptide, an HIV peptide, and an HTLV peptide; (ii) a truncated human CD47 domain and at least one p15E peptide, an HIV peptide, and an HTLV peptide; (iii) a truncated human CD47 domain and at least one HIV peptide; (iv) a truncated human CD47 domain and at least an HTLV peptide; (v) a sequence having at least 95% sequence identity to one or more of SEQ ID NOs: 256, 259, 262, 265, 268, 271; or (vi) a viral UL18 protein; at least one immunosuppressive effector exerts an inhibitory effect on undesired cytotoxic activity of the inhibitory cells; A genetically engineered immune cell population, wherein the genetically engineered immune cells exhibit one or more of increased expansion capacity, increased cytotoxicity against target cells, and increased persistence compared to cells that do not contain said immunosuppressive effector.

35. 35. The genetically engineered immune cell population of claim 34, comprising one or more of genetically engineered NK cells and genetically engineered T cells.

36. 36. The genetically engineered immune cell population of claim 34 or 35, wherein at least one immunosuppressive effector is bound to the outer cell membrane of the immune cell, and wherein at least one immunosuppressive effector comprises a transmembrane protein, and the transmembrane protein is selected from CD8α, CD4, CD3ε, CD3γ, CD3δ, CD3ζ, CD28, CD137, glycophorin A, glycophorin D, a nicotinic acetylcholine receptor, a GABA receptor, FcεRIγ, and a T cell receptor.

37. 37. The genetically engineered immune cell population of any one of claims 34-36, wherein the immunosuppressive effector comprises one or more peptides having at least 95% sequence identity to one or more of SEQ ID NOs: 199-215, 219, 220, 223, 225, 228, 230, 235, 238, 240, 243, 250, or 253.

38. A method for producing a genetically engineered immune cell population, comprising: contacting a population of immune cells with a polynucleotide encoding a cytotoxic receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex; contacting the immune cell population with a further polynucleotide encoding at least one immunosuppressive effector comprising: (i) two or more of a truncated human CD47, a p15E peptide, an HIV peptide, and an HTLV peptide; (ii) a truncated human CD47 domain and at least one p15E peptide, an HIV peptide, and an HTLV peptide; (iii) a truncated human CD47 domain and at least one HIV peptide; (iv) a truncated human CD47 domain and at least an HTLV peptide; (v) a sequence having at least 95% sequence identity to one or more of SEQ ID NOs: 256, 259, 262, 265, 268, 271; or (vi) a viral UL18 protein; at least one immunosuppressive effector exerts an inhibitory effect on the cytotoxic activity of the suppressor cell; The method, wherein the engineered immune cells exhibit one or more of increased expansion capability, increased cytotoxicity against target cells, and increased persistence compared to cells that do not contain the at least one immunosuppressive effector.

39. A method for reducing fratricide in a mixed population of genetically engineered immune cells, comprising: contacting a first subpopulation of immune cells from the mixed immune cell population with a polynucleotide encoding at least one immunosuppressive effector; expression of at least one immunosuppressive effector by a first subpopulation of immune cells reduces the suppressive activity of a second subpopulation of immune cells that has tropism for the first subpopulation of immune cells; and contacting a second subpopulation from the mixed immune cell population with a polynucleotide encoding at least an additional immunosuppressive effector; expression of at least a further immunosuppressive effector by the second subpopulation of immune cells comprises decreasing the suppressive activity of the first subpopulation of immune cells having tropism for the second subpopulation of immune cells; The method, wherein expression of the immunosuppressive effector by the first subpopulation and the second subpopulation reduces fratricide in the genetically engineered immune cell population.

40. 1. A method for engineering a genetically engineered immune cell population, comprising: (a) genetically editing a mixed population of immune cells, including NK cells and T cells, to reduce expression of human leukocyte antigens (HLA) on the surface of the immune cells; The reduced expression of HLA on the surface of immune cells reduces T cell-mediated cytotoxicity against the edited immune cell population; The reduced expression of HLA on the surface of the cells sensitizes the edited immune cell population to NK-mediated cytotoxicity against the edited immune cells; and (b) genetically engineering the edited cells to express one or more immunosuppressive effectors that reduce NK-mediated cytotoxicity against a mixed population of immune cells; the one or more immunosuppressive effectors comprise one or more of a viral immunosuppressive peptide, a viral protein that is an HLA homolog, HLA-E, HLA-G, a human protein or fragment thereof that reduces phagocytosis of cells, a chimeric construct comprising a viral immunosuppressive peptide and a human protein or fragment thereof that reduces phagocytosis of cells, or a combination thereof; The reduced T cell mediated cytotoxicity reduces engineered T cell mediated fratricidal cytotoxicity against the engineered NK cells, and upon administration to a subject, reduces host T cell mediated cytotoxicity against the engineered NK cells; The method, wherein the reduced NK cell mediated cytotoxicity reduces engineered NK cell mediated fratricidal cytotoxicity against engineered T cells and upon administration reduces host NK cell mediated cytotoxicity against the engineered NK cells and engineered T cells.

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