Modulation of immune cell gene expression

Abstract

Several embodiments of the methods and compositions disclosed herein relate to immune cells in which the expression of one or more genes or proteins is modulated (e.g., knocked down or knocked out). In several embodiments, the immune cells are also engineered to express chimeric receptors (e.g., chimeric antigen receptors). In several embodiments, the immune cells having modulated gene expression exhibit enhanced expansion, cytotoxicity against target cells, and / or persistence after administration to a subject, or reduced potential side effects when, for example, the cells are administered to a subject.

Description

NKT 118W0 PATENTMODULATION OF IMMUNE CELL GENE EXPRESSIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No. 63 / 724729, filed November 25, 2024, United States Provisional Application No. 63 / 735015, filed December 17, 2024, and United States Provisional Application No. 63 / 745989, filed January 16, 2025, the entire contents of each of which is incorporated by reference herein.FIELD

[0002] Several embodiments disclosed herein relate to methods and compositions comprising immune cells in which the expression of one or more genes or proteins is modulated (e.g., knocked down or knocked out). In several embodiments, the immune cells are also engineered to express chimeric receptors (e.g., chimeric antigen receptors). In several embodiments, the immune cells having modulated gene expression exhibit enhanced expansion, cytotoxicity against target cells, and / or persistence after administration to a subject, or reduced potential side effects when, for example, the cells are administered to a subject.BACKGROUND

[0003] Immunotherapy presents a new technological advancement in the treatment of diseases wherein immune cells are engineered to express certain targeting and / or effector molecules that specifically identify and react to diseased or damaged cells. However, treatment of diseases by immunotherapy can be impeded by expression of certain genes that interfere or reduce the efficacy of therapy. One approach to overcome this challenge is to downregulate the expression of such genes in immune cells, thereby increasing the expansion, persistence, and / or cytotoxicity against aberrant cells of interest. Provided herein are compositions, methods and uses of immune cells wherein expression of one or more genes or proteins have been modulated to enhance the function of immune cells.INCORPORATION BY REFERENCE OF MATERIAL IN SEQUENCE LISTING FILE

[0004] This application incorporates by reference the material contained in the Sequence Listing XML file being submitted concurrently herewith: File name: NKT.l 18WO_ST26.xml; created on November 20, 2025 and is 218,956 bytes in size.SUMMARY

[0005] Provided herein is an immune cell having an inhibited expression of at least two target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12. PRDM1 and PTPN2. In several embodiments, there is provided an immune cell having an inhibited expression of at least three target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In several embodiments, there is provided an immune cell having an inhibited expression of at least four target proteins selected from the group consisting of ADAM 17, B2M. Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2. ICAM3, MED12, PRDM1 and PTPN2. In several embodiments, the immune cell has inhibited expression of between two and four target proteins selected from the group consisting of ADAMI 7. B2M. Cbl-b, CD58, CIITA, CIS, FAS, FASL, 1CAM1, 1CAM2, 1CAM3, MED12. PRDM1 and PTPN2. In several embodiments, the immune cell has inhibited expression of two target proteins selected from the group consisting of ADAM 17, B2M, Cbl- b. CD58, CIITA, CIS, FAS, FASL, ICAML ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In several embodiments, the immune cell has inhibited expression of three target proteins selected from the group consisting of ADAM17, B2M. Cbl-b, CD58. CIITA. CIS, FAS, FASL. ICAM1, ICAM2. ICAM3, MED12. PRDM1 and PTPN2. In several embodiments, the immune cell has inhibited expression of four target proteins selected from the group consisting of ADAMI 7, B2M, Cbl-b, CD58, CIITA. CIS, FAS, FASL. ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2.

[0006] In several embodiments, ADAMI 7 expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nucleases as disclosed herein, using one of the following ADAM17-specific guide RNAs: SEQ ID NOS: 127-132, or 216. In some embodiments, the expression of ADAM17 is inhibited using a plurality of guide RNAs selected from SEQ ID NO: 127-132, or 216. In some embodiments, an ADAM17-specific guide RNA comprises the sequence set forth in any one of SEQ ID NOS: 127-132, or 216.

[0007] In several embodiments, B2M expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nucleases as disclosed herein, using one of the following B2M- specific guide RNAs: SEQ ID NOS: 164-173. In some embodiments, tire expression of B2M is inhibited using a plurality of guide RNAs selected from SEQ ID NO: 164-173. In some embodiments, a B2M- specific guide RNA comprises the sequence set forth in any one of SEQ ID NOS: 164-173.

[0008] In several embodiments, CD58 expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nucleases as disclosed herein, using the following CD58-specific guide RNA: SEQ ID NO: 217. In some embodiments, the expression of CD58 is inhibited using a guide RNA of SEQ ID NO: 217. In some embodiments, a CD58-specific guide RNA comprises the sequence set forth in SEQ ID NO: 217.

[0009] In several embodiments. CISH expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nucleases as disclosed herein, using one of the following CISH-specific guide RNAs: SEQ ID NOS: 187-196. In some embodiments, the expression of CISH is inhibited using a plurality of guide RNAs selected from SEQ ID NO: 187-196. In some embodiments, a CISH-specific guide RNA comprises the sequence set forth in any one of SEQ ID NOS: 187-196.

[0010] In several embodiments, CBLB expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or oilier guided nucleases as disclosed herein, using one of the following CBLB- specific guide RNAs: SEQ ID NOS: 174-186. In some embodiments, the expression of CBLB is inhibited using a plurality of guide RNAs selected from SEQ ID NO: 174-186. In some embodiments, a CBLB-specific guide RNA comprises the sequence set forth in any one of SEQ ID NOS: 174-186.

[0011] In several embodiments. FAS expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nucleases as disclosed herein, using one of the following FAS- specific guide RNAs: SEQ ID NOS: 201-204. or 215. In some embodiments, the expression of FAS is inhibited using a plurality of guide RNAs selected from SEQ ID NO: 201-204. or 215. In some embodiments, a FAS-specific guide RNA comprises the sequence set forth in any one of SEQ ID NOS: 201-204, or 215.

[0012] In several embodiments, FASLG expression is inhibited using a Crispr-Cas mediated approach (e.g.. Cas9). or other guided nucleases as disclosed herein, using one of the following FASLG- specific guide RNAs: SEQ ID NOS: 205-207. In some embodiments, the expression of FASLG is inhibited using a plurality of guide RNAs selected from SEQ ID NO: 205-207. In some embodiments, a FASL-specific guide RNA comprises the sequence set forth in any one of SEQ ID NOS: 205-207.

[0013] In several embodiments, ICAM1 expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nucleases as disclosed herein, using the ICAMl-specific guide RNA: SEQ ID NO: 218. In some embodiments, the expression of ICAM1 is inhibited using a guide RNA of SEQ ID NO: 218. In some embodiments, an ICAMl-specific guide RNA comprises the sequence set forth in SEQ ID NO: 218.

[0014] In several embodiments, ICAM2 expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nucleases as disclosed herein, using one of the following ICAM2- specific guide RNAs: SEQ ID NOS: 219-221. In some embodiments, the expression of ICAM2 is inhibited using a plurality of guide RNAs selected from SEQ ID NO: 219-221. In some embodiments, a ICAM2-specific guide RNA comprises the sequence set forth in any one of SEQ ID NOS: 219-221.

[0015] In several embodiments, ICAM3 expression is inhibited using a Crispr-Cas mediated approach (e g., Cas9), or other guided nuclease as disclosed herein, using a specific guide RNA. In some embodiments, the expression of ICAM3 is inhibited using a plurality of guide RNAs.

[0016] In several embodiments, MED12 expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nucleases as disclosed herein, using one of the following MED12- specific guide RNAs: SEQ ID NOS: 133-142. In some embodiments, the expression of MED12 is inhibited using a plurality of guide RNAs selected from SEQ ID NOS: 133-142. In some embodiments, a MED12-specific guide RNA comprises the sequence set forth in any one of SEQ ID NOS: 133-142.

[0017] In several embodiments, PTPN2 expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nucleases as disclosed herein, using one of the following PTPN2- specific guide RNAs: SEQ ID NOS: 208-211. In some embodiments, the expression of PTPN2 is inhibited using a plurality of guide RNAs selected from SEQ ID NOS: 208-211. In some embodiments, a PTPN2-specific guide RNA comprises the sequence set forth in any one of SEQ ID NOS: 208-211.

[0018] In several embodiments, PRDM1 expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nucleases as disclosed herein, using one of the following PRDM1 -specific guide RNAs: SEQ ID NOS: 197-200. In some embodiments, the expression of PRDMlis inhibited using a plurality of guide RNAs selected from SEQ ID NOS: 197-200. In some embodiments, a PRDM1 -specific guide RNA comprises the sequence set forth in any one of SEQ ID NOS: 197-200.

[0019] In several embodiments, CI1TA expression is inhibited using a Crispr-Cas mediated approach (e.g., Cas9). or other guided nucleases as disclosed herein, with the use of CIITA-specific guide RNAs. In some embodiments, the expression of CIITA is inhibited using a plurality of guide RNAs.

[0020] In several embodiments, there is provided an immune cell having an inhibited expression of at least two target proteins selected from the group consisting of ADAM17, B2M, Cbl- b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2, wherein the immune cell comprises a genomic disruption within a target sequence of the gene encoding each of the at least two target proteins. In several embodiments, the immune cell comprises an inhibited expression of two target proteins selected from the group consisting of ADAM 17, B2M, Cbl-b, CD58, CIITA, CIS, FAS. FASL, ICAM1. 1CAM2, 1CAM3, MED12. PRDM1 and PTPN2; and the immune cell comprises a genomic disruption with a target sequence of the gene encoding each of the tw o target proteins. In several embodiments, there is provided an immune cell having an inhibited expression of at least three target proteins selected from the group consisting of ADAM 17. B2M, Cbl-b. CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2, wherein the immune cell comprises a genomic disruption within a target sequence of the gene encoding each of the at least three target proteins. In several embodiments, the immune cell comprises an inhibited expression of three target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2; and the immune cell comprises a genomic disruption with a target sequence of die gene encoding each of the three target proteins. In several embodiments, there is provided an immune cell having an inhibited expression of at least four target proteins selected from the group consisting of ADAMI 7, B2M, Cbl-b, CD58. CIITA, CIS, FAS, FASL, ICAM1. ICAM2, ICAM3, MED12. PRDM1 and PTPN2, wherein the immune cell comprises a genomic disruption within a target sequence of the gene encoding each of the at least four target proteins. In several embodiments, the immune cell comprises an inhibited expression of fourtarget proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2; and the immune cell comprises a genomic disruption with a target sequence of the gene encoding each of the four target proteins. In several embodiments, the target proteins comprise ADAM 17. In several embodiments, the target proteins comprise B2M. In several embodiments, the target proteins comprise CD58. In several embodiments, the target proteins comprise CIS. In several embodiments, the target proteins comprise CBLB. In several embodiments, the target proteins comprise Cbl-b and CIS. In several embodiments, the target proteins comprise FAS. In several embodiments, the target proteins comprise FAS and CIS. In several embodiments, the target proteins comprise FAS and MED 12. In several embodiments, the target proteins comprise FASL. In several embodiments, the target proteins comprise FASL and CIS. In several embodiments, the target proteins comprise FASL and MED 12. In several embodiments, the target proteins comprise 1CAM1. In several embodiments, the target proteins comprise 1CAM2. In several embodiments, the target proteins comprise ICAM3. In several embodiments, the target proteins comprise MED12. In several embodiments, the target proteins comprise PTPN2. In several embodiments, the target proteins comprise CIS. In several embodiments, the target proteins comprise MED12. In several embodiments, the target proteins comprise PTPN2 and MED12. In several embodiments, the target proteins comprise PRDM1. In several embodiments, the target proteins comprise PRDM1 and CIS. In several embodiments, the target proteins comprise PRDM1 and MED12. In several embodiments, the target proteins comprise CIITA. In several embodiments, the target proteins comprise ICAM1 and ICAM2. In several embodiments, the target proteins comprise ICAM1 and CD58. In several embodiments, the target proteins comprise ICAM1 and FAS. In several embodiments, the target proteins comprise ICAM2 and CD58. In several embodiments, the target proteins comprise ICAM2 and FAS. In several embodiments, the target proteins comprise ICAM1, ICAM2 and CD58. In several embodiments, the target proteins comprise ICAM1, ICAM2 and FAS. In several embodiments, the target proteins comprise ICAM1, CD58 and FAS. In several embodiments, the target proteins comprise ICAM2, CD58 and FAS. In several embodiments, the target proteins comprise ICAM1, ICAM2, FAS and CD58.

[0021] In several embodiments, there is provided an immune cell, wherein the immune cell comprises at least two synthetic shRNA sequences. In several embodiments, the synthetic shRNA sequences are expressed in the immune cell. In several embodiments, the synthetic shRNA sequences reduce expression of at least two target proteins selected from the group consisting of ADAMI 7, B2M, Cbl-b, CD58, CIITA, CIS. FAS, FASL, ICAM1, ICAM2. ICAM3, MED12, PRDM1 and PTPN2. In several embodiments, the synthetic shRNA sequences expressed in the immune cell reduce expression of two target proteins in the immune cell. In several embodiments, the synthetic shRNA sequences expressed in the immune cell reduce expression of (a) MED12 and PTPN2 or (b) MED12 and FAS. In several embodiments, the synthetic shRNA sequences expressed in the immune cell reduce expression of MED 12 and PTPN2. In several embodiments, the synthetic shRNA sequences expressed in theimmune cell reduce expression of MED12 and FAS. In several embodiments, there is provided an immune cell, wherein the immune cell comprises at least three synthetic shRNA sequences. In several embodiments, the synthetic shRNA sequences are expressed in the immune cell. In several embodiments, the synthetic shRNA sequences reduce expression of at least three target proteins selected from the group consisting of ADAMI 7, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In several embodiments, the synthetic shRNA sequences expressed in the immune cell reduce expression of three target proteins in the immune cell. In several embodiments, there is provided an immune cell, wherein the immune cell comprises at least four synthetic shRNA sequences. In several embodiments, the synthetic shRNA sequences are expressed in the immune cell. In several embodiments, the synthetic shRNA sequences reduce expression of at least four target proteins selected from the group consisting of ADAM 17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, 1CAM1, ICAM2. 1CAM3, MED12. PRDM1 and PTPN2. In several embodiments, the synthetic shRNA sequences expressed in the immune cell reduce expression of four target proteins in the immune cell. In several embodiments, there is provided an immune cell, wherein the immune cell comprises at least five synthetic shRNA sequences. In several embodiments, the synthetic shRNA sequences are expressed in the immune cell. In several embodiments, the synthetic shRNA sequences reduce expression of at least five target proteins selected from the group consisting of ADAMI 7, B2M, Cbl-b. CD58, CIITA, CIS, FAS, FASL. ICAM1, ICAM2, ICAM3. MED12, PRDM1 and PTPN2. In several embodiments, the synthetic shRNA sequences expressed in the immune cell reduce expression of five target proteins in the immune cell.

[0022] In several embodiments, there is provided an immune cell that expresses a polynucleotide, wherein the polynucleotide comprises (a) a nucleic acid sequence encoding a chimeric receptor; and (b) at least two shRNA sequences, wherein the shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least two target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In several embodiments, the immune cell further expresses a nucleic acid sequence encoding a membrane-bound interleukin- 15 (mbIL15). In several embodiments, the polynucleotide further comprises (c) a nucleic acid sequence encoding a membrane-bound interleukin- 15 (mbIL15). In several embodiments, there is provided an immune cell that expresses a polynucleotide, wherein the polynucleotide comprises (a) a nucleic acid sequence encoding a chimeric receptor; (b) at least two shRNA sequences, wherein the shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least two target proteins selected from the group consisting of ADAM17. B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2; and (c) anucleic acid sequence encoding a membrane -bound interleukin- 15 (mbIL15). In some embodiments, at least two shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least two, at least three, or at least four target proteins. In some embodiments, two shRNA sequences in the polynucleotide reduce expression of the sametarget protein by binding to the same or an adjacent region within the target nucleic acid. In some embodiments, two shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to different regions within the target nucleic acid. In some embodiments, two shRNA sequences in the polynucleotide reduce expression of different target proteins. In some embodiments, two shRNA sequences in the polynucleotide are separated by a linker. In some embodiments, two shRNA sequences in the polynucleotide are contiguous. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA- based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0023] In several embodiments, there is provided an immune cell that expresses a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence encoding at least two shRNA sequences, wherein the shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least two target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In some embodiments, two shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to the same or an adjacent region within the target nucleic acid. In some embodiments, two shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to different regions within the target nucleic acid. In some embodiments, two shRNA sequences in the polynucleotide reduce expression of different target proteins. In some embodiments, two shRNA sequences in the polynucleotide are separated by a linker. In some embodiments, two shRNA sequences in the polynucleotide are contiguous. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA- based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0024] In several embodiments, there is provided an immune cell that expresses a polynucleotide, wherein the polynucleotide comprises (a) a nucleic acid sequence encoding a chimeric receptor; (b) at least three shRNA sequences, wherein the shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least three target proteins selected from the group consisting of ADAM17, B2M. Cbl-b, CD58. CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED 12, PRDM1 and PTPN2; and (c) a nucleic acid sequence encoding a membrane-bound interleukin- 15 (mbIL15). In some embodiments, at least two shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least two. at least three, or at least four target proteins. In some embodiments, three shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to the same or an adjacent region within the target nucleic acid. In some embodiments, three shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to different regions within the target nucleic acid. In some embodiments, three shRNA sequences in the polynucleotide reduce expression of different target proteins. In someembodiments, three shRNA sequences in the polynucleotide are separated by a linker. In some embodiments, three shRNA sequences in the polynucleotide are contiguous. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA-based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0025] In several embodiments, there is provided an immune cell that expresses a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence encoding at least three shRNA sequences, wherein the shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least three target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58. CIITA, CIS, FAS. FASL, ICAM1, ICAM2. ICAM3, MED12, PRDM1 and PTPN2. In some embodiments, three shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to the same or an adjacent region within the target nucleic acid. In some embodiments, three shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to different regions within the target nucleic acid. In some embodiments, three shRNA sequences in the polynucleotide reduce expression of different target proteins. In some embodiments, three shRNA sequences in the polynucleotide are separated by a linker. In some embodiments, three shRNA sequences in the polynucleotide are contiguous. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA-based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0026] In several embodiments, there is provided an immune cell that expresses a polynucleotide, wherein the polynucleotide comprises (a) a nucleic acid sequence encoding a chimeric receptor; (b) at least four shRNA sequences, wherein the shRNA sequences are expressed in die immune cell and the shRNA sequences reduce expression of at least four target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2; and (c) anucleic acid sequence encoding a membrane -bound interleukin- 15 (mbIL15). In some embodiments, at least two shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least two, at least three, or at least four target proteins. In some embodiments, four shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to the same or an adjacent region within the target nucleic acid. In some embodiments, four shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to different regions within the target nucleic acid. In some embodiments, four shRNA sequences in the polynucleotide reduce expression of different target proteins. In some embodiments, four shRNA sequences in the polynucleotide are separated by a linker. In some embodiments, four shRNA sequences in the polynucleotide are contiguous. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA-based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0027] In several embodiments, there is provided an immune cell that expresses a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence encoding at least four shRNA sequences, wherein the shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least four target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58. CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In some embodiments, four shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to the same or an adjacent region within the target nucleic acid. In some embodiments, four shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to different regions within the target nucleic acid. In some embodiments, four shRNA sequences in the polynucleotide reduce expression of different target proteins. In some embodiments, four shRNA sequences in the polynucleotide are separated by a linker. In some embodiments, four shRNA sequences in the polynucleotide are contiguous. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA- based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0028] In several embodiments, there is provided an immune cell that expresses a polynucleotide, wherein the polynucleotide comprises (a) a nucleic acid sequence encoding a chimeric receptor; (b) at least five shRNA sequences, wherein the shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least five target proteins selected from the group consisting of ADAM17, B2M. Cbl-b, CD58, CIITA. CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2; and (c) anucleic acid sequence encoding a membrane -bound interleukin- 15 (mbIL15). In some embodiments, at least two shRNA sequences are expressed in the immune cell and die shRNA sequences reduce expression of at least tw o. at least three, or at least four target proteins. In some embodiments, five shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to the same or an adjacent region within the target nucleic acid. In some embodiments, five shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to different regions within the target nucleic acid. In some embodiments, five shRNA sequences in the polynucleotide reduce expression of different target proteins. In some embodiments, five shRNA sequences in the polynucleotide are separated by a linker. In some embodiments, five shRNA sequences in the polynucleotide are contiguous. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA- based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0029] In several embodiments, there is provided an immune cell that expresses a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence encoding at least fiveshRNA sequences, wherein the shRNA sequences are expressed in the immune cell and the shRNA sequences reduce expression of at least five target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In some embodiments, five shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to the same or an adjacent region within the target nucleic acid. In some embodiments, five shRNA sequences in the polynucleotide reduce expression of the same target protein by binding to different regions within the target nucleic acid. In some embodiments, five shRNA sequences in the polynucleotide reduce expression of different target proteins. In some embodiments, five shRNA sequences in the polynucleotide are separated by a linker. In some embodiments, five shRNA sequences in the polynucleotide are contiguous. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA- based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0030] In several embodiments, there is provided a polynucleotide, wherein the polynucleotide comprises at least two shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAM17, B2M, CD58. CISH, Cbl-b, FAS, FASL. ICAM1, ICAM2, ICAM3. MED12, CIITA, PRDM1 and PTPN2. In several embodiments, there is provided a polynucleotide, wherein the polynucleotide comprises at least two shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAM17, B2M, CD58, CISH, Cbl-b, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, CIITA, PRDM1 and PTPN2, a chimeric receptor sequence and a membrane-bound interleukin- 15 (mbIL15) sequence. In several embodiments, there is provided a polynucleotide, wherein the polynucleotide comprises two shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAM17, B2M, CD58, CISH, Cbl-b, FAS, FASL. ICAM1, ICAM2, ICAM3, MED12. CIITA, PRDM1 and PTPN2. In several embodiments, there is provided a polynucleotide, wherein the polynucleotide comprises two shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAM17, B2M. CD58, CISH. Cbl-b, FAS, FASL. ICAM1, ICAM2. ICAM3, MED 12, CIITA, PRDM1 and PTPN2, a chimeric receptor sequence and a membranebound interleukin- 15 (mbIL15) sequence. In several embodiments, there is provided a polynucleotide, wherein die polynucleotide comprises three shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAMI 7, B2M, CD58, CISH, Cbl-b, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12. CIITA, PRDM1 and PTPN2. In several embodiments, there is provided apolynucleotide, wherein the polynucleotide comprises three shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAM 17, B2M, CD58, CISH, Cbl-b, FAS, FASL, ICAM1, ICAM2, ICAM3, MED 12, CIITA, PRDM1 and PTPN2, a chimeric receptor sequence and a membranebound interleukin- 15 (mbIL15) sequence. In several embodiments, there is provided a polynucleotide, wherein the polynucleotide comprises four shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAM17, B2M, CD58, CISH, Cbl-b. FAS. FASL, ICAM1. ICAM2, ICAM3. MED12, CIITA. PRDM1 and PTPN2. In several embodiments, there is provided a polynucleotide, wherein the polynucleotide comprises four shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAM 17, B2M. CD58, CISH. Cbl-b, FAS, FASL, ICAM1, ICAM2, ICAM3, MED 12, CIITA, PRDM1 and PTPN2, a chimeric receptor sequence and a membranebound interleukin- 15 (mbIL15) sequence. In several embodiments, there is provided a polynucleotide, wherein the polynucleotide comprises five shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAM17, B2M, CD58, CISH, Cbl-b, FAS, FASL. ICAM1, ICAM2, ICAM3, MED12. CIITA, PRDM1 and PTPN2. In several embodiments, there is provided a polynucleotide, wherein the polynucleotide comprises five shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAM17, B2M. CD58, CISH. Cbl-b, FAS, FASL. ICAM1, ICAM2. ICAM3, MED 12, CIITA, PRDM1 and PTPN2, a chimeric receptor sequence and a membranebound interleukin- 15 (mbIL15) sequence. In several embodiments, there is provided a polynucleotide, wherein the polynucleotide comprises six shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAMI 7, B2M, CD58, CISH, Cbl-b, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12. CIITA, PRDM1 and PTPN2. In several embodiments, there is provided a polynucleotide, wherein the polynucleotide comprises six shRNA sequences, wherein each of shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ADAM17. B2M, CD58, CISH, Cbl-b. FAS, FASL, ICAM1, ICAM2, ICAM3, MED 12, CIITA, PRDM1 and PTPN2, a chimeric receptor sequence and a membranebound interleukin-15 (mbIL15) sequence. In several embodiments, the polynucleotide further comprises a chimeric receptor sequence. In several embodiments, the polynucleotide further comprises a membrane-bound interleukin- 15 (mbIL15) sequence. In some embodiments, each of the shRNA sequences in the polynucleotide are separated by a linker. In some embodiments, each of the shRNA sequences in the polynucleotide are contiguous. In some embodiments, each of the shRNA sequencesis a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA-based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0031] In some embodiments, each of the shRNA sequences are miRNA-based shRNAs (shRNA-miRs) comprised within a single polynucleotide. In some embodiments, miRNA-based shRNAs are obtained by combining miRNA scaffolds into a chimeric cluster for delivering multiple shRNA sequences.

[0032] In several embodiments, there is provided an immune cell having an inhibited expression of at least two target proteins selected from the group consisting of ADAM 17, B2M, Cbl-b, CD58, CIITA. CIS, FAS, FASL. ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In several embodiments, there is provided an immune cell having an inhibited expression of at least three target proteins selected from the group consisting of ADAMI 7. B2M. Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3. MED12. PRDM1 and PTPN2. In several embodiments, there is provided an immune cell having an inhibited expression of at least four target proteins selected from the group consisting of ADAM17, B2M. Cbl-b, CD58. CIITA. CIS, FAS, FASL. ICAM1, ICAM2. ICAM3, MED12. PRDM1 and PTPN2. In several embodiments, there is provided an immune cell having an inhibited expression of at least five target proteins selected from the group consisting of ADAM 17, B2M, Cbl-b. CD58, CIITA. CIS, FAS. FASL, ICAM1, ICAM2, ICAM3. MED12, PRDM1 and PTPN2. In several embodiments, there is provided an immune cell having an inhibited expression of at least six target proteins selected from the group consisting of ADAM17. B2M. Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2. ICAM3, MED12. PRDM1 and PTPN2. In several embodiments, there is provided a composition comprising a population of immune cells as disclosed herein.

[0033] In several embodiments, there is provided a method of treating a disease or condition in a subject comprising administering to the subject the immune cells as disclosed herein. In several embodiments, there is provided a method of treating a disease or condition in a subject comprising administering to the subject a composition as disclosed herein. In several embodiments, there is provided a method of treating a disease or condition in a subject comprising administering to the subject a pharmaceutical composition as disclosed herein. In several embodiments, there is provided a method of treating a disease or condition in a subject comprising administering the polynucleotide as disclosed herein.

[0034] In several embodiments, there is provided a use of a population of immune cells as disclosed herein in a medicament or in the manufacture of a medicament for the treatment of a disease or condition in a subject in need thereof. In several embodiments, there is provided a use of a polynucleotide as disclosed herein in the manufacture of a medicament for the treatment of a disease or condition in a subject in need thereof. In several embodiments, there is provided a use of a composition as disclosed herein in the manufacture of a medicament for the treatment of a disease or condition in a subject in need thereof. In several embodiments, there is provided a use of a pharmaceutical compositionas disclosed herein in the manufacture of a medicament for the treatment of a disease or condition in a subject in need thereof.

[0035] In several embodiments, there is provided a population of immune cells as disclosed herein for use in the treatment of a disease or condition in a subject in need thereof. In several embodiments, there is provided a composition as disclosed herein for use in the treatment of a disease or condition in a subject in need thereof. In several embodiments, there is provided a pharmaceutical composition as disclosed herein for use in the treatment of a disease or condition in a subject in need thereof.

[0036] In several embodiments, the disease or condition comprises an infectious disease, an autoimmune disease, a cancer, or a tumor. In several embodiments, the disease or condition comprises an infectious disease. In several embodiments, the disease or condition comprises an autoimmune disease. In several embodiments, the disease or condition comprises a cancer. In several embodiments, the disease or condition comprises a tumor.

[0037] Also provided herein is a population of immune cells having inhibited expression of a target protein selected from the group consisting of FAS, PRDM1. PTPN2. or any combination thereof. In some embodiments, the immune cells have inhibited expression of a target protein selected from the group consisting of FAS, PRDM1, and PTPN2. In some embodiments, the immune cells have inhibited expression of FAS. In some embodiments, the immune cells have inhibited expression of PRDM1. In some embodiments, the immune cells have inhibited expression of PTPN2.

[0038] Also provided herein is a population of immune cells having inhibited expression of FAS. Also provided herein is a population of immune cells having inhibited expression of PRDM1. Also provided herein is a population of immune cells having inhibited expression of PTPN2.

[0039] Also provided herein is a population of immune cells: having inhibited expression of a target protein selected from the group consisting of FAS, PRDM1, PTPN2, or any combination thereof; expressing a chimeric antigen receptor that binds to an antigen expressed by or associated with cells of a disease or condition; and expressing a membrane-bound interleukin- 15 (mbIL15). In some embodiments, the immune cells have inhibited expression of FAS. In some embodiments, the immune cells have inhibited expression of PRDM1. In some embodiments, the immune cells have inhibited expression of PTPN2. Also provided herein is a population of natural killer (NK) cells: having inhibited expression of PRDM1; expressing a chimeric antigen receptor that binds to an antigen expressed by or associated with cells of a disease or condition; and expressing a membrane-bound interleukin- 15 (mbIL15).

[0040] In some embodiments, the immune cells comprise T cells, natural killer (NK) cells, or T cells and NK cells. In some embodiments, the immune cells comprise T cells. In some embodiments, the immune cells comprise NK cells. In some embodiments, the immune cells comprise T cells and NK cells.

[0041] Also provided herein is a composition comprising any population of immune cells described herein. In some embodiments, the composition comprises a pharmaceutically acceptable excipient. Also provided herein is a pharmaceutical composition comprising any population of immune cells described herein.

[0042] Also provided herein is a method of increasing the expansion of a population of immune cells, wherein the method comprises introducing into a population of immune cells an inhibitory nucleic acid molecule that reduces the expression of a target protein selected from the group consisting of FAS, PRDM1, PTPN2, or any combination thereof. In some embodiments, the inhibitory nucleic acid molecule reduces the expression of a target protein selected from the group consisting of FAS, PRDM1, and PTPN2. In some embodiments, the inhibitory nucleic acid molecule reduces the expression of FAS. In some embodiments, the inhibitory nucleic acid molecule reduces the expression of PRDM1. In some embodiments, the inhibitory nucleic acid molecule reduces the expression of PTPN2.

[0043] Also provided herein is a method of increasing the expansion of a population of natural killer (NK) cells, wherein the method comprises introducing into a population of NK cells an inhibitory nucleic acid molecule that reduces the expression of PRDM1. wherein the NK cells express a chimeric receptor. Also provided herein is a method of increasing the expansion of a population of natural killer (NK) cells, wherein the method comprises introducing into a population of NK cells: an inhibitory nucleic acid molecule that reduces the expression of PRDM1; and a nucleic acid molecule that encodes a chimer receptor.

[0044] In some embodiments, the method increases the ex vivo or in vitro expansion of the population of immune cells. In some embodiments, the method increases the expansion of the population of immune cells as compared to a population of immune cells not introduced with the inhibitory nucleic acid molecule. In some embodiments, the method increases the expansion of the population of immune cells by at least about 100%, 200%, 300%, 400%, or 500%.

[0045] Also provide herein is a method of increasing the in vivo persistence of a population of immune cells, wherein the method comprises introducing into a population of immune cells an inhibitory nucleic acid molecule that reduces the expression of a target protein selected from the group consisting of FAS, PRDM1, PTPN2, or any combination thereof. In some embodiments, the inhibitory nucleic acid molecule reduces the expression of a target protein selected from the group consisting of FAS, PRDM1, and PTPN2. In some embodiments, the inhibitory nucleic acid molecule reduces the expression of FAS. In some embodiments, the inhibitory nucleic acid molecule reduces the expression of PRDM1. In some embodiments, the inhibitory nucleic acid molecule reduces the expression of PTPN2.

[0046] Also provided herein is a method of increasing the in vivo persistence of a population of natural killer (NK) cells, wherein the method comprises introducing into a population of NK cells an inhibitory nucleic acid molecule that reduces the expression of PRDM1, wherein the NK cells expressa chimeric receptor. Also provided herein is a method of increasing the in vivo persistence of a population of natural killer (NK) cells, wherein the method comprises introducing into a population of NK cells: an inhibitory nucleic acid molecule that reduces the expression of PRDM1 ; and a nucleic acid molecule that encodes a cliimer receptor. In some embodiments, the NK cells express a membranebound interleukin- 15 (mbIL15). In some embodiments, the method includes introducing into the NK cells a nucleic acid molecule that encodes a membrane -bound interleukin- 15 (mbIL15).

[0047] In some embodiments, the in vivo persistence of the population of immune cells as compared to a population of immune cells not introduced with the inhibitory nucleic acid molecule. In some embodiments, the method increases the in vivo persistence of the population of immune cells by at least about 100%, 200%, 300%, 400%. or 500%.

[0048] Also provided herein is a population of immune cells produced by a method provided herein. Also provided herein is a method for treating a subject having a disease or condition, wherein the method comprises administering to the subject a population of immune cells, a composition, or a pharmaceutical composition as provided herein. Also provided herein is a use of a population of immune cells, a composition, or a pharmaceutical composition as provided herein as a medicament or in the manufacture of a medicament for treatment of a subject having a disease or condition, such as a an infectious disease, an autoimmune disease, a cancer, or a tumor.

[0049] Provided herein is a method of increasing the in vivo persistence of immune cells, the method comprising administering to a subject having a disease or condition allogeneic immune cells having inhibited expression of a target protein selected from the group consisting of FAS, PRDM1, PTPN2, or any combination thereof. In some embodiments, the allogeneic immune cells have inhibited expression of FAS. In some embodiments, the allogeneic immune cells have inhibited expression of PRDM1. In some embodiments, the allogeneic immune cells have inhibited expression of PTPN2. In some embodiments, the allogeneic immune cells express a chimeric receptor. In some embodiments, the allogeneic immune cells comprise T cells, natural killer (NK) cells, or T cells and NK cells. In some embodiments, the allogeneic immune cells are natural killer (NK) cells.

[0050] In some embodiments, the population of immune cells comprises T cells, natural killer (NK) cells, or T cells and NK cells. In some embodiments the population of immune cells is a population of T cells. In some embodiments the population of immune cells is a population of natural killer (NK) cells. In some embodiments the population of immune cells is a population of T and NK cells. In some embodiments, the population of immune cells express a chimeric receptor. In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In some embodiments, the CAR binds to an antigen expressed by or associated with cells of a disease or condition. In some embodiments, the population of immune cells expresses a membrane-bound interleukin- 15 (mbIL15).

[0051] In some embodiments, the immune cells are allogeneic to the subject. In several embodiments, the disease or condition comprises an infectious disease, an autoimmune disease, a cancer, or a tumor. In several embodiments, the disease or condition comprises an infectious disease.In several embodiments, the disease or condition comprises an autoimmune disease. In several embodiments, the disease or condition comprises a cancer. In several embodiments, the disease or condition comprises a tumor.

[0052] Provided herein is a method of increasing the in vivo persistence of allogeneic immune cells, the method comprising administering to a subject having a disease or condition allogeneic immune cells having inhibited expression of a target protein selected from the group consisting of ICAM1, ICAM2, and CD58.

[0053] Provided herein is a method of increasing the in vivo persistence of allogeneic immune cells, the method comprising administering to a subject having a disease or condition allogeneic immune cells having inhibited expression of a target protein selected from the group consisting of ICAM1, ICAM2, FAS and CD58.

[0054] Also provided herein is a method reducing the cytotoxicity of a subject’s immune cells against allogeneic immune cells, the method comprises administering to a subject having a disease or condition allogeneic immune cells having inhibited expression of a target protein selected from the group consisting of ICAM1, ICAM2, and CD58.

[0055] Also provided herein is a method reducing the cytotoxicity of a subject’s immune cells against allogeneic immune cells, the method comprises administering to a subject having a disease or condition allogeneic immune cells having inhibited expression of a target protein selected from the group consisting of ICAM1, ICAM2, FAS and CD58.

[0056] In some embodiments, the target protein comprises ICAM1. In some embodiments, the target protein comprises ICAM2. In some embodiments, the target protein comprises CD58. In some embodiments, the target protein comprises FAS. In some embodiments, the target protein comprises ICAM1 and CD58. In some embodiments, the target proteins are ICAM1 and CD58. In some embodiments, the target protein comprises ICAM1 and FAS. In some embodiments, the target proteins arc ICAM1 and FAS. In some embodiments, the target protein comprises ICAM2 and CD58. In some embodiments, the target proteins are ICAM2 and CD58. In some embodiments, the target protein comprises ICAM2 and FAS. In some embodiments, the target proteins are ICAM2 and FAS. In some embodiments, the target protein comprises ICAM1 and ICAM2. In some embodiments, the target proteins are ICAM1 and ICAM2. In some embodiments, the target protein comprises CD58 and FAS. In some embodiments, the target proteins are CD58 and FAS. In some embodiments, the target protein comprises ICAM1, ICAM2, and CD58. In some embodiments, the target proteins are ICAM1, ICAM2, and CD58. In some embodiments, the target protein comprises ICAM1, ICAM2, FAS and CD58. In some embodiments, the target proteins are ICAM1, ICAM2, FAS and CD58.

[0057] In some embodiments, the allogeneic immune cells comprise natural killer (NK) cells or T cells. In some embodiments, the allogeneic immune cells comprise natural killer (NK) cells. In some embodiments, the allogeneic immune cells comprise T cells. In some embodiments, the allogeneic immune cells comprise natural killer (NK) cells and T cells. In some embodiments, the allogeneicimmune cells are natural killer (NK) cells. In some embodiments, the allogeneic immune cells express a recombinant receptor. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR) that binds to an antigen expressed by or associated with cells of tire disease or condition. In some embodiments, the disease or condition is a tumor, a cancer, an autoimmune disease, or an infectious disease.

[0058] In some embodiments, the autoimmune disease is selected from the group consisting of chronic inflammatory demyelinating polyneuropathy (CIDP), IgA nephropathy (IgAN), ankylosing spondylitis (AS), antiphospholipid syndrome (APS), autoimmune encephalitis (AE), autoimmune hepatitis (AIH), bullous pemphigoid (BP). Crohn’s disease, chronic graft-versus-host-disease (cGvHD), cold agglutinin disease (CAD). IgG4-related disease (IgG4-RD). neuromyelitis optica spectrum disorder (NMOSD), pemphigus vulgaris (PV). primary biliary cholangitis (PBC), primary membranous nephropathy (pMN), primary progressive multiple sclerosis (PPMS), primary sclerosing cholangitis (PSC), rheumatoid arthritis (RA). Sjogren’s syndrome, and warm autoimmune hemolytic anemia (wAIHA), or any combination thereof. In some embodiments, the autoimmune disease comprises chronic inflammatory demyelinating polyneuropathy (CIDP). In some embodiments, the autoimmune disease comprises IgA nephropathy (IgAN). In some embodiments, the autoimmune disease comprises ankylosing spondylitis (AS), autoimmune hepatitis (AIH), chronic graft-versus-host-disease (cGvHD), cold agglutinin disease (CAD). IgG4-related disease (IgG4-RD), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC). or any combination thereof. In some embodiments, the autoimmune disease comprises ankylosing spondylitis (AS). In some embodiments, the autoimmune disease comprises autoimmune hepatitis (AIH). In some embodiments, the autoimmune disease comprises chronic graft-versus-host-disease (cGvHD). In some embodiments, the autoimmune disease comprises cold agglutinin disease (CAD). In some embodiments, the autoimmune disease comprises IgG4-related disease (IgG4-RD). In some embodiments, the autoimmune disease comprises primary biliary cholangitis (PBC). In some embodiments, the autoimmune disease comprises primary membranous nephropathy (pMN). In some embodiments, the autoimmune disease comprises primary sclerosing cholangitis (PSC). In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus ery thematosus (SLE), lupus nephritis (LN), scleroderma, rheumatoid arthritis (RA), myasthenia gravis (MG), multiple sclerosis (MS), NMDA / NMDAR encephalitis, transverse myelitis, neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein antibody disease (MOGAD), myelin oligodendrocyte glycoprotein spectrum disorder (MOGSD), idiopathic inflammatory myopathy (IIM; also known as myositis), Sjogren’s disease, pemphigus vulgaris, bullous pemphigoid (BP), membranous nephropathy (MN), immune thrombocytopenia (ITP), Hashimoto’ disease, Grave’s disease, insulin resistance, type I diabetes, antiphospholipid syndrome, vasculitis, anti-neutrophilic cytoplasmic antibodies (ANCA) vasculitis (AAV), and anti-synthetase syndrome (ASSD). In some embodiments, the autoimmune disease comprises idiopathic inflammatory myopathy (IIM). multiple sclerosis (MS), myasthenia gravis (MG).rheumatoid arthritis (RA), scleroderma, thyroid disease, type 1 diabetes, and vasculitis, or any combination thereof. In some embodiments, the autoimmune disease is selected from the group consisting of SLE, LN, scleroderma, MG. IIM, and vasculitis. In some embodiments, the autoimmune disease comprises Evans syndrome, autoimmune podocytopathies, scleritis, uveitis, mixed connective tissue disease (MCTD), juvenile dennatomyositis, primary systemic Sjogren’s disease, or any combination thereof. In some embodiments, the autoimmune disease comprises Evans syndrome. In some embodiments, the autoimmune disease comprises an autoimmune podocytopathy. In some embodiments, the autoimmune disease comprises non-infectious scleritis. In some embodiments, the autoimmune disease comprises non-infectious uveitis. In some embodiments, the autoimmune disease comprises mixed connective tissue disease (MCTD). In some embodiments, the autoimmune disease comprises juvenile dennatomyositis. In some embodiments, the autoimmune disease comprises primary systemic Sjogren’s disease.

[0059] In some embodiments, the autoimmune disease is refractory to one or more prior lines of therapy.

[0060] In several embodiments, the disease or condition being treated by administration of a population of immune cells as disclosed herein comprises a disease or condition expressing or associated with a NKG2D ligand. In several embodiments, the disease or condition being treated by administration of a population of immune cells as disclosed herein comprises a disease or condition expressing or associated with CD 19. In several embodiments, the disease or condition being treated by administration of a population of immune cells as disclosed herein comprises a disease or condition expressing or associated with CD70. In several embodiments, the disease or condition being treated by administration of a population of immune cells as disclosed herein comprises a disease or condition expressing or associated with BCMA.

[0061] In several embodiments, the immune cells comprise natural killer (NK) cells and / or T cells. In several embodiments, the immune cells comprise natural killer (NK) cells. In several embodiments, the immune cells comprise T cells. In several embodiments, the immune cells comprise NK cells and T cells. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are T cells. In several embodiments, the immune cells are allogeneic to the subject. In some embodiments, the immune cells are obtained from a donor that does not have cancer. In some embodiments, the immune cells are obtained from a donor that does not have an autoimmune disease. In several embodiments, the immune cells are autologous to the subject.

[0062] In several embodiments, expression of one or more genes or proteins in immune cells is modulated using an RNA-guided endonuclease. In several embodiments, expression of one or more genes or proteins in immune cells is modulated using an inhibitory nucleic acid molecule (e.g.. RNA interfering agent). In several embodiments, expression of one or more genes or proteins in immune cells is modulated using short hairpin RNA (shRNA). In several embodiments, expression of one or more genes or proteins in immune cells is modulated using small interfering RNA (siRNA). In severalembodiments, expression of one or more genes or proteins in immune cells is modulated using microRNA (miRNA). In several embodiments, expression of one or more genes or proteins in immune cells is modulated by more than one inhibitory nucleic acid molecule (e.g., RNA interfering agent).

[0063] In some embodiments, the one or more inhibitory nucleic acid molecule(s) is encoded by an expression vector. In some embodiments, the expression vector comprises a polynucleotide comprising one or more inhibitory nucleic acid molecule(s) that can reduce expression of a target protein. In some embodiments, the expression vector comprises a polynucleotide comprising two inhibitory nucleic acid molecule(s) that reduce expression of a target protein. In some embodiments, the expression vector comprises a polynucleotide comprising three inhibitory nucleic acid molecule(s) that reduce expression of a target protein. In some embodiments, the expression vector comprises a polynucleotide comprising four inhibitory nucleic acid molecule(s) that reduce expression of a target protein. In some embodiments, the expression vector comprises a polynucleotide comprising five inhibitory nucleic acid molecule(s) that reduce expression of a target protein. In some embodiments, the expression vector comprises a polynucleotide comprising six inhibitory nucleic acid molecule(s) that reduce expression of a target protein. In some embodiments, the polynucleotide comprising more than one inhibitory nucleic acid molecule reduces expression of the same target protein. In some embodiments, the polynucleotide comprising more than one inhibitory nucleic acid molecule reduces expression of the same target protein by binding to the same or an adjacent region within the target nucleic acid. In some embodiments, the polynucleotide comprising more than one inhibitory nucleic acid molecule reduces expression of the same target protein by binding to different regions within the target nucleic acid. In some embodiments, the polynucleotide comprising more than one inhibitory nucleic acid molecule reduces expression of different target proteins. In some embodiments, the polynucleotide comprising more than one inhibitory nucleic acid molecule is separated by a linker. In some embodiments, the inhibitory' nucleic acid molecule is shRNA. In some embodiments, the inhibitory nucleic acid molecule is siRNA. In some embodiments, the inhibitory nucleic acid molecule is miRNA. In some embodiments, each of the shRNA sequences are miRNA-based shRNAs (shRNA- miRs) comprised within a single polynucleotide. In some embodiments, miRNA-based shRNAs are obtained by combining efficient miRNA scaffolds into a chimeric cluster for delivering multiple shRNA sequences.

[0064] In several embodiments, a vector comprising any of the polynucleotides disclosed herein is provided.

[0065] In several embodiments, the immune cell expresses a chimeric receptor. In several embodiments, the chimeric receptor comprises an extracellular ligand binding domain, a transmembrane domain, and an intracellular signaling region. In some embodiments, the extracellular ligand binding domain targets an antigen expressed by the diseased cells.

[0066] In several embodiments, the extracellular ligand binding domain targets an antigen selected from BCMA. a NKG2D ligand. CD 19, or CD70. In several embodiments, the extracellularligand binding domain targets a BCMA antigen. In several embodiments, the extracellular ligand binding domain targets an NKG2D ligand. In several embodiments, the extracellular ligand binding domain targets a CD 19 antigen. In several embodiments, the extracellular ligand binding domain targets a CD70 antigen.

[0067] In several embodiments, the extracellular ligand binding domain targets an antigen selected from BAFF-R, BCMA, CD20, CD22, CD27, CD28, CD33, CD38, CD45, CD47. CD54, CD56, CD81, CD117, CD138, CD200, FcRH5, GPRC5D, or SLAMF7. In some embodiments, the extracellular ligand binding domain targets a BAFF-R antigen. In some embodiments, the extracellular ligand binding domain targets a BCMA antigen. In some embodiments, the extracellular ligand binding domain targets a CD20 antigen. In some embodiments, the extracellular ligand binding domain targets a CD22 antigen. In some embodiments, the extracellular ligand binding domain targets a CD27 antigen. In some embodiments, the extracellular ligand binding domain targets a CD28 antigen. In some embodiments, the extracellular ligand binding domain targets a CD38 antigen. In some embodiments, the extracellular ligand binding domain targets a CD45 antigen. In some embodiments, the extracellular ligand binding domain targets a CD47 antigen. In some embodiments, the extracellular ligand binding domain targets a CD54 antigen. In some embodiments, the extracellular ligand binding domain targets a CD56 antigen. In some embodiments, the extracellular ligand binding domain targets a CD81 antigen. In some embodiments, the extracellular ligand binding domain targets a CD 117 antigen. In some embodiments, the extracellular ligand binding domain targets a CD138 antigen. In some embodiments, the extracellular ligand binding domain targets a CD200 antigen. In some embodiments, the extracellular ligand binding domain targets an FcRH5 antigen. In some embodiments, the extracellular ligand binding domain targets a GPRC5D antigen. In some embodiments, the extracellular ligand binding domain targets a SLAMF7 antigen.

[0068] In several embodiments, the transmembrane domain comprises CD8, CD28, or a portion thereof, optionally wherein the transmembrane domain comprises CD8a or a portion thereof. In several embodiments, the transmembrane domain comprises CD8 or a portion thereof. In several embodiments, the transmembrane domain comprises CD28, or a portion thereof. Depending on the embodiment the transmembrane domain optionally comprises CD8a or a portion thereof, in combination with CD8 or CD28.

[0069] In several embodiments, the intracellular signaling region comprises a primary signaling domain and a co-stimulatory signaling domain. In some embodiments, the primary signaling domain comprises a CD3zeta domain. In some embodiments, the co-stimulatory signaling domain comprises an intracellular signaling domain of 0X40, 4- IBB, CD28. or a signaling portion thereof. In some embodiments, the co-stimulatory signaling domain comprises an intracellular signaling domain of 0X40 or a signaling portion thereof. In some embodiments, the co-stimulatory signaling domain comprises an intracellular signaling domain of 4-1BB or a signaling portion thereof. In someembodiments, the co-stimulatory signaling domain comprises an intracellular signaling domain of CD28 or a signaling portion thereof.

[0070] In several embodiments, the immune cell is engineered to express a membrane bound IL- 15 (mbIL15). In some such embodiments, the chimeric receptor and the mbIL15 are encoded by the same nucleic acid molecule. In several embodiments, the nucleic acid sequences encoding the chimeric receptor and the mbIL15 are separated by a nucleic acid sequence encoding a 2A peptide. In some such embodiments, wherein the chimeric receptor and the mbIL15 are encoded by the same nucleic acid molecule, the nucleic acid sequences encoding the chimeric receptor and the mbIL15 are separated by a nucleic acid sequence encoding a 2 A peptide.

[0071] In some embodiments, the one or more inhibitory nucleic acid molecule(s) and the chimeric receptor are comprised within the same polynucleotide. In some embodiments, the one or more shRNA(s) and the chimeric receptor are comprised within the same polynucleotide. In some embodiments, the one or more inhibitory nucleic acid molecule(s) and the chimeric receptor are comprised in different polynucleotides. In some embodiments, the one or more shRNA(s) and the chimeric receptor are comprised in different polynucleotides. In some embodiments, the one or more shRNA(s) and the chimeric receptor are expressed bicistronically. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0072] In some embodiments, the one or more inhibitory nucleic acid molecule(s), the chimeric receptor and a membrane-bound interleukin- 15 (mbIL15) are comprised within the same polynucleotide. In some embodiments, the one or more shRNA(s), the chimeric receptor and a membrane-bound interleukin- 15 (mbIL15) are comprised within the same polynucleotide. In some embodiments, the one or more inhibitory nucleic acid molecule(s), the chimeric receptor and a membrane-bound interleukin- 15 (mbIL15) are comprised in different polynucleotides. In some embodiments, the one or more shRNA(s), the chimeric receptor and a membrane-bound interleukin- 15 (mbIL15) arc comprised in different polynucleotides. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0073] In some such embodiments, the immune cells are modified within an additional target sequence in a target gene to yield reduced levels of expression of a protein encoded by the target gene, as compared to an immune cell not modified within the additional target sequence.

[0074] In additional embodiments, there is provided a population of immune cells that express a chimeric receptor comprising an extracellular ligand binding domain, a transmembrane domain, and an intracellular signaling region. In some embodiments, the extracellular ligand binding domain targets an antigen expressed by cells of a disease or condition. In some embodiments, the immune cells are modified within a target sequence in a target gene selected from, ADAM 17, B2M, CBLB, CD58, CUT A, CISH. FAS, FASLG. ICAM1, ICAM2, ICAM3, MED12. PRDM1, PTPN2 or any combination thereof. In some embodiments, the modification yields a reduced expression and / or function of the protein encoded by the target gene, as compared to an immune cell not modified within the targetsequence in the target gene. In some embodiments, the immune cells are modified at an additional target sequence within a target gene to yield reduced levels of expression of the protein encoded by the target gene, as compared to an immune cell not modified at the additional target sequence.

[0075] In several embodiments, the modification to the nucleic acid sequence encoding the target protein is made using an RNA-guided endonuclease. In several embodiments, the modification to the nucleic acid sequence encoding the target protein is made using a Crispr / Cas9 system.

[0076] In several embodiments, the expression of one or more genes or proteins in immune cells is modulated using an RNA interfering agent. In several embodiments, the expression of one or more genes or proteins in immune cells is modulated using short hairpin RNA (shRNA). In several embodiments, the expression of one or more genes or proteins in immune cells is modulated using small interfering RNA (siRNA). In several embodiments, expression of one or more genes or proteins in immune cells is modulated using microRNA (miRNA).

[0077] In several embodiments, the immune cells comprise Natural Killer (NK) cells. T cells, induced pluripotent stem cells (iPSCs), iPSC-derived NK cells, iPSC-derived T cells, NK-92 cells, or any combination thereof. In several embodiments, the immune cells comprise Natural Killer (NK) cells. In several embodiments, the immune cells comprise T cells. In several embodiments, the immune cells comprise iPSCs. In several embodiments, the immune cells comprise iPSC-derived NK cells. In several embodiments, the immune cells comprise iPSC-derived T cells. In several embodiments, the immune cells comprise Natural Killer (NK) cells and T cells.

[0078] In several embodiments, the immune cells comprise a mixture of NK cells and T cells or a mixture of iPSC-derived NK cells and T cells. In several embodiments, the immune cells comprise a mixture of iPSC-derived NK cells and / or iPSC-derived T cells.

[0079] In several embodiments, the immune cells provided for herein exhibit enhanced expansion capability, enhanced cytotoxicity against target tumor cells, enhanced persistence, or any combination thereof, as compared to immune cells that do not comprise a modulation of expression of one or more genes or proteins.

[0080] In several embodiments, there is provided a method of inhibiting expression of at least two target proteins in an immune cell. In several embodiments, the method comprises introducing into the immune cell at least two shRNA sequences. In some embodiments, the shRNA sequences are expressed in the immune cell and reduce expression of at least two target proteins in the immune cell. In some embodiments, the at least tw o target proteins are selected from the group consisting of ADAM17, B2M, Cbl-b, CD58. CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3. MED12, PRDM1 and PTPN2. In several embodiments, the shRNA sequences expressed in the immune cell reduce expression of two target proteins in the immune cell. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA- based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0081] In several embodiments, there is provided a method of inhibiting expression of at least three target proteins in an immune cell. In several embodiments, the method comprises introducing into the immune cell at least three shRNA sequences. In some embodiments, the shRNA sequences are expressed in the immune cell and reduce expression of at least three target proteins in the immune cell. In some embodiments, the at least three target proteins are selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In several embodiments, the shRNA sequences expressed in the immune cell reduce expression of three target proteins in the immune cell. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA- based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0082] In several embodiments, there is provided a method of inhibiting expression of at least four target proteins in an immune cell. In several embodiments, the method comprises introducing into the immune cell at least four shRNA sequences. In some embodiments, the shRNA sequences are expressed in the immune cell and reduce expression of at least four target proteins in the immune cell. In some embodiments, the at least four target proteins are selected from the group consisting of ADAM17. B2M. Cbl-b, CD58, CIITA, CIS, FAS, FASL. ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In several embodiments, the shRNA sequences expressed in the immune cell reduce expression of four target proteins in the immune cell. In some embodiments, each of the shRNA sequences is a microRNA-based shRNA (shRNA miR). In some embodiments, each of the microRNA- based shRNA (shRNA miR) is comprised within a single polynucleotide. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0083] In several embodiments, the target proteins comprise ADAM 17. In several embodiments, the target proteins comprise B2M. In several embodiments, the target proteins comprise CD58. In several embodiments, the target proteins comprise FAS. In several embodiments, the target proteins comprise CIS. In several embodiments, the target proteins comprise CIS and MED 12. In several embodiments, the target proteins comprise Cbl-b. In several embodiments, the target proteins comprise Cbl-b and CIS. In several embodiments, the target proteins comprise FAS. In several embodiments, the target proteins comprise FAS and CIS. In several embodiments, the target proteins comprise FAS and MED 12. In several embodiments, the target proteins comprise FASL. In several embodiments, the target proteins comprise FASL and CIS. In several embodiments, the target proteins comprise FASL and MED12. In several embodiments, the target proteins comprise ICAM1. In several embodiments, the target proteins comprise ICAM1 and MED 12. In several embodiments, the target proteins comprise ICAM1 and PTPN2. In several embodiments, the target proteins comprise ICAM1 and PRDM1. In several embodiments, the target proteins comprise ICAM2. In several embodiments, the target proteins comprise ICAM2 and MED 12. In several embodiments, the target proteins comprise ICAM2 and PTPN2. In several embodiments, the target proteins comprise ICAM2 and PRDM1. Inseveral embodiments, the target proteins comprise ICAM3. In several embodiments, the target proteins comprise ICAM3 and MED12. In several embodiments, the target proteins comprise ICAM3 and PTPN2. In several embodiments, the target proteins comprise ICAM3 and PRDM1. In several embodiments, the target proteins comprise MED 12. In several embodiments, the target proteins comprise PTPN2. In several embodiments, the target proteins comprise PTPN2 and MED 12. In several embodiments, the target proteins comprise PTPN2 and PRDM1. In several embodiments, the target proteins comprise PRDM1. In several embodiments, the target proteins comprise PRDM1 and CIS. In several embodiments, the target proteins comprise PRDM1 and MED12. In several embodiments, the target proteins comprise CIITA. In several embodiments, the target proteins comprise ICAM1 and ICAM2. In several embodiments, the target proteins comprise ICAM1 and CD58. In several embodiments, the target proteins comprise ICAM1 and FAS. In several embodiments, the target proteins comprise ICAM2 and CD58. In several embodiments, the target proteins comprise ICAM2 and FAS. In several embodiments, the target proteins comprise FAS and CD58. In several embodiments, the target proteins comprise ICAM1, ICAM2 and CD58. In several embodiments, the target proteins comprise ICAM1, ICAM2 and FAS. In several embodiments, the target proteins comprise ICAM1, FAS and CD58. In several embodiments, the target proteins comprise ICAM2. FAS and CD58 In several embodiments, the target proteins comprise ICAM1. ICAM2, FAS and CD58. In some embodiments, all the shRNA sequences are miRNA-based shRNAs (shRNA-miRs) comprised within a single polynucleotide. In some embodiments. miRNA-based shRNAs are obtained by combining miRNA scaffolds into a chimeric cluster for delivering multiple shRNA sequences. In several embodiments, the shRNA sequences disclosed herein are synthetic shRNA sequences.

[0084] In several embodiments, there is provided a method of inhibiting expression of a FAS gene in a natural killer (NK) cell. In several embodiments, the method comprises administering to the NK cell, a CRISPR / Cas system comprising a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the FAS gene; and a Cas molecule; or a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the FAS gene; and a second nucleotide sequence encoding a Cas molecule. In several embodiments, the method comprises administering to the NK cell a CRISPR / Cas system comprising a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the FAS gene and a Cas molecule. In several embodiments, the method comprises administering to the NK cell a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the FAS gene; and a second nucleotide sequence encoding a Cas molecule. In several embodiments, the target sequence of the FAS gene comprises SEQ ID NOS: 201-204. or 215.

[0085] In several embodiments, there is provided a method of inhibiting expression of the PTPN2 gene in a natural killer (NK) cell. In several embodiments, the method comprises administeringto the NK cell, a CRISPR / Cas system comprising a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the PTPN2 gene; and a Cas molecule; or a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the PTPN2 gene; and a second nucleotide sequence encoding a Cas molecule. In several embodiments, the method comprises administering to the NK cell a CRISPR / Cas system comprising a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the PTPN2 gene; and a Cas molecule. In several embodiments, the method comprises administering to the NK cell a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the PTPN2 gene; and a second nucleotide sequence encoding a Cas molecule. In several embodiments, the target sequence of the PTPN2 gene comprises SEQ ID NOS: 208-211.

[0086] In several embodiments, there is provided a method of inhibiting expression of the ICAM1 gene in a natural killer (NK) cell. In several embodiments, the method comprises administering to the NK cell, a CRISPR / Cas system comprising a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the ICAM1 gene; and a Cas molecule; or a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the ICAM1 gene; and a second nucleotide sequence encoding a Cas molecule. In several embodiments, the method comprises administering to the NK cell a CRISPR / Cas system comprising a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the ICAM1 gene; and a Cas molecule. In several embodiments, the method comprises administering to the NK cell a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the ICAM1 gene; and a second nucleotide sequence encoding a Cas molecule. In several embodiments, the target sequence of the ICAM1 gene comprises SEQ ID NO:218.

[0087] In several embodiments, there is provided a method of inhibiting expression of the ICAM2 gene in a natural killer (NK) cell. In several embodiments, the method comprises administering to the NK cell, a CRISPR / Cas system comprising a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the ICAM2 gene; and a Cas molecule; or a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the ICAM2 gene; and a second nucleotide sequence encoding a Cas molecule. In several embodiments, the method comprises administering to the NK cell a CRISPR / Cas system comprising a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the ICAM2 gene; and a Cas molecule. In several embodiments, the method comprises administering to the NK cell a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising atargeting domain that is complementary with a target sequence of the ICAM2 gene; and a second nucleotide sequence encoding a Cas molecule. In several embodiments, the target sequence of the ICAM2 gene comprises SEQ ID NOS: 219-221.

[0088] In several embodiments, there is provided a method of inhibiting expression of the CD58 gene in a natural killer (NK) cell. In several embodiments, the method comprises administering to the NK cell, a CRISPR / Cas system comprising a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the CD58 gene; and a Cas molecule; or a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the CD58 gene; and a second nucleotide sequence encoding a Cas molecule. In several embodiments, the method comprises administering to the NK cell a CRISPR / Cas system comprising a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the CD58 gene; and a Cas molecule. In several embodiments, the method comprises administering to the NK cell a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the CD58 gene; and a second nucleotide sequence encoding a Cas molecule. In several embodiments, the target sequence of the CD58 comprises SEQ ID NO: 217.

[0089] In several embodiments, a composition comprising a plurality of the immune cells as disclosed herein is provided. In several embodiments, a composition comprising a plurality of the immune cells as disclosed herein and a pharmaceutically acceptable excipient is provided. In several embodiments, a pharmaceutical composition comprising a plurality of the immune cells as disclosed herein is provided. In several embodiments, a pharmaceutical composition comprising a plurality of the immune cells as disclosed herein and a pharmaceutically acceptable excipient is provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 shows a non-limiting embodiment of a schematic workflow for genetic editing, transduction, expansion, and analysis of immune cells.

[0091] Figures 2A and 2B show the cytotoxicity of natural killer (NK) cells expressing a BCMA CAR and edited at the indicated gene targets against Daudi target cells when co-cultured at an effector-to-target cell ratio (E:T) of 1 : 1 or 1 :2, respectively, for 14 days (arrows indicate rechallenge with target cells; EP Control: mock-electroporated and mock-transduced NK cells; EP Control BCMA CAR: mock-electroporated, BCMA CAR-transduced NK cells).

[0092] Figure 2C shows the persistence of NK cells expressing a BCMA CAR and edited at the indicated gene targets when cultured in the presence of interleukin 2 (IL2) for 14 days (EP Control: mock -electroporated and mock-transduced NK cells; EP Control BCMA CAR: mock-electroporated, BCMA CAR-transduced NK cells).

[0093] Figures 3A and 3B show the cytotoxicity of NK cells expressing a BCM A CAR and edited at the indicated gene targets against Daudi target cells when co-cultured at an E:T of 1:1 or 1:2, respectively, for 12 days (arrows indicate rechallenge with target cells; EP Control: mock- electroporated and mock-transduced NK cells; EP Control BCMA CAR: mock-electroporated, BCMA CAR-transduced NK cells).

[0094] Figure 3C shows the persistence of NK cells expressing a BCMA CAR and edited at the indicated gene targets when cultured with Daudi target cells for 17 days in the absence of interleukin-2 (IL-2; EP Control: mock-electroporated and mock-transduced NK cells; EP Control BCMA CAR: mock-electroporated, BCMA CAR-transduced NK cells).

[0095] Figures 4A and 4B show the cytotoxicity of NK cells expressing a CD 19 CAR and edited at the indicated gene targets against Nalm6 target cells when co-cultured at an E:T of 1 :2 or 1 :4, respectively, for 5 days (arrows indicate rechallenge with target cells; EP Control: mock-electroporated and mock -transduced NK cells; EP Control CD 19 CAR: mock -electroporated, CD 19 CAR-transduced NK cells).

[0096] Figure 4C shows the cytotoxicity of NK cells expressing a CD 19 CAR and edited at the indicated gene targets against Nalm6 target cells when co-cultured at an E:T of 1 :2 for 18 days (arrows indicate rechallenge with target cells; EP Control CD 19 CAR: mock-electroporated, CD 19 CAR-transduced NK cells).

[0097] Figures 5A and 5B show the cytotoxicity of NK cells expressing a NKG2D CAR and edited at the indicated gene targets against HL60 or Mohn 13 target cells, respectively, when co-cultured at an E:T of 1:2 for 6 days (arrows indicate rechallenge with target cells; EP Control: mock- electroporated and mock-transduced NK cells; EP Control NKG2D CAR: mock-electroporated, NKG2D CAR-transduced NK cells).

[0098] Figure 6A shows the cytotoxicity of NK cells expressing a BCMA CAR and edited at the indicated gene targets against RPMI8226 target cells when co-culturcd at an E:T of 1:2 for 18 days (arrows indicate rechallenge with target cells; EP Control BCMA CAR: mock-electroporated, BCMA CAR-transduced NK cells).

[0099] Figure 6B shows the cytotoxicity of NK cells expressing a BCMA CAR and edited at the indicated gene targets against Daudi target cells when co-cultured at an E:T of 1:2 for 24 days (arrows indicate rechallenge with target cells; EP Control BCMA CAR: mock-electroporated, BCMA CAR-transduced NK cells).

[0100] Figure 6C shows the cytotoxicity of NK cells expressing a NKG2D CAR and edited at the indicated gene targets against Molml3 target cells when co-cultured at an E:T of 1:2 for 6 days (arrows indicate rechallenge with target cells; EP Control: mock-electroporated and mock-transduced NK cells; EP Control NKG2D CAR: mock-electroporated, NKG2D CAR-transduced NK cells).

[0101] Figure 7A shows the expansion of NK cells expressing a CD19 CAR and edited at the indicated gene targets, with fold expansion normalized to cell number at day 14 for each group andassessed through day 62 (EP Control: mock-electroporated and mock-transduced NK cells; EP Control CD19 CAR: mock-electroporated, CD19 CAR-transduced NK cells).

[0102] Figure 7B shows the cytotoxicity of NK cells expressing a CD19 CAR and edited at the indicated gene targets against Nalm6 target cells when co-cultured at an E:T of 1:2 for 11 days (arrows indicate rechallenge with target cells; EP Control CD 19 CAR: mock-electroporated, CD 19 CAR-transduced NK cells).

[0103] Figure 7C shows the persistence of NK cells expressing a CD19 CAR and edited at the indicated gene targets when cultured for 54 days in the absence of IL-2 (EP Control: mock- electroporated and mock-transduced NK cells; EP Control CD19 CAR: mock-electroporated, CD19 CAR-transduced NK cells).

[0104] Figure 8A shows the persistence of NK cells expressing a NKG2D CAR and edited at the indicated gene targets when cultured with target cells for 21 days in tire absence of IL-2 (EP Control: mock-electroporated and mock-transduced NK cells; EP Control CD19 CAR: mock-electroporated. CD19 CAR-transduced NK cells).

[0105] Figure 8B shows the cytotoxicity of NK cells edited at the indicated gene targets against HL60 target cells when co-cultured at an E:T of 1:2 for 7 days (arrows indicate rechallenge with target cells).

[0106] Figure 9A shows in vivo tumor control as assessed by bioluminescence imaging (BLI) in NOD scid gamma (NSG) mice inoculated with HL60 target cells on days -1, 7, and 17 and injected with vehicle or NK cells expressing a NKG2D CAR and edited at the indicated gene targets on day 0 (EP Control NKG2D CAR: mock-electroporated, NKG2D CAR-transduced NK cells).

[0107] Figure 9B shows the in vivo peripheral persistence of NKG2D CAR NK cells in selected groups of mice from Figure 9A, as assessed by the number of CAR+CD56+ cells per 10,000 live leukocy tes (EP Control NKG2D CAR: mock-electroporated, NKG2D CAR-transduced NK cells).

[0108] Figure 9C shows in vivo tumor control as assessed by biolumincsccncc imaging (BLI) m NOD scid gamma (NSG) mice inoculated with HL60 target cells on days -1, 13, and 25 and injected with vehicle or NK cells expressing a NKG2D CAR and edited at the indicated gene targets on day 0 (EP Control NKG2D CAR: mock-electroporated, NKG2D CAR-transduced NK cells).

[0109] Figure 9D shows in vivo tumor control as assessed by bioluminescence imaging (BLI) in NOD scid gamma (NSG) mice inoculated with Nalm6 target cells on days -3 and 10, and injected with vehicle or NK cells expressing a CD 19 CAR and edited at the indicated gene targets on day 0 (EP Control CD19 CAR: mock-electroporated, CD19 CAR-transduced NK cells).

[0110] Figure 9E shows the in vivo peripheral persistence of CD19 CAR NK cells in mice from Figure 9A, as assessed by the number of CAR+CD56+ cells per 10.000 live leukocytes (EP Control CD19 CAR: mock -electroporated, CD19 CAR-transduced NK cells).

[0111] Figures 10A and 10B show the cytotoxicity of donor NK cells against K562 target cells edited at the indicated gene targets when co-cultured at an E:T of 1:2 for 5 days or 70 hours, respectively.

[0112] Figure 11 shows day 11 expression of target genes by NK cells edited at the indicated targets (EP Control: mock-electroporated, mock-transduced NK cells; EP Control CD19 CAR: mock- electroporated, CD 19 CAR-transduced NK cells).

[0113] Figures 12A and 12B show the percentage of CD3+ T cells expressing CD25 following coculture of T cells or peripheral blood mononuclear cells (PBMCs), respectively, with allogeneic NK cells edited at the indicated targets (EP Control: mock-electroporated, mock-transduced NK cells; EP Control CD 19 CAR: mock-electroporated, CD 19 CAR-transduced NK cells; TA: T Cell TransAct™ reagent).

[0114] Figures 12C and 12D show the percentage of CD3+ T cells expressing CD25 following coculture of T cells or peripheral blood mononuclear cells (PBMCs), respectively, with autologous NK cells edited at the indicated targets (EP Control: mock-electroporated, mock-transduced NK cells; EP Control CD19 CAR: mock-electroporated, CD19 CAR-transduced NK cells; TA: T Cell TransAct™ reagent).

[0115] Figure 13A shows CellTrace™ Far Red (CTFR) fluorescence in T cells following coculture with allogeneic or autologous NK cells edited at the indicated targets (EP Control: mock- electroporated. mock-transduced NK cells; EP Control CD19 CAR: mock-electroporated, CD19 CAR- transduced NK cells; TA: T Cell TransAct™ reagent).

[0116] Figure 13B shows CellTrace™ Far Red (CTFR) fluorescence in PBMCs following coculture with allogeneic or autologous NK cells edited at the indicated targets (EP Control: mock- electroporated, mock-transduced NK cells; EP Control CD19 CAR: mock-electroporated, CD19 CAR- transduced NK cells; TA: T Cell TransAct™ reagent).DETAILED DESCRIPTION

[0117] Some embodiments of the methods and compositions provided herein relate to immune cells in which the expression of one or more genes or proteins is modulated (e.g., knocked down or knocked out) and use of the same in immunotherapy. In several embodiments, the immune cells are also engineered to express chimeric receptors (e.g., chimeric antigen receptors). As used herein, the term ‘’chimeric receptor complexes” shall be given its ordinary' meaning and shall also refer to (unless otherwise indicated), chimeric antigen receptors (CAR) and chimeric receptors (also called activating chimeric receptors in the case of NKG2D chimeric receptors). Some embodiments include methods of use of the compositions or cells as a medicament e.g., in immunotherapy, such as for the treatment of a cancer or an autoimmune disease. Some embodiments include methods of use of the compositions or cells in immunotherapy, such as for the treatment of an autoimmune disease.

[0118] While autologous CAR T cell therapies have been developed and shown to exhibit substantial in vivo persistence and efficacy, the majority of patients treated with autologous CAR T cell therapy experience cytokine release syndrome (CRS) and / or a neurotoxicity. Further, autologous CAR T cell therapies face numerous challenges, including the need for leukapheresis and manufacture of a conforming CAR T cell product from patients that are often extremely sick, heavily pre-treated, or both. Manufacturing a sufficient number of CAR T cells from such patients can be difficult, or in some cases, impossible. In addition, a potential patient may not survive the length of time it takes to manufacture the final CAR T cell product from the T cells obtained from the patient.

[0119] By contrast, NK cell therapies, including allogeneic NK cell therapies manufactured from healthy donors, can obviate many of these challenges. For example, manufacturing success rates for allogeneic CAR NK cells may be higher due to better quality of incoming donor cells. Allogeneic CAR NK cell therapies can also be provided when a patient is in need, without having to wait for the patient’s own cells to be manufactured. Thus, allogeneic NK cell therapies are being investigated for use as off-the-shelf products. Despite the potential benefits offered by NK cells, they have not been shown to persist in vivo to the same extent as T cells. Solutions are therefore needed to overcome this challenge. Described herein are modulation of the expression of one or more genes or proteins in NK cells that increase the persistence, efficacy (e.g.. cytotoxicity), or both, of NK cells. Embodiments of such NK cells include compositions and methods of using the same to treat or inhibit a disease or condition (e.g., infectious disease, autoimmune disease, tumor, or cancer) in a subject. For example, experiments described herein found that disruption of particular genes, including e.g., ADAM17, B2M, CBLB, CD58, CIITA, CIS, FAS, FASLG, ICAM1, ICAM2, ICAM3, MED12, PRDM1 or PTPN2, or a combination thereof, imparted surprisingly beneficial effects to NK cells, including enhanced cytotoxicity'. These results were observed in NK cells expressing CARs targeting different antigens (e.g., BCMA, NKG2D or CD19). Without wishing to be bound by theory, these findings are consistent with an observ ation that such gene edits impart benefits to immune cells, including CAR-cxprcssing immune cells (e.g., NK cells), regardless of the particular antigen targeted by the CAR.

[0120] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. Cell Therapy and Engineering Cells

[0121] Some embodiments of the methods and compositions provided herein relate to a cell such as an immune cell. In some embodiments, the level of gene or protein expression in an immune cell is modulated (e.g., reduced). In some embodiments, an immune cell is engineered to express a chimeric receptor that binds to an antigen. In some embodiments, the immune cell expresses a chimeric receptor and is engineered to express reduced levels of one or more genes. In some embodiments, the immune cell expresses a chimeric receptor and is engineered to express reduced levels of one or more proteins. In some embodiments, the immune cells that are engineered to express a chimeric receptorthat binds to an antigen also express a membrane -bound IL15 (mbIL15). The recombinant receptor, such as a CAR, generally includes an extracellular antigen-binding domain specific to the antigen (e.g., CD 19), which is linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s).

[0122] For example, an immune cell, such as a T cell, may be engineered to include a chimeric receptor such as a CD19-directed chimeric receptor, or engineered to include a nucleic acid encoding said chimeric receptor as described herein. In some embodiments, a NK cell is engineered to express a chimeric receptor that binds to an antigen (e.g., an antigen expressed by a cancer cell, e.g., CD19). Additional embodiments relate to engineering a second set of cells to express another cytotoxic receptor complex, such as an NKG2D chimeric receptor complex as disclosed herein. Thus, in some embodiments, combinations or compositions comprising two different types of immune cells, (e.g., T cells and NK cells) are contemplated. In some embodiments, the engineered T cells and the engineered NK cells express the same chimeric receptor. In some embodiments, the engineered T cells and the engineered NK cells express different chimeric receptors. In some embodiments, the engineered T cells and the engineered NK cells express chimeric receptors that bind to the same antigen (e.g.. different epitopes of the same antigen). In some embodiments, the engineered T cells and the engineered NK cells express chimeric receptors that bind different antigens.

[0123] Also provided are compositions comprising any of the immune cells described herein. In some aspects, the immune cells disclosed herein are provided as pharmaceutical compositions and formulations suitable for administration to a subject, such as for cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, uses of the compositions for treatment of subjects, and uses of the compositions in the manufacture of medicaments for treating subjects.

[0124] Additional embodiments relate to the further modulation of the expression of one or more genes or proteins in immune cells, such as NK cells (e.g., donor NK cells), to increase persistence and / or potency of NK cells. Still additional embodiments relate to the further genetic manipulation of immune cells (e.g., donor NK) to reduce, disrupt, minimize and / or eliminate the ability of endogenous immune cells to exert cytotoxicity against the manipulated immune cells.

[0125] Cellular immunotherapy has enabled approaches that harness certain aspects of the immune system to fight various diseases (e.g., autoimmune diseases, cancers, etc.). In some cases, a patient’s own immune cells are modified to specifically eradicate that patient’s disease. 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.

[0126] To facilitate cellular immunotherapies, provided herein are polynucleotides (e.g., encoding chimeric receptors), polypeptides (e.g., chimeric receptors), and vectors that encode chimeric antigen receptors (CAR) that comprise a target binding moiety (e.g., an extracellular binder of a ligand, or an antigen-directed chimeric receptor, expressed by a diseased or infected cell) and an intracellularsignaling region. For example, some embodiments include a polynucleotide, polypeptide, or vector that encodes, for example a chimeric antigen receptor directed against an antigen, for example, CD 19, CD123, CD70, Her2, mesothelin, Claudin 6, BCMA, or EGFR, among others, to facilitate targeting of an immune cell to a cancer and exerting cytotoxic effects on the cancer cell. Some embodiments include a polynucleotide, polypeptide, or vector that encodes, for example a chimeric antigen receptor directed against an antigen associated with an autoimmune disease, for example, BAFF-R, BCMA, CD 19, CD20, CD22, CD27. CD28, CD33, CD38, CD45, CD47, CD54, CD56, CD81, CD117, CD138, CD200, FcRH5, GPRC5D or SLAMF7. Also provided are engineered immune cells (e.g.. T cells or NK cells) expressing such CARs. In some embodiments, a chimeric antigen receptor binds to ligands of NKG2D. In some embodiments, a chimeric antigen receptor binds to CD 19. In some embodiments, a chimeric antigen receptor binds to CD70. In some embodiments, a chimeric antigen receptor binds to BCMA. In some embodiments, the chimeric antigen receptor binds to BAFF-R. In some embodiments, the chimeric antigen receptor binds to CD20. In some embodiments, the chimeric antigen receptor binds to CD22. In some embodiments, the chimeric antigen receptor binds to CD27. In some embodiments, the chimeric antigen receptor binds to CD28. In some embodiments, the chimeric antigen receptor binds to CD38. In some embodiments, the chimeric antigen receptor binds to CD45. In some embodiments, the chimeric antigen receptor binds to CD47. In some embodiments, the chimeric antigen receptor binds to CD54. In some embodiments, the chimeric antigen receptor binds to CD56. In some embodiments, the chimeric antigen receptor binds to CD81. In some embodiments, the chimeric antigen receptor binds to CD117. In some embodiments, the chimeric antigen receptor binds to CD138. In some embodiments, the chimeric antigen receptor binds to CD200. In some embodiments, the chimeric antigen receptor binds to FcRH5. In some embodiments, the chimeric antigen receptor binds to GPRC5D. In some embodiments, the chimeric antigen receptor binds to SLAMF7. an intracellular signaling region

[0127] Also provided are chimeric receptors that comprise an antigen-binding domain and an intracellular signaling region. For example, some embodiments include a chimeric receptor directed against an antigen (e.g., CD 19, BCMA, NKG2D or CD70). Also provided are immune cells (e.g., NK cells) genetically engineered to express such chimeric receptors. In some embodiments, the immune cells are genetically modulated to express a reduced level of ADAM 17, B2M, CBLB, CD58, CIITA, CIS, FAS. FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 or PTPN2.

[0128] To facilitate cellular therapies, there are also provided herein polynucleotides (e.g., encoding chimeric receptors), polypeptides (e.g., chimeric receptors), and vectors that encode chimeric receptors that comprise a target binding moiety (e.g., an extracellular binder of a ligand expressed by a diseased or infected cell, including a cancer cell) and an intracellular signaling region. For example, some embodiments include a polynucleotide, polypeptide, or vector that encodes, for example an activating chimeric receptor comprising an NKG2D extracellular domain that is directed against a tumor marker, for example, MICA, MICB, ULBP1, ULBP2. ULBP3, ULBP4, ULBP5. or ULBP6.among others, to facilitate targeting of an immune cell to a cancer and exerting cytotoxic effects on the cancer cell. In some embodiments, the chimeric receptor comprises an extracellular domain of NKG2D.

[0129] In some embodiments, the composition (e.g., genetically manipulated or modified NK cell composition) for use in accordance with the provided methods includes administering manipulated or modified NK cells expressing recombinant receptors (e.g. CAR) designed to recognize and / or specifically bind to an antigen associated with the autoimmune disease. In particular embodiments, the antigen that is bound or recognized by the recombinant receptor (e.g. CAR) is an antigen expressed by cancer cells. In particular embodiments, the antigen that is bound or recognized by the recombinant receptor (e.g. CAR) is an antigen expressed by cells implicated in the pathogenesis of an autoimmune disease. In particular embodiments, the antigen that is bound or recognized by the recombinant receptor (e.g. CAR) is an antigen expressed by B cells. In particular embodiments, the antigen that is bound or recognized by the recombinant receptor (e.g. CAR) is CD19. In some embodiments, binding to the antigen results in a response, such as an immune response against such antigen. In some embodiments, binding to the antigen results in the reduction or depletion of cells expressing the antigen (e.g., B cells expressing CD 19, or a subset thereof). For example, binding to the antigen may reduce or deplete peripheral B cells in a subject being treated. The reduction or depletion of B cells may correspondingly reduce the level and / or activity of an autoantibody in the subject.

[0130] In some embodiments, the genetically manipulated or modified cells contain or are engineered to contain the recombinant receptor, such as a chimeric antigen receptor (CAR). The recombinant receptor, such as a CAR. generally includes an extracellular antigen-binding domain specific to the antigen (e.g., CD19), which is linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). In some aspects, the genetically manipulated or modified NK cells are provided as pharmaceutical compositions and formulations suitable for administration to a subject, such as for cell therapy .

[0131] Also provided arc therapeutic methods for administering the cells and compositions to subjects, uses of the compositions for treatment of subjects, and uses of the compositions in the manufacture of medicaments for treating subjects. Also provided are uses of the vectors and polynucleotides in the manufacture of medicaments for treating subjects.A. Chimeric Antigen Receptors

[0132] Among the provided recombinant receptors are chimeric antigen receptors (CARs). Among the provided recombinant receptors, e.g., CD19-directed CARs, are chimeric receptors that specifically bind to CD19, such as receptors comprising an anti-CD19 antibody, e.g.. antibody fragment. Also provided are immune cells (e.g., NK cells) expressing the recombinant receptors and uses thereof in treatment of diseases and condition, such as autoimmune diseases. The chimeric receptors, such as CARs, generally include an extracellular antigen-binding domain that includes an anti-CD19 antibody. Such recombinant receptors include antibodies (including antigen-bindingfragments thereof) that specifically bind to CD19 proteins, such as human CD19 protein (e.g., SEQ ID NO:1). In some embodiments, the antibodies include those that are multi-domain antibodies, such as those containing VH and VL domains. In some embodiments, the antibodies include a variable heavy chain and a variable light chain, such as scFvs. Among the provided anti-CD19 antibodies are human and humanized antibodies.

[0133] Also among the provided recombinant receptors are chimeric receptors that specifically bind to BCMA, such as receptors comprising an anti-BCMA antibody, e.g., antibody fragment. Among the antigen receptors are chimeric antigen receptors (CARs). Also provided are immune cells (e.g., NK cells) expressing the recombinant receptors and uses thereof in treatment of diseases and condition, such as cancer or autoimmune diseases. The chimeric receptors, such as CARs, generally include an extracellular antigen-binding domain that includes an anti-BCMA antibody. Such recombinant receptors include antibodies (including antigen-binding fragments thereof) that specifically bind to BCMA proteins, such as human BCMA protein (e.g.. SEQ ID NO:222).

[0134] Also among the provided recombinant receptors are chimeric receptors that specifically bind to CD70, such as receptors comprising an anti-CD70 antibody, e.g.. antibody fragment. Among the antigen receptors are chimeric antigen receptors (CARs). Also provided are immune cells (e g., NK cells) expressing the recombinant receptors and uses thereof in treatment of diseases and condition, such as cancer or autoimmune diseases. The chimeric receptors, such as CARs, generally include an extracellular antigen-binding domain that includes an anti-CD70 antibody. Such recombinant receptors include antibodies (including antigen-binding fragments thereof) that specifically bind to CD70 proteins, such as human CD70 protein (e.g., SEQ ID NO:223).

[0135] The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies. chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherw ise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof. IgM, IgE, IgA, and IgD.

[0136] The terms "complementarity determining region,” and “CDR.” synonymous with “hypervariable region” or “HVR.” are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and / or binding affinity. Ingeneral, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR- H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).

[0137] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including the Kabat numbering scheme (Sequences of Proteins of Immunological Interest, 1987 and 1991, NIH, Bethesda, MD), the Chothia numbering scheme (Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al.. 1989, Nature 342:878-883). the Contact numbering scheme (MacCallum et al.. J. Mol. Biol. 262:732-745 (1996), “Antibody -antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732- 745), the AbM numbering scheme (Martin et al.. Proc. Natl. Acad. Sci., 86:9268-9272; 1989). the IMGT numbering scheme (the international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005). and the Aho numbering scheme (Honegger and Pluckthun. J. Mol. Biol. 309(3): 657-670; 2001).

[0138] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex cry stal structures and is similar in many respects to the Chothia numbering scheme.

[0139] Table 1, below, lists non-limiting position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located between CDR-L1 and CDR- L2, and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35 A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies betw een H32 and H34, depending on the length of the loop.:Kabat et al., "Sequences of Proteins of Immunological Interest," (1991) 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD2Al-Lazikani ct al., J. Mol. Biol. (1997) 273(4):927-48

[0140] Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., “CDR-H1, CDR-H2), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VH or VL amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g.. CDR-H3) within the variable region, as defined by any of the aforementioned schemes. In some embodiments, specific CDR sequences are specified.

[0141] Likewise, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR-H1, FR-H2). of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR or FR is given.

[0142] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0143] Among the provided antibodies are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and a variable light chain region, such as scFvs.

[0144] In some embodiments, the antibodies are synthetically-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with tw o or more antibodyregions or chains joined by synthetic linkers, e.g., peptide linkers, and / or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.

[0145] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human or humanized single-domain antibody.

[0146] A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g.. to restore or improve antibody specificity or affinity.

[0147] Among the provided anti-CD19 antibodies are human antibodies. A “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibodyencoding sequences, including human antibody libraries. The term excludes humanized forms of non- human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. The term includes antigen-binding fragments of human antibodies.

[0148] Among the provided antibodies are monoclonal antibodies, including monoclonal antibody fragments. The term “monoclonal antibody ” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.c., the individual antibodies comprising die population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.

[0149] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and CD19-binding peptides, may include amino acid residues including natural and / or non-natural amino acid residues. The terms alsoinclude post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity.

[0150] The antigen-binding domain may be or comprise any antibody (e.g., anti-CD19 antibody). CD19-binding domains are known and described in the art, including any of those as described in PCT Application Nos. PCT / US2015 / 024671, PCT / US2018 / 029107,PCT / US2020 / 020824, PCT / US2020 / 033559, PCT / IB2021 / 060213. and PCT / CN2021 / 106892. each of which is expressly incorporated by reference in its entirety.

[0151] Provided herein are recombinant receptors (e.g.. CARs) comprising any of the CD19 antibodies or binding domains described herein. The extracellular antigen-binding domain generally is linked to an intracellular signaling domain comprising intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR. In some embodiments, the extracellular antigen-binding domain of a CAR is linked to an intracellular signaling domain by a transmembrane domain. Thus, in some embodiments, the CD19-binding molecule (e.g., antibody) is linked to a transmembrane and intracellular signaling domain. In some embodiments, a CAR comprises an extracellular antigenbinding domain that binds to CD 19, a transmembrane domain, and an intracellular signaling domain comprising a co-stimulatory signal region and a primary signaling domain (e.g.. CD3zeta). Additional CD19-directed CARs are known and described in the art, including any of those as described in Kalos et al., Sci Transl Med 3:95ra73 (2011); Porter et al.. NEJM 365:725-733 (2011); Grupp et al., NEJM 368: 1509-1518 (2013); and PCT Application Nos. PCT / US2015 / 024671, PCT / US2018 / 029107, PCT / US2020 / 020824, and PCT / CN2021 / 106892 all of which are expressly incorporated by reference in their entirety.

[0152] Provided herein are recombinant receptors (e.g., CARs) comprising any of the CD70 antibodies or binding domains described herein. The extracellular antigen-binding domain generally is linked to an intracellular signaling domain comprising intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR. In some embodiments, the extracellular antigen-binding domain of a CAR is linked to an intracellular signaling domain by a transmembrane domain. Thus, in some embodiments, the CD70-binding molecule (e.g., antibody) is linked to a transmembrane and intracellular signaling domain. In some embodiments, a CAR comprises an extracellular antigenbinding domain that binds to CD 70, a transmembrane domain, and an intracellular signaling domain comprising a co-stimulatory signal region and a primary signaling domain (e.g., CD3zeta). CD70- binding domains or CD70-directed CARs are known and described in the art, including any of those as described in PCT Application No. PCT / US2021 / 036879, which is expressly incorporated by reference in its entirety.

[0153] Provided herein are recombinant receptors (e.g., CARs) comprising any of the BCMA antibodies or binding domains described herein. The extracellular antigen-binding domain generally is linked to an intracellular signaling domain comprising intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR. In some embodiments, the extracellular antigen-binding domain of a CAR is linked to an intracellular signaling domain by a transmembrane domain. Thus, in some embodiments, the BCMA-binding molecule (e.g., antibody) is linked to a transmembrane and intracellular signaling domain. In some embodiments, a CAR comprises an extracellular antigenbinding domain that binds to BCMA. a transmembrane domain, and an intracellular signaling domain comprising a co-stimulatory signal region and a primary signaling domain (e.g., CD3zeta). BCMA- binding domains or BCMA-directed CARs are known and described in the art, including any of those as described in PCT Application No. PCT / US2022 / 073567, which is expressly incorporated by reference in its entirety.

[0154] Provided herein are recombinant receptors (e.g., CARs) comprising any of the NKG2D antibodies or binding domains described herein. The extracellular antigen-binding domain generally is linked to an intracellular signaling domain comprising intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR. In some embodiments, the extracellular antigen-binding domain of a CAR is linked to an intracellular signaling domain by a transmembrane domain. Thus, in some embodiments, the NKG2D-binding molecule (e.g., antibody) is linked to a transmembrane and intracellular signaling domain. In some embodiments, a CAR comprises an extracellular antigenbinding domain that binds to NKG2D, a transmembrane domain, and an intracellular signaling domain comprising a co-stimulatory’ signal region and a primary’ signaling domain (e.g., CD3zeta). NKG2D ligand-binding domains or NKG2D ligand-directed CARs are known and described in the art, including any of those as described in PCT Application No. PCT / US2018 / 024650, which is expressly incorporated by reference in its entirety.

[0155] In several embodiments, an antigen binding protein directed against CD38 (also known as ADP-ribosyl cyclase 1, cADPr hydrolase 1, Cyclic ADP-ribose hydrolase 1, or T 10) is provided. According to one embodiment, the CD38 antigen binding protein is an antigen binding domain of a CAR which comprises a transmembrane domain, a signaling domain, and optionally a co-stimulatory domain as disclosed herein. In several embodiments, the antigen binding protein binds to an epitope of the human CD38, and in particular to an epitope of the extracellular domain of the human CD38.

[0156] In several embodiments, the CD38 binding protein comprises an scFv comprising a light chain variable region (VL domain) and heavy chain variable region (VH domain). In several embodiments, the VH domain comprises a complementarity -determining region 1 (CDR-H1), a CDR- H2. and a CDR-H3. In several embodiments, the VL domain comprises a complementarity -determining region 1 (CDR-L1). a CDR-L2, and a CDR-L3. In several embodiments, the anti-CD38 VL domaincomprises the sequence of SEQ ID NO: 2, or an amino acid sequence with 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 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 2. In several embodiments, the anti-CD38 VL domain comprises the sequence of SEQ ID NO: 2. In some embodiments, the VL comprises a CDR-L1, a CDR-L2, and a CDR-L3 as comprised in the sequence set forth in SEQ ID NO:2. In several embodiments, the anti- CD38 VH domain comprises the sequence of SEQ ID NO: 3, or an amino acid sequence with 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 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 3. In several embodiments, the anti-CD38 VH domain comprises the sequence of SEQ ID NO: 3. In some embodiments, the VH domain comprises a CDR- Hl. a CDR-H2. and a CDR-H3 as comprised in the sequence set forth in SEQ ID NO:3. In several embodiments, the anti-CD38 binding protein is an scFv that comprises the sequence of SEQ ID NO: 4. or an amino acid sequence with 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 97%. at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 4. In several embodiments, the anti-CD38 binding protein is an scFv that comprises the sequence of SEQ ID NO: 4. In several embodiments, the anti-CD38 CAR comprises the sequence of SEQ ID NO: 5, or an amino acid sequence with 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 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 5. In several embodiments, the anti-CD38 CAR comprises the sequence of SEQ ID NO: 5. In several embodiments, the anti-CD38 binding protein comprises at least one CDR from SEQ ID NO: 6-11 or a CDR having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to any of SEQ ID NO: 6-11. In several embodiments, the antigen binding protein is affinity matured to enhance binding to CD38. In several embodiments, the nucleotide sequence encoding the antigen binding protein is codon-optimized for humans to enhance expression and / or stability of the protein. CD38-b inding domains or CD38-directed CARs are known and described in the art, including any of those as described in PCT Application No. PCT / US2024 / 021673, which is expressly incorporated by reference in its entirety.

[0157] In several embodiments, an antigen binding protein directed against GPRC5D is provided. According to one embodiment, the GPRC5D antigen binding protein is an antigen binding domain of a CAR which comprises a transmembrane domain, a signaling domain, and optionally a costimulatory domain as disclosed herein. In several embodiments, the antigen binding protein binds to an epitope of the human GPRC5D. In several embodiments, the GPRC5D antigen binding domain comprises a VL domain and / or VH domain. In several embodiments, the GPRC5D antigen binding domain comprises a VL domain and a VH domain. In several embodiments, the VH domain comprisesa complementarity-determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3. In several embodiments, the VL domain comprises a complementarity -determining region 1 (CDR-L1), a CDR- L2, and a CDR-L3. In several embodiments, the GPRC5D antigen binding domain is an scFv comprising the amino acid sequence of any one of SEQ ID NOs: 12-21, or an amino acid sequence with 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 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to any of SEQ ID NOs: 12-21. In several embodiments, the GPRC5D antigen binding domain is an scFv comprising the amino acid sequence of any one of SEQ ID NOs: 12-21. In several embodiments, the antigen binding protein is affinity matured to enhance binding to GPRC5D. In several embodiments, the nucleotide sequence encoding the antigen binding protein is codon-optimized for humans to enhance expression and / or stability of the protein.

[0158] In several embodiments, an antigen binding protein directed against CD 138 is provided. In several embodiments, the anti-CD138 binding protein comprises a VL domain and / or VH domain. In several embodiments, the anti-CD138 binding protein comprises a VL domain and a VH domain. In several embodiments, the VH domain comprises a complementarity -determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3. In several embodiments, the VL domain comprises a complementarity -determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3. In several embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence with 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 97%. at least about 98%. or at least about 99%, sequence identity, homolog}' and / or functional equivalence to SEQ ID NO: 22. In several embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the VL domain comprises a CDR-L1. a CDR-L2, and a CDR-L3 as comprised in SEQ ID NO:22. In several embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence with 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 97%, at least about 98%, or at least about 99%, sequence identity, homolog}' and / or functional equivalence to SEQ ID NO: 23. In several embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the VH domain comprises a CDR-H1, a CDR-H2, and a CDR-H3 as comprised in SEQ ID NO:23. In several embodiments, the anti-CD138 binding protein comprises at least one CDR from SEQ ID NO: 24-29 or a CDR having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to any of SEQ ID NO: 24-29. In several embodiments, the anti-CD138 binding protein is an scFv comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence with 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 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 30. In several embodiments, the anti-CD138 binding protein is an scFv comprising the amino acid sequenceof SEQ ID NO: 30. In several embodiments, the anti-CD138 binding protein is integrated into a CAR which comprises a transmembrane domain, a signaling domain, and optionally a co-stimulatory domain as disclosed herein comprising tire amino acid sequence of SEQ ID NO: 31 or 32, or an amino acid sequence with 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 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 31 or 32. In several embodiments, the CAR comprises the sequence of SEQ ID NO:31. In several embodiments, the CAR comprises the sequence of SEQ ID NO:32. In several embodiments, the antigen binding protein is affinity matured to enhance binding to CD 138. In several embodiments, the nucleotide sequence encoding the antigen binding protein is codon-optimized for humans to enhance expression and / or stability of the protein.

[0159] In several embodiments, an antigen binding protein directed against DLL3 is provided. In several embodiments, the anti-DLL3 binding protein is an antigen binding domain of a CAR which comprises a transmembrane domain, a signaling domain, and optionally a co-stimulatory domain as disclosed herein. In several embodiments, the anti-DLL3 binding protein comprises a VL domain and a VH domain. In several embodiments, the VH domain comprises a complementarity -determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3. In several embodiments, the VL domain comprises a complementarity -determining region 1 (CDR-L1), a CDR-L2, and a CDR-L3. In several embodiments, the anti-DLL3 antigen binding protein comprises a VH domain comprising the amino acid sequence of any of SEQ ID NOs: 33-44, or a sequence having at least about 95%, at least about 96%. at least about 97%, at least about 98%, or at least about 99%, sequence identity', homology and / or functional equivalence to any of SEQ ID NO: 33-44. In several embodiments, the anti-DLL3 antigen binding protein comprises a VH domain comprising the amino acid sequence of any of SEQ ID NOs: 33-44. In some embodiments, the VH comprises a CDR-H1, a CDR-H2, and a CDR-H3 as comprised in any one of SEQ ID NO:33-44. In several embodiments, the anti-DLL3 antigen binding protein comprises a VL domain comprising the amino acid sequence of any of SEQ ID NOs: 45-56, or a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to any of SEQ ID NO: 45-56. In several embodiments, the anti-DLL3 antigen binding protein comprises a VL domain comprising the amino acid sequence of any of SEQ ID NOs: 45-56. In some embodiments, the VL domain comprises a CDR- Ll, a CDR-L2, and a CDR-L3 as comprised in any one of SEQ ID NO:45-56. In several embodiments, the anti-DLL3 binding protein comprises a polypeptide that targets DLL3 and comprises the amino acid sequence of any of SEQ ID NO: 57-58, or a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homolog}- and / or functional equivalence to any of SEQ ID NO: 57-58. In several embodiments, the anti-DLL3 binding protein comprises an scFv comprising the sequence of any of SEQ ID NO: 59-62, or a sequence having at least about 95%, at least about 96%. at least about 97%, at least about 98%. or at least about 99%. sequence identity, homology and / or functional equivalence to any of SEQ ID NO: 59-62. In severalembodiments, the anti-DLL3 binding protein comprises an scFv comprising the sequence of any of SEQ ID NO: 59-62. In several embodiments, the antigen binding protein is affinity matured to enhance binding to DLL3. In several embodiments, the nucleotide sequence encoding the antigen binding protein is codon-optimized for humans to enhance expression and / or stability of the protein.

[0160] In several embodiments, an antigen binding protein directed against the epidermal growth factor receptor (EGFR) is provided. In several embodiments, the anti-EGFR binding protein is an antigen binding domain of a CAR which comprises a transmembrane domain, a signaling domain, and optionally a co-stimulatory domain as disclosed herein. In several embodiments, the anti-EGFR binding protein comprises a VH domain comprising the amino acid sequence of any of SEQ ID NO: 63-64, or a sequence having at least about 95%, at least about 96%, at least about 97%. at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to any of SEQ ID NO: 63-64. In several embodiments, the anti-EGFR binding protein comprises a VH domain comprising the amino acid sequence of any of SEQ ID NO: 63-64. In some embodiments, the VH domain comprises a CDR-H1, a CDR-H2, and a CDR-H3 as comprised in SEQ ID NO:63 or 64. In several embodiments, the anti-EGFR binding protein comprises a VL domain comprising the amino acid sequence of any of SEQ ID NO: 65-66, or a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or frmctional equivalence to any of SEQ ID NO: 65-66. In several embodiments, the anti-EGFR binding protein comprises a VL domain comprising the amino acid sequence of any of SEQ ID NO: 65-66. In some embodiments, the VL domain comprises a CDR-L1, a CDR-L2, and a CDR-L3 as comprised in SEQ ID NO: 65 or 66. In several embodiments, the anti-EGFR binding protein is an scFv comprising the amino acid sequence of any of SEQ ID NOs: 67-72, or a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to any of SEQ ID NO: 67-72. In several embodiments, the anti-EGFR binding protein is an scFv comprising the amino acid sequence of any of SEQ ID NOs: 67- 72. In several embodiments, the anti-EGFR binding protein is incorporated into a CAR having the sequence of any of SEQ ID NOs: 73-76, or a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to any of SEQ ID NO: 73-76. In some embodiments, the CAR comprises the sequence of any one of SEQ ID NOS:73-76. In several embodiments, the antigen binding protein is affinity matured to enhance binding to the EGFR. In several embodiments, the nucleotide sequence encoding the antigen binding protein is codon-optimized for humans to enhance expression and / or stability of the protein.

[0161] In several embodiments, an antigen binding protein directed against PSMA is provided. In several embodiments, the anti-PSMA binding protein is an antigen binding domain of a CAR which comprises a transmembrane domain, a signaling domain, and optionally a co-stimulatory domain as disclosed herein. In several embodiments, the anti-PSMA binding protein comprises a VL domaincomprising the amino acid sequence of SEQ ID NO: 77, or a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 77 In several embodiments, the anti-PSMA binding protein comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the VL domain comprises a CDR-L1, a CDR-L2, and a CDR-L3 as comprised in SEQ ID NO:77. In several embodiments, the anti-PSMA binding protein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 78 or a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 78. In several embodiments, the anti-PSMA binding protein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the VH domain comprises a CDR-H1. a CDR-H2, and a CDR-H3 as comprised in SEQ ID NO:78. In several embodiments, the anti-PSMA binding protein comprises an scFv comprising the amino acid sequence of SEQ ID NO: 80. or a sequence having at least about 95%. at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 80. In several embodiments, the anti-PSMA binding protein comprises an scFv comprising the amino acid sequence of SEQ ID NO: 80. In several embodiments, the anti-PSMA binding protein comprises an antibody comprising the amino acid sequence of SEQ ID NO: 79, or a sequence having at least about 95%, at least about 96%, at least about 97%. at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 79. In several embodiments, the anti-PSMA binding protein comprises an antibody comprising the amino acid sequence of SEQ ID NO: 79. In several embodiments, the antigen binding protein is affinity matured to enhance binding to PSMA. In several embodiments, the nucleotide sequence encoding die antigen binding protein is codon-optimized for humans to enhance expression and / or stability of the protein.

[0162] In several embodiments, an antigen binding protein directed against FLT3 is provided. In several embodiments, the anti-FLT3 binding protein is an antigen binding domain of a CAR which comprises a transmembrane domain, a signaling domain, and optionally a co -stimulatory domain as disclosed herein. In several embodiments, the anti-FLT3 binding protein comprises one or more CDRs from the VL domain and / or VH domain selected from SEQ ID NOs: 81-89, or a CDR having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. sequence identity, homology and / or functional equivalence to any of SEQ ID NO: 81-89. In several embodiments, the anti-FLT3 binding protein comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 90, or a sequence having at least about 95%. at least about 96%, at least about 97%, at least about 98%. or at least about 99%. sequence identity, homology and / or functional equivalence to SEQ ID NO: 90. In several embodiments, the anti-FLT3 binding protein comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, the VL domain comprises a CDR-L1, a CDR-L2, and a CDR-L3 as comprised in SEQ ID NO:90. In severalembodiments, the anti-FLT3 binding protein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 91, or a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or functional equivalence to SEQ ID NO: 91. In several embodiments, the anti-FLT3 binding protein comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 91. In some embodiments, the VH domain comprises a CDR-H1, a CDR-H2, and a CDR-H3 as comprised in SEQ ID NO:91. In several embodiments, the antigen binding protein is affinity matured to enhance binding to FLT3. In several embodiments, the nucleotide sequence encoding the antigen binding protein is codon-optimized for humans to enhance expression and / or stability of the protein.

[0163] In several embodiments, an antigen binding protein directed against KREMEN2 is provided. In several embodiments, the anti-KREMEN2 binding protein is an antigen binding domain of a CAR which comprises a transmembrane domain, a signaling domain, and optionally a co- stimulatoiy domain as disclosed herein. In several embodiments, the anti-KREMEN2 binding protein comprises a VL domain comprising the amino acid sequence of any of SEQ ID NOs:92-96, or a sequence having at least about 95%, at least about 96%. at least about 97%, at least about 98%. or at least about 99%. sequence identity, homology and / or functional equivalence to any of SEQ ID NOs: 92-96. In several embodiments, the anti-KREMEN2 binding protein comprises a VL domain comprising the amino acid sequence of any of SEQ ID NOs: 92-96. In some embodiments, the VL domain comprises a CDR-L1, a CDR-L2, and a CDR-L3 as comprised in any one of SEQ ID NO: 92- 96. In several embodiments, the anti-KREMEN2 binding protein comprises a VH domain comprising the amino acid sequence of any of SEQ ID NOs: 97-100, or a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99%, sequence identity, homology and / or functional equivalence to any of SEQ ID NOs: 97-100. In several embodiments, tire anti-KREMEN2 binding protein comprises a VH domain comprising the amino acid sequence of any of SEQ ID NOs: 97-100. In some embodiments, the VH domain comprises a CDR-H1, a CDR-H2, and a CDR-H3 as comprised in any one of SEQ ID NO: 97-100. In several embodiments, the anti- KREMEN2 binding protein is an antibody comprising the amino acid sequence of SEQ ID NO: 96, or a sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and / or fimctional equivalence to SEQ ID NO: 96. In several embodiments, the anti-KREMEN2 binding protein is an antibody comprising the amino acid sequence of SEQ ID NO: 96. In several embodiments, the antigen binding protein is affinity matured to enhance binding to KREMEN2. In several embodiments, the nucleotide sequence encoding the antigen binding protein is codon-optimized for humans to enhance expression and / or stability of the protein.

[0164] In some embodiments, the transmembrane domain is fused to the extracellular domain. The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-boundor transmembrane protein. Transmembrane regions include those derived from (e.g., comprising at least the transmembrane region(s) of) CD3, CD4, CD5, CD8, CD9, CD 16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof. Alternatively, the transmembrane domain in some embodiments is synthetic.

[0165] In several embodiments, the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein normally expressed on both T cells and NK cells. In several embodiments, the transmembrane domain comprises CD8alpha (CD8a). In several embodiments, the transmembrane domain comprises a CD8 (e.g., CD8a) hinge and a CD8 (e.g., CD8a) transmembrane region.

[0166] In several embodiments, the transmembrane domain comprises a hinge, e.g. a CD8a hinge. In several embodiments, the sequence encoding the CD8a hinge is truncated or modified. In some embodiments, the CD8a hinge is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 101. In several embodiments, the CD8a hinge comprises the nucleic acid sequence of SEQ ID NO: 101. In several embodiments, the CD8a hinge is truncated or modified. In some embodiments, the CD8a hinge has at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 102. In several embodiments, the hinge of CD8a comprises the amino acid sequence of SEQ ID NO: 102.

[0167] In several embodiments, the transmembrane domain comprises a CD8a transmembrane region. In several embodiments, the CD8a transmembrane region is truncated or modified. In some embodiments, the CD8a transmembrane region is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. or at least 95% sequence identity to the sequence of SEQ ID NO: 103. In several embodiments, the CD8a transmembrane region is encoded by a nucleic acid sequence of SEQ ID NO:103. In several embodiments, the CD8a transmembrane region is truncated or modified. In some embodiments, the CD8a transmembrane region has at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 104. In several embodiments, the CD8a transmembrane region comprises the amino acid sequence of SEQ ID NO: 104.

[0168] Thus, in several embodiments, the CD8 transmembrane domain is truncated or modified and is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 105. In several embodiments, the CD8 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 105. In some embodiments, the CD8 transmembrane domain is truncated or modified and comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 106. In several embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 106.

[0169] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain or a fragment thereof. In several embodiments, the CD28 transmembrane domain is truncated or modified. In some embodiments, the CD28 transmembrane domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 107. In several embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 107.

[0170] The receptor, e.g.. the CAR, generally includes an intracellular signaling domain comprising intracellular signaling components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, upon ligation of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g.. NK cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of an immune cell (e.g., NK cell) such as cytolytic activity and / or secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact ininiunostiniiilatorv chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain includes the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects, also those of coreceptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.

[0171] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the receptor. T cell activation is in some aspects described as being mediated by two classes of cy toplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the receptor includes one or both of such signaling components.

[0172] In some aspects, the receptor includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta. CD3 epsilon, CD5, CD22, CD79a. CD79b. and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3zeta.

[0173] For example, immune cells engineered according to several embodiments disclosed herein may comprise at least one subunit of the CD3 T cell receptor complex (or a fragment thereof). In several embodiments, the signaling domain comprises the CD3 zeta subunit. In several embodiments, the CD3zeta can be truncated or modified. In some embodiments, the CD3zeta is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 108. In several embodiments, the CD3zeta is encoded by the nucleic acid sequence of SEQ ID NO: 108. In several embodiments, the CD3zeta is truncated or modified. In some embodiments, the CD3zeta comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 109. In several embodiments, the CD3zeta comprises the amino acid sequence of SEQ ID NO: 109.

[0174] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling region, such as an intracellular signaling region of CD28, 4-1BB, 0X40. DAP10. ICOS, or any combination thereof. In some embodiments, the intracellular signaling domain comprises an intracellular signaling region of CD28. In some embodiments, the intracellular signaling domain comprises an intracellular signaling region of 4-1BB. In some embodiments, the intracellular signaling domain comprises an intracellular signaling region of 0X40. In some embodiments, the intracellular signaling domain comprises an intracellular signaling region of DAP 10. In some embodiments, the intracellular signaling domain comprises an intracellular signaling region of ICOS. In some embodiments, the intracellular signaling domain does not include DAP10 and / or DAP12. In some embodiments, the intracellular signaling domain does not include DAP10. In some embodiments, the intracellular signaling domain does not include DAP12. In some aspects, the same receptor includes both a CD3zeta and a costimulatory signaling region. Thus, in some embodiments, the intracellular signaling domain of the recombinant receptor, such as CAR, comprises a CD3zeta intracellular domain and a costimulatory signaling region.

[0175] In several embodiments, the intracellular signaling domain comprises an intracellular signaling region of 0X40. In several embodiments, the 0X40 intracellular signaling region is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 110. In several embodiments, the 0X40 intracellular signaling region is encoded by the nucleic acid sequence of SEQ ID NO: 110. In several embodiments, the 0X40 intracellular signaling region comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 111. In several embodiments, the 0X40 intracellular signaling region comprises the amino acid sequence of SEQ ID NO: 111. In several embodiments, 0X40 is used as the sole intracellular signaling component in the construct. However, in several embodiments, 0X40 can be used with one or more other components. For example.combinations of 0X40 and CD3zeta are used in some embodiments. In some embodiments, the intracellular signaling domain comprises an 0X40 costimulatory signaling region linked to CD3zeta.

[0176] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising the sequence set forth in SEQ ID NO: 112, a CD8alpha transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 104, an 0X40 intracellular signaling region comprising the amino acid sequence set forth in SEQ ID NO: 111, and a CD3zeta domain comprising the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, the CAR comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 113. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 113.

[0177] By way of further example, combinations of CD28, 0X40. 4-1BB and / or CD3zeta are used in some embodiments.

[0178] In several embodiments, the intracellular signaling domain comprises an intracellular signaling region of 4-1BB. In several embodiments, the 4-1BB intracellular signaling region is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 114. In several embodiments, the 4- IBB intracellular signaling region is encoded by the nucleic acid sequence of SEQ ID NO: 114. In several embodiments, the 4-1BB intracellular signaling region comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%. at least 85%, at least 90%. or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 115. In several embodiments, the 4-1BB intracellular signaling region comprises the amino acid sequence of SEQ ID NO: 115. In several embodiments, 4-1BB is used as the sole intracellular signaling component in the construct, however, in several embodiments, 4- IBB can be used with one or more other components. For example, combinations of 4- IBB and CD3zeta are used in some embodiments. In some embodiments, the intracellular signaling domain comprises a 4- IBB costimulatory signaling region linked to CD3zcta. By way of further example, combinations of CD28, 0X40, 4- IBB and / or CD3zeta are used in some embodiments.

[0179] In several embodiments, the intracellular signaling domain comprises an intracellular signaling region of CD28. In several embodiments, the CD28 intracellular signaling region comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 116. In several embodiments, the CD28 intracellular signaling region comprises the amino acid sequence of SEQ ID NO: 116. In several embodiments, CD28 is used as the sole intracellular signaling component in the construct, however, in several embodiments, CD28 can be used with one or more other components. For example, combinations of CD28 and CD3zeta are used in some embodiments. In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory signaling region linked to CD3zeta. By way of further example, combinations of CD28, 0X40, 4-1BB and / or CD3zeta are used in someembodiments. In several embodiments, an expression vector, such as a MSCV-IRES-GFP plasmid, a non-limiting example of which is provided in SEQ ID NO: 117, is used to express any of the chimeric antigen receptors provided for herein.

[0180] In any of the provided embodiments, die nucleic acid encoding the chimeric receptor, or a portion thereof, is codon-optimized for expression in humans. In some embodiments, the polynucleotides are optimized, or contain certain features designed for optimization, such as for codon usage, to reduce RNA heterogeneity and / or to modify, e.g., increase or render more consistent among cell product lots, expression, such as surface expression, of the encoded receptor. In some embodiments, polynucleotides, encoding chimeric receptors, are modified as compared to a reference polynucleotide, such as to remove cryptic or hidden splice sites, to reduce RNA heterogeneity. In some embodiments, polynucleotides, encoding chimeric receptors, are codon optimized, such as for expression in a mammalian, e.g., human, cell such as in a human T cell. In some aspects, the modified polynucleotides result in improved, e.g., increased or more uniform or more consistent level of, expression, e.g.. surface expression, when expressed in a cell.B. Engineered Cells

[0181] Also provided are methods, nucleic acids, compositions, and kits, for producing the genetically engineered immune cells (e.g., NK cells). In some aspects, the genetic engineering involves introduction of a nucleic acid encoding the genetically engineered component or other component for introduction into the cell, such as a component encoding a gene-disrupting protein or nucleic acid. Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and / or function of transferred cells; genes to provide a genetic marker for selection and / or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo. i. Vectors and Methods for Genetic Engineering

[0182] Also provided are methods, polynucleotides, compositions, and kits, for expressing the antigen-binding molecules, including recombinant receptors (e.g., CARs) comprising the binding molecules, and for producing the genetically engineered immune cells (e.g., NK cells) expressing such binding molecules. In some embodiments, one or more binding molecules, including recombinant receptors (e.g., CARs) can be genetically engineered into cells or a plurality of cells. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into the cell, such as by retroviral transduction, transfection, or transformation.

[0183] Also provided are polynucleotides encoding the antibodies and chimeric antigen receptors and / or portions, e.g., chains, thereof. Among the provided polynucleotides are those encoding the chimeric antigen receptors (e.g., antigen-binding fragment) described herein. Also provided arepolynucleotides encoding one or more antibodies and / or portions thereof, e.g., those encoding one or more of the antibodies (e.g., antigen-binding fragment) described herein and / or other antibodies and / or portions thereof, e.g., antibodies and / or portions thereof that binds other target antigens. The polynucleotides may include those encompassing natural and / or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications. The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide" may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, and include, but are not limited to, DNA, RNA, or PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide. Also provided are polynucleotides that have been optimized for codon usage.

[0184] Also provided are vectors containing the polynucleotides, such as any of the polynucleotides described herein, and cells containing the vectors, e.g.. for producing the antibodies or antigen-binding fragments thereof. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a lentiviral vector. Also provided are methods for producing the antibodies or antigen-binding fragments thereof. The nucleic acid may encode an amino acid sequence comprising the VL domain and / or an amino acid sequence comprising the VH domain of the antibody (e.g., the light and / or heavy chains of the antibody). The nucleic acid may encode one or more amino acid sequence comprising the VL domain and / or an amino acid sequence comprising the VH domain of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such polynucleotides are provided. In a further embodiment, a host cell comprising such polynucleotides is provided. In another such embodiment, a host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL domain of the antibody and an amino acid sequence comprising the VH domain of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL domain of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH domain of the antibody. In some embodiments, a host cell comprises (e.g., has been transformed with) one or more vectors comprising one or more nucleic acid that encodes one or more an amino acid sequence comprising one or more antibodies and / or portions thereof, e.g., antigen-binding fragments thereof. In some embodiments, one or more such host cells are provided. In some embodiments, a composition containing one or more such host cells are provided. In some embodiments, the one or more host cells can express different antibodies, or the same antibody. In some embodiments, each of the host cells can express more than one antibody.

[0185] Also provided are methods of making the chimeric antigen receptors. For recombinant production of the chimeric receptors, a nucleic acid sequence encoding a chimeric receptor antibody, e.g., as described herein, may be isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid sequences may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). In some embodiments, a method of making the chimeric antigen receptor is provided, wherein the method comprises culturing a host cell comprising a nucleic acid sequence encoding the antibody, as provided above, under conditions suitable for expression of the receptor. In particular examples, immune cells, such as human immune cells are used to express the provided polypeptides encoding chimeric antigen receptors. In some examples, the immune cells are NK cells including primary NK cells.

[0186] In some embodiments, gene transfer is accomplished by transduction of the immune cells (e.g., activated immune cells), and expansion in culture to numbers sufficient for clinical applications. In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g.. antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions. Non-limiting examples of methods include those for transfer of polynucleotides encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.

[0187] In some embodiments, recombinant polynucleotides are transferred into immune cells (e.g., NK cells) using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant polynucleotides are transferred into immune cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors. In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g.. a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or human immunodeficiency virus type 1 (HIV-1). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically arc amphotropic, meaning drat they arc capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and / or env sequences. A number of illustrative retroviral systems have been described. Methods of lentiviral transduction are known and described in the art.

[0188] Among additional polynucleotides, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and / or function of transferred cells; genes to provide a genetic marker for selection and / or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo.

[0189] In some cases, the polynucleotide containing nucleic acid sequences encoding the chimeric receptor, e.g., chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signalpeptide. In some aspects, a non-limiting example of a signal peptide comprises a CD8 alpha (CD8a) signal peptide. In some aspects, a non-limiting example of a signal peptide comprises a CD8 alpha (CD8a) signal peptide set forth in SEQ ID NO: 118.

[0190] In some embodiments the vector or construct can contain promoter and / or enhancer or regulatory elements to regulate expression of the encoded recombinant receptor. In some examples the promoter and / or enhancer or regulatory elements can be condition-dependent promoters, enhancers, and / or regulatory elements. In some examples these elements drive expression of the transgene.

[0191] In some embodiments, the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such nucleic acid molecules, e.g., transcripts, can be multicistronic (bicistronic or tricistronic). For example, in some embodiments, transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows co-expression of gene products (e.g. encoding a chimeric receptor and membrane-bound interleukin- 15) by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding a chimeric receptor and membrane-bound interleukin- 15) separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A cleavage sequences) or a protease recognition site. The ORF thus encodes a single polypeptide, which, either during (in the case of T2A) or after translation, is cleaved into the individual proteins. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation betw een the end of the 2A sequence and the next peptide downstream. Many 2A elements are known. Examples of 2A peptides that can be used in the methods and polynucleotides disclosed herein, without limitation, 2A peptides from the foot-and- mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A, e.g. SEQ ID NO: 120, encoded by SEQ ID NO: 119), and porcine teschovirus-1 (P2A). In some embodiments, the one or more different or separate promoters drive the expression of a nucleic acid molecule encoding a binding molecule, e.g., recombinant receptor and a nucleic acid encoding membrane-bound interleukin- 15. ii. Interleukin- 15

[0192] In several embodiments, any of the immune cells as provided herein are engineered to express interleukin 15 (IL 15). In some aspects, the IL 15 is a membrane-bound form of IL15. Thus, in several embodiments, any of the immune cells as provided herein are engineered to express a membrane-bound interleukin 15 (mbIL15). In such embodiments, mbIL15 expression on the immune cell (e.g., NK cell) enhances the cytotoxic effects of the engineered cell by enhancing the proliferation and / or longevity of the cells. In some embodiments, the IL15 is expressed from a separate cassette on the construct comprising any one of the CARs disclosed herein. In some embodiments, the IL 15 is expressed from the same cassette as any one of the CARs disclosed herein.

[0193] In some embodiments, the chimeric receptor and IL 15 are separated by a nucleic acid sequence encoding a cleavage site, for example, a proteolytic cleavage site or a T2A, P2A, E2 A, or F2A self-cleaving peptide cleavage site. In some embodiments, the chimeric receptor and IL15 are separated by a T2A peptide (e.g., SEQ ID NO: 120, encoded by SEQ ID NO: 119). In some embodiments, the IL15 is a membrane-bound IL15 (mbIL15). In some embodiments, the mbIL15 comprises a native IL15 sequence, such as a human native IL15 sequence (e.g., SEQ ID NO: 122, encoded by SEQ ID NO: 121). In some embodiments, the mbIL15 comprises a native IL15 sequence, such as a human native IL15 sequence (e.g., SEQ ID NO: 122, encoded by SEQ ID NO: 121), and at least one transmembrane domain (e.g., CD8a). In several embodiments, IL15 is encoded by the nucleic acid sequence of SEQ ID NO: 121. In several embodiments, IL15 can be truncated or modified, such that it is encoded by a nucleic acid sequence that has at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 121. In several embodiments, the IL15 comprises the amino acid sequence of SEQ ID NO: 122. In several embodiments, the IL15 is truncated or modified, such that it has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 122.

[0194] Thus, in some embodiments, any of the CARs as described herein are encoded by the same nucleic acid sequence as a mbIL15. In some embodiments, a nucleic acid sequence encoding the CAR and a nucleic acid sequence encoding the mbIL15 are separated by a T2A-encoding sequence (e.g., SEQ ID NO: 119). In some embodiments, any of the engineered cells as described herein express a CD19-targeting recombinant receptor (e.g., CAR) and a mbIL15.

[0195] In some embodiments, the mbIL15 is membrane-bound by virtue of the fusion of IL 15 to a transmembrane domain. Thus, in some embodiments, mbIL15 comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a hinge and / or a transmembrane region. In some embodiments, the transmembrane domain comprises a hinge and a transmembrane region. In some embodiments, the hinge is a CD8a hinge sequence (e.g., SEQ ID NO: 102). In some embodiments, the transmembrane region is a CD8a transmembrane region (e.g., SEQ ID NO: 104). In some embodiments, the mbIL15 comprises a native IL 15 sequence, such as a human native IL 15 sequence, and at least one transmembrane domain (e.g., CD8a transmembrane domain). In some embodiments, the CD8a transmembrane domain comprises the sequence of SEQ ID NO: 106. In several embodiments, the mbIL15 is truncated or modified such that it comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%. at least 85%, at least 90%. or at least 95% sequency identity to the amino acid sequence of SEQ ID NO: 123. In several embodiments, the mbIL15 comprises the amino acid sequence of SEQ ID NO:123. Membrane-bound IL15 sequences are described in PCT publications WO 2018 / 183385 and WO 2020 / 056045, each of which is hereby expressly incorporated by reference in its entirety.iii. Cell Types

[0196] Some embodiments of the methods and compositions provided herein relate to a cell such as an immune cell. In some embodiments, an immune cell is modulated to reduce expression of a target gene or target protein. In some embodiments, an immune cell is also engineered to express a chimeric receptor that binds to an antigen.

[0197] Genetic engineering has enabled approaches to be developed that harness certain aspects of the immune system to fight disease. In some cases, a healthy donor’s immune cells are modified to specifically eradicate cells of, or associated with, a disease. 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.

[0198] To facilitate immunotherapies for treatment of diseases (e.g., autoimmune diseases or cancers), there are provided for herein polynucleotides, polypeptides, and vectors that encode chimeric antigen receptors (CAR) that comprise a target binding moiety (e.g., an antigen expressed by a B cell) and a cytotoxic signaling complex. For example, some embodiments include a polynucleotide, polypeptide, or vector that encodes, for example a chimeric antigen receptor directed against a target antigen, to facilitate targeting of an immune cell to diseased cells expressing the target antigen. Methods of treating diseases (e.g., infectious diseases, autoimmune diseases, cancers, and tumors) and other uses of such cells for immunotherapy are also provided for herein. Also provided are engineered immune cells (e.g., NK cells) expressing such chimeric receptors.

[0199] In several embodiments, cells of the immune system are engineered to have enhanced cytotoxic effects against target cells. For example, a cell of the immune system may be engineered to include a CAR as described herein. A cell of the immune system may also be modulated to reduce expression of a target gene or a target protein. In several embodiments, white blood cells or leukocytes, are used, since their native function is to defend the body against growth of abnormal cells and infectious disease. There are a variety of types of white bloods cells that serve specific roles in the human immune system and are therefore a preferred starting point for the engineering of cells disclosed herein. White blood cells include granulocytes and agranulocytes (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 those that follow or are otherwise described herein may be modulated to express reduced levels of a gene or protein and / or engineered to include a CAR or a nucleic acid encoding the CAR. In several embodiments, the immune cells are engineered to express (e.g., co-express) a membrane-bound interleukin 15 (mbIL15). In some embodiments, the immune cells engineered to express a CAR are engineered to bicistronically express a mbIL15 domain. a. Monocytes

[0200] In some embodiments, the immune cells comprise monocytes. Monocytes are a subtype of leukocyte. Monocytes can differentiate into macrophages and myeloid lineage dendritic cells.Monocytes are associated with the adaptive immune system and serve the main functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of uptake of cellular material, or entire cells, followed by digestion and destruction of the engulfed cellular material.

[0201] In some embodiments, a monocyte is positive for cell surface expression of a marker selected from among the group consisting of CCR2, CCR5, CDllc, CD14, CD16, CD62L, CD68+, CX3CR1, or HLA-DR, or any combination thereof. In some embodiments, a monocyte is positive for cell surface expression of CD14. In some embodiments, a monocyte is positive for cell surface expression of CCR2. In some embodiments, a monocyte is positive for cell surface expression of CCR5. In some embodiments, a monocyte is positive for cell surface expression of CD62L.

[0202] In several embodiments, monocytes are used in connection with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions described herein relate to a monocyte that includes an antigen-directed CAR, or a nucleic acid encoding the antigen-directed CAR. In some embodiments, the monocytes express a CAR that binds to an antigen, for example, CD19, CD123, CD70, Her2, mesothelin, Claudin 6, BCMA, or EGFR. In some embodiments, the monocytes express a CAR that binds to an antigen, for example, BCMA. CD 19, CD20. CD22, or GPRC5D.

[0203] Some embodiments of the methods and compositions described herein relate to a monocyte that has been modulated to reduce expression of a target gene or a target protein.

[0204] In some embodiments, the monocytes are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, the monocytes engineered to express a chimeric receptor are also engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain. Thus, in some embodiments, the monocytes are engineered to bicistronically express the chimeric receptor and mbIL15.

[0205] In some embodiments, the monocytes are allogeneic cells. In some embodiments, the monocy tes arc obtained from a donor who docs not have cancer. b. Lymphocytes

[0206] In some embodiments, the immune cells comprise lymphocytes. Lymphocytes, the other primary sub-type of leukocyte include T cells (cell-mediated, cytotoxic adaptive immunity), natural killer cells (cell-mediated, cytotoxic innate immunity), and B cells (humoral, antibody-driven adaptive immunity)- While B cells are engineered according to several embodiments, disclosed herein, several embodiments also relate to engineered T cells or engineered NK cells (mixtures of T cells and NK cells are used in some embodiments, either from the same donor, or different donors). Thus, in some embodiments, the immune cells comprise T cells. In some embodiments, the immune cells comprise NK cells. In some embodiments, the immune cells comprise T cells and NK cells. In some embodiments, the immune cells comprise B cells.

[0207] In several embodiments, lymphocytes are used in connection with one or more additional engineered cells as disclosed herein.

[0208] In some embodiments, the lymphocytes express a CAR that binds to an antigen, for example, CD19, CD123, CD70, Her2, mesothelin, Claudin 6, BCMA, or EGFR. In some embodiments, the lymphocytes express a CAR that binds to an antigen, for example, BCMA, CD19, CD20. CD22, or GPRC5D.

[0209] Some embodiments of the methods and compositions described herein relate to a lymphocyte that has been modulated to reduce expression of a target gene or a target protein.

[0210] In some embodiments, the lymphocytes are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, the lymphocytes engineered to express a chimeric receptor are also engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain. Thus, in some embodiments, lymphocytes are engineered to bicistronically express the chimeric receptor and mbIL15.

[0211] In some embodiments, the lymphocytes are allogeneic cells. In some embodiments, the lymphocytes are obtained from a donor who does not have cancer. In some embodiments, the monocytes are obtained from a donor who does not have an autoimmune disease. c. T Cells

[0212] In some embodiments, the immune cells comprise T cells. T cells are distinguishable from other lymphocytes sub-types (e.g.. B cells orNK cells) based on the presence of a T-cell receptor on tire cell surface.

[0213] T cells can be divided into various different subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cell, mucosal associated invariant T cells and gamma delta T cells. In some embodiments, a specific subtype of T cell is engineered. In some embodiments, a T cell is positive for cell surface expression of a marker selected from among the group consisting of CD3, CD4, and / or CD8. In some embodiments, a T cell is positive for cell surface expression of CD3. In some embodiments, a T cell is positive or cell surface expression of CD4. In some embodiments, a T cell is positive or cell surface expression of CD8.

[0214] In some embodiments, CD3+ T cells are engineered. In some embodiments, CD4+ T cells are engineered. In some embodiments, CD8+ T cells are engineered. In some embodiments, regulatory' T cells are engineered. In some embodiments, gamma delta T cells are engineered. In some embodiments, a mixed pool of T cell subtypes is engineered. For example, in some embodiments, CD4+ and CD8+ T cells are engineered. In some embodiments, there is no specific selection of a type of T cells to be engineered to express the chimeric receptor complexes disclosed herein. In several embodiments, specific techniques, such as use of cytokine stimulation are used to enhance expansion / collection of T cells with a specific marker profile. For example, in several embodiments,activation of certain human T cells, e.g. CD4+ T cells, CD8+ T cells is achieved through use of CD3 and / or CD28 as stimulatory' molecules.

[0215] In several embodiments, there is provided a method of treating or preventing cancer, autoimmune or an infectious disease, comprising administering a therapeutically effective amount of T cells expressing the chimeric receptor complex and / or a homing moiety as described herein. In several embodiments, there is provided a method of treating, or preventing cancer, autoimmune or an infectious disease, comprising administering T cells expressing a chimeric receptor complex as described herein. In several embodiments, the engineered T cells are autologous cells, while in some embodiments, the T cells are allogeneic cells. In some embodiments, the T cells are allogeneic cells. In some embodiments, the T cells are obtained from a donor who does not have cancer. In some embodiments, the T cells are obtained from a donor who does not have an autoimmune disease. In some embodiments, the T cells are obtained from a donor who does not have an infectious disease.

[0216] In some embodiments, the T cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, the T cells engineered to express a CAR are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain. Thus, in some embodiments, the T cells are engineered to bicistronically express the CAR and mbIL15.

[0217] Several embodiments of the methods and compositions disclosed herein relate to T cells engineered to express a CAR that targets an antigen, for example, CD19, CD123. CD70, Her2, mesothelin, Claudin 6, BCMA, NKG2D, or EGFR. In some embodiments, the T cells express a CAR that binds to an antigen, for example, BCMA, CD19, CD20. CD22, or GPRC5D. In some embodiments, the T cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, the T cells engineered to express a chimeric receptor are also engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain. Thus, in some embodiments, the T cells arc engineered to bicistronically express the chimeric receptor and mbIL15.

[0218] Some embodiments of the methods and compositions described herein relate to a T cell that has been modulated to reduce expression of a target gene or a target protein.

[0219] In some embodiments, the immune cells comprise T cells and NK cells (either from the same donor or from different donors). d. NK Cells

[0220] In some embodiments, the immune cells comprise natural killer (NK) cells. In several embodiments, there is provided a method of treating, or preventing cancer, autoimmune disease or an infectious disease, comprising administering a therapeutically effective amount of natural killer (NK) cells expressing the chimeric receptor complex and / or a homing moiety as described herein. In several embodiments, there is provided a method of treating or preventing a cancer comprising administering natural killer (NK) cells expressing a chimeric receptor complex as described herein. In severalembodiments, there is provided a method of treating, or preventing an autoimmune disease comprising administering natural killer (NK) cells expressing a chimeric receptor complex as described herein. In several embodiments, there is provided a method of treating, inhibiting, or preventing an infectious disease comprising administering natural killer (NK) cells expressing a chimeric receptor complex as described herein. In several embodiments, the engineered NK cells are autologous cells, while in some embodiments, the NK cells are allogeneic cells. In some embodiments, the NK cells are derived from a donor who does not have an autoimmune disease. In some embodiments, the NK cells are derived from a donor who does not have a B cell-mediated disease. In some embodiments, the NK cells are derived from a donor who does not have cancer. In some embodiments, the NK cells are derived from a donor who does not have an infectious disease. In some embodiments, the NK cells are derived from a donor who does not have an autoimmune disease, such as SLE. In some embodiments, the NK cells are derived from a donor who does not have the disease to be treated.

[0221] In some embodiments, the NK cells are derived from peripheral blood mononuclear cells (PBMCs), e.g., of a donor. In some embodiments, the NK cells are not derived from cord blood. In some embodiments, the NK cells are not derived from induced pluripotent stem cells (iPSCs).

[0222] In several embodiments, NK cells are preferred because the natural cytotoxic potential of NK cells is relatively high. In several embodiments, it is unexpectedly beneficial that the engineered cells disclosed herein can further upregulate the cytotoxic activity of NK cells, leading to an even more effective activity' against target cells (e.g., tumor or other diseased cells).

[0223] In some embodiments, a NK cell is positive for cell surface expression of a marker selected from among the group consisting of CCR7, CD16, CD56, CD57, CD11, CX3CR1. a Killer Ig- like receptor (KIR), NKp30, NKp44, and NKp46, or any combination thereof. In some embodiments, aNK cell is positive for cell surface expression of CD 16. In some embodiments, a NK cell is positive for cell surface expression of CD56. In some embodiments, a NK cell is positive for cell surface expression of a Killer Ig-likc receptor.

[0224] Some embodiments of the methods and compositions described herein relate to NK cells engineered to express a CAR that targets an antigen, for example, CD19, CD123, CD70, Her2, mesothelin, Claudin 6, BCMA, or EGFR, among any of the others disclosed herein, and optionally a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, NK cells express a CAR that binds to CD 19. In some embodiments, NK cells express a CAR that binds to CD70. In some embodiments, NK cells express a CAR that binds to BCMA. Several embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express an activating chimeric receptor that targets a ligand on a diseased cell, for example, MICA, MICB. ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6 (among others) and optionally a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments. NK cells express a chimeric receptor that binds to a NKG2D ligand. In some embodiments. NK cells express a chimeric receptor comprising an extracellular domain of NKG2D.

[0225] In any of the provided embodiments, die NK cells are engineered to express a CAR that binds to an antigen other than CD19, e.g., an antigen associated with an autoimmune disease. For example, in some embodiments, the genetically engineered NK cells express a CAR that binds to an antigen selected from the group consisting of BAFF-R, BCMA, CD20, CD22, CD27, CD28, CD33, CD38, CD45, CD47, CD54, CD56, CD81, CD117, CD138, CD200, FcRH5, GPRC5D, and SLAMF7. Thus, in some aspects, the NK cells express a CAR that binds to BAFF-R. In some aspects, the NK cells express a CAR that binds to BCMA. In some aspects, the NK cells express a CAR that binds to CD20. In some aspects, the NK cells express a CAR that binds to CD22. In some aspects, the NK cells express a CAR that binds to CD27. In some aspects, the NK cells express a CAR that binds to CD28. In some aspects, the NK cells express a CAR that binds to CD33. In some aspects, the NK cells express a CAR that binds to CD38. In some aspects, the NK cells express a CAR that binds to CD45. In some aspects, the NK cells express a CAR that binds to CD47. In some aspects, the NK cells express a CAR that binds to CD54. In some aspects, the NK cells express a CAR that binds to CD56. In some aspects, the NK cells express a CAR that binds to CD81. In some aspects, the NK cells express a CAR that binds to CD117. In some aspects, the NK cells express a CAR that binds to CD 138. In some aspects, the NK cells express a CAR that binds to CD200. In some aspects, the NK cells express a CAR that binds to FcRH5. In some aspects, the NK cells express a CAR that binds to GPRC5D. In some aspects, the NK cells express a CAR that binds to SLAMF7.

[0226] In any of the provided embodiments, the NK cells engineered to express a CD 19 CAR are further engineered to express a CAR that binds to an antigen other than CD 19. In some embodiments, the antigen is associated with an autoimmune disease. For example, in some embodiments, the genetically engineered NK cells also express a CAR that binds to an antigen selected from the group consisting of BAFF-R, BCMA, CD20. CD22, CD27, CD28, CD33, CD38. CD45, CD47, CD54, CD56, CD81, CD117, CD138, CD200, FcRH5, GPRC5D, and SLAMF7. Thus, in some aspects, the NK cells arc engineered to express an anti-CD19 CAR as provided herein and a CAR that binds to any one of BAFF-R, BCMA, CD20, CD22, CD27, CD28, CD33, CD38, CD45, CD47, CD54, CD56, CD81, CD117, CD138, CD200, FcRH5, GPRC5D, or SLAMF7. For example, in some embodiments, the NK cells are engineered to express a CAR that binds to CD19 and a CAR that binds to BCMA. Anti-BCMA CARs are known in the art and include any of those described in PCT Application No. PCT / US2022 / 073567, which is expressly incorporated by reference in its entirety. In some embodiments, the NK cells are engineered to express a CAR that binds to CD19 and a CAR that binds to BAFF-R. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to CD20. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to CD22. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to CD27. In some embodiments, the NK cells are engineered to express a CAR that binds to CD19 and a CAR that binds to CD28. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR thatbinds to CD33. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to CD38. In some embodiments, the NK cells are engineered to express a CAR that binds to CD19 and a CAR that binds to CD45. In some embodiments, the NK cells are engineered to express a CAR that binds to CD19 and a CAR that binds to CD47. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to CD54. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to CD56. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to CD81. In some embodiments, the NK cells are engineered to express a CAR that binds to CD19 and a CAR that binds to CD117. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to CD 138. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to CD200. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to FcRH5. In some embodiments, the NK cells are engineered to express a CAR that binds to CD 19 and a CAR that binds to GPRC5D. In some embodiments, the NK cells are engineered to express a CAR that binds to CD19 and a CAR that binds to SLAMF7.

[0227] Some embodiments of the methods and compositions described herein relate to a NK cell that has been modulated to reduce expression of a target gene or a target protein.

[0228] In some embodiments, the NK cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, the NK cells engineered to express a chimeric receptor are also engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain. Thus, in some embodiments, the NK cells are engineered to bicistronically express the chimeric receptor and mbIL15.

[0229] In some embodiments, the NK cells are derived from cell line NK-92. NK-92 cells are derived from NK cells, but lack major inhibitory receptors displayed by normal NK cells, while retaining the majority of activating receptors. Sonic embodiments of NK-92 cells described herein related to NK-92 cell engineered to silence certain additional inhibitory receptors, for example, SMAD3, allowing for upregulation of interferon-y (IFNy), granzyme B, and / or perforin production. Additional information relating to the NK-92 cell line is disclosed in WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044, both of which are expressly incorporated by reference in their entireties.

[0230] In some embodiments, the NK cells are used in combination with T cells. Thus, in some embodiments, the immune cells comprise T cells and NK cells (either from the same donor or from different donors). NK-92 cells are used, in several embodiments, 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 NK cells as disclosed herein. In an additional embodiment, NK-92 cells are used in combination with T cells as disclosed herein.e. Hematopoietic Stem Cells

[0231] In some embodiments, hematopoietic stem cells (HSCs) are used in the methods of cellular therapy disclosed herein. In several embodiments, the cells are engineered to express a homing moiety and / or a chimeric receptor complex. In several embodiments, the cells are engineered to express a chimeric receptor complex. HSCs are used, in several embodiments, to leverage their ability to engraft for long-term blood cell production, which could result in a sustained source of targeted anti-cancer effector cells, for example to combat cancer remissions. In several embodiments, this ongoing production helps to offset anergy or exhaustion of other cell types, for example due to the tumor microenvironment.

[0232] In some embodiments, a HSC is positive for cell surface expression of a marker selected from among the group consisting of CD34, CD59, and CD90. In some embodiments, a HSC is positive for cell surface expression of CD34. In some embodiments, a HSC is positive for cell surface expression of CD59. In some embodiments, a HSC is positive for cell surface expression of CD90.

[0233] In several embodiments allogeneic HSCs are used, while in some embodiments, autologous HSCs are used. In several embodiments, HSCs are used in combination with one or more additional engineered cell type disclosed herein.

[0234] Some embodiments of the methods and compositions described herein relate to a stem cell, such as a hematopoietic stem cell engineered to express a CAR that targets an antigen, for example, CD19, CD123, CD70, Her2, mesothelin, Claudin 6, BCMA, or EGFR, among any of the others disclosed herein, and optionally a membrane-bound interleukin 15 (mbIL15) domain. Several embodiments of the methods and compositions disclosed herein relate to hematopoietic stem cells engineered to express an activating chimeric receptor that targets a ligand on a diseased cell, for example, MICA, MICB. ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6 (among others) and optionally a membrane-bound interleukin 15 (mbIL15) domain. Several embodiments of the methods and compositions disclosed herein relate to hematopoietic stem cells engineered to express a chimeric receptor that targets an antigen associated with an autoimmune disease. For example, in some embodiments, the genetically engineered hematopoietic stem cells express a CAR that binds to an antigen selected from the group consisting of BAFF-R. BCMA, CD20, CD22, CD27, CD28, CD33, CD38, CD45. CD47, CD54, CD56. CD81, CD117, CD138, CD200, FcRH5. GPRC5D. and SLAMF7 and optionally a membrane -bound interleukin 15 (mbIL15) domain.

[0235] Some embodiments of the methods and compositions described herein relate to a HSC that has been modulated to reduce expression of a target gene or a target protein.

[0236] In some embodiments, the HSCs are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, the HSCs engineered to express a chimeric receptor are also engineered to also express (e g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain. Thus, in some embodiments, the HSCs are engineered to bicistronically express the chimeric receptor and mbIL15.f. Induced Pluripotent Stem Cells

[0237] In some embodiments, immune cells are derived (differentiated) from pluripotent stem cells (PSCs). In some embodiments, immune cells (e.g.. NK and / or T cells) derived from induced pluripotent stem cells (iPSCs) are used in the method of immunotherapy disclosed herein. For example, in some embodiments, NK cells are derived from iPSCs. In some embodiments, induced pluripotent stem cells (iPSCs) are used in the method of immunotherapy disclosed herein. iPSCs are used, in several embodiments, to leverage their ability to differentiate and derive into non-pluripotent cells, including, but not limited to, CD34 cells, hemogenic endothelium cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors. T cells, NKT cells, NK cells, and B cells comprising one or several genetic modifications at selected sites through differentiating iPSCs or less differentiated cells comprising the same genetic modifications at the same selected sites. In several embodiments, the iPSCs are used to generate iPSC-derived NK or T cells. In several embodiments, the iPSCs are used to generate iPSC-derived NK cells. In several embodiments, tire iPSCs are used to generate iPSC-derived T cells.

[0238] In several embodiments, the cells are engineered to express a homing moiety and / or a chimeric receptor complex. In several embodiments, the cells arc engineered to express a chimeric receptor complex. In several embodiments, iPSCs are used in combination with one or more additional engineered cell type disclosed herein.

[0239] Several embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express a chimeric receptor that targets an antigen, for example, CD19, CD123, CD70, Her2, mesothelin, Claudin 6, BCMA, or EGFR. In some embodiments, the iPSCs engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain. Some embodiments of the methods and compositions described herein relate to a stem cell, such as an induced pluripotent stem cell engineered to express a CAR that targets an antigen, for example, CD19, CD123, CD70. Her2. mesothelin, Claudin 6. BCMA, or EGFR, among any of the others disclosed herein, and optionally a membrane-bound interleukin 15 (mbIL15) domain.

[0240] Several embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express an activating chimeric receptor that targets a ligand on a diseased cell, for example, MICA, MICB. ULBP1, ULBP2, ULBP3, ULBP4. ULBP5. or ULBP6 (among others). In some embodiments, the iPSCs engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain. Several embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express an activating chimeric receptor that targets a ligand on a diseased cell, for example, MICA. MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5. or ULBP6 (among others) and optionally a membrane-bound interleukin 15 (mbIL15) domain.

[0241] Several embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express a chimeric receptor that targets an antigen associated with an autoimmune disease. For example, in some embodiments, the genetically engineered induced pluripotent stem cells also express a CAR that binds to an antigen selected from the group consisting of BAFF-R, BCMA, CD20, CD22, CD27, CD28, CD33, CD38, CD45, CD47, CD54, CD56, CD81, CD117, CD138, CD200, FcRH5, GPRC5D, and SLAMF7 and optionally a membrane-bound interleukin 15 (mbIL15) domain.

[0242] In several embodiments, the engineered iPSCs are differentiated into NK, T, or other immune cells, such as for use in a composition or method provided herein. In several embodiments, the engineered iPSCs are differentiated into NK cells. In several embodiments, the engineered iPSCs are differentiated into T cells. In several embodiments, the engineered iPSCs are differentiated into NK and T cells.C. Preparation of Cells for Genetic Engineering

[0243] In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for introduction of the recombinant receptor (e.g., CAR) may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the sample is an apheresis (e.g.., leukapheresis) sample.

[0244] In some embodiments, the subject from which the cells are isolated is one not having the disease (e.g., autoimmune disease) or in need of a cell therapy or not to which a cell therapy will be administered. In some embodiments, the cells are isolated from a subject that is different than the subject in need of a cell therapy or to which a cell therapy will be administered. Thus, in some embodiments, the cells are allogeneic to the subject to whom they are administered.

[0245] In some embodiments, the subject from which the cells are isolated is one having the disease (e.g., autoimmune disease) or in need of a cell therapy or to which a cell therapy will be administered. In some embodiments, the cells are isolated from the subject to which a cell therapy will be administered. Thus, in some embodiments, the cells arc autologous to the subject to whom they arc administered.

[0246] The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0247] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis (e.g., a leukapheresis) product. In some embodiments, the cells are isolated from an apheresis (e.g., leukapheresis) sample. Non-limitingsamples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. In some embodiments, the cells are derived from PBMCs. In some embodiments, the cells are not derived from cord blood. In some embodiments, the cells are not derived from induced pluripotent stem cells (iPSCs). Samples include, in the context of cell therapy, e.g.. adoptive cell therapy, samples from autologous and allogeneic sources.

[0248] The cells in some embodiments are primary cells, e.g., primary' human cells. In some embodiments, the cells are immune cells, e.g. primary NK cells. In some embodiments, the cells are NK cells derived from PBMCs. In some embodiments, the cells are allogeneic NK cells derived from PBMCs of a donor. Thus, in some embodiments, the NK cells are not derived from cord blood or iPSCs.

[0249] In some embodiments, isolation of the cells includes one or more preparation and / or non affmity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0250] In some examples, cells from the circulating blood of a subject are obtained, e.g.. by apheresis (e.g.. leukapheresis). The samples, in some aspects, contain lymphocytes, including NK cells, T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects contain cells other than red blood cells and platelets.

[0251] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity -based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0252] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is availablethat specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0253] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0254] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell ty pes can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0255] For example, in some aspects, NK cells or specific subpopulations thereof, such as cells positive or expressing high levels of one or more surface markers, e.g.. CD56+, CCR7+, CD16+, CD57+, CD11+, CX3CR1+, a Killer Ig-like receptor (KIR) +, NKp30+, NKp44+, or NKp46+ NK cells, are isolated by positive or negative selection techniques. In some aspects, NK cells are isolated by positive selection for CD56. For example, CD56+ NK cells can be positively selected using anti-CD56 conjugated magnetic beads.

[0256] In some embodiments, the cells (e.g., NK cells) are expanded in culture prior to, during, and / or following genetic engineering. In some embodiments, the cells are expanded in culture prior to genetic engineering. In some embodiments, the cells arc expanded in culture following genetic engineering. In some embodiments, the cells are expanded in culture prior to and following genetic engineering. Methods for expanding cells are known in the art and include any of those described in US Patent Nos. 7,435,596 and 8,026,097; and Patent Application Nos. PCT / SG2018 / 050138; PCT / US2020 / 044033; PCT / US2021 / 071330; and PCT / US2022 / 074164, all of which are expressly incorporated by reference in their entireties.

[0257] In some embodiments, expanding the cells in culture comprises co-culturing the cells with feeder (e.g., stimulatory) cells. Thus, in some embodiments, the cells are expanded in culture prior to genetic engineering by co-culturing the cells with feeder cells. In some embodiments, the feeder cells express IL15 (e.g.. membrane-bound IL 15) and 4-1BBL. In some embodiments, the feeder cells express membrane-bound interleukin 15 (mbIL15) and 4-1BBL. In some embodiments, the feeder cells do not express MHCI molecules. In some embodiments, the feeder cells do not express MHCII molecules. In some embodiments, the feeder cells are immune cells. In some embodiments, the feeder cells are K562cells. Engineered feeder cells are disclosed in, for example, International Patent Application PCT / SG2018 / 050138, which is expressly incorporated by reference in its entirety. In some embodiments, following genetic engineering, the cells are allowed to further expand in culture.

[0258] In some embodiments, expanding the cells in culture comprises culturing the cells in the presence of IL2, IL12, and / or IL18. In some embodiments, the cells are cultured in the presence of IL2. In some embodiments, the cells are cultured in the presence of IL12 and IL18. In some embodiments, the cells are cultured in the presence of IL2. IL12, and IL18.

[0259] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, engineering, and / or expansion. In some aspects, the cells are cryopreserved after engineering. In some aspects, such as when the cells are further expanded in culture after genetic engineering, the cells are crvoprcscrvcd after the further expansion. In some embodiments, the cells are suspended in a freezing solution. In some embodiments, a composition provided herein is cryopreserved (e.g., prior to infusion into a subject). In some embodiments, a composition (e.g., a pharmaceutical composition) provided herein comprises a cryopreservative. Any of a variety of known freezing solutions and parameters in some aspects may be used.D. Genetic Modulation of Immune Cells

[0260] As discussed above, a variety7of cell types can be utilized in cellular immunotherapy. Further, as elaborated in more detail below, and shown in the Examples, modifications can be made to these cells to enhance one or more aspects of their expansion (e.g.. ex vivo or in vitro), efficacy (e.g., cytotoxicity) and / or persistence, such as in vivo persistence (e.g., active life span). As discussed herein, in several embodiments immune cells are used for immunotherapy. Provided are methods and uses of genetically modulated immune cells, including engineered immune cells that are modulated, and / or compositions thereof. In some aspects, the immune cells are genetically modulated to increase or decrease expression of a target protein. In some aspects, the immune cells are genetically modulated to increase expression of a target protein. In some aspects, the immune cells arc genetically modulated to decrease expression of a target protein. In some aspects, the methods comprise genetically modulating die immune cells, such as to increase or decrease expression of a target protein. In some aspects, die methods comprise genetically modulating the immune cells to increase expression of a target protein. In some aspects, the methods comprise genetically modulating the immune cells to decrease expression of a target protein. Expression of a target protein can be reduced by disrupting a gene (a target gene) encoding the target protein or a portion thereof. Expression of a target protein can also be reduced by provision of an inhibitory nucleic acid molecule that binds to mRNA encoding the target protein or a portion thereof.

[0261] As discussed below, in several embodiments, genetic modulation is employed to reduce or knockout expression of target proteins, for example by disrupting the underlying gene encoding the protein. Alternatively, in some embodiments, expression of a target protein is reduced byan inhibitor}' nucleic acid molecule, such as one that is complementary to. targets, inhibits and / or binds a gene (a target gene) encoding the target protein or a portion thereof. In some of any such embodiments, the inhibitory nucleic acid molecule includes an RNA interfering agent. In some of any such embodiments, the inhibitory nucleic acid molecule is or contains or encodes a small interfering RNA (siRNA), a microRNA-adapted shRNA, a short hairpin RNA (shRNA), a hairpin siRNA, a precursor microRNA (pre-miRNA) or a microRNA (miRNA). In some of any such embodiments, the inhibitory nucleic acid molecule contains a sequence complementary to a target protein-encoding nucleic acid. In some of any such embodiments, the inhibitory nucleic acid molecule contains an antisense oligonucleotide complementary to a target protein-encoding nucleic acid.

[0262] In some embodiments, the genetically modulated cells express a chimeric receptor. It is contemplated that the cells can be genetically modulated at any point prior to, during, and / or after engineering of the cells to express a chimeric receptor. It is contemplated that the cells can be introduced with an inhibitory nucleic acid molecule to modulate gene or protein expression at any point prior to. during, and / or after engineering of the cells to express a chimeric receptor.

[0263] In some embodiments, the immune cells can be introduced with an inhibitory nucleic acid molecule and a nucleic acid sequence encoding a chimeric receptor at the same time. In some embodiments, an inhibitory nucleic acid molecule sequence and a nucleic acid sequence encoding a chimeric receptor are comprised within a single polynucleotide.

[0264] In some embodiments, the genetically modulated cells express a membrane-bound interleukin 15 (mbIL15). In some embodiments, the immune cells can be introduced with an inhibitory nucleic acid molecule, a nucleic acid sequence encoding a chimeric receptor and a nucleic acid sequence encoding a membrane-bound interleukin 15 (mbIL15) at the same time. In some embodiments, an inhibitory nucleic acid molecule sequence, a nucleic acid sequence encoding a chimeric receptor and a nucleic acid sequence encoding a mbIL15 are comprised within a single polynucleotide. In some embodiments, the immune cells arc introduced with at least one inhibitor}' nucleic acid molecule, a nucleic acid sequence encoding a chimeric receptor, and a nucleic acid sequence encoding a membranebound interleukin 15 (mbIL15) at the same time.

[0265] In some embodiments, the immune cells are introduced with an shRNA, a nucleic acid sequence encoding a chimeric receptor and a nucleic acid sequence encoding a membrane-bound interleukin 15 (mbIL15) at the same time. In some embodiments, the shRNA, the nucleic acid sequence encoding a chimeric receptor and the nucleic acid sequence encoding a mbIL15 are comprised within a single polynucleotide. In some embodiments, the immune cells are introduced with at least one shRNA, a nucleic acid sequence encoding a chimeric receptor and a nucleic acid sequence encoding a a membrane-bound interleukin 15 (mbIL15) at the same time. In some embodiments, the at least one shRNA. a nucleic acid sequence encoding a chimeric receptor and a nucleic acid sequence encoding a mbIL15 are comprised within a single polynucleotide.

[0266] For example, in some embodiments, the cells are genetically modulated prior to engineering of the cells to express mbIL15. In some embodiments, the cells are genetically modulated concurrent with engineering of the cells to express mbIL15. In some embodiments, the cells are genetically modulated after engineering of the cells to express mbIL15. In some embodiments, die cells are genetically modulated prior to engineering of the cells to express a recombinant receptor. In some embodiments, the cells are genetically modulated concurrent with engineering of the cells to express a recombinant receptor. In some embodiments, the cells are genetically modulated after engineering of the cells to express a recombinant receptor. In several embodiments, engineering and gene modulation are substantially contemporaneous.

[0267] In several embodiments, gene modulation reduces transcription of a target gene 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%. or more (including any amount between those listed). In several embodiments, gene modulation reduces transcription of a target gene by at least about 30%. In several embodiments, gene modulation reduces transcription of a target gene by at least about40%. In several embodiments, gene modulation reduces transcription of a target gene by at least about50%. In several embodiments, gene modulation reduces transcription of a target gene by at least about60%. In several embodiments, gene modulation reduces transcription of a target gene by at least about70%. In several embodiments, gene modulation reduces transcription of a target gene by at least about80%. In several embodiments, gene modulation reduces transcription of a target gene by at least about90%. In several embodiments, the gene is completely knocked out, such that transcription of the target gene is undetectable.

[0268] In several embodiments, gene modulation reduces expression of a 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%, or more (including any amount between those listed). In several embodiments, gene modulation reduces expression of a target protein by at least about 30%. In several embodiments, gene modulation reduces expression of a target protein by at least about 40%. In several embodiments, gene modulation reduces expression of a target protein by at least about 50%. In several embodiments, gene modulation reduces expression of a target protein by at least about 60%. In several embodiments, gene modulation reduces expression of a target protein by at least about 70%. In several embodiments, gene modulation reduces expression of a target protein by at least about 80%. In several embodiments, gene modulation reduces expression of a target protein by at least about 90%. In several embodiments, the gene is completely knocked out, such that expression of the target protein is undetectable.

[0269] In several embodiments, an inhibitory nucleic acid molecule (e.g., an RNA interfering agent) reduces expression of a 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%. or more (including any amount between those listed). In several embodiments, theinhibitory nucleic acid molecule reduces expression of a target protein by at least about 30%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 40%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 50%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 60%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 70%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 80%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of a target protein by at least about 90%. In several embodiments, the inhibitory nucleic acid molecule eliminates expression of the target protein, such that it is undetectable.

[0270] In some embodiments, the inhibitory nucleic acid molecule is fully complementary to the host or target protein coding nucleic acid sequence. In some embodiments, the inhibitory nucleic acid molecule is partially complementary to host or target protein coding nucleic acid sequence. In some embodiments, more than one inhibitory nucleic acid molecule(s) is used to reduce expression of a target protein. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense strand having a sequence complementary to the host or target nucleic acid and a sense strand comprising a sequence capable of pairing with the antisense strand.

[0271] In some embodiments, the one or more inhibitory nucleic acid molecule(s) is encoded by an expression vector. In some embodiments, the expression vector comprises a polynucleotide comprising one or more inhibitory nucleic acid molecule(s) that can reduce expression of a target protein. In some embodiments, the expression vector comprises a polynucleotide comprising two inhibitory nucleic acid molecule(s) that can reduce expression of a target protein. In some embodiments, the expression vector comprises a polynucleotide comprising three inhibitory nucleic acid molecule(s) that can reduce expression of a target protein. In some embodiments, the expression vector comprises a polynucleotide comprising four inhibitory nucleic acid molcculc(s) that can reduce expression of a target protein. In some embodiments, the expression vector comprises a polynucleotide comprising five inhibitory nucleic acid molecule(s) that can reduce expression of a target protein. In some embodiments, the expression vector comprises a polynucleotide comprising six inhibitory nucleic acid molecule(s) that can reduce expression of a target protein. In some embodiments, the polynucleotide comprising more than one inhibitory' nucleic acid molecule reduces expression of the same target protein. In some embodiments, the polynucleotide comprising more than one inhibitory nucleic acid molecule reduces expression of the same target protein by binding to the same region within the target nucleic acid. In some embodiments, the polynucleotide comprising more than one inhibitory nucleic acid molecule reduces expression of the same target protein by binding to different regions within the target nucleic acid. In some embodiments, the polynucleotide comprising more than one inhibitory nucleic acid molecule reduces expression of different target proteins. In some embodiments, the polynucleotide comprising more than one inhibitory nucleic acid molecule is separated by a linker.In some embodiments, the one or more inhibitory nucleic acid molecules, chimeric receptor and a membrane-bound interleukin 15 (mbIL15) are encoded by a single expression vector. In some embodiments, the expression vector comprises a polynucleotide comprising one or more inhibitory nucleic acid molecules, chimeric receptor and a membrane -bound interleukin 15 (mbIL15). In some embodiments, the one or more shRNAs, chimeric receptor and a membrane-bound interleukin 15 (mbIL15) are encoded by a single expression vector. In some embodiments, the expression vector comprises a polynucleotide comprising one or more shRNAs. chimeric receptor and a membrane-bound interleukin 15 (mbIL15).

[0272] In several embodiments, gene modulation is used to “knock in” or otherwise increase transcription of a target gene. In several embodiments, transcription of a target gene is increased 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%. or more (including any amount between those listed). In several embodiments, transcription of a target gene is increased by at least about 30%. In several embodiments, transcription of a target gene is increased by at least about 40%. In several embodiments, transcription of a target gene is increased by at least about 50%. In several embodiments, transcription of a target gene is increased by at least about 60%. In several embodiments, transcription of a target gene is increased by at least about 70%. In several embodiments, transcription of a target gene is increased by at least about 80%. In several embodiments, transcription of a target gene is increased by at least about 90%. In several embodiments, transcription of a target gene is increased by at least about 100%.

[0273] In several embodiments, gene modulation is used to “knock in” or otherwise enhance expression of a target protein. In several embodiments, expression of a target protein can be enhanced 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%, or more (including any amount between those listed). In several embodiments, expression of a target protein is increased by at least about 30%. In several embodiments, expression of a target protein is increased by at least about 40%. In several embodiments, expression of a target protein is increased by at least about 50%. In several embodiments, expression of a target protein is increased by at least about 60%. In several embodiments, expression of a target protein is increased by at least about 70%. In several embodiments, expression of a target protein is increased by at least about 80%. In several embodiments, expression of a target protein is increased by at least about 90%. In several embodiments, expression of a target protein is increased by at least about 100%.

[0274] Unless indicated otherwise to the contrary, the sequences provided for guide RNAs (gRNAs) that are recited using deoxyribonucleotides refer to the target DNA sequence (which is complementary to the corresponding non-target DNA sequence to which the gRNA binds) and shall be considered as also referencing those guides used in practice (e.g.. employing ribonucleotides, where the ribonucleotide uracil is used in lieu of deoxyribonucleotide thymine or vice-versa where thymine isused in lieu of uracil, wherein both are complementary base pairs to adenine when reciting either an RNA or DNA sequence). In other words, the sequences provided for particular gRNAs provided herein are identical to the gRNA sequences used in practice, except that the gRNA sequences include uracil in lieu of thymine. For example, a gRNA with the sequence ATGCTCAATGCGTC (SEQ ID NO: 124) shall also refer to the following sequence AUGCUCAAUGCGUC (SEQ ID NO: 125) or a gRNA with sequence AUGCUCAAUGCGUC (SEQ ID NO: 125) shall also refer to the following sequence ATGCTCAATGCGTC (SEQ ID NO: 124). Further, the non-target DNA sequence to which a particular gRNA sequence binds is complementary to the sequence of the particular gRNA. For example, a gRNA with the provided sequence of ATGCTCAATGCGTC (SEQ ID NO: 124) binds to a non-target DNA sequence of TACGAGTTACGCAG (SEQ ID NO: 126). In this situation, the corresponding target DNA sequence, which is complementary to the non-target DNA sequence, is ATGCTCAATGCGTC (SEQ ID NO: 124).

[0275] In several embodiments, gene modulation of the immune cells also provides unexpected enhancement in the expansion, persistence and / or cytotoxicity of the edited immune cell. As disclosed herein, engineered cells (e.g.. those expressing a CAR) may also be modulated, the combination of which provides for a robust cell for immunotherapy. In several embodiments, the modulations allow for unexpectedly improved NK cell expansion, persistence and / or cytotoxicity. In several embodiments, knockout of gene expression in NK cells removes a potent negative regulator or other suppressor of signaling and / or activity of NK cells, thereby disinhibiting the NK cells and allowing for one or more of enhanced NK cell homing, NK cell migration, activation of NK cells, expansion, cytotoxicity and / or persistence. Additionally, in several embodiments, the modulation enhances NK and / or T cell function in the otherwise suppressive tumor microenvironment.

[0276] In several embodiments, genetic modulation (whether knock out or knock in) of any of the target genes disclosed herein, is accomplished through targeted introduction of DNA breakage, and a subsequent DNA repair mechanism. In several embodiments, double strand breaks of DNA arc repaired by non-homologous end joining (NHEJ), wherein enzymes are used to directly join the DNA ends to one another to repair the break. NHEJ is an error-prone process. In general, in the absence of a repair template, the NHEJ process re-ligates the ends of the cleaved DNA strands, which frequently results in nucleotide deletions and insertions at the cleavage site. In several embodiments, however, double strand breaks (DSB) are repaired by homology directed repair (HDR), which is advantageously more accurate, thereby allowing sequence specific breaks and repair. HDR uses a homologous sequence as a template for regeneration of missing DNA sequences at the break point, such as a vector with the desired genetic elements (e.g.. an insertion element to disrupt the coding sequence of a gene) within a sequence that is homologous to the flanking sequences of a double strand break. This will result in the desired change (e.g., insertion) being inserted at the site of the DSB. The HDR pathway can occur by way of the canonical HDR pathway or the alternative HDR pathway. Unless otherwiseindicated, the term “HDR’‘ or “homology -directed repair” as used herein encompasses both canonical HDR and alternative HDR.

[0277] Canonical HDR or “canonical homology-directed repair” or cHDR,” are used interchangeably, and refers to tire process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, such as a sister chromatid; or an exogenous nucleic acid, such as a donor template). Canonical HDR typically acts when there has been a significant resection at the DSB, forming at least one single-stranded portion of DNA. In a normal cell, canonical HDR typically involves a series of steps such as recognition of the break, stabilization of the break, resection, stabilization of single-stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation. The canonical HDR process requires RAD51 and BRCA2. and the homologous nucleic acid, e.g., repair template, is typically double-stranded. In canonical HDR, a double-stranded polynucleotide, e.g., a double-stranded repair template, is introduced, which comprises a sequence that is homologous to the targeting sequence, and which will either be directly integrated into the targeting sequence or will be used as a template to insert the sequence, or a portion the sequence, of the repair template into the target gene. After resection at the break, repair can progress by different pathways, e.g.. by the double Holliday junction model (also referred to as the double strand break repair, or DSBR, pathway), or by the synthesis-dependent strand annealing (SDSA) pathway.

[0278] In the double Holliday junction model, strand invasion occurs by the two single stranded overhangs of the targeting sequence to the homologous sequences in the double-stranded polynucleotide, e.g., double stranded donor template, which results in the formation of an intermediate with two Holliday junctions. The junctions migrate as new DNA is synthesized from the ends of the invading strand to fill the gap resulting from the resection. The end of the newly synthesized DNA is ligated to the resected end, and the junctions are resolved, resulting in the insertion at the targeting sequence, or a portion of the targeting sequence that includes the gene variant. Crossover with the polynucleotide, e.g., repair template, may occur upon resolution of the junctions.

[0279] In the SDSA pathway, only one single stranded overhang invades the polynucleotide, e.g., donor template, and new DNA is synthesized from the end of the invading strand to fill the gap resulting from resection. The newly synthesized DNA then anneals to the remaining single stranded overhang, new DNA is synthesized to fill in the gap, and tire strands are ligated to produce the modified DNA duplex.

[0280] Alternative HDR, or “alternative homology -directed repair,” or “alternative HDR,” are used interchangeably, and refers, in some embodiments, to the process of repairing DNA damage using a homologous nucleic acid (e.g.. an endogenous homologous sequence, such as a sister chromatid; or an exogenous nucleic acid, such as a repair template). Alternative HDR is distinct from canonical HDR in that the process utilizes different pathways from canonical HDR, and can be inhibited by the canonical HDR mediators. RAD51 and BRCA2. Moreover, alternative HDR is also distinguished by the involvement of a single-stranded or nicked homologous nucleic acid template, e.g.. repair template.whereas canonical HDR generally involves a double-stranded homologous template. In the alternative HDR pathway, a single strand template polynucleotide, e.g., repair template, is introduced. A nick, single strand break, or DSB at the cleavage site, for altering a desired target site, e.g., a gene variant in a target gene, is mediated by a nuclease molecule, and resection at the break occurs to reveal single stranded overhangs. Incorporation of the sequence of the template polynucleotide, e.g.. repair template, to alter the target site of the DNA typically occurs by the SDSA pathway, as described herein. In some embodiments, HDR is carried out by introducing, into a cell, one or more agent(s) capable of inducing a DSB, and a repair template, e.g.. a single-stranded oligonucleotide. The introducing can be carried out by any suitable delivery. The conditions under which HDR is allowed to occur can be any conditions suitable for carrying out HDR in a cell.

[0281] In several embodiments, gene modulation is accomplished by one or more of a variety of engineered nucleases. In several embodiments, restriction enzymes are used, particularly when double strand breaks are desired at multiple regions. In several embodiments, a bioengineered nuclease is used. Depending on the embodiment, one or more of a Zinc Finger Nuclease (ZFN), transcription-activator like effector nuclease (TALEN), meganuclease and / or clustered regularly interspaced short palindromic repeats (CRISPR / Cas9) system are used to specifically edit the genes encoding one or more of the TCR subunits.

[0282] Meganucleases are characterized by their capacity' to recognize and cut large DNA sequences (from 14 to 40 base pairs). In several embodiments, a meganuclease from the LAGLID ADG family is used, and is subjected to mutagenesis and screening to generate a meganuclease variant that recognizes a unique sequence(s), such as a specific site in a TCR subunit (e.g., TRAC), or CISH, or any other target gene disclosed herein. Target sites in a TCR subunit can readily be identified. Further information of target sites within a region of the TCR can be found in US Patent Publication No. 2018 / 0325955, and US Patent Publication No. 2015 / 0017136, each of which is expressly incorporated by reference in its entirety. In several embodiments, two or more mcganuclcascs, or functions fragments thereof, are fused to create a hybrid enzyme that recognize a desired target sequence within die target gene (e.g., CISH).

[0283] In contrast to meganucleases, ZFNs and TALEN function based on a non-specific DNA cutting catalytic domain which is linked to specific DNA sequence recognizing peptides such as zinc fingers or transcription activator-like effectors (TALEs). Desirably, the ZFNs and TALENs allow sequence-independent cleavage of DNA, with 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 part of each finger is responsible for the specific recognition of the DNA sequence. While the sequences recognized by ZFNs are relatively short, (e.g., ~3 base pairs), in several embodiments, combinations of 2, 3, 4. 5, 6, 7, 8. 9, 10 or more zinc fingers whose recognition sites have been characterized are used, thereby allowing targeting of specific sequences, such as a portion of the TCR (or an immune checkpoint). The combined ZFNs are thenfused with the catalytic domain(s) of an endonuclease, such as FokI (optionally a FokI heterodimer), to induce a targeted DNA break. Additional information on uses of ZFNs to edit a TCR subunit and / or immune checkpoints can be found in US Patent No. 9,597,357, which is expressly incorporated by reference in its entirety .

[0284] Transcription activator-like effector nucleases (TALENs) are specific DNA-binding proteins that feature an array of 33 or 34-amino acid repeats. Like ZFNs, TALENs are a fusion of a DNA cutting domain of a nuclease to TALE domains, which allow for sequence-independent introduction of double stranded DNA breaks with highly precise target site recognition. TALENs can create double strand breaks at the target site that can be repaired by error-prone non-homologous endjoining (NHEJ), resulting in gene disruptions through the introduction of small insertions or deletions. Advantageously, TALENs are used in several embodiments, at least in part due to their higher specificity in DNA binding, reduced off-target effects, and ease in construction of the DNA-binding domain.

[0285] CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are genetic elements that bacteria use as protection against viruses. The repeats are short sequences that originate from viral genomes and have been incorporated into the bacterial genome. Cas (CRISPR associated proteins) process these sequences and cut matching viral DNA sequences. By introducing plasmids containing Cas genes and specifically constructed CRISPRs into eukaryotic cells, the eukaryotic genome can be cut at any desired position. Additional information on CRISPR can be found in US Patent Publication No. 2014 / 0068797, which is expressly incorporated by reference in its entirety . In several embodiments, CRISPR is used to manipulate the gene(s) encoding a target gene to be knocked out or knocked in, for example CISH. TGFBR2, TCR. B2M, CIITA, CD47, HLA-E, etc. In several embodiments, CRISPR is used to edit one or more of the TCRs of a T cell and / or the genes encoding one or more immune checkpoints. In several embodiments, the immune checkpoint is selected from one or more of CTL A4 or PD 1. In several embodiments, CRISPR is used to truncate one or more of TCRa, TCR , TCRy, or TCR5. In several embodiments, a TCR is truncated without impacting the function of the CD3z signaling domain of the TCR.

[0286] Depending on the embodiment and which target gene is to be edited, a Class 1 or Class 2 Cas is used. In several embodiments, a Class 1 Cas is used, and the Cas type is selected from the following types: I, IA, IB. IC. ID. IE, IF, IU, III, IIIA, IIIB, IIIC, HID, IV IVA, or IVB, and combinations thereof. In several embodiments, the Cas is selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b. Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3. GSU0054, CaslO. Csm2, Cmr5, Casio, Csxl l, CsxlO, and Csfl, and combinations thereof. In several embodiments, a Class 2 Cas is used and the Cas type is selected from the following types: II, HA, IIB, IIC, V. or VI, and combinations thereof. In several embodiments, the Cas is selected from the group consisting of Cas9, Csn2, Cas4, Casl2a (previously known as Cpfl), C2cl, C2c3, Casl3a (previously known as C2c2), Casl3b. Casl3c. CasX, and CasY and combinations thereof. In some embodiments, the Cas is Cas9. In someembodiments, class 2 CasX is used, wherein CasX is capable of forming a complex with a guide nucleic acid and wherein the complex can bind to a target DNA, and wherein the target DNA comprises a nontarget strand and a target strand. In some embodiments, class 2 CasY is used, wherein CasY is capable of binding and modifying a target nucleic acid and / or a polypeptide associated with target nucleic acid.

[0287] RNA interference (RNAi) is a posttranscriptional gene silencing process mediated by double stranded RNA (dsRNA). RNAi is based on sequence-specific degradation of the host mRNA by introducing dsRNAs that are identical to the target sequence (Fire A. et al., Nature. (1998). 391 :806- 811). Teclmiques for RNAi are well known in the art and are described in, for example, Lehner et al., (Briefings in Functional Genomics and Proteomics (2004), 3(1) 68-83), Reynolds (Nat Biotechnol. (2004), 22(3):326-30), Downward (BMJ. (2004), 328(7450): 1245-8). RNAi uses small double-stranded RNA (dsRNA) molecules as triggers to direct homology -dependent control of gene activity. One fonn of RNAi involves the transfection of chemically synthesized short interfering RNA oligonucleotides (siRNAs) directly into the cytosol of the cells. Another form of RNAi involves the use of short hairpin RNAs (shRNAs) synthesized within the cell by DNA vector-mediated production. Like siRNAs, shRNAs may be transfected as plasmid vectors encoding shRNAs transcribed by RNA pol III or modified pol II promoters, but can also be delivered into mammalian cells through infection of the cell with virally produced vectors. While the first form of RNAi delivers the siRNA duplex directly to the cytosol, shRNAs are capable of DNA integration and consist of two complementary 19-22 bp RNA sequences linked by a short loop of 4-11 nt similar to the hairpin found in naturally occurring miRNA (Moore C.B., et al. Methods Mol Biol. (2010). 629:141-158).

[0288] RNAi involving the use of short hairpin RNAs (shRNAs) comprises a two-step mechanism for RNAi-induced gene silencing (Kim D. H. et al., Biotechniques. (2008) 44(5):613-616). The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), that are around 21 to 25 nucleotides long, by an RNase Ill-like activity of Dicer. In the second step, the siRNAs join an RNase complex, RISC (RNA-induccd silencing complex), which acts on the cognate mRNA and degrades it (Agrawal N et al., Microbiol Mol Biol Rev. (2003) 67(4):657-685). In mammalian cells, siRNAs can also be synthesized by chemical or biochemical methods. Dicer is complexed with RNA- binding proteins, the TAR-RNA-b inding protein (TRBP), PACT, and Ago-2, which are involved in the hand-off of siRNAs to the RNA-induced silencing complex (RISC). While siRNAs loaded into RISC are double-stranded, Ago-2 cleaves and releases the “passenger” strand, leading to an activated form of RISC with a single-stranded “guide” RNA molecule that directs the specificity of the target recognition by intermolecular base pairing (Kim D. H. et al., Biotechniques. (2008) 44(5):613— 616). In some embodiments, the guide strand of RNA is referred to as the antisense strand of RNA. In some embodiments, the passenger strand of RNA is referred to as the sense strand of RNA.

[0289] Another form of RNAi involves the microRNAs (miRNAs). miRNAs are endogenous substrates for the RNAi machinery (Kim D. H. et al., Biotechniques. (2008) 44(5):613— 616). They are initially expressed as long primary transcripts (pri-miRNAs). which are processed withinthe nucleus into 60-70 bp hairpins by the microprocessor complex, consisting of Drosha and DGCR8 into pre-miRNAs (Lee Yet al. Nature. (2003), 425:415-419: Han J, et al.. Genes Dev. (2004), 18:3016- 3027). The pre-miRNAs are further processed in the cytoplasm by Dicer and one of the two strands is loaded into RISC, presumably via interaction with one of the Dicer accessory proteins (Lee Y, et al., EMBO J. (2006);2 5:522-532).

[0290] Given that Dicer is responsible for processing shRNAs into siRNAs, the use of pre- miRNA secondary structures as a scaffold for siRNA production was contemplated (Zeng Y, et al., Molecular cell. 2002; 9: 1327-1333). The first generation of shRNAs (pre-miRNA-like shRNA) was characterized by the use of RNA polymerase III promoters (Pol III). Mimicking the structures of pri- miRNA opened the door to designing the second generation of shRNAs (pri-miRNA-like shRNA) driven by RNA polymerase II promoters (Pol II) (Stegmeier Fet al.. Proceedings of the National Academy of Sciences of the United States of America. (2005);102:13212-13217; Silva lM et al. Nature genetics. (2005);37: 1281-1288). miRNA-based shRNA refers to a short hairpin RNA (shRNA) that is designed to mimic the structure and processing pathway of a miRNA, allowing for more efficient and controlled gene silencing by leveraging the cell's endogenous miRNA machinery to process the shRNA into a functional silencing molecule (Choi JG et al., Mol Ther. (2015); 23(2):310-20). The use of pol II promoters for shRNA expression requires embedding of the shRNA sequences into flanking regions that are typically derived from endogenous miRNA precursors. shRNAs flanked by a miRNA scaffold mimic the structure of endogenous miRNAs and are termed miRNA-based shRNAs (shRNA-miRs). (Guda S et al., Molecular Therapy (2015), 23(9): 1465-1474; McBride. I.L. et al.. Proc Natl Acad Sci USA, 105 (2008), pp. 5868-5873; Zeng Y et al., Mol Cell, 9 (2002), pp. 1327-1333). Choi showed that multiple shRNAs can be used to simultaneously suppress multiple genes at once, for example, multiplexing seven shRNA-miRs to suppress HIV replication (Choi IG et al., 2015).

[0291] In some embodiments, the dsRNA comprises at least 18, 30, 50, 75, or 100 or more contiguous nucleic acids of the nucleic acid sequence of a gene or host mRNA, and / or complements thereof, which may be in sense and / or antisense orientation. In some embodiments, the dsRNAs are processed by Dicer into short double stranded fragments known as small interfering RNAs (siRNAs) with a guide (antisense) strand and a passenger (sense) strand. As used herein, the antisense strand or the guide strand is the sequence of the siRNA that is complementary with the host mRNA sequence. In some embodiments, the antisense strand or the guide strand is fully complementary to the host mRNA sequence. In some embodiments, the antisense strand or the guide strand is partially complementary to the host mRNA sequence. As used herein, the sense strand or the passenger strand is the sequence of the siRNA which is complementary to the guide strand or the antisense strand. In some embodiments, the sense strand or the passenger strand is fully complementary to the antisense strand or the guide strand. In some embodiments, the sense strand or the passenger strand is partially complementary to the antisense strand or the guide strand.i. Targets for Gene Modulation

[0292] As discussed above, gene modulation can be used to disrupt a target gene (or genes) to enhance the expansion, cytotoxicity, or persistence of immune cells, such as NK cells. In some embodiments, inhibitory nucleic acid molecules can be used to reduce expression of a target gene (or genes) or target protein (or proteins), such as by causing degradation of target protein-encoding RNA.

[0293] In some embodiments, immune cells are genetically modulated at ADAM 17, B2M, CBLB, CD58, CIITA, CIS, FAS, FASLG, ICAM1, ICAM2, ICAM3. MED12, PRDM1 or PTPN2 or any combination thereof. In some embodiments, immune cells are genetically modulated at ADAM 17. In some embodiments, immune cells are genetically modulated at B2M. In some embodiments, immune cells are genetically modulated at CBLB. In some embodiments, immune cells are genetically modulated at CD58. In some embodiments, immune cells are genetically modulated at CIITA. In some embodiments, immune cells are genetically modulated at CIS. In some embodiments, immune cells are genetically modulated at FAS. In some embodiments, immune cells are genetically modulated at FASLG. In some embodiments, immune cells are genetically modulated at ICAM1. In some embodiments, immune cells are genetically modulated at ICAM2. In some embodiments, immune cells are genetically modulated at ICAM3. In some embodiments, immune cells are genetically modulated at MED12. In some embodiments, immune cells arc genetically modulated at PRDM1. In some embodiments, immune cells are genetically modulated at PTPN2.

[0294] In some embodiments, the target protein comprises ADAM 17, B2M, Cbl-b, CD58, CIITA, CIS. FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 or PTPN2, or any combination thereof. In some embodiments, the target protein comprises ADAMI 7. In some embodiments, the target protein comprises B2M. In some embodiments, the target protein comprises Cbl-b. In some embodiments, the target protein comprises CD58. In some embodiments, the target protein comprises CIITA. In some embodiments, the target protein comprises CIS. In some embodiments, the target protein comprises FAS. In some embodiments, the target protein comprises FASL. In some embodiments, the target protein comprises ICAM1. In some embodiments, the target protein comprises ICAM2. In some embodiments, the target protein comprises ICAM3. In some embodiments, the target protein comprises MED12. In some embodiments, the target protein comprises PRDM1. In some embodiments, the target protein comprises PTPN2.

[0295] In some embodiments, the target protein is a single target protein selected from the group consisting of ADAM17. B2M. Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAML ICAM2. ICAM3, MED 12, PRDM1 and PTPN2.

[0296] In some embodiments, the target proteins comprise two different target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58. CIITA, CIS, FAS, FASL, ICAM1, ICAM2. ICAM3, MED12, PRDM1 and PTPN2. In some embodiments, the target proteins are two different target proteins selected from the group consisting of ADAM17, B2M. Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In some embodiments, thetarget proteins comprise three different target proteins selected from the group consisting of ADAM 17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In some embodiments, the target proteins are three different target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2. In some embodiments, the target proteins comprise four different target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS, FAS, FASL, ICAM1, ICAM2, ICAM3, MED12. PRDM1 and PTPN2. In some embodiments, the target proteins are four different target proteins selected from the group consisting of ADAM17, B2M, Cbl-b, CD58, CIITA, CIS. FAS, FASL, ICAM1, ICAM2, ICAM3, MED12, PRDM1 and PTPN2.

[0297] In some embodiments, the target proteins comprise ADAM 17 and B2M. In some embodiments, the target proteins comprise ADAM17 and Cbl-b. In some embodiments, the target proteins comprise ADAM17 and CD58. In some embodiments, the target proteins comprise ADAM17 and CIITA. In some embodiments, the target proteins comprise ADAMI 7 and CIS. In some embodiments, the target proteins comprise ADAMI 7 and FAS. In some embodiments, the target proteins comprise ADAM 17 and FASL. In some embodiments, the target proteins comprise ADAM 17 and ICAM1. In some embodiments, the target proteins comprise ADAM17 and ICAM2. In some embodiments, the target proteins comprise ADAM 17 and ICAM3. In some embodiments, the target proteins comprise ADAM17 and MED12. In some embodiments, the target proteins comprise ADAM17 and PTPN2. In some embodiments, the target proteins comprise ADAM17 and PRDM1. In some embodiments, the target proteins comprise B2M and Cbl-b. In some embodiments, the target proteins comprise B2M and CD58. In some embodiments, the target proteins comprise B2M and CIITA. In some embodiments, the target proteins comprise B2M and CIS. In some embodiments, the target proteins comprise B2M and FAS. In some embodiments, the target proteins comprise B2M and FASL. In some embodiments, the target proteins comprise B2M and ICAM1. In some embodiments, the target proteins comprise B2M and IC AM2. In some embodiments, the target proteins comprise B2M and ICAM3. In some embodiments, the target proteins comprise B2M and MED12. In some embodiments, the target proteins comprise B2M and PTPN2. In some embodiments, the target proteins comprise B2M and PRDM1. In some embodiments, the target proteins comprise Cbl-b and CD58.I11 some embodiments, the target proteins comprise Cbl-b and CIITA. In some embodiments, the target proteins comprise Cbl-b and CIS. In some embodiments, the target proteins comprise Cbl-b and FAS. In some embodiments, the target proteins comprise Cbl-b and FASL. In some embodiments, the target proteins comprise Cbl-b and ICAM1. In some embodiments, the target proteins comprise Cbl-b and ICAM2. In some embodiments, the target proteins comprise Cbl-b and ICAM3. In some embodiments, the target proteins comprise Cbl-b and MED12. In some embodiments, the target proteins comprise Cbl-b and PTPN2. In some embodiments, the target proteins comprise Cbl-b and PRDM1. In some embodiments, the target proteins comprise CD58 and CIITA. In some embodiments, the target proteins comprise CD58 and CIS. In some embodiments, the target proteins comprise CD58 and FAS. In someembodiments, the target proteins comprise CD58 and FASL. In some embodiments, the target proteins comprise CD58 and ICAM1. In some embodiments, the target proteins comprise CD58 and ICAM2. In some embodiments, the target proteins comprise CD58 and ICAM3. In some embodiments, the target proteins comprise CD58 and MED12. In some embodiments, the target proteins comprise CD58 and PTPN2. In some embodiments, the target proteins comprise CD58 and PRDM1. In some embodiments, the target proteins comprise CIITA and CIS. In some embodiments, the target proteins comprise CIITA and FAS. In some embodiments, the target proteins comprise CIITA and FASL. In some embodiments, the target proteins comprise CIITA and ICAM1. In some embodiments, the target proteins comprise CIITA and ICAM2. In some embodiments, the target proteins comprise CIITA and ICAM3. In some embodiments, the target proteins comprise CIITA and MED 12. In some embodiments, the target proteins comprise CIITA and PTPN2. In some embodiments, the target proteins comprise CIITA and PRDM1. In some embodiments, the target proteins comprise CIS and FAS. In some embodiments, the target proteins comprise CIS and FASL. In some embodiments, the target proteins comprise CIS and ICAM1. In some embodiments, the target proteins comprise CIS and ICAM2. In some embodiments, the target proteins comprise CIS and ICAM3. In some embodiments, the target proteins comprise CIS and MED 12. In some embodiments, the target proteins comprise CIS and PTPN2. In some embodiments, the target proteins comprise CIS and PRDM1. In some embodiments, the target proteins comprise FAS and FASL. In some embodiments, the target proteins comprise FAS and ICAM1. In some embodiments, the target proteins comprise FAS and ICAM2. In some embodiments, the target proteins comprise FAS and ICAM3. In some embodiments, the target proteins comprise FAS and MED 12. In some embodiments, the target proteins comprise FAS and PTPN2. In some embodiments, the target proteins comprise FAS and PRDM1. In some embodiments, the target proteins comprise FASL and ICAM1. In some embodiments, the target proteins comprise FASL and ICAM2. In some embodiments, the target proteins comprise FASL and ICAM3. In some embodiments, the target proteins comprise FASL and MED12. In some embodiments, the target proteins comprise FASL and PTPN2. In some embodiments, the target proteins comprise FASL and PRDM1. In some embodiments, the target proteins comprise ICAM1 and ICAM2. In some embodiments, the target proteins comprise ICAM1 and ICAM3. In some embodiments, the target proteins comprise ICAM1 and MED12. In some embodiments, the target proteins comprise ICAM1 and PTPN2. In some embodiments, the target proteins comprise ICAM1 and PRDM1. In some embodiments, the target proteins comprise ICAM2 and ICAM3. In some embodiments, the target proteins comprise ICAM2 and MED 12. In some embodiments, the target proteins comprise ICAM2 and PTPN2. In some embodiments, the target proteins comprise ICAM2 and PRDM1. In some embodiments, the target proteins comprise ICAM3 and MED 12. In some embodiments, the target proteins comprise ICAM3 and PTPN2. In some embodiments, the target proteins comprise ICAM3 and PRDM1. In some embodiments, the target proteins comprise MED12 and PTPN2. In some embodiments, the target proteins comprise MED12 and PRDM1. In some embodiments, the target proteins comprise PTPN2 and PRDM1.

[0298] In some embodiments, the target proteins are ADAM 17 and B2M. In In some embodiments, the target proteins are ADAM 17 and Cbl-b. In some embodiments, the target proteins are ADAM17 and CD58. In some embodiments, the target proteins are ADAM17 and CIITA. In some embodiments, the target proteins are ADAM 17 and CIS. In some embodiments, the target proteins are ADAM 17 and FAS. In some embodiments, the target proteins are ADAM 17 and FASL. In some embodiments, the target proteins are ADAM17 and ICAM1. In some embodiments, the target proteins are ADAM 17 and ICAM2. In some embodiments, the target proteins are ADAM 17 and ICAM3. In some embodiments, the target proteins are ADAMI 7 and MED12. In some embodiments, the target proteins are ADAM17 and PTPN2. In some embodiments, the target proteins are ADAM17 and PRDM1. In some embodiments, the target proteins are B2M and Cbl-b. In some embodiments, the target proteins are B2M and CD58. In some embodiments, the target proteins are B2M and CIITA. In some embodiments, the target proteins are B2M and CIS. In some embodiments, the target proteins are B2M and FAS. In some embodiments, the target proteins are B2M and FASL. In some embodiments, the target proteins are B2M and ICAM1. In some embodiments, the target proteins are B2M and ICAM2. In some embodiments, the target proteins are B2M and ICAM3. In some embodiments, the target proteins are B2M and MED 12. In some embodiments, the target proteins are B2M and PTPN2. In some embodiments, the target proteins are B2M and PRDM1. In some embodiments, the target proteins are Cbl-b and CD58. In some embodiments, the target proteins are Cbl-b and CIITA. In some embodiments, the target proteins are Cbl-b and CIS. In some embodiments, the target proteins are Cbl- b and FAS. In some embodiments, the target proteins are Cbl-b and FASL. In some embodiments, the target proteins are Cbl-b and ICAM1. In some embodiments, the target proteins are Cbl-b and ICAM2. In some embodiments, the target proteins are Cbl-b and ICAM3. In some embodiments, the target proteins are Cbl-b and MED 12. In some embodiments, the target proteins are Cbl-b and PTPN2. In some embodiments, the target proteins are Cbl-b and PRDM 1. In some embodiments, the target proteins arc CD58 and CIITA. In some embodiments, the target proteins arc CD58 and CIS. In some embodiments, the target proteins are CD58 and FAS. In some embodiments, the target proteins are CD58 and FASL. In some embodiments, the target proteins are CD58 and ICAM1. In some embodiments, the target proteins are CD58 and ICAM2. In some embodiments, the target proteins are CD58 and ICAM3. In some embodiments, the target proteins are CD58 and MED12. In some embodiments, the target proteins are CD58 and PTPN2. In some embodiments, the target proteins are CD58 and PRDM1. In some embodiments, the target proteins are CIITA and CIS. In some embodiments, the target proteins are CIITA and FAS. In some embodiments, the target proteins are CIITA and FASL. In some embodiments, the target proteins are CIITA and ICAM1. In some embodiments, the target proteins are CIITA and ICAM2. In some embodiments, the target proteins are CIITA and ICAM3. In some embodiments, the target proteins are CIITA and MED12. In some embodiments, the target proteins are CIITA and PTPN2. In some embodiments, the target proteins are CIITA and PRDM1. In some embodiments, the target proteins are CIS and FAS. In some embodiments.the target proteins are CIS and FASL. In some embodiments, the target proteins are CIS and ICAM1. In some embodiments, the target proteins are CIS and ICAM2. In some embodiments, the target proteins are CIS and ICAM3. In some embodiments, the target proteins are CIS and MED12. In some embodiments, the target proteins are CIS and PTPN2. In some embodiments, the target proteins are CIS and PRDM1. In some embodiments, the target proteins are FAS and FASL. In some embodiments, the target proteins are FAS and ICAM1. In some embodiments, the target proteins are FAS and ICAM2. In some embodiments, the target proteins are FAS and ICAM3. In some embodiments, the target proteins are FAS and MED 12. In some embodiments, the target proteins are FAS and PTPN2. In some embodiments, the target proteins are FAS and PRDM1. In some embodiments, the target proteins are FASL and ICAM1. In some embodiments, the target proteins are FASL and ICAM2. In some embodiments, the target proteins are FASL and ICAM3. In some embodiments, the target proteins are FASL and MED 12. In some embodiments, the target proteins are FASL and PTPN2. In some embodiments, the target proteins are FASL and PRDM1. In some embodiments, the target proteins are ICAM1 and ICAM2. In some embodiments, the target proteins are ICAM1 and ICAM3. In some embodiments, the target proteins are ICAM1 and MED12. In some embodiments, the target proteins are ICAM1 and PTPN2. In some embodiments, the target proteins are ICAM1 and PRDM1. In some embodiments, the target proteins are ICAM2 and ICAM3. In some embodiments, the target proteins are ICAM2 and MED 12. In some embodiments, the target proteins are ICAM2 and PTPN2. In some embodiments, the target proteins are ICAM2 and PRDM1. In some embodiments, the target proteins are ICAM3 and MED12. In some embodiments, the target proteins are ICAM3 and PTPN2. In some embodiments, the target proteins are ICAM3 and PRDM1. In some embodiments, the target proteins are MED 12 and PTPN2. In some embodiments, the target proteins are MED 12 and PRDM1. In some embodiments, the target proteins are PTPN2 and PRDM1.

[0299] By way of non-limiting example, in several embodiments, a disintegrin and mctalloprotcasc domain (ADAM) family member, in particular embodiments ADAM 17, is a target of gene modulation. ADAM 17, located on chromosome 2, is implicated in antibody -dependent cell- mediated cytotoxicity (ADCC), which is a key mechanism of action in anti-tumor responses. CD16A is a membrane-bound protein expressed by NK cells and a receptor for the Fc portion of IgGs. While engagement of CD16A (e.g., by antibody -coated target cells) triggers NK cell-mediated ADCC, CD16A is rapidly downregulated after NK cell activation by cleavage from the NK cell surface (either in vivo or in vitro, for example by PMA). ADAM 17 is believed to be the primary protease responsible for cleavage of CD16A from the NK cell surface; inhibition of ADAM17 (such as by disruption in ADAM 17 expression) reduces, ameliorates, or otherwise inhibits the cleavage of CD 16 A, which allows ADCC to continue as an operative anti-tumor pathway (Wu et al., J Leukoc Biol (2019) 105(6): 1297- 1303). Moreover. CD62 ligand (CD62L) is a substrate of ADAM 17, and the disruption of expression of ADAM 17 functions, in several embodiments, to stabilize CD62L expression. CD62L, an L-se lectin molecule mediates homing of leukocytes to lymphoid organs. CD56dimCD62L+ cells represent aunique subset of mature, polyfunctional NK cells that affect the magnitude of the local NK cell response, in particular, by the ability to produce IFN-y after cytokine stimulation, proliferate in vivo during viral infection, and kill target cells upon engagement of activating receptors. Thus, stabilizing CD62L may, in several embodiments, further enhance NK cell function.

[0300] In several embodiments, gene modulation reduces transcription of ADAMI 7 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%. or more (including any amount between those listed). In several embodiments, gene modulation reduces transcription of ADAMI 7 by at least about 30%, In several embodiments, gene modulation reduces transcription of ADAM17 by at least about 40%, In several embodiments, gene modulation reduces transcription of ADAM 17 by at least about50%, In several embodiments, gene modulation reduces transcription of ADAM17 by at least about60%, In several embodiments, gene modulation reduces transcription of ADAM 17 by at least about70%, In several embodiments, gene modulation reduces transcription of ADAM 17 by at least about80%, In several embodiments, gene modulation reduces transcription of ADAM 17 by at least about90%.

[0301] In several embodiments, gene modulation reduces expression of a 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%, or more (including any amount between those listed). In several embodiments, gene modulation reduces expression of ADAM17 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%. or more (including any amount between those listed). In several embodiments, gene modulation reduces expression of ADAM 17 protein by at least about 30%, In several embodiments, gene modulation reduces expression of ADAM 17 protein by at least about 40%, In several embodiments, gene modulation reduces expression of ADAM 17 protein by at least about 50%, In several embodiments, gene modulation reduces expression of ADAM 17 protein by at least about 60%, In several embodiments, gene modulation reduces expression of ADAM 17 protein by at least about 70%, In several embodiments, gene modulation reduces expression of ADAMI 7 protein by at least about 80%, In several embodiments, gene modulation reduces expression of ADAM 17 protein by at least about 90%.

[0302] In several embodiments, ADAM 17 expression is disrupted and / or knocked out using a Crispr-Cas mediated approach (e.g., Cas9). or other guided nuclease as disclosed elsewhere herein, with the use of one more of ADAM17-specific guide RNAs. In several embodiments, a guide RNA (gRNA) comprising the sequence of any of SEQ ID NOS: 127-132, or 216 is used to disrupt (e.g., reduce expression ol) the ADAM17 gene.

[0303] In several embodiments, expression of AD AM 17 protein is reduced by an inhibitory nucleic acid molecule, such as one that is complementary to. targets, inhibits and / or binds a ADAM 17 protein-encoding nucleic acid or a portion thereof. In some of any such embodiments, theinhibitory nucleic acid molecule includes an RNA interfering agent. In some of any such embodiments, the inhibitory nucleic acid is or contains or encodes a small interfering RNA (siRNA), a microRNA- adapted shRNA, a short hairpin RNA (shRNA), a hairpin siRNA, a precursor microRNA (pre-miRNA) or a microRNA (miRNA). In some of any such embodiments, the inhibitory nucleic acid molecule contains a sequence complementary to the ADAM 17 protein-encoding nucleic acid. In some of any such embodiments, the inhibitory' nucleic acid molecule contains an antisense oligonucleotide complementary to ADAM 17 protein-encoding nucleic acid.

[0304] In several embodiments, an inhibitory nucleic acid molecule (e.g., an RNA interfering agent) reduces expression of ADAM 17 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%. or more (including any amount between those listed). In several embodiments, the inhibitory nucleic acid molecule reduces expression of ADAM 17 protein by at least about 30%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of ADAM 17 protein by at least about 40%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of ADAM 17 protein by at least about 50%. In several embodiments, the inhibitory' nucleic acid molecule reduces expression of ADAMI 7 protein by at least about 60%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of ADAMI 7 protein by at least about 70%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of ADAM 17 protein by at least about 80%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of ADAM 17 protein by at least about 90%. In several embodiments, the inhibitory nucleic acid molecule eliminates expression of ADAM17 protein, such that it is undetectable.

[0305] In some embodiments, the expression of ADAM 17 protein is reduced by more than one inhibitory' nucleic acid molecule, such as ones that are complementary to, target, inhibit and / or bind a ADAM 17 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind the same region of the ADAM 17 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind different regions of the ADAM 17 protein-encoding nucleic acid or a portion thereof.

[0306] In several embodiments, an inhibitory nucleic acid molecule (e.g., air RNA interfering agent) reduces transcription of ADAM17 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%, or more (including any amount between those listed). In several embodiments, the inhibitory nucleic acid molecule reduces transcription of ADAMI 7 by at least about 30%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of ADAM 17 by at least about 40%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of AD AM 17 by at least about 50%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of ADAM 17 by at least about 60%. In several embodiments, the inhibitory nucleic acid molecule reducestranscription of ADAM17 by at least about 70%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of ADAM 17 by at least about 80%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of ADAMI 7 by at least about 90%. In several embodiments, the inhibitory nucleic acid molecule eliminates transcription of ADAM 17, such that it is undetectable.

[0307] In some embodiments, the transcription of ADAM 17 is reduced by more than one inhibitory nucleic acid molecule, such as ones that are complementary to, target, inhibit and / or bind a ADAM 17 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind the same region of the ADAM 17 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind different regions of the ADAM 17 protein-encoding nucleic acid or a portion thereof.

[0308] Cells require many different types of molecular complexes to achieve the cellular processes of transcription and translation. These complexes, made of multiple, sometimes differing, subunits have the capacity to impart cell-specific functions, depending on their assembly and activity. One such molecular complex is the Mediator complex, which is expressed and required in cells where genes are actively being expressed, such as immune cells, like NK cells. It primarily functions as a “molecular bridge” that anchors two regions of otherwise unconnected DNA within the cell. For example, it can link a promotor and an enhancer, to physically localize the various elements and associated transcription factors required for expression of genes transcribed by RNA polymerases. Mediator complex subunit 12 (MED 12) is one part of the four-part cyclin dependent kinase (CDK) module of Mediator along with MED13, cyclin-dependent kinase 8 (CDK8) and cyclin C (CCNC). Mutations in MED12 have been associated with lymphoproliferative disorders (Kainpjarvi et al., Oncotarget (2015) 6(3): 1884-88). More recently, targeted deletion of MED12, CCNC, or CDK8 in human CAR T cells was observed to increase proliferation, cytokine production, and antitumor activity. In particular, MED 12 deficient T cells exhibited changes at genes regulating effector T cell differentiation. See Freitas et al., Cancer Res (2022) 82(12_suppl):2822; and Freitas et al., Science (2022) 378(6620):eabn5647. It is contemplated that reductions in MED12, MED13, CDK8 and / or CCNC will provide edited cells, such as NK cells, with an enhanced persistence, allowing (when engineered in accordance with embodiments provided for herein) enhanced cytotoxicity against target tumor cells.

[0309] In several embodiments, gene modulation reduces transcription of MED 12 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%, or more (including any amount between those listed), In several embodiments, gene modulation reduces transcription of MED 12 by at least about 30%, In several embodiments, gene modulation reduces transcription of MED 12 by at least about 40%, In several embodiments. gene modulation reduces transcription of MED 12 by at least about 50%, Inseveral embodiments, gene modulation reduces transcription of MED 12 by at least about 60%, In several embodiments, gene modulation reduces transcription of MED12 by at least about 70%, In several embodiments, gene modulation reduces transcription of MED 12 by at least about 80%, In several embodiments, gene modulation reduces transcription of MED12 by at least about 90%.

[0310] In several embodiments, gene modulation can reduce expression of a 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%. or more (including any amount between those listed). In several embodiments, gene modulation reduces expression of MED12 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%, or more (including any amount between those listed). In several embodiments, gene modulation reduces expression of MED 12 protein by at least about 30%, In several embodiments, gene modulation reduces expression of MED 12 protein by at least about 40%, In several embodiments, gene modulation reduces expression of MED 12 protein by at least about 50%, In several embodiments, gene modulation reduces expression of MED 12 protein by at least about 60%, In several embodiments, gene modulation reduces expression of MED12 protein by at least about 70%, In several embodiments, gene modulation reduces expression of MED 12 protein by at least about 80%, In several embodiments, gene modulation reduces expression of MED12 protein by at least about 90%.

[0311] In several embodiments, MED 12 expression is disrupted and / or knocked out using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nuclease as disclosed elsewhere herein, with the use of one more of the following MED12-specific guide RNAs: SEQ ID NOS 133-142 (see e.g., Table 2). In several embodiments, a guide RNA (gRNA) comprising the sequence of any of SEQ ID NOS:133-142 is used to disrupt (e.g., reduce expression of) the MED12 gene. Non-limiting examples of guide RNAs to reduce and / or eliminate MED12 expression are provided below in Table 2.Table 2: MED 12 Guide RNAs

[0312] In several embodiments, expression of MED 12 protein is reduced by an inhibitory nucleic acid molecule, such as one that is complementary to, targets, inhibits and / or binds a MED 12 protein-encoding nucleic acid or a portion thereof. In some of any such embodiments, the inhibitory nucleic acid molecule includes an RNA interfering agent. In some of any such embodiments, the inhibitor}’ nucleic acid is or contains or encodes a small interfering RNA (siRNA), a microRNA-adapted shRNA, a short hairpin RNA (shRNA), a hairpin siRNA, a precursor microRNA (pre-miRNA) or amicroRNA (miRNA). In some of any such embodiments, the inhibitory nucleic acid molecule contains a sequence complementary to the MED 12 protein-encoding nucleic acid. In some of any such embodiments, the inhibitory nucleic acid molecule contains an antisense oligonucleotide complementary to MED 12 protein-encoding nucleic acid.

[0313] In several embodiments, an inhibitory nucleic acid molecule (e.g., an RNA interfering agent) reduces expression of MED12 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%. or more (including any amount between those listed). In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 12 protein by at least about 30%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 12 protein by at least about 40%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 12 protein by at least about 50%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 12 protein by at least about 60%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 12 protein by at least about 70%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 12 protein by at least about 80%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 12 protein by at least about 90%. In several embodiments, the inhibitory nucleic acid molecule eliminates expression of MED 12 protein, such that it is undetectable.

[0314] In some embodiments, the expression of MED 12 protein is reduced by more than one inhibitory nucleic acid molecule, such as ones that are complementary to, target, inhibit and / or bind a MED 12 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory’ nucleic acid molecule is complementary to, target, inhibit and / or bind the same region of the MED 12 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind different regions of the MED 12 protcin-cncoding nucleic acid or a portion thereof

[0315] In several embodiments, an inhibitory nucleic acid molecule (e.g., an RNA interfering agent) reduces transcription of MED 12 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%, or more (including any amount between those listed). In several embodiments, the inhibitory nucleic acid molecule reduces transcription of MED12 by at least about 30%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of MED 12 by at least about 40%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of MED 12 by at least about 50%. In several embodiments, the inhibitory' nucleic acid molecule reduces transcription of MED 12 by at least about 60%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of MED 12 by at least about 70%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of MED 12 by at least about 80%. In several embodiments, the inhibitory nucleic acidmolecule reduces transcription of MED12 by at least about 90%. In several embodiments, the inhibitory nucleic acid molecule eliminates transcription of MED12, such that it is undetectable.

[0316] In some embodiments, the transcription of MED 12 is reduced by more than one inhibitory nucleic acid molecule, such as ones that are complementary to, target, inhibit and / or bind a MED 12 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind the same region of the MED 12 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind different regions of the MED 12 protein-encoding nucleic acid or a portion thereof.

[0317] In several embodiments, gene modulation reduces transcription of MED 13 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%. or more (including any amount between those listed). In several embodiments, gene modulation reduces transcription of MED 13 by at least about 30%. In several embodiments, gene modulation reduces transcription of MED 13 by at least about 40%, In several embodiments. gene modulation reduces transcription of MED 13 by at least about 50%. In several embodiments, gene modulation reduces transcription of MED 13 by at least about 60%. In several embodiments, gene modulation reduces transcription of MED 13 by at least about 70%. In several embodiments, gene modulation reduces transcription of MED 13 by at least about 80%, In several embodiments, gene modulation reduces transcription of MED 13 by at least about 90%.

[0318] In several embodiments, gene modulation reduces expression of a target protein by about 30%. about 40%, about 50%, about 60%, about 70%, about 75%. about 80%, about 85%, about90%. about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed). In several embodiments, gene modulation reduces expression of MED13 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%, or more (including any amount between those listed). In several embodiments, gene modulation reduces expression of MED 13 protein by at least about 30%, In several embodiments, gene modulation reduces expression of MED 13 protein by at least about 40%, In several embodiments, gene modulation reduces expression of MED 13 protein by at least about 50%, In several embodiments, gene modulation reduces expression of MED 13 protein by at least about 60%, In several embodiments, gene modulation reduces expression of MED13 protein by at least about 70%, In several embodiments, gene modulation reduces expression of MED 13 protein by at least about 80%, In several embodiments, gene modulation reduces expression of MED 13 protein by at least about 90%.

[0319] In several embodiments, MED13 expression is disrupted and / or knocked out using a Crispr-Cas mediated approach (e.g., Cas9). or other guided nuclease as disclosed elsewhere herein, with the use of one more of the following MED 13 -specific guide RNAs: SEQ ID NOS: 143-146. In several embodiments, a guide RNA (gRNA) comprising the sequence of any of SEQ ID NOS: 143-146 is used to disrupt (e.g.. reduce expression of) the MED 13 gene.

[0320] In several embodiments, expression of MED 13 protein is reduced by an inhibitory nucleic acid molecule, such as one that is complementary to, targets, inhibits and / or binds a MED13 protein-encoding nucleic acid or a portion thereof. In some of any such embodiments, the inhibitory nucleic acid molecule includes an RNA interfering agent. In some of any such embodiments, the inhibitory nucleic acid is or contains or encodes a small interfering RNA (siRNA), a microRNA-adapted shRNA, a short hairpin RNA (shRNA), a hairpin siRNA, a precursor microRNA (pre-miRNA) or a microRNA (miRNA). In some of any such embodiments, the inhibitory nucleic acid molecule contains a sequence complementary to the MED 13 protein-encoding nucleic acid. In some of any such embodiments, the inhibitory nucleic acid molecule contains an antisense oligonucleotide complementary to MED 13 protein-encoding nucleic acid.

[0321] In several embodiments, an inhibitory nucleic acid molecule (e.g., an RNA interfering agent) reduces expression of MED13 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%. or more (including any amount between those listed). In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 13 protein by at least about 30%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 13 protein by at least about 40%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 13 protein by at least about 50%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 13 protein by at least about 60%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 13 protein by at least about 70%. In several embodiments, the inhibitory nucleic acid molecule reduces expression of MED 13 protein by at least about 80%. In several embodiments, the inhibitory' nucleic acid molecule reduces expression of MED13 protein by at least about 90%. In several embodiments, the inhibitory nucleic acid molecule eliminates expression of MED13 protein, such that it is undetectable.

[0322] In some embodiments, the expression of MED 13 protein is reduced by more than one inhibitory nucleic acid molecule, such as ones that are complementary to, target, inhibit and / or bind a MED 13 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind the same region of the MED 13 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind different regions of the MED 13 protein-encoding nucleic acid or a portion thereof.

[0323] In several embodiments, an inhibitory nucleic acid molecule (e.g., an RNA interfering agent) reduces transcription of MED 13 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%, or more (including any amount between those listed). In several embodiments, the inhibitory nucleic acid molecule reduces transcription of MED13 by at least about 30%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of MED 13 by at least about 40%. In severalembodiments, the inhibitory nucleic acid molecule reduces transcription of MED 13 by at least about 50%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of MED 13 by at least about 60%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of MED13 by at least about 70%. In several embodiments, the inhibitory' nucleic acid molecule reduces transcription of MED13 by at least about 80%. In several embodiments, the inhibitory nucleic acid molecule reduces transcription of MED 13 by at least about 90%. In several embodiments, the inhibitory nucleic acid molecule eliminates transcription of MED13, such that it is undetectable.

[0324] In some embodiments, the transcription of MED 13 is reduced by more than one inhibitory nucleic acid molecule, such as ones that are complementary to, target, inhibit and / or bind a MED 13 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind the same region of the MED 13 protein-encoding nucleic acid or a portion thereof. In some embodiments, more than one inhibitory nucleic acid molecule is complementary to, target, inhibit and / or bind different regions of the MED 13 protein-encoding nucleic acid or a portion thereof.

[0325] In several embodiments, gene modulation reduces transcription of CDK8 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%, or more (including any amount between those listed). In several embodiments, gene modulation reduces transcription of CDK8 by at least about 30%, In several embodiments, gene modulation reduces transcription of CDK8 by at least about 40%. In several embodiments, gene modulation reduces transcription of CDK8 by at least about 50%. In several embodiments, gene modulation reduces transcription of CDK8 by at least about 60%, In several embodiments, gene modulation reduces transcription of CDK8 by at least about 70%, In several embodiments, gene modulation reduces transcription of CDK8 by at least about 80%, In several embodiments, gene modulation reduces transcription of CDK8 by at least about 90%.

[0326] In several embodiments, gene modulation reduces expression of a target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%. about 97%, about 98%, about 99%, or more (including any amount between those listed). In several embodiments, gene modulation reduces expression of CDK8 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%, or more (including any amount between those listed). In several embodiments, gene modulation reduces expression of CDK8 protein by at least about 30%, In several embodiments, gene modulation reduces expression of CDK8 protein by at least about 40%, In several embodiments, gene modulation reduces expression of CDK8 protein by at least about 50%, In several embodiments, gene modulation reduces expression of CDK8 protein by at least about 60%, In several embodiments, gene modulation reduces expression of CDK8 protein by at least about 70%, In several embodiments, gene modulation reduces expression of CDK8 protein by at least about 80%, In several embodiments, gene modulation reduces expression of CDK8 protein by at least about 90%.

[0327] In several embodiments, CDK8 expression is disrupted and / or knocked out using a Crispr-Cas mediated approach (e.g., Cas9), or other guided nuclease as disclosed elsewhere herein, with the use of one more of the following CDK8-specific guide RNAs: SEQ ID NOS: 147-153 (see e.g., Table 3). In several embodiments, a guide RNA (gRNA) comprising the sequence of any of SEQ ID NOS: 147- 153 is used to disrupt (e.g., reduce expression of) the CDK8 gene. Non-limiting examples of guide RNAs to reduce and / or eliminate CDK8 expression are provided below in Table 3.Table 3: CDK8 Guide RNAs

[0328] In several embodiments, expression of CDK8 protein is reduced by an inhibitory nucleic acid molecule, such as one that is complementary to, targets, inhibits and / or binds a CDK8 protein-encoding nucleic acid or a portion thereof. In some of any such embodiments, the inhibitory nucleic acid molecule includes an RNA interfering agent. In some of any such embodiments, the inhibitory nucleic acid is or contains or encodes a small interfering RNA (siRNA), a microRNA-adapted shRNA, a short hairpin RNA (shRNA), a hairpin siRNA, a precursor microRNA (pre-miRNA) or a microRNA (miRNA). In some of any such embodiments, the inhibitory nucleic acid molecule contains a sequence complementary to the CDK8 protein-encoding nucleic acid. In some of any such embodiments, the inhibitory nucleic acid molecule contains an antisense oligonucle...

Claims

WHAT IS CLAIMED IS:

1. Ail immune cell having inhibited expression of at least two target proteins selected from the group consisting of ICAM1. CD58, ADAM 17, B2M, CIS, Cbl-b, FAS, FASL, ICAM2, ICAM3, MED 12, CUT A, PRDM1 and PTPN2.

2. The immune cell of claim 1, wherein the immune cell comprises inhibited expression of two target proteins selected from the group consisting of ICAM1. CD58, ADAM17, B2M, CIS. Cbl- b, FAS, FASL. ICAM2, ICAM3, MED12. CIITA, PRDM1 and PTPN2.

3. An immune cell having inhibited expression of at least three target proteins selected from the group consisting of ICAM1, ICAM2, CD58, ADAM17, B2M, CIS, Cbl-b, FAS, FASL, ICAM3. MED12, CIITA. PRDM1 and PTPN2.

4. The immune cell of claim 3, wherein the immune cell comprises inhibited expression of three target proteins selected from the group consisting of ICAM1, ICAM2, CD58, ADAMI 7, B2M, CIS, Cbl-b, FAS, FASL, ICAM3. MED12, CIITA, PRDM1 and PTPN2.

5. An immune cell having inhibited expression of at least four target proteins selected from the group consisting of ICAM1, ICAM2, CD58, FAS, ADAM17, B2M, CIS, Cbl-b, FASL, ICAM3, MED 12, CIITA, PRDM1 and PTPN2.

6. The immune cell of claim 5, wherein the immune cell comprises inhibited expression of four target proteins selected from the group consisting of ICAM1, ICAM2. CD58, FAS, ADAMI 7, B2M, CIS, Cbl-b, FASL, ICAM3, MED12, CIITA, PRDM1 and PTPN2.

7. An immune cell having inhibited expression of at least two target proteins selected from the group consisting of ICAM1, CD58, ADAM 17, B2M, CIS. Cbl-b, FAS, FASL, ICAM2, ICAM3, MED12, CIITA, PRDM1 and PTPN2, wherein the immune cell comprises a genomic disruption within a target sequence of the gene encoding each of the at least two target proteins.

8. The immune cell of claim 7. wherein the immune cell comprises (a) inhibited expression of two target proteins selected from the group consisting ofICAML CD58, ADAM17, B2M, CIS, Cbl-b, FAS, FASL. ICAM2, ICAM3, MED 12. CIITA, PRDM1 and PTPN2; and (b) the immune cell comprises a genomic disruption with a target sequence of the gene encoding each of the tw o target proteins.

9. An immune cell having inhibited expression of at least three target proteins selected from the group consisting of ICAM1, ICAM2. CD58, ADAM17, B2M, CIS, Cbl-b, FAS, FASL, ICAM3. MED12, CIITA, PRDM1 and PTPN2. wherein the immune cell comprises a genomic disruption w ithin a target sequence of the gene encoding each of the at least three target proteins.

10. The immune cell of claim 9, wherein the immune cell comprises (a) inhibited expression of three target proteins selected from the group consisting of ICAM1, ICAM2, CD58, ADAM17. B2M, CIS, Cbl-b, FAS, FASL, ICAM3, MED12, CIITA, PRDM1 and PTPN2; and (b) theimmune cell comprises a genomic disruption with a target sequence of the gene encoding each of the three target proteins.

11. An immune cell having inhibited expression of at least four target proteins selected from the group consisting of ICAM1, ICAM2, CD58. FAS. ADAMI 7, B2M, CIS, Cbl-b, FASL, ICAM3, MED12, CIITA, PRDM1 and PTPN2, wherein the immune cell comprises a genomic disruption within a target sequence of the gene encoding each of the at least four target proteins.

12. The immune cell of claim 11, wherein the immune cell comprises (a) inhibited expression of four target proteins selected from the group consisting of ICAM1, ICAM2, CD58. FAS, ADAM17, B2M, CIS, Cbl-b, FASL, ICAM3. MED12, CIITA, PRDM1 and PTPN2; and (b) the immune cell comprises a genomic disruption with a target sequence of the gene encoding each of the four target proteins.

13. An immune cell comprising at least two synthetic shRNA sequences, wherein the synthetic shRNA sequences are expressed in the immune cell and the synthetic shRNA sequences reduce expression of at least two target proteins selected from the group consisting of ICAM1, CD58, ADAM 17. B2M. CIS, Cbl-b, FAS, FASL. ICAM2, ICAM3, MED 12. CIITA, PRDM1 and PTPN2.

14. The immune cell of claim 13, wherein the synthetic shRNA sequences expressed in the immune cell reduce expression of two target proteins in the immune cell.

15. The immune cell of claim 13, wherein the synthetic shRNA sequences expressed in the immune cell reduce expression of (a) ICAM1 and CD58; (b) MED12 and PTPN2; or (c) MED12 and FAS.

16. An immune cell expressing at least three synthetic shRNA sequences, wherein die synthetic shRNA sequences are expressed in the immime cell and the synthetic shRNA sequences reduce expression ofat least three target proteins selected from the group consisting ofICAMI, ICAM2, CD58, ADAM17, B2M, CIS. Cbl-b, FAS, FASL, ICAM3, MED12, CIITA, PRDM1 and PTPN2.

17. The immune cell of claim 16, wherein the synthetic shRNA sequences expressed in the immune cell reduce expression of three target proteins in the immune cell.

18. An immune cell expressing at least four synthetic shRNA sequences, wherein the synthetic shRNA sequences are expressed in the immune cell and the synthetic shRNA sequences reduce expression of at least four target proteins selected from the group consisting ofICAMI, ICAM2, CD58, FAS. ADAMI 7, B2M, CIS, Cbl-b, FASL, ICAM3, MED12, CIITA, PRDM1 and PTPN2.

19. The immune cell of claim 18, wherein the synthetic shRNA sequences expressed in the immune cell reduce expression of four target proteins in the immune cell.

20. The immune cell of claim 18. wherein the target proteins comprise ADAM17.

21. The immune cell of claim 18. wherein the target proteins comprise Cbl-b.

22. The immune cell of claim 18. wherein the target proteins comprise CIS.

23. The immune cell of claim 18. wherein the target proteins comprise MED 12.

24. The immune cell of claim 18. wherein the target proteins comprise PRDM1.

25. The immune cell of claim 18, wherein the target proteins comprise PTPN2.

26. The immune cell of claim 18, wherein the target proteins comprise MED 12 andPRDM1.

27. The immune cell of claim 18, wherein the target proteins comprise MED 12 andPTPN2.

28. The immune cell of claim 18, wherein the target proteins comprise ICAM1 andICAM2.

29. The immune cell of claim 18, wherein the target proteins comprise ICAM1 and CD58.

30. The immune cell of claim 18, wherein the target proteins comprise ICAM2 and CD58.

31. The immune cell of claim 18, wherein the target proteins comprise ICAM1, ICAM2, and CD58.

32. The immune cell of any one of claims 1-31. wherein the immune cell expresses a chimeric receptor.

33. The immune cell of any one of claims 1-32, wherein the immune cell expresses a membrane -bound interleukin- 15 (mbIL15).

34. The immune cell of claim 32 or claim 33, wherein the synthetic shRNA sequences and a nucleic acid sequence encoding the chimeric receptor are comprised within the same polynucleotide.

35. The immune cell of any one of claims 32-34, wherein the synthetic shRNA sequences and the chimeric receptor are expressed bicistronically.

36. The immune cell of any one of claims 33-35, wherein the nucleic acid sequence encoding the chimeric receptor and a nucleic acid sequence encoding the mbIL15 are comprised within the same polynucleotide.

37. The immune cell of any one of claims 33-36, wherein the nucleic acid sequences encoding the chimeric receptor and die mbIL15 are expressed bicistronically.

38. An immune cell expressing a polynucleotide, wherein the polynucleotide comprises:(a) a nucleic acid sequence encoding a chimeric receptor; and(b) at least two synthetic shRNA sequences, wherein the synthetic shRNA sequences are expressed in the immune cell and the synthetic shRNA sequences reduce expression of at least two target proteins selected from the group consisting of ICAM1, CD58, ADAM17, B2M, CIS, Cbl-b, FAS, FASL, ICAM2, ICAM3, MED12. CIITA, PRDM1 and PTPN2.

39. The immune cell of claim 36, the polynucleotide further comprising a nucleic acid sequence encoding a membrane-bound interleukin- 15 (mbIL15).

40. An immune cell expressing a polynucleotide, wherein the polynucleotide comprises:(a) a nucleic acid sequence encoding a chimeric receptor;(b) at least two synthetic shRNA sequences, wherein the synthetic shRNA sequences are expressed in the immune cell and the synthetic shRNA sequences reduce expression of at least twotarget proteins selected from the group consisting of ICAM1, CD58, ADAM17, B2M, CIS, Cbl-b, FAS, FASLICAM2, ICAM3, MED12, CIITA, PRDM1 and PTPN2; and(c) a membrane-bound interleukin- 15 (mbIL15).

41. The immune cell of any one of claims 38-40, wherein the at least two synthetic shRNA sequences are expressed in the immune cell and the synthetic shRNA sequences reduce expression of at least two, at least three, or at least four target proteins.

42. The immune cell of any one of claims 32-41. wherein the chimeric receptor is a chimeric antigen receptor (CAR).

43. The immune cell of any one of claims 13-42, wherein each of the synthetic shRNA sequences is a micro RNA-based shRNA (shRNA miR).

44. The immune cell of any one of claims 13-43, wherein all of the shRNA sequences are comprised within a single polynucleotide.

45. The immune cell of claim 42 or claim 43, wherein each of the synthetic shRNA sequences is a shRNA miR comprised within a single polynucleotide.

46. A method of inhibiting expression of at least two target proteins in an immune cell, the method comprising introducing into the immune cell at least two synthetic shRNA sequences, wherein:(i) the synthetic shRNA sequences are expressed in the immune cell and reduce expression of at least two target proteins in the immune cell; and(ii) the at least two target proteins are selected from the group consisting of ICAM1, CD58, ADAM17. B2M, CIS, Cbl-b, FAS, FASL. ICAM2, ICAM3, MED12. CIITA, PRDM1 and PTPN2.

47. The method of claim 46. wherein the synthetic shRNA sequences expressed in the immune cell reduce expression of two target proteins in the immune cell.

48. A method of inhibiting expression of at least three target proteins in an immune cell, the method comprising introducing into the immune cell at least three synthetic shRNA sequences, wherein:(i) the synthetic shRNA sequences are expressed in the immune cell and reduce expression of at least three target proteins in the immune cell; and(ii) the at least three target proteins are selected from the group consisting of ICAM1, ICAM2, CD58, ADAM17, B2M, CIS, Cbl-b, FAS, FASL, ICAM3, MED12, CIITA, PRDM1 and PTPN2.

49. The method of claim 48, wherein the synthetic shRNA sequences expressed in the immune cell reduce expression of three target proteins in the immune cell.

50. A method of inhibiting expression of at least four target proteins in an immune cell, the method comprising introducing into the immune cell at least four synthetic shRNA sequences, wherein:(i) the synthetic shRNA sequences are expressed in the immune cell and reduce expression of at least four target proteins in the immune cell; and(ii) the at least four target proteins are selected from the group consisting of ICAM1, ICAM2, CD58, FAS. ADAM17, B2M, CIS, Cbl-b, FASL, ICAM3, MED12, CIITA, PRDM1 and PTPN2.

51. The method of claim 50, wherein the synthetic shRNA sequences expressed in the immune cell reduce expression of four target proteins in the immune cell.

52. The method of claim 46, wherein the target proteins comprise ADAMI 7.

53. The method of claim 46, wherein the target proteins comprise Cbl-b.

54. The method of claim 46, wherein the target proteins comprise CIS.

55. The method of claim 46, wherein the target proteins comprise MED 12.

56. The method of claim 46, wherein the target proteins comprise PRDM1.

57. The method of claim 46, wherein the target proteins comprise PTPN2.

58. The method of claim 46, wherein the target proteins comprise MED12 and PRDM1.

59. The method of claim 46, wherein the target proteins comprise MED12 and PTPN2.

60. The method of claim 46, wherein the target proteins comprise ICAM1 and ICAM2.

61. The method of claim 46, wherein the target proteins comprise ICAM1 and CD58.

62. The method of claim 46, wherein the target proteins comprise ICAM2 and CD58.

63. The method of claim 46, wherein the target proteins comprise ICAM1. ICAM2, and CD58.

64. The method of any one of claims 46-63, wherein each of the synthetic shRNA sequences is a micro RNA-based shRNA (shRNA miR).

65. The method of any one of claims 46-64, wherein all of the synthetic shRNA sequences are comprised within a single polynucleotide.

66. The method of claim 64 or claim 65, wherein each of the synthetic shRNA sequences is a shRNA miR comprised with a single polynucleotide.

67. The method of claim 66, wherein: (a) the immune cell expresses a chimeric receptor; and / or (b) the single polynucleotide further encodes a chimeric receptor.

68. The immune cell of any one of claims 1-45 or the method of any one of claims 46-67, wherein the immune cell is a natural killer (NK) cell, a T cell or combination of NK and T cells.

69. The immune cell of any one of claims 1-45 or the method of any one of claims 46-67, wherein the immune cell is a natural killer (NK) cell.

70. A method of inhibiting expression of the FAS gene in a natural killer (NK) cell, the method comprising administering to the NK cell:(i) a CRISPR / Cas system comprising: (a) a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the FAS gene; and (b) a Cas molecule; or(ii) a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary- with a target sequence of the FAS gene; and (b) a second nucleotide sequence encoding a Cas molecule.

71. The method of claim 70, wherein the target sequence of the FAS gene comprises SEQ ID NO: 201-204 or 215.

72. A method of inhibiting expression of the PRDM1 gene in a natural killer (NK) cell, the method comprising administering to the NK cell:(i) a CRISPR / Cas system comprising: (a) a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the PRDM1 gene: and (b) a Cas molecule; or(ii) a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the PRDM1 gene; and (b) a second nucleotide sequence encoding a Cas molecule.

73. The method of claim 72. wherein the target sequence of the PRDM1 gene comprises SEQ ID NO: 197-200.

74. A method of inhibiting expression of the PTPN2 gene in a natural killer (NK) cell, the method comprising administering to the NK cell:(i) a CRISPR / Cas system comprising: (a) a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the PTPN2 gene; and (b) a Cas molecule; or(ii) a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the PTPN2 gene; and (b) a second nucleotide sequence encoding a Cas molecule.

75. The method of claim 74, wherein the target sequence of the PTPN2 gene comprises SEQ ID NQ:208-211.

76. A method of inhibiting expression of the ICAM1 gene in a natural killer (NK) cell, the method comprising administering to the NK cell:(i) a CRISPR / Cas system comprising: (a) a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the ICAM1 gene; and (b) a Cas molecule; or(ii) a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the ICAM1 gene; and (b) a second nucleotide sequence encoding a Cas molecule77. The method of claim 76, wherein the target sequence of the ICAM1 gene comprises SEQ ID NO:218.

78. A method of inhibiting expression of the ICAM2 gene in a natural killer (NK) cell, the method comprising administering to the NK cell:(i) a CRISPR / Cas system comprising: (a) a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the ICAM2 gene; and (b) a Cas molecule; or(ii) a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the ICAM2 gene; and (b) a second nucleotide sequence encoding a Cas molecule.

79. The method of claim 78, wherein tire target sequence of the ICAM2 gene comprises SEQ ID NO:219-221.

80. A method of inhibiting expression of the CD58 gene in a natural killer (NK) cell, the method comprising administering to the NK cell:(i) a CRISPR / Cas system comprising: (a) a guide RNA (gRNA) molecule comprising a targeting domain that is complementary with a target sequence of the CD58 gene; and (b) a Cas molecule; or(ii) a nucleic acid composition comprising: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of the CD58 gene; and (b) a second nucleotide sequence encoding a Cas molecule.

81. The method of claim 80, wherein the target sequence of the CD58 gene comprises SEQ ID NO:217.

82. The method of any one of claims 70-81, wherein the Cas molecule is a Cas9 molecule or a Casl2a molecule.

83. The method of claim 82, wherein the Cas molecule is Cas9.

84. An immune cell produced by the method of any one of claims 46-83.

85. A polynucleotide comprising: (a) at least two synthetic shRNA sequences, wherein each of synthetic shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ICAM1, CD58, ADAM 17, B2M, CISH, Cbl-b, FAS, FASL. ICAM2, ICAM3, MED12, CIITA, PRDM1 and PTPN2; and (b) a nucleic acid sequence encoding a chimeric receptor.

86. The polynucleotide of claim 85, comprising tw o synthetic shRNA sequences, wherein each of the synthetic shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ICAM1, CD58, ADAM 17, B2M, CISH, Cbl-b, FAS, FASL, ICAM2, ICAM3, MED12, CIITA, PRDM1 and PTPN2.

87. The poly nucleotide of claim 85, comprising three sy nthetic shRNA sequences, wherein each of the synthetic shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ICAM1, ICAM2, CD58, ADAM17, B2M, CISH, Cbl-b, FAS, FASL, ICAM3, MED12, CIITA, PRDM1 and PTPN2.

88. The polynucleotide of claim 85. comprising four synthetic shRNA sequences, wherein each of the synthetic shRNA sequences is complementary to a messenger RNA (mRNA) sequence encoding a different target protein selected from the group consisting of ICAM1, ICAM2, CD58. FAS, ADAM17, B2M, CISH, Cbl-b. FASL, ICAM3, MED12. CIITA. PRDM1 and PTPN2.

89. The polynucleotide of any one of claims 85-88, wherein each of the synthetic shRNA sequences is a micro RNA-based shRNA (shRNA miR).

90. The polynucleotide of any one of claims 85-89, further comprising (c) a nucleic acid sequence encoding a membrane -bound interleukin- 15 (mbIL15).

91. A composition comprising a plurality of tire immune cells of any one of claims 1-45 and 84 or the polynucleotide of any one of claims 85-90.

92. A pharmaceutical composition comprising a plurality of the immune cells of any one of claims 1-45 and 84, or the polynucleotide of any one of claims 85-90, and a pharmaceutically acceptable excipient.

93. A method for treating a subject having a disease or condition, wherein the method comprises administering to the subject a plurality of the immune cells of any one of claims 1-45 and 84, the polynucleotide of any one of claims 85-90, the composition of claim 91, or the pharmaceutical composition of claim 92.

94. The method of claim 93, wherein the disease or condition comprises an infectious disease, a cancer, a tumor, or an autoimmune disease, optionally wherein the disease or condition is a cancer or an autoimmune disease.

95. The method of claim 93 or claim 94. wherein the immune cells are natural killer (NK) cells.

96. The method of any one of claims 93-95, wherein the immune cells are allogeneic to the subject.

97. Use of the immune cell of any one of claims 1-45 and 84, the polynucleotide of any one of claims 85-90, the composition of claim 91, or the pharmaceutical composition of claim 92 in the manufacture of a medicament for treatment of a subject having a disease or condition.

98. The use of claim 97, wherein the disease or condition comprises an infectious disease, a cancer, a tumor, or an autoimmune disease, optionally wherein the disease or condition is a cancer or an autoimmune disease.

99. The use of claim 97 or claim 98, wherein the immune cells are natural killer (NK) cells.

100. The use of any one of claims 97-99, wherein the immune cells are allogeneic to the subject.

101. The immune cell of any one of claims 1-45 and 84, the polynucleotide of any one of claims 85-90, the composition of claim 91, or the pharmaceutical composition of claim 92 for use in treating a subject having a disease or condition.

102. The immune cell, composition, or pharmaceutical composition for use of claim 101, wherein the disease or condition comprises an infectious disease, a cancer, a tumor, or an autoimmune disease, optionally wherein the disease or condition is a cancer or an autoimmune disease.

103. The immune cell, composition, or pharmaceutical composition for use of claim 101 or claim 102, wherein the immune cells are natural killer (NK) cells.

104. The immune cell, composition, or pharmaceutical composition for use of any one of claims 101-103, wherein the immune cells are allogeneic to the subject.

105. A population of immune cells having inhibited expression of a target protein selected from the group consisting of FAS, PRDM1, PTPN2. or any combination thereof.

106. The population of immune cells of claim 105, wherein the immune cells have inhibited expression of a target protein selected from the group consisting of FAS, PRDM1, and PTPN2.

107. The population of immune cells of claim 105 or claim 106, wherein the immune cells have inhibited expression of PRDM1.

108. A composition comprising the population of immune cells of any one of claims 105- 107.

109. A pharmaceutical composition the population of immune cells of any one of claims 105-107 a pharmaceutically acceptable excipient.

110. A method of increasing the expansion of a population of immune cells, wherein the method comprises introducing into a population of immune cells an inhibitory nucleic acid molecule that reduces the expression of a target protein selected from the group consisting of FAS, PRDM1, PTPN2, or any combination thereof.

111. The method of claim 110, wherein the inhibitory nucleic acid molecule reduces the expression of a target protein selected from the group consisting of FAS, PRDM1. and PTPN2, optionally wherein the inhibitory nucleic acid molecule reduces the expression of PRDM1.

112. The method of claiml 10 or claim 111, wherein the method increases the ex vivo or in vitro expansion of the population of immune cells.

113. The method of any one of claims 110-112, wherein the method increases the expansion of the population of immune cells as compared to a population of immune cells not introduced with the inhibitory nucleic acid molecule, optionally wherein the method increases the expansion of the population of immune cells by at least about 100%, 200%, 300%, 400%, or 500%.

114. A method of increasing the in vivo persistence of a population of immune cells, wherein the method comprises introducing into a population of immune cells an inhibitory' nucleic acid molecule that reduces the expression of a target protein selected from the group consisting of FAS. PRDM1, PTPN2, or any combination thereof.

115. The method of claim 114, wherein the inhibitory nucleic acid molecule reduces the expression of a target protein selected from the group consisting of FAS, PRDM1, and PTPN2, optionally wherein the inhibitory nucleic acid molecule reduces the expression of PRDM1.

116. The method of claim 115, wherein the method increases the in vivo persistence of the population of immune cells as compared to a population of immune cells not introduced with the inhibitory nucleic acid molecule, optionally wherein the method increases the in vivo persistence of the population of immune cells by at least about 100%, 200%. 300%, 400%, or 500%.

117. The population of immune cells of any one of claims 105-107, the composition of claim 108, the pharmaceutical composition of claim 109, or the method of any one of claims 110-116, wherein die population of immune cells comprises T cells, natural killer (NK) cells, or T cells and NK cells.

118. The population of immune cells of any one of claims 105-107, the composition of claim 108, the pharmaceutical composition of claim 109, or the method of any one of claims 110-117, wherein the population of immune cells is a population of natural killer (NK) cells.

119. The population of immune cells of any one of claims 105-107, 117. and 118, the composition of any one of claims 108. 117, and 118. the pharmaceutical composition of any one of claims 109, 117, and 118. or the method of any one of claims 110-118, wherein the population of immune cells express a chimeric receptor, optionally a chimeric antigen receptor (CAR).

120. The population of immune cells of any one of claims 105-107 and 117-119, the composition of any one of claims 108 and 117-119, the pharmaceutical composition of any one of claims 109 and 117-119. or the method of any one of claims 110-119, wherein the population of immune cells expresses a membrane-bound interleukin- 15 (mbIL15).

121. A population of immune cells produced by the method of any one of claims 110-120.

122. A method for treating a subject having a disease or condition, wherein the method comprises administering to the subject the population of immune cells of any one of claims 105-107 and 117-121, the composition of any one of claims 108 and 117-120. or the pharmaceutical composition of any one of claims 109 and 117-120.

123. Use of the population of immune cells of any one of claims 105-107 and 117-121, the composition of any one of claims 108 and 117-120, or the pharmaceutical composition of any one of claims 109 and 117-120 in the manufacture of a medicament for treatment of a subject having a disease or condition.

124. A method of increasing die in vivo persistence of allogeneic immune cells, the method comprising administering to a subject having a disease or condition allogeneic immune cells having inhibited expression of a target protein selected from the group consisting of ICAM1, ICAM2, and CD58.

125. A method of reducing the cytotoxicity of a subject’s immune cells against allogeneic immune cells, the method comprises administering to a subject having a disease or condition allogeneic immune cells having inhibited expression of a target protein selected from the group consisting of ICAM1, ICAM2, and CD58.

126. The method of claim 124 or claim 125, wherein the target protein comprises ICAM1.

127. The method of any one of claims 124-126. wherein the target protein comprises CD58.

128. The method of any one of claims 124-127, wherein the target proteins are ICAM1 and CD58.

129. The method of any one of claims 124-128. wherein the allogeneic immune cells comprise natural killer (NK) cells and / or T cells.

130. The method of any one of claims 124-129, wherein the allogeneic immune cells are natural killer (NK) cells.

131. The method of any one of claims 124-130, wherein the allogeneic immune cells express a recombinant receptor.

132. The method of claim 131, wherein the recombinant receptor is a chimeric antigen receptor (CAR) that binds to an antigen expressed by or associated with cells of the disease or condition.

133. The method of any one of claims 122 and 124-132 or the use of claim 123, wherein the disease or condition is a tumor, a cancer, an autoimmune disease, or an infectious disease.

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