Genetically modified natural killer cells for CD70-directed cancer immunotherapy

Abstract

Some embodiments of the methods and compositions disclosed herein relate to immune cells that have been engineered to express chimeric antigen receptors (CARs) and / or genetically modified to reduce potential side effects of cellular immunotherapy. Some embodiments relate to genetic modifications to immune cells, such as natural killer (NK) cells, to reduce, substantially reduce, or eliminate expression of markers by the immune cells that would otherwise cause self-targeting by those CARs. In some embodiments, the CAR targets CD70 and, in some embodiments, is used in renal cell carcinoma immunotherapy.

Description

[Technical Field]

[0001] Related Projects This application claims priority to U.S. Provisional Patent Application No. 63 / 038,645, filed June 12, 2020, U.S. Provisional Patent Application No. 63 / 090,041, filed October 9, 2020, U.S. Provisional Patent Application No. 63 / 141,411, filed January 25, 2021, and U.S. Provisional Patent Application No. 63 / 201,490, filed April 30, 2021, the entire contents of each of which are incorporated herein by reference.

[0002] Field Some embodiments disclosed herein relate to methods and compositions comprising genetically engineered cells for cancer immunotherapy, particularly cells engineered to reduce expression of specific markers that are also present on target cells. In some embodiments, the present disclosure relates to cells engineered to express chimeric antigen receptors and have reduced expression of one or more markers that increase efficacy and / or reduce potential side effects when the cells are used in cancer immunotherapy. [Background technology]

[0003] As more is learned about different cancers and what properties cancerous cells have that can be used to specifically distinguish them from healthy cells, therapeutic agents are being developed that exploit the distinctive features of cancerous cells. Immunotherapy using engineered immune cells is one approach to treating cancer.

[0004] Incorporating information by reference in an ASCII text file This application incorporates by reference the Sequence Listing contained in the following ASCII text file, filed simultaneously: Filename: NKT.056WO_ST25.txt; created on June 10, 2021, and is 1,550,527 bytes in size. Summary of the Invention [Problem to be solved by the invention]

[0005] Immunotherapy offers a new technological advance in disease treatment, where immune cells are engineered to express specific targeting and / or effector molecules that specifically identify and react to diseased or damaged cells. This represents a promising advance, at least in part, due to the possibility of specifically targeting diseased or damaged cells, as opposed to more traditional approaches such as chemotherapy, in which all cells are affected, with the desired outcome being the survival of sufficient healthy cells to sustain patient survival. One immunotherapy approach is the recombinant expression of chimeric receptors in immune cells to achieve targeted recognition and destruction of the desired abnormal cells.

[0006] In some instances, immune cell populations for immunotherapy may express one or more endogenous markers that overlap in scope with those expressed by tumor cell populations. Targeting such common markers can limit the efficacy of therapeutic cells to the extent that they target both the tumor population and other members of the therapeutic cell population. Thus, in some embodiments, a genetically engineered immune cell population, such as a natural killer (NK) cell, a T cell, or a combination thereof, for cancer immunotherapy is provided, comprising a plurality of culture-expanded immune cells engineered to express a chimeric antigen receptor (CAR) comprising a tumor-binding domain, a transmembrane domain, and a cytotoxic signaling complex, wherein the tumor-binding domain targets CD70, and the immune cells have been gene-edited to express reduced levels of CD70 compared to unedited culture-expanded immune cells, the reduced CD70 expression being engineered via editing of the endogenous CD70 gene.

[0007] In some embodiments, cells are gene-edited to express reduced levels of cytokine-inducible SH2-containing (CIS) protein encoded by the CISH gene compared to unedited cells. In some embodiments, reduced (e.g., reduced, eliminated, or otherwise undetectable) CIS expression is achieved through editing of the CIS gene. Such editing confers one or more of the following to the edited cells compared to cells expressing natural levels of CIS: increased expansion capacity, increased cytotoxicity against target cells, and increased persistence. In some embodiments, further editing is performed on the cells, such as editing to reduce the expression level of an adenosine receptor. In some embodiments, the reduction in adenosine receptor expression is achieved by editing one or more genes encoding the adenosine receptor, resulting in one or more of increased expansion capacity, increased cytotoxicity against target cells, and increased persistence compared to cells expressing the adenosine receptor at natural levels. In some embodiments, the editing and manipulation of the cell work in concert, in that a polynucleotide encoding a CAR is inserted into the edited gene. However, in some embodiments, the editing site does not comprise a polynucleotide encoding a CAR.

[0008] In some embodiments, the tumor-binding domain of the CAR comprises a heavy chain variable region (VH), wherein the VH is encoded by a polynucleotide comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the polynucleotides in SEQ ID NOs: 1104, 1053, 1091, 1047, 1106, 1052, 1077, 1064, 1098, and 1088. In some embodiments, the tumor-binding domain of the CAR comprises a light chain variable region (VL), wherein the VL is encoded by a polynucleotide comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the polynucleotides in SEQ ID NOs: 1178, 1127, 1165, 1121, 1180, 1126, 1151, 1138, 1171, and 1162. In some embodiments, the tumor-binding domain comprises a single-chain variable fragment (scFv), and the scFv is encoded by a polynucleotide comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the polynucleotides of SEQ ID NOs: 104, 53, 91, 47, 106, 52, 77, 64, 98, and 88.

[0009] In some embodiments, the tumor-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3, and wherein CDR-H1 is at least 80%, at least 85%, at least 90%, or at least identical to one or more sequences selected from SEQ ID NOs: 494, 443, 481, 437, 496, 442, 467, 454, 488, and 478. CDR-H2 comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more sequences selected from SEQ ID NOs: 568, 517, 555, 511, 570, 516, 541, 528, 562, and 552; and CDR-H3 comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more sequences selected from SEQ ID NOs: 642, 591, 629, 585, 644, 590, 615, 602, 636, and 626. CDR-L1 comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with one or more sequences selected from SEQ ID NOs: 734, 683, 721, 677, 736, 682, 707, 694, 728, and 718; CDR-L2 comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with one or more sequences selected from SEQ ID NOs: 808, 757, 795, 751, 810, 75 CDR-L3 comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with one or more sequences selected from SEQ ID NOs: 882, 831, 869, 825, 884, 830, 855, 842, 876, and 855.

[0010] In some embodiments, the tumor-binding domain comprises a VH, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the amino acid sequences of SEQ ID NOs: 956, 905, 943, 899, 958, 904, 929, 916, 950, and 940. In some embodiments, the tumor-binding domain comprises a VL, wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the amino acid sequences of SEQ ID NOs: 1030, 979, 1017, 973, 1032, 978, 1003, 990, 1024, and 1014.

[0011] In some embodiments, the tumor-binding domain comprises an scFv, wherein the scFv comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the amino acids in SEQ ID NOs: 296, 245, 283, 239, 298, 244, 269, 256, 290, 280.

[0012] In some embodiments, immune cells are engineered to express membrane-bound IL-15 (mbIL15). In some embodiments, mbIL15 is bicistronically encoded on the polynucleotide encoding the CAR. In some embodiments, the polynucleotides encoding the CAR and mbIL15 comprise a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the polynucleotides set forth in SEQ ID NOs: 204, 153, 191, 147, 206, 152, 177, 164, 198, and 188. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 379, 328, 366, 322, 381, 327, 352, 339, 373, and 363. In some embodiments, mbIL15 is encoded by a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:1188.

[0013] In some embodiments, the cytotoxic signaling complex comprises an OX-40 subdomain and a CD3 zeta subdomain. In some embodiments, the OX40 subdomain is encoded by a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the CD3 zeta subdomain is encoded by a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 7.

[0014] In some embodiments, expression of CIS by edited cells is substantially reduced compared to cells that are not edited for CISH. In some embodiments, edited cells do not express detectable levels of CIS protein. In some embodiments, expression of adenosine receptors is substantially reduced compared to cells that are not edited for adenosine receptors. In some embodiments, edited cells do not express detectable levels of adenosine receptors. In some embodiments, the edited adenosine receptors include one or more of the A2A adenosine receptor, the A2B adenosine receptor, the A3 adenosine receptor, or the A1 adenosine receptor. In some embodiments, the edited adenosine receptor includes the A2A adenosine receptor (A2AR). In some embodiments, the cells are further gene-edited to express reduced levels of one or more of transforming growth factor beta receptor (TGFBR), beta-2 microglobulin (B2M), CIITA (class II major histocompatibility complex transactivator), natural killer group 2, member A (NKG2A) receptor, Cbl proto-oncogene B protein encoded by the CBLB gene, tripartite motif-containing protein 29 encoded by the TRIM29 gene, and suppressor of cytokine signaling 2 protein encoded by the SOCS2 gene, compared to unedited NK cells. In some embodiments, gene editing to reduce or induce expression is performed using a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system comprises a Cas selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, CasX, CasY, and combinations thereof. In one embodiment, the Cas is Cas9 (which may be a Cas9 with reduced activity).

[0015] In some embodiments, Cas is guided to the CD70 gene by one or more guide RNAs that have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123. In some embodiments, Cas is guided to the CISH gene by one or more guide RNAs that have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 134. In some embodiments, Cas is guided to the adenosine receptor gene by one or more guide RNAs that have at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 396, SEQ ID NO: 397, or SEQ ID NO: 398. In some embodiments, the Cas is guided to the TGFBR2 gene by one or more guide RNAs having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134.

[0016] In some embodiments, gene editing to reduce or induce expression is performed using zinc finger nucleases (ZFNs). In other embodiments, gene editing to reduce or induce expression is performed using transcription activator-like effector nucleases (TALENs).

[0017] In some embodiments, the engineered and edited immune cells comprise NK cells, hi some embodiments, the engineered and edited immune cells consist of or consist essentially of NK cells.

[0018] In some embodiments, provided herein are methods for treating cancer in a subject, comprising administering to the subject a genetically engineered and edited immune cell population, e.g., an NK cell population, as provided herein. In some embodiments, the cancer is renal cell carcinoma or metastasis from renal cell carcinoma. Also provided herein is the use of genetically engineered and edited immune cells, e.g., NK cells, as provided herein, in the treatment of cancer. Further provided herein is the use of genetically engineered and edited immune cells, e.g., NK cells, as provided herein, in the manufacture of a medicament for treating cancer.

[0019] Further provided herein is a method of treating cancer in a subject, comprising administering to the subject a genetically engineered immune cell population comprising a plurality of immune cells, such as NK cells, T cells, or a combination thereof, expanded in culture, wherein the plurality of NK cells are engineered to express a chimeric antigen receptor (CAR) comprising a tumor-binding domain, a transmembrane domain, and a cytotoxic signaling complex, wherein the tumor-binding domain targets CD70, and the cells have been gene-edited to express reduced levels of CD70 compared to non-edited cells expanded in culture, wherein the reduced CD70 expression has been engineered via editing of the endogenous CD70 gene.

[0020] In some embodiments, the cells have been gene-edited to express reduced levels of cytokine-inducible SH2-containing (CIS) protein encoded by the CISH gene compared to unedited cells, the reduced CIS expression being engineered via editing of the CIS gene, and the gene-edited cells exhibit one or more of increased expansion capacity, increased cytotoxicity against target cells, and increased persistence compared to cells expressing native levels of CIS. In some embodiments, the cells have also been gene-edited to express reduced expression of an adenosine receptor, the reduced adenosine receptor expression being achieved via editing of a gene encoding the adenosine receptor, and the gene-edited cells exhibit one or more of increased expansion capacity, increased cytotoxicity against target cells, and increased persistence compared to cells expressing native levels of the adenosine receptor.

[0021] In some embodiments, the tumor-binding domain comprises a heavy chain variable region (VH), wherein the VH is encoded by a polynucleotide comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the polynucleotides of SEQ ID NOs: 1104, 1053, 1091, 1047, 1106, 1052, 1077, 1064, 1098, and 1088; and the tumor-binding domain comprises a light chain variable region (VL), wherein the VL is encoded by a polynucleotide comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the polynucleotides of SEQ ID NOs: 1178, 1127, 1165, 1121, 1180, 1126, 1151, 1138, 1171, and 1162.

[0022] In some embodiments of these methods, the tumor-binding domain comprises a single-chain variable fragment (scFv), and the scFv is encoded by a polynucleotide comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the polynucleotides of SEQ ID NOs: 104, 53, 91, 47, 106, 52, 77, 64, 98, and 88.In some embodiments of these methods, the tumor-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3, and wherein CDR-H1 is at least 80%, at least 85%, at least 90%, or at least 90% identical to one or more sequences selected from SEQ ID NOs: 494, 443, 481, 437, 496, 442, 467, 454, 488, and 478. or a sequence having at least 95% sequence identity to one or more sequences selected from SEQ ID NOs: 568, 517, 555, 511, 570, 516, 541, 528, 562, and 552; CDR-H3 comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more sequences selected from SEQ ID NOs: 642, 591, 629, 585, 644, 590, 615, 602, 636, and 626. CDR-L1 comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with one or more of the sequences selected from SEQ ID NOs: 734, 683, 721, 677, 736, 682, 707, 694, 728, and 718; CDR-L2 comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with one or more of the sequences selected from SEQ ID NOs: 808, 757, 795, 751, 810, 756, 781, 768, 802, and 792; and CDR-L3 comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with one or more sequences selected from SEQ ID NOs: 882, 831, 869, 825, 884, 830, 855, 842, 876, and 855.

[0023] In some embodiments of these methods, the tumor-binding domain comprises a VH, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the amino acid sequences of SEQ ID NOs: 956, 905, 943, 899, 958, 904, 929, 916, 950, and 940; and the tumor-binding domain comprises a VL, wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one or more of the amino acid sequences of SEQ ID NOs: 1030, 979, 1017, 973, 1032, 978, 1003, 990, 1024, and 1014. In some embodiments of these methods, the tumor-binding domain comprises an scFv, and the scFv comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, or at least 95% identity with one or more of the amino acid sequences of SEQ ID NOs: 296, 245, 283, 239, 298, 244, 269, 256, 290, and 280.

[0024] In some embodiments of these methods, the chimeric antigen receptor comprises an OX40 subdomain and a CD3 zeta subdomain, and the cells are engineered to express membrane-bound IL-15 (mbIL15). In some embodiments, mbIL15 is bicistronically encoded on the polynucleotide encoding the CAR. In some embodiments, the polynucleotides encoding the CAR and mbIL15 comprise a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with one or more of the polynucleotides set forth in SEQ ID NOs: 204, 153, 191, 147, 206, 152, 177, 164, 198, and 188. In some embodiments, the CAR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with one or more of the amino acid sequences set forth in SEQ ID NOs: 379, 328, 366, 322, 381, 327, 352, 339, 373, and 363. In some embodiments, the OX40 subdomain is encoded by a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5, the CD3 zeta subdomain is encoded by a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:7, and mbIL15 is encoded by a sequence having at least 80%, at least 85%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:1188.

[0025] In some embodiments, the expression of CIS is substantially reduced compared to cells that have not been edited for CISH, and / or the cells do not express detectable levels of CIS protein. In some embodiments, the expression of adenosine receptors is substantially reduced compared to cells that have not been edited for adenosine receptors, and / or the cells do not express detectable levels of adenosine receptors. In some embodiments, the adenosine receptors include A2A adenosine receptors, A2B adenosine receptors, A3 adenosine receptors, or A1 adenosine receptors. In some embodiments, the gene editing is performed using a CRISPR-Cas system, wherein the Cas comprises a Cas9 enzyme. In some embodiments, the engineered and edited immune cells include NK cells. In some embodiments, the engineered and edited immune cells consist of, or consist essentially of, NK cells.

[0026] Also provided herein is a polynucleotide encoding an anti-CD70 chimeric antigen receptor, wherein the CAR comprises an anti-CD70 binding domain, wherein the anti-CD70 binding domain is encoded by a polynucleotide comprising a sequence having at least 95% sequence identity to one or more of SEQ ID NOs: 36-120, 221-229, 1038-1111, 1112-1185, and / or comprises an amino acid sequence having at least 95% sequence identity to one or more of the amino acid sequences of SEQ ID NOs: 230-312, 890-963, 964-1037, or a portion thereof that is capable of generating a cytotoxic signal upon binding to CD70 on a target cell. In some embodiments, the polynucleotide further encodes an OX40 domain and a CD3 zeta domain, wherein the OX40 subdomain is encoded by a sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:5, and the CD3 zeta subdomain is encoded by a sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:7. In some embodiments, the polynucleotide further encodes mbIL15, wherein mbIL15 is encoded by a sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:1188. In some embodiments, one or more of SEQ ID NOs:36-120, 221-229, 1038-1111, or 1112-1185, the polynucleotide encoding OX40, the polynucleotide encoding CD3 zeta, and the polynucleotide encoding mbIL15 are arranged in a 5' to 3' direction within the polynucleotide.

[0027] Further provided herein is a method for increasing the persistence of an immune cell population for use in cancer immunotherapy, comprising identifying a target marker on a tumor to be treated, determining whether an immune cell population engineered to express a CAR that binds to the target marker also endogenously expresses the target marker, editing the genome of the immune cell population to disrupt the gene encoding the endogenous target marker, and engineering the immune cell population to express the CAR, wherein disruption of the endogenous expression of the target marker by the immune cells reduces the ability of the CAR to bind to the endogenous target marker on the immune cells, thereby increasing the persistence of the immune cell population. In some embodiments, the immune cells are NK cells, T cells, or a combination thereof, the target marker is CD70, and the gene editing is performed using the CRISPR-Cas system.

[0028] In some embodiments, the method further comprises using a CRISPR-Cas system to disrupt expression of a cytokine-inducible SH2-containing (CIS) protein encoded by a CISH gene, and / or comprises using a CRISPR-Cas system to disrupt expression of an adenosine receptor, wherein the adenosine receptor comprises an A2A adenosine receptor, an A2B adenosine receptor, an A3 adenosine receptor, and / or an A1 adenosine receptor.

[0029] In some embodiments, provided herein is an anti-CD70 chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD70 binding domain, an OX40 domain, and a CD3 zeta domain, wherein the anti-CD70 CAR is encoded by a polynucleotide having at least 85%, at least 90%, or at least 95% sequence identity to one or more of SEQ ID NOs: 138-220. Also provided herein is an anti-CD70 chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD70 binding domain, an OX40 domain, and a CD3 zeta domain, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to one or more of the amino acid sequences of SEQ ID NOs: 313-395, or a portion thereof that is capable of generating a cytotoxic signal upon binding to CD70 on a target cell.

[0030] Further provided herein is an anti-CD70 binding domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1 comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 428-501; CDR-H2 comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 502-575; and CDR-H3 comprises a sequence selected from SEQ ID NOs: 576-64. CDR-L1 comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 668-741; CDR-L2 comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 742-815; and CDR-L3 comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 816-889. In some embodiments, the heavy chain variable domain is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any sequence selected from SEQ ID NOs: 1038-1111. In some embodiments, the light chain variable domain is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any sequence selected from SEQ ID NOs: 1112-1185. Depending on the embodiment, the anti-CD70 binding domain is an antibody, a Fab' fragment, a F(ab')2 fragment, or an scFv. In some embodiments, provided herein is a CAR comprising an anti-CD70 binding domain disclosed herein. In some embodiments, the CAR further comprises an OX40 subdomain and a CD3 zeta subdomain.In some embodiments, cells such as immune cells are provided that comprise the anti-CD70 binding domain or CAR provided herein. In some embodiments, the cells comprise, consist of, or essentially consist of NK cells. In some embodiments, the cells are gene-edited to express reduced levels of CISH, adenosine receptor, A2A adenosine receptor, A2B adenosine receptor, A3 adenosine receptor, A1 adenosine receptor, A2AR, TGFBR, B2M, CIITA, NKG2A, CBLB, TRIM29, SOCS2, SMAD3, MAPKAPK3, CEACAM1, or DDIT4, or any combination thereof, compared to unedited cells. Also provided is a method for treating cancer in a subject, comprising administering to the subject an anti-CD70 binding domain, CAR, or cell as provided herein. Also provided is the use of the anti-CD70 binding domain, CAR, or cell provided herein in the treatment of cancer and / or the manufacture of a medicament for the treatment of cancer.

[0031] Also provided is a genetically engineered immune cell population for cancer immunotherapy, comprising a plurality of immune cells expanded in culture, wherein the plurality of immune cells are engineered to express a chimeric antigen receptor (CAR) comprising a tumor-binding domain that targets CD70, a transmembrane domain, and a cytotoxic signaling complex, and the immune cells are gene-edited to express reduced levels of CD70 compared to non-edited immune cells expanded in culture, wherein the reduced CD70 expression is engineered via editing of the endogenous CD70 gene. In some embodiments, the immune cell population comprises, consists of, or consists essentially of a NK cell population.

[0032] Also provided in some embodiments is a method of generating a genetically engineered immune cell population for cancer immunotherapy, comprising: engineering an immune cell population to express a CAR that binds to a target marker, wherein at least a portion of the immune cell population endogenously expresses the target marker; and editing the genome of the immune cell population to disrupt a gene encoding the endogenous target marker, wherein disruption of the endogenous expression of the target marker by the immune cells reduces the ability of the CAR to bind to the endogenous target marker on the immune cells. In some embodiments, the immune cell population comprises, consists of, or consists essentially of an NK cell population.

[0033] In some embodiments, provided herein are genetically engineered natural killer (NK) cell populations for cancer immunotherapy, comprising a plurality of NK cells expanded in culture, wherein the plurality of NK cells are engineered to express a chimeric antigen receptor (CAR) comprising a tumor-binding domain, a transmembrane domain, and a cytotoxic signaling complex, wherein the tumor-binding domain targets CD70. In some embodiments, the NK cells have been gene-edited to express reduced levels of CD70 compared to non-edited NK cells expanded in culture, wherein the reduced CD70 expression has been engineered via editing of the endogenous CD70 gene.

[0034] In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject a genetically engineered immune cell population comprising a plurality of NK cells expanded in culture, wherein the plurality of NK cells are engineered to express a chimeric antigen receptor (CAR) comprising a tumor-binding domain, a transmembrane domain, and a cytotoxic signaling complex, wherein the tumor-binding domain targets CD70, and the chimeric antigen receptor comprises an OX40 subdomain and a CD3 zeta subdomain, and wherein the NK cells have been gene-edited to express reduced levels of CD70 compared to non-edited NK cells expanded in culture, wherein the reduced CD70 expression has been engineered via editing of the endogenous CD70 gene.

[0035] In some embodiments, the chimeric antigen receptor cytotoxicity signaling complex comprises an OX40 subdomain and a CD3 zeta subdomain. In some embodiments, the cells are also engineered to express membrane-bound IL-15.

[0036] In some embodiments, NK cells are genetically edited to express reduced levels of cytokine-inducible SH2-containing (CIS) protein encoded by the CISH gene compared to non-genetically engineered NK cells, where the reduced CIS expression is engineered via editing of the CISH gene, and the genetically engineered NK cells exhibit one or more of increased expansion capacity, increased cytotoxicity against target cells, and increased persistence compared to NK cells expressing native levels of CIS. In some embodiments, the CISH editing substantially reduces expression of CIS compared to cells not edited for CISH. In some embodiments, the edited cells do not express detectable levels of CIS.

[0037] In some embodiments, NK cells are gene-edited to express reduced levels of adenosine receptors compared to non-manipulated NK cells, and the reduced adenosine receptor expression is engineered via editing of the adenosine receptor-encoding gene, and the genetically engineered NK cells exhibit one or more of increased expansion capacity, increased cytotoxicity against target cells, and increased persistence compared to NK cells expressing natural levels of adenosine receptors. In some embodiments, editing the adenosine receptor-encoding gene substantially reduces adenosine receptor expression compared to cells not edited for adenosine receptors. In some embodiments, the edited cells do not express detectable levels of adenosine receptors. Depending on the embodiment, the edited gene may encode the A2A adenosine receptor, the A2B adenosine receptor, the A3 adenosine receptor, or the A1 adenosine receptor. In some embodiments, the edited gene encodes the A2A adenosine receptor (A2AR). In some embodiments, more than one adenosine receptor is edited.

[0038] In some embodiments, the genes encoding CIS and adenosine receptors are edited to substantially reduce the expression of CIS and adenosine receptors compared to cells that are not edited for CIS and adenosine receptors. In some embodiments, the edited cells do not express detectable levels of CIS or adenosine receptors.

[0039] In some embodiments, the tumor-binding domain is encoded by a polynucleotide comprising a sequence having at least 85%, at least 90%, or at least 95% sequence identity to one or more of the polynucleotides set forth in SEQ ID NOs: 36-120, 221-229, 1038-1111, 1112-1185, and / or comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to one or more of the amino acid sequences set forth in SEQ ID NOs: 230-312, 890-963, 964-1037. In some embodiments, the OX40 subdomain is encoded by a sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the OX40 subdomain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the CD3 zeta subdomain is encoded by a sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the CD3 zeta subdomain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:8.

[0040] In some embodiments, provided herein is an anti-CD70 binding domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3; and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 428-501; CDR-H2 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 502-575; and CDR-H3 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 576-649, CDR-L1 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 668-741, CDR-L2 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 742-815, and CDR-L3 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 816-889. In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 90%, 95%, 99%, or 100% sequence identity to any sequence selected from SEQ ID NOs: 890-963. In some embodiments, the light chain variable region comprises an amino acid sequence having at least 95%, 99%, or 100% sequence identity to any sequence selected from SEQ ID NOs: 964 to 1037. In some embodiments, 1) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 890, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 964; 2) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 891, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 965; 3) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 892, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 966;4) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 893, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 967; 5) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 894, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 968; 6) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 895, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 969. 7) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 896 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 970; 8) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 897 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 971; 9) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 898 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 971 9) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 899, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 973; 11) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 900, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 974; 12) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 901. 11) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 975; 12) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 902, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 976; 13) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 902, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 976; 14) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 903, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 977;15) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 904, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 978; 16) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 905, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 979; 17) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 906, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 980. 18) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 907 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 981; 19) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 908 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 982; 20) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 909. 19) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 910, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 984; 20) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 911, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 985; 21) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 910, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 984; 22) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 911, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 985; 23) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 912. 23) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 986; 24) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 913, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 987; 25) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 914, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 988;26) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 915, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 989; 27) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 916, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 990; 28) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 917, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 991. 29) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 918 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 992; 30) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 919 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 993; 31) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 920. 31) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 921, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 995; 32) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 921, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 995; 33) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 922, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 996; 34) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 923 34) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 997; 35) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 924, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 998; 36) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 925, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 999;37) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 926, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1000; 38) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 927, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1001; 39) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 928, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1002. 40) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 929 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1003; 41) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 930 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1004; 42) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 931. 41) the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1005; 42) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 932, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1006; 43) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 932, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1006; 44) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 933, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1007; 45) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 934. 46) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 935 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1009; 47) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 936 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1010;48) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 937, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1011; 49) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 938, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1012; 50) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 939, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 939. 51) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 940, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1014; 52) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 941, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 942; 53) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 942 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1016; 54) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 943 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1017; 55) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 944. 55) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 945, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1019; 56) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 945, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1019; 57) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 946, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1020; 58) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 947, 59) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 948 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1022; 60) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 949 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1023; 61) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 949 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1023; 62) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 951 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1025; 63) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 952 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1026;64) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 953, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1027; 65) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 954, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1028; 66) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 955, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1029. 67) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 956, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1030; 68) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 957, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1031; 69) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 958, and the light chain 70) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 959, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1033; 71) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 960, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1034; 72) the heavy chain variable region comprises CDR-H1 within SEQ ID NO: 961. , CDR-H2, CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1035; 73) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 962, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1036; and / or 74) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 963, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1037;

[0041] In some embodiments, 1) the heavy chain variable region comprises SEQ ID NO:890 and the light chain variable region comprises SEQ ID NO:964; 2) the heavy chain variable region comprises SEQ ID NO:891 and the light chain variable region comprises SEQ ID NO:965; 3) the heavy chain variable region comprises SEQ ID NO:892 and the light chain variable region comprises SEQ ID NO:966; 4) the heavy chain variable region comprises SEQ ID NO:893 and the light chain variable region comprises SEQ ID NO:967; 5) the heavy chain variable region comprises SEQ ID NO:894 and the light chain variable region comprises SEQ ID NO:968; 6) the heavy chain variable region comprises SEQ ID NO:895 and the light chain variable region comprises SEQ ID NO:969; 7) the heavy chain 8) the heavy chain variable region comprises SEQ ID NO:897 and the light chain variable region comprises SEQ ID NO:971; 9) the heavy chain variable region comprises SEQ ID NO:898 and the light chain variable region comprises SEQ ID NO:972; 10) the heavy chain variable region comprises SEQ ID NO:899 and the light chain variable region comprises SEQ ID NO:973; 11) the heavy chain variable region comprises SEQ ID NO:900 and the light chain variable region comprises SEQ ID NO:974; 12) the heavy chain variable region comprises SEQ ID NO:901 and the light chain variable region comprises SEQ ID NO:975; 13) the heavy chain variable region comprises SEQ ID NO:902 14) the heavy chain variable region comprises SEQ ID NO: 903 and the light chain variable region comprises SEQ ID NO: 977; 15) the heavy chain variable region comprises SEQ ID NO: 904 and the light chain variable region comprises SEQ ID NO: 978; 16) the heavy chain variable region comprises SEQ ID NO: 905 and the light chain variable region comprises SEQ ID NO: 979; 17) the heavy chain variable region comprises SEQ ID NO: 906 and the light chain variable region comprises SEQ ID NO: 980; 18) the heavy chain variable region comprises SEQ ID NO: 907 and the light chain variable region comprises SEQ ID NO: 981; 19) the heavy chain variable region comprises SEQ ID NO: 908 and the light chain variable region comprises 20) the heavy chain variable region comprises SEQ ID NO:909 and the light chain variable region comprises SEQ ID NO:983; 21) the heavy chain variable region comprises SEQ ID NO:910 and the light chain variable region comprises SEQ ID NO:984; 22) the heavy chain variable region comprises SEQ ID NO:911 and the light chain variable region comprises SEQ ID NO:985; 23) the heavy chain variable region comprises SEQ ID NO:912 and the light chain variable region comprises SEQ ID NO:986; 24) the heavy chain variable region comprises SEQ ID NO:913 and the light chain variable region comprises SEQ ID NO:987; 25) the heavy chain variable region comprises SEQ ID NO:914 and the light chain variable region comprises SEQ ID NO:988;26) the heavy chain variable region comprises SEQ ID NO: 915 and the light chain variable region comprises SEQ ID NO: 989; 27) the heavy chain variable region comprises SEQ ID NO: 916 and the light chain variable region comprises SEQ ID NO: 990; 28) the heavy chain variable region comprises SEQ ID NO: 917 and the light chain variable region comprises SEQ ID NO: 991; 29) the heavy chain variable region comprises SEQ ID NO: 918 and the light chain variable region comprises SEQ ID NO: 992; 30) the heavy chain variable region comprises SEQ ID NO: 919 and the light chain variable region comprises SEQ ID NO: 993; 31) the heavy chain variable region comprises SEQ ID NO: 920 and the light chain variable region comprises SEQ ID NO: 994; 32) the heavy chain variable region comprises SEQ ID NO: 31) the heavy chain variable region comprises SEQ ID NO: 921 and the light chain variable region comprises SEQ ID NO: 995; 32) the heavy chain variable region comprises SEQ ID NO: 922 and the light chain variable region comprises SEQ ID NO: 996; 33) the heavy chain variable region comprises SEQ ID NO: 922 and the light chain variable region comprises SEQ ID NO: 996; 34) the heavy chain variable region comprises SEQ ID NO: 923 and the light chain variable region comprises SEQ ID NO: 997; 35) the heavy chain variable region comprises SEQ ID NO: 924 and the light chain variable region comprises SEQ ID NO: 998; 36) the heavy chain variable region comprises SEQ ID NO: 925 and the light chain variable region comprises SEQ ID NO: 999; 37) the heavy chain variable region comprises SEQ ID NO: 926 and the light chain variable region comprises SEQ ID NO: 1000; 38) the heavy chain variable region comprises SEQ ID NO: 927 and the light chain variable region comprises SEQ ID NO: 929 39) the heavy chain variable region comprises SEQ ID NO:928 and the light chain variable region comprises SEQ ID NO:1002; 40) the heavy chain variable region comprises SEQ ID NO:929 and the light chain variable region comprises SEQ ID NO:1003; 41) the heavy chain variable region comprises SEQ ID NO:930 and the light chain variable region comprises SEQ ID NO:1004; 42) the heavy chain variable region comprises SEQ ID NO:931 and the light chain variable region comprises SEQ ID NO:1005; 43) the heavy chain variable region comprises SEQ ID NO:932 and the light chain variable region comprises SEQ ID NO:1006; 44) the heavy chain variable region comprises SEQ ID NO:933 and the light chain variable region comprises SEQ ID NO: 1007; 45) the heavy chain variable region comprises SEQ ID NO:934 and the light chain variable region comprises SEQ ID NO:1008; 46) the heavy chain variable region comprises SEQ ID NO:935 and the light chain variable region comprises SEQ ID NO:1009; 47) the heavy chain variable region comprises SEQ ID NO:936 and the light chain variable region comprises SEQ ID NO:1010; 48) the heavy chain variable region comprises SEQ ID NO:937 and the light chain variable region comprises SEQ ID NO:1011; 49) the heavy chain variable region comprises SEQ ID NO:938 and the light chain variable region comprises SEQ ID NO:1012; 50) the heavy chain variable region comprises SEQ ID NO:939 and the light chain variable region comprises SEQ ID NO:1013;51) the heavy chain variable region comprises SEQ ID NO:940 and the light chain variable region comprises SEQ ID NO:1014; 52) the heavy chain variable region comprises SEQ ID NO:941 and the light chain variable region comprises SEQ ID NO:1015; 53) the heavy chain variable region comprises SEQ ID NO:942 and the light chain variable region comprises SEQ ID NO:1016; 54) the heavy chain variable region comprises SEQ ID NO:943 and the light chain variable region comprises SEQ ID NO:1017; 55) the heavy chain variable region comprises SEQ ID NO:944 and the light chain variable region comprises SEQ ID NO:1018; 56) the heavy chain variable region comprises SEQ ID NO:945 and the light chain variable region comprises SEQ ID NO:1019; 7) the heavy chain variable region comprises SEQ ID NO: 946 and the light chain variable region comprises SEQ ID NO: 1020; 58) the heavy chain variable region comprises SEQ ID NO: 947 and the light chain variable region comprises SEQ ID NO: 1021; 59) the heavy chain variable region comprises SEQ ID NO: 948 and the light chain variable region comprises SEQ ID NO: 1022; 60) the heavy chain variable region comprises SEQ ID NO: 949 and the light chain variable region comprises SEQ ID NO: 1023; 61) the heavy chain variable region comprises SEQ ID NO: 950 and the light chain variable region comprises SEQ ID NO: 1024; 62) the heavy chain variable region comprises SEQ ID NO: 951 and the light chain variable region comprises SEQ ID NO: 1025; 63) 64) the heavy chain variable region comprises SEQ ID NO: 953 and the light chain variable region comprises SEQ ID NO: 1027; 65) the heavy chain variable region comprises SEQ ID NO: 954 and the light chain variable region comprises SEQ ID NO: 1028; 66) the heavy chain variable region comprises SEQ ID NO: 955 and the light chain variable region comprises SEQ ID NO: 1029; 67) the heavy chain variable region comprises SEQ ID NO: 956 and the light chain variable region comprises SEQ ID NO: 1030; 68) the heavy chain variable region comprises SEQ ID NO: 957 and the light chain variable region comprises SEQ ID NO: 1031; 69) the heavy chain 70) the heavy chain variable region comprises SEQ ID NO:959 and the light chain variable region comprises SEQ ID NO:1033; 71) the heavy chain variable region comprises SEQ ID NO:960 and the light chain variable region comprises SEQ ID NO:1034; 72) the heavy chain variable region comprises SEQ ID NO:961 and the light chain variable region comprises SEQ ID NO:1035; 73) the heavy chain variable region comprises SEQ ID NO:962 and the light chain variable region comprises SEQ ID NO:1036; and / or 74) the heavy chain variable region comprises SEQ ID NO:963 and the light chain variable region comprises SEQ ID NO:1037.

[0042] In some embodiments, the heavy chain variable region further comprises FW-H1, FW-H2, FW-H3, and FW-H4, and the light chain variable region further comprises FW-L1, FW-L2, FW-L3, wherein FW-H1 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 399-402; FW-H2 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 403-406; FW-H3 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 407-422; and FW-H4 comprises at least one sequence identical to a sequence selected from SEQ ID NOs: 423-427. FW-L1 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 650 to 653; FW-L2 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 654 to 657; FW-L3 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 658 to 661; and / or FW-L4 comprises a sequence having at least 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 662 to 667. In some embodiments, the heavy chain variable domain is encoded by a nucleic acid sequence having at least 90%, 95%, 99%, or 100% sequence identity to any sequence selected from SEQ ID NOs: 1038-1111. In some embodiments, the light chain variable domain is encoded by a nucleic acid sequence having at least 90%, 95%, 99%, or 100% sequence identity to any sequence selected from SEQ ID NOs: 1112-1185. In some embodiments, the anti-CD70 binding domain is an antibody, Fab' fragment, F(ab')2 fragment, or scFv. In some embodiments, one or more anti-CD70 binding domains disclosed above are incorporated into a CAR.In some embodiments, such a CAR further comprises an OX40 subdomain and a CD3 zeta subdomain (or any signaling / co-stimulatory domain disclosed herein). In some embodiments, the CAR consists of, or consists essentially of, a CD70 binding domain, a transmembrane domain / hinge, an OX40 domain, and a CD3 zeta domain disclosed herein. In some embodiments, the OX40 subdomain comprises an amino acid sequence having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:6. In some embodiments, the OX40 subdomain is encoded by a sequence having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:5. In some embodiments, the CD3 zeta subdomain comprises an amino acid sequence having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:8. In some embodiments, the CD3 zeta subdomain is encoded by a sequence having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:7. In some embodiments, the CAR comprises at least two anti-CD70 binding domains, and the CAR is a multivalent CAR. In some embodiments, the multivalent CAR comprises at least two anti-CD70 binding domains, and the CAR is a bivalent CAR. In some embodiments, the bivalent CAR comprises a first anti-CD70 binding domain and a second anti-CD70 binding domain, each comprising: a) a heavy chain variable region comprising the sequence of SEQ ID NO: 923 and a light chain variable region comprising the sequence of SEQ ID NO: 997; b) a heavy chain variable region comprising the sequence of SEQ ID NO: 949 and a light chain variable region comprising the sequence of SEQ ID NO: 1023; c) a heavy chain variable region comprising the sequence of SEQ ID NO: 950 and a light chain variable region comprising the sequence of SEQ ID NO: 1024; d) a heavy chain variable region comprising the sequence of SEQ ID NO: 952 and a light chain variable region comprising the sequence of SEQ ID NO: 1026; and / or e) a heavy chain variable region comprising the sequence of SEQ ID NO: 953 and a light chain variable region comprising the sequence of SEQ ID NO: 1027. In some embodiments, a cell is provided that comprises an anti-CD70 binding domain and / or a CAR disclosed above. In some embodiments, another CAR is engineered into the cell.In some embodiments, the CAR does not target an NKG2D ligand. In some embodiments, the CAR does not target CD19. In some embodiments, the cell is an immune cell. In some embodiments, the cell is an NK cell. In some embodiments, the cell is used in combination with another cell type (e.g., an engineered T cell). In some embodiments, the immune cell is not a T cell, a gamma T cell, or a delta gamma T cell. In some embodiments, the cell is gene-edited to express reduced levels of CISH, adenosine receptor, A2A adenosine receptor, A2B adenosine receptor, A3 adenosine receptor, A1 adenosine receptor, A2AR, TGFBR, B2M, CIITA, NKG2A, CBLB, TRIM29SOCS2, SMAD3, MAPKAPK3, CEACAM1, or DDIT4, or any combination thereof, compared to unengineered cells. In some embodiments, the cells are gene-edited with one or more guide RNAs having at least 90% or at least 95% sequence identity to SEQ ID NOs: 1190-1201. In some embodiments, the NK cells are gene-edited to express reduced levels of SMAD3, MAPKAPK3, CEACAM1, or DDIT4, or any combination thereof, compared to non-genetically engineered NK cells. In some embodiments, the NK cells are gene-edited with one or more guide RNAs having at least 90% or at least 95% sequence identity to SEQ ID NOs: 1190-1201. In some embodiments, the selected gene may not be disrupted in the engineered cells. For example, in one embodiment, the immune cells have not undergone a disruption of the T cell receptor alpha constant (TRAC) gene. In one embodiment, the immune cells have not undergone a disruption of the B2M gene. In one embodiment, the immune cells have not undergone a disruption of MHC class I.

[0043] In some embodiments, provided are methods of treating cancer in a subject comprising administering to the subject one or more anti-CD70 binding domains described above (or elsewhere herein). In some embodiments, provided is use of an anti-CD70 binding domain described above (or elsewhere herein) for treating cancer and / or in the manufacture of a medicament for treating cancer.

[0044] In some embodiments, the NK cells disclosed herein are engineered to express interleukin-15 (IL15, IL-15). In some embodiments, the IL15 is membrane-bound IL15 (mbIL15). In some embodiments, the mbIL15 comprises a native IL15 sequence and at least one transmembrane domain. In some embodiments, the native IL15 sequence is a human native IL15 sequence. In some embodiments, the native IL15 sequence is encoded by a sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 11. In some embodiments, the native IL15 sequence comprises a peptide sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the mbIL15 is encoded by a sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1188. In some embodiments, the mbIL15 comprises a peptide sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1189. In some embodiments, mbIL15 is optionally bicistronically encoded on the polynucleotide encoding the CAR. In some embodiments, the CAR is encoded by a polynucleotide having at least 85%, at least 90%, or at least 95% sequence identity to one or more of the polynucleotides of SEQ ID NOs: 138-220, or a portion thereof (e.g., the portion excluding the mbIL15 sequence and / or the self-cleaving peptide sequence), and / or comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to one or more of the amino acid sequences of SEQ ID NOs: 313-395, or a portion thereof (e.g., the portion excluding the mbIL15 sequence and / or the self-cleaving peptide sequence).

[0045] In some embodiments, expression of CIS is substantially reduced compared to non-engineered NK cells, hi some embodiments, the NK cells do not express detectable levels of CIS protein.

[0046] In some embodiments, the NK cells are further engineered to express reduced levels of transforming growth factor beta receptor (TGFBR) compared to non-engineered NK cells, reduced levels of beta-2 microglobulin (B2M) compared to non-engineered NK cells, reduced levels of CIITA (class II major histocompatibility complex transactivator) compared to non-engineered NK cells, reduced levels of natural killer group 2, member A (NKG2A) receptor compared to non-engineered NK cells, reduced levels of Cbl proto-oncogene B protein encoded by the CBLB gene compared to non-engineered NK cells, reduced levels of tripartite motif-containing protein encoded by the TRIM29 gene compared to non-engineered NK cells, and reduced levels of the protein encoded by the SOCS2 gene compared to non-engineered NK cells. the NK cells are genetically engineered to express reduced levels of suppressor of cytokine signaling 2 protein, compared to non-engineered NK cells, genetically engineered to express reduced levels of SMAD3 protein encoded by the mother's decapentapentapentreg homolog 3 (SMAD3) gene, compared to non-engineered NK cells, genetically engineered to express reduced levels of MAPKAPK3 protein encoded by the MAP kinase-activated protein kinase 3 (MAPKAPK3) gene, compared to non-engineered NK cells, genetically engineered to express reduced levels of CEACAM1 protein encoded by the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) gene, compared to non-engineered NK cells, genetically engineered to express reduced levels of DDIT4 protein encoded by the DNA damage-inducible transcription factor 4 (DDIT4) gene, compared to non-engineered NK cells, genetically engineered to express CD47, and / or genetically engineered to express HLA-E, or any combination thereof.In some embodiments, the NK cells are further gene edited to disrupt expression of at least one immune checkpoint protein by the NK cells, in some embodiments, the at least one immune checkpoint protein is selected from CTLA4, PD-1, lymphocyte activation gene (LAG-3), NKG2A receptor, KIR2DL-1, KIR2DL-2, KIR2DL-3, KIR2DS-1 and / or KIR2DA-2, and combinations thereof.

[0047] In some embodiments, gene editing to decrease or increase expression is performed using a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system includes a Cas selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, CasX, and CasY, and combinations thereof. In some embodiments, the Cas is Cas9.

[0048] According to some embodiments, the CRISPR-Cas system comprises a Cas selected from Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, and combinations thereof. In some embodiments, the CD70 gene is edited using one or more guide RNAs having at least 95% sequence identity to SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123. In some embodiments, the CISH gene is edited using one or more guide RNAs having at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 134. In some embodiments, the TGFBR2 gene is edited using one or more guide RNAs that have at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 134.

[0049] In some embodiments, gene editing to reduce or induce expression is performed using zinc finger nucleases (ZFNs). In some embodiments, gene editing to reduce or induce expression is performed using transcription activator-like effector nucleases (TALENs).

[0050] Depending on the method, the cancer being treated is renal cell carcinoma or metastasis from renal cell carcinoma.

[0051] In some embodiments, the methods disclosed herein further comprise optionally administering a plurality of engineered T cells, wherein the T cells are engineered to express a CAR. In some embodiments, the CAR expressed by the T cells has a directionality for CD70.

[0052] In some embodiments, a polynucleotide encoding an anti-CD70 chimeric antigen receptor is provided, wherein the CAR comprises an anti-CD70 binding domain, wherein the anti-CD70 binding domain is encoded by a polynucleotide comprising a sequence having at least 95% sequence identity to one or more of SEQ ID NOs: 38-120, 221-229, 1038-1111, 1112-1185, and / or comprises an amino acid sequence having at least 95% sequence identity to one or more of the amino acid sequences of SEQ ID NOs: 230-312, 890-963, 964-1037. In some embodiments, the CAR comprises an OX40 subdomain encoded by a sequence having at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 5. In some embodiments, the OX40 subdomain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the CAR comprises a CD3 zeta domain encoded by a sequence having at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 7. In some embodiments, the CD3 zeta subdomain comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, a polynucleotide encoding mbIL15 is further provided, wherein mbIL15 is encoded by a sequence having at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 1188. In some embodiments, mbIL15 comprises an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1189. In some embodiments, one or more of SEQ ID NOs: 38-120, 221-229, 1038-1111, 1112-1185, a polynucleotide encoding OX40, a polynucleotide encoding CD3 zeta, and a polynucleotide encoding mbIL15 are arranged in a 5' to 3' direction within the polynucleotide.

[0053] Also provided herein is a method of increasing the persistence of an immune cell population used in cancer immunotherapy, comprising identifying a target marker on the tumor to be treated, determining whether the immune cell population to be engineered to express a CAR that binds to the target marker endogenously expresses the target marker, editing the genome of the immune cell population to disrupt the gene encoding the endogenous target marker, and engineering the immune cell population to express the CAR, wherein disruption of the endogenous expression of the target marker by the immune cells reduces the ability of the CAR to bind to the endogenous target marker on the immune cells, thereby increasing the persistence of the immune cell population.

[0054] In some embodiments, the immune cells are NK cells, T cells, or a combination thereof. In some embodiments, the target marker is CD70. In some embodiments, the gene editing is performed using a CRISPR-Cas system, where Cas is optionally guided to the endogenous gene by one or more of SEQ ID NOs: 121-123. In some embodiments, the CRISPR-Cas system disrupts expression of a cytokine-inducible SH2-containing (CIS) protein encoded by the CISH gene. In some embodiments, Cas is guided to the endogenous gene by one or more of SEQ ID NOs: 130-134. In some embodiments, an adenosine receptor, such as A2AR, is edited. In some embodiments, the CRISPR-Cas system is used to edit a gene encoding an adenosine receptor. In some embodiments, Cas is guided to an endogenous gene by one or more of SEQ ID NOs: 396-398. In some embodiments, SMAD3 is edited. In some embodiments, the CRISPR-Cas system is used to edit a gene encoding SMAD3. In some embodiments, Cas is guided to the endogenous gene by one or more of SEQ ID NOs: 1190-1192. In some embodiments, MAPKAPK3 is edited. In some embodiments, a CRISPR-Cas system is used to edit the gene encoding MAPKAPK3. In some embodiments, Cas is guided to the endogenous gene by one or more of SEQ ID NOs: 1193-1195. In some embodiments, CEACAM1 is edited. In some embodiments, a CRISPR-Cas system is used to edit the gene encoding CEACAM1. In some embodiments, Cas is guided to the endogenous gene by one or more of SEQ ID NOs: 1196-1198. In some embodiments, DDIT4 is edited. In some embodiments, a CRISPR-Cas system is used to edit the gene encoding DDIT4. In some embodiments, Cas is guided to the endogenous gene by one or more of SEQ ID NOs: 1199-1201.In some embodiments, a combination of one or more of the above genes is edited (optionally in combination with other genes that are edited as disclosed elsewhere herein).

[0055] In some embodiments, provided herein is an anti-CD70 chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD70 binding domain, an OX40 domain, and a CD3 zeta domain, and wherein the anti-CD70 CAR is encoded by a polynucleotide having at least 80%, 85%, 90%, or 95% sequence identity to one or more of SEQ ID NOs: 138-220, which also bicistronicly encodes mbIL15.

[0056] In some embodiments, provided herein is an anti-CD70 chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD70 binding domain, an OX40 domain, and a CD3 zeta domain, and wherein the anti-CD70 CAR comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to one or more of the amino acid sequences of SEQ ID NOs: 313-395, or a portion thereof (e.g., a portion excluding the mbIL15 sequence and / or a self-cleaving peptide sequence).

[0057] Some embodiments relate to methods comprising administering the immune cells described herein to a subject in need thereof, wherein the subject has cancer, hi some embodiments, the administration treats, inhibits, or prevents the progression of the cancer.

[0058] Some embodiments provide for the use of the cells, anti-CD70 scFvs, anti-CD70 CARs, and / or polynucleotides or amino acid sequences disclosed herein in the treatment or prevention of cancer.Some embodiments provide for the use of the cells, anti-CD70 scFvs, anti-CD70 CARs, and / or polynucleotides or amino acid sequences disclosed herein in the manufacture of a medicament for the treatment or prevention of cancer. [Brief explanation of the drawings]

[0059] [Figure 1] FIG. 1 shows a non-limiting schematic diagram of a tumor-tropic chimeric antigen receptor. [Figure 2] FIG. 2 shows a further non-limiting schematic diagram of a tumor-tropic chimeric antigen receptor. [Figure 3] FIG. 3 shows a further non-limiting schematic diagram of a tumor-tropic chimeric antigen receptor. [Figure 4] FIG. 4 shows a further non-limiting schematic diagram of a tumor-tropic chimeric antigen receptor. [Figure 5] FIG. 5 shows a further non-limiting schematic diagram of a tumor-tropic chimeric antigen receptor. [Figure 6] FIG. 6 shows a non-limiting schematic diagram of a tumor-tropic chimeric antigen receptor directed against non-limiting examples of tumor markers. [Figure 7] FIG. 7 shows a further non-limiting schematic diagram of a tumor-tropic chimeric antigen receptor directed against non-limiting examples of tumor markers. [Figure 8] 8A-8C show flow cytometry data regarding the extent of CD70 expression by natural killer cells. [Figure 9] FIG. 9 shows a non-limiting schematic process flow for the generation of engineered NK cells for use in therapeutic methods for cancer treatment, according to some embodiments disclosed herein. [Figure 10] 10A-10B show schematic diagrams of non-limiting examples of protocols for CRISPR-based modification and culture of engineered NK cells according to some embodiments disclosed herein. [Figure 11]Figures 11A-11D show flow cytometry data for CRISPR-mediated knockdown of CD70. Figure 11A shows data for CD70 expression on NK cells from a first donor after CRISPR-mediated knockdown using three different guide RNAs. Figure 11B shows corresponding control data for the first donor. Figure 11C shows data for CD70 expression on NK cells from a second donor after CRISPR-mediated knockdown using three different guide RNAs. Figure 11D shows corresponding control data for the first donor. These experiments were performed using the KD7 protocol. [Figure 12] Figures 12A-12E show flow cytometry data for CRISPR-mediated knockdown of CD70 expression on NK cells from additional donors. Figure 12A shows CD70 expression using guide RNA 1. Figure 12B shows CD70 expression using guide RNA 2. Figure 12C shows CD70 expression using guide RNA 3. Figure 12D shows CD70 expression by non-electroporated NK cells. Figure 12E shows unstained NK cells. These experiments were performed using the KD7 protocol. [Figure 13] Figures 13A-13D show flow cytometry data for CRISPR-mediated knockdown of CD70 expression on NK cells from the same donor as Figure 12, but using combinations of guide RNAs. Figure 13A shows CD70 expression using guide RNAs 1+2. Figure 13B shows CD70 expression using guide RNAs 1+3. Figure 13C shows CD70 expression using guide RNAs 2+3. Figures 13D and 13E are the same controls as those shown in Figures 12D and 12E. These experiments were performed using the KD7 protocol. [Figure 14]Figures 14A-14F show flow cytometry data for CD70 expression 14 days after CRISPR-mediated CD70 knockout (21 days total). Figure 14A shows CD70 expression using guide RNA 1. Figure 14B shows CD70 expression using guide RNA 2. Figure 14C shows CD70 expression using guide RNA 3. Figure 14D shows CD70 expression by non-electroporated NK cells. Figure 14E shows unstained NK cells. These experiments were performed using the KD7 protocol, with cells cultured in low IL-2 medium from day 11 to day 21. Figure 14F shows a further non-limiting gene editing process. [Figure 15] Figures 15A-15D show flow cytometry data for CD70 expression 14 days after CRISPR-mediated CD70 knockout (21 days total), using NK cells from the same donor as Figures 12-14, but using guide RNA combinations. Figure 15A shows CD70 expression using guide RNAs 1+2. Figure 15B shows CD70 expression using guide RNAs 1+3. Figure 15C shows CD70 expression using guide RNAs 2+3. Figures 15D and 15E are the same controls as those shown in Figures 14D and 14E. These experiments were performed using the KD7 protocol, with cells cultured in low IL-2 medium from days 11 to 21. [Figure 16] Figures 16A-16C show flow cytometry data for knockdown of CD70 expression in two different donors. For these experiments, CRISPR-mediated CD70 knockout was performed prior to NK cell expansion using the KD0 protocol, and data are shown from day 5 after electroporation. Figure 16A shows data from the first donor using each of the three guide RNAs alone. Figure 16B shows CD70 expression using the same individual guide RNAs but with NK cells from a different donor. Figure 16C shows control data (donor 2). [Figure 17]Figures 17A-17C show flow cytometry data for knockdown of CD70 expression in the same two donors as in Figure 16, with expression assessed after 8 days (day 13 of the KD0 protocol). Figure 17A shows data from the first donor using each of the three guide RNAs alone. Figure 17B shows CD70 expression using the same individual guide RNAs but with NK cells from a different donor. Figure 17C shows control data (donor 2). [Figure 18] Figures 18A-18D show data on cytotoxicity against CD70 knockout NK cells (CD70-KO-NK) derived from two donors. Figure 18A shows the cytotoxicity of CD70-KO-NK cells (generated using individual guide RNAs 1, 2, or 3 on NK cells derived from the first donor) against REH cells that do not express CD27, the ligand for CD70, at the indicated effector:target ratios. Figure 18B shows the cytotoxicity of CD70-KO-NK cells derived from donor 1 against Jurkat cells that express CD27. Figure 18C shows the cytotoxicity of CD70-KO-NK cells (NK cells derived from the second donor) against REH cells. Figure 18D shows the cytotoxicity of CD70-KO-NK cells derived from donor 2 against Jurkat cells. Cytotoxicity was assessed on day 14. [Figure 19] 19A-19B show a schematic timeline for the protocol to genetically engineer and expand NK cells, and data regarding NK cell expansion. [Figure 20] Figures 20A-20C show flow cytometry data after CRISPR gene editing of NK cells. Figure 20A shows CD70 expression by NK cells using three different guide RNAs. Figure 20B shows CD70 expression by NK cells from a second donor, edited using the same guide RNA. Figure 20C shows related control data. [Figure 21]Figures 21A-21B show flow cytometry data after CRISPR gene editing of Jurkat cells. Figure 21A shows CD70 expression by Jurkat cells from the first donor after editing with three different combinations of guide RNAs. Figure 21B shows control data. [Figure 22] Figures 22A-22D show flow cytometry data after CRISPR gene editing of Jurkat cells. Figure 22A shows the sample protocol. Figure 22B shows CD70 expression by Jurkat cells after CRISPR editing using three different guide RNA sets, where the cells are maintained in culture after gene editing. Figure 22C shows CD70 expression by Jurkat cells after CRISPR editing using the same guide RNA set, but the cells are frozen after gene editing and then thawed for flow analysis. Figure 22D shows control data. [Figure 23] Figure 23 shows a schematic of a non-limiting embodiment of a gene editing construct for CRISPR modification (knock-in) of the endogenous CD70 locus in NK cells to insert a CD70-directed CAR construct into the endogenous locus. [Figure 24]Figures 24A-24F present data assessing engineered expression of CD70 on Jurkat cells and Jurkat cells subjected to CRISPR gene editing. The left panel of each figure is a negative control, and the center panel is a control using secondary antibody only. Figure 24A shows data regarding CD70 and GFP expression on native Jurkat cells. The right panel of Figure 24A shows that native Jurkat cells express relatively low levels of CD70. Figure 24B shows CD70 expression in Jurkat cells engineered to express elevated markers (e.g., for sustained signaling and CD70 CAR screening purposes). As seen in the right panel of Figure 24B, CD70 expression is significantly increased on these Jurkat cells (approximately 85% of cells are positive for human CD70 and GFP (used as a marker for transduced cells)). Figure 24C shows the reduction of Jurkat native CD70 expression using first guide RNA and CRISPR gene editing. Figure 24D shows that engineered constitutive expression of CD70 on Jurkat cells is maintained in the face of CRISPR gene editing to reduce CD70. Figures 24E and 24F show similar data regarding the maintenance of CD70 expression on engineered Jurkat cells. [Figure 25] Figure 25 shows a table summarizing the CD70 expression data (MFI) for the various conditions shown in Figures 24A-24F. The data show that the engineered CD70-expressing Jurkat cells approach even higher constitutive expression of CD70 than 786-O cells, a known high-expressing cell line. [Figure 26]Figures 26A-26C relate to the expression of anti-CD70 CAR constructs on Jurkat cells. As non-limiting examples of anti-CD70 CARs, Jurkat cells were transduced with NK71 or NK72 constructs (schematically shown in Figure 6). The left panels of Figures 26A-26C are negative controls, the middle panels are secondary antibody controls, and the right panels show data for signals detected with an anti-Flag antibody. Figure 26A shows control data for the detection of Flag expression on Jurkat cells. In some embodiments, a Flag tag is used to detect expression of the CAR construct. In some embodiments, a Flag tag is not included. Figure 26B shows expression of an NK71 anti-CD70 CAR construct, and Figure 26C shows expression of an NK72 anti-CD70 CAR construct by Jurkat cells. [Figure 27]Figures 27A-27F show data on the ability of anti-CD70 CAR-expressing Jurkat cells to bind to human CD70. Figure 27A shows data from native Jurkat cells. Migration from left to right is a negative control, a control for an antibody against human Fc, binding when 2 μg / ml of CD70-hFc complex is incubated with Jurkat cells, binding detected with human Fc-APC antibody, and binding when 10 μg / ml of CD70-hFc complex is incubated with Jurkat cells, binding detected with human Fc-APC antibody. Figure 27B shows data from native Jurkat cells. The shift from left to right is a control for an antibody against mouse Fc, which binds when 2 μg / ml of CD70-mouse Fc complex is incubated with Jurkat cells, and binding is detected with mouse Fc-APC antibody; the shift from left to right is a control for an antibody against mouse Fc, which binds when 10 μg / ml of CD70-mouse Fc complex is incubated with Jurkat cells, and binding is detected with mouse Fc-APC antibody, and Flag tag (present in the NK71 / 72 CAR construct but not in native Jurkat cells). Figure 27C shows data from NK71-expressing Jurkat cells. The shift from left to right is a negative control for an antibody against human Fc, which binds when 2 μg / ml of CD70-hFc complex is incubated with Jurkat cells, and binding is detected with human Fc-APC antibody; the shift from left to right is a control for an antibody against human Fc, which binds when 2 μg / ml of CD70-hFc complex is incubated with Jurkat cells, and binding is detected with human Fc-APC antibody; the shift from left to right is a control for an antibody against human Fc, which binds when 10 μg / ml of CD70-hFc complex is incubated with Jurkat cells, and binding is detected with human Fc-APC antibody. Figure 27D shows data from NK71-expressing Jurkat cells. The shift from left to right is a control for antibodies against mouse Fc, binding when 2 μg / ml of CD70-mouse Fc complex is incubated with Jurkat cells, binding detected with mouse Fc-APC antibody, binding when 10 μg / ml of CD70-mouse Fc complex is incubated with Jurkat cells, binding detected with mouse Fc-APC antibody, and Flag tag (present in the NK71 / 72 CAR construct but not in native Jurkat cells). Figure 27E shows data from Jurkat cells expressing NK72.The shift from left to right is a negative control, a control for antibodies against human Fc, binding when 2 μg / ml of CD70-hFc complex is incubated with Jurkat cells, binding detected with human Fc-APC antibody, and binding when 10 μg / ml of CD70-hFc complex is incubated with Jurkat cells, binding detected with human Fc-APC antibody. Figure 27F shows data from Jurkat cells expressing NK72. The shift from left to right is a control for antibodies against mouse Fc, binding when 2 μg / ml of CD70-mouse Fc complex is incubated with Jurkat cells, binding detected with mouse Fc-APC antibody, and binding when 10 μg / ml of CD70-mouse Fc complex is incubated with Jurkat cells, binding detected with mouse Fc-APC antibody and Flag tag (present in the NK71 / 72 CAR construct but not in native Jurkat cells). [Figure 28] Figures 28A-28E show data on CRISPR-mediated gene editing of two targets in NK cells. Figure 28A shows a non-limiting example of an electroporation protocol. In these figures, expression of NKG2A (in combination with CD70) and TGFBR2 (in combination with CD70) was assessed. Knockout of CD70 expression is discussed in more detail below. Figure 28B shows expression of NKG2A after NK cells were exposed to CRISPR / guide RNA targeting NKG2A. Control data are shown in Figure 28C. Figure 28D shows expression of TGFBR2 after NK cells were exposed to CRISPR / guide RNA targeting TGFBR2. Figure 28E shows control data. [Figure 29]Figures 29A-29J show data on the expression of CAR constructs 4 days (11 days after electroporation) after CRISPR-mediated gene editing of two targets in NK cells and subsequent transduction with a CAR-encoding vector. Figure 29A shows the expression of a non-limiting embodiment of a CAR targeting CD70 after NK cells are exposed to CD70 gRNA. Figure 29B shows knockdown of CD70 expression on NK cells via editing with CD70 gRNA. Figure 29C shows anti-CD70 CAR expression on NK cells after NK cells are exposed to CD70 and CISH gRNA. Figure 29D shows knockdown of CD70 expression on NK cells via editing with CD70 gRNA and CISH gRNA. Figure 29E shows anti-CD70 CAR expression on NK cells after NK cells are exposed to CD70 and NKG2A gRNA. Figure 29F shows knockdown of CD70 expression on NK cells via editing with CD70 gRNA and NKG2A gRNA. Figure 29G shows anti-CD70 CAR expression on NK cells after exposure to CD70 and TGFBR2 gRNA. Figure 29H shows knockdown of CD70 expression on NK cells via editing with CD70 and TGFBR2 gRNA. Figure 29I shows mock control data for anti-CD70 expression on NK cells. Figure 29J shows mock control data for CD70 expression on NK cells. [Figure 30]Figures 30A-30J show data on the expression of CAR constructs 11 days (18 days after electroporation) after CRISPR-mediated gene editing of two targets in NK cells and subsequent transduction with a CAR-encoding vector. Figure 30A shows the expression of a non-limiting embodiment of a CAR targeting CD70 after NK cells are exposed to a CD70 gRNA. Figure 30B shows knockdown of CD70 expression on NK cells via editing with a CD70 gRNA. Figure 30C shows anti-CD70 CAR expression on NK cells after NK cells are exposed to CD70 and CISH gRNA. Figure 30D shows knockdown of CD70 expression on NK cells via editing with a CD70 gRNA and CISH gRNA. Figure 30E shows anti-CD70 CAR expression on NK cells after NK cells are exposed to CD70 and NKG2A gRNA. Figure 30F shows knockdown of CD70 expression on NK cells via editing with CD70 gRNA and NKG2A gRNA. Figure 30G shows anti-CD70 CAR expression on NK cells after exposure to CD70 and TGFBR2 gRNA. Figure 30H shows knockdown of CD70 expression on NK cells via editing with CD70 gRNA and TGFBR2 gRNA. Figure 30I shows mock control data for anti-CD70 expression on NK cells. Figure 30J shows mock control data for CD70 expression on NK cells. [Figure 31]Figures 31A-31C show summary expression data for a first non-limiting embodiment of an anti-CD70 CAR on NK cells subjected to gene editing knockdown of one or more targets, assessed 11 days post-transduction. Figure 31A shows expression levels of a first non-limiting anti-CD70 CAR (NK71) on NK cells treated with CD70 gRNA, CD70 and CISH gRNA, CD70 and NKG2A gRNA, CD70 and TGFBR2 gRNA, or GFP. Data are the percentage of NK cells positive for the FLAG tag in the NK71 CAR construct (some embodiments do not use a FLAG tag). Figure 31B shows the underlying raw mean fluorescence intensity (MFI) data. Figure 31C shows data regarding the degree of CD70 knockout in NK cells with the indicated gRNAs. [Figure 32] Figures 32A-32C show summary expression data for a second non-limiting embodiment of an anti-CD70 CAR on NK cells subjected to gene editing knockdown of one or more targets, assessed 11 days post-transduction. Figure 32A shows expression levels of a first non-limiting anti-CD70 CAR (NK72) on NK cells treated with CD70 gRNA, CD70 and CISH gRNA, CD70 and NKG2A gRNA, CD70 and TGFBR2 gRNA, or GFP. Data are the percentage of NK cells positive for the FLAG tag in the NK72 CAR construct (some embodiments do not use a FLAG tag). Figure 32B shows the underlying raw mean fluorescence intensity (MFI) data. Figure 32C shows data regarding the degree of CD70 knockout in NK cells with the indicated gRNAs. [Figure 33]Figures 33A-33B show data regarding changes in NK cell proliferation based on knockout of different targets in NK cells, where the NK cells are engineered to express an anti-CD70 CAR. Figure 33A shows data showing that knockdown of CD70 expression and engineered expression of an anti-CD70 CAR (this experiment used the non-limiting NK71 construct), as well as knockdown of CISH expression, results in increased NK cell proliferation (compared to knockdown of CD70 alone). Similar data is shown when CD70 and TGFRB2 are knocked out. In contrast, knockdown of NKG2A results in decreased proliferation (compared to CD70 knockout alone). Figure 33B shows corresponding data on proliferation when NK cells are subjected to the same gene editing procedure but transduced with a construct encoding a non-limiting example anti-CD70 CAR, NK72. [Figure 34] Figures 34A-34B show data on NK cell survival based on knockout of different targets in NK cells, engineering the NK cells to express an anti-CD70 CAR. Figure 34A shows survival data for NK cells engineered to express the non-limiting NK71 anti-CD70 CAR for 35 days (post-electroporation). As shown, double knockout of CD70 and TGFBR2 results in slightly improved survival over the experimental time course (compared to CD70 knockout). Double knockout of CD70 and CISH results in significantly increased survival, with an approximately two-fold increase in survival (compared to CD70 alone). Knockout of NKG2A results in decreased survival, with the NK population gradually declining from day 21 to day 35. The decreased survival of this group makes the CD70-CISH double knockout nearly three times that of the NKG2A group. Figure 34B shows a similar trend between groups for the non-limiting NK72 anti-CD70 CAR. These data also suggest that, according to some embodiments, the NK71 construct provides increased survival compared to the NK72 construct. [Figure 35]Figures 35A-35D show data on the cytotoxicity of NK cells expressing an anti-CD70 CAR and subjected to knockout of various NK cell regulatory targets. Figure 35A shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK71 anti-CD70 CAR against 786-O cells, which express high levels of CD70, at an E:T ratio of 1:1. Figure 35B shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK71 anti-CD70 CAR against 786-O cells at an E:T ratio of 1:2. Figure 35C shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK72 anti-CD70 CAR against 786-O cells at an E:T ratio of 1:1. Figure 35B shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK72 anti-CD70 CAR against 786-O cells at an E:T ratio of 1:2. Data were collected 7 days after transduction. [Figure 36] Figures 36A-36D show data regarding the cytotoxicity of NK cells expressing an anti-CD70 CAR and subjected to knockout of various NK cell regulatory targets. Figure 36A shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK71 anti-CD70 CAR on ACHN cells expressing low levels of CD70 at an E:T ratio of 1:1. Figure 36B shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK71 anti-CD70 CAR on ACHN cells at an E:T ratio of 1:2. Figure 36C shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK72 anti-CD70 CAR on ACHN cells at an E:T ratio of 1:1. Figure 36D shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK72 anti-CD70 CAR on ACHN cells at an E:T ratio of 1:2. Data were collected 7 days post-transduction. [Figure 37]Figures 37A-37B show data on the cytotoxicity of NK cells expressing an anti-CD70 CAR and subjected to knockout of various NK cell regulatory targets. Figure 37A shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK71 anti-CD70 CAR against 786-O cells, which express high levels of CD70, at an E:T ratio of 1:2. Figure 37B shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK71 anti-CD70 CAR against 786-O cells at an E:T ratio of 1:4. Data were collected 14 days post-transduction. [Figure 38] Figures 38A-38B show data on the cytotoxicity of NK cells expressing an anti-CD70 CAR and subjected to knockout of various NK cell regulatory targets. Figure 38A shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK71 anti-CD70 CAR against ACHN cells expressing low levels of CD70 at an E:T ratio of 1:1. Figure 38B shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK71 anti-CD70 CAR against ACHN cells at an E:T ratio of 1:2. Data were collected 14 days post-transduction. [Figure 39] Figures 39A-39B show data on the cytotoxicity of NK cells expressing an anti-CD70 CAR and subjected to knockout of various NK cell regulatory targets. Figure 39A shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK72 anti-CD70 CAR against 786-O cells, which express high levels of CD70, at an E:T ratio of 1:2. Figure 39B shows the cytotoxic effect of engineered NK cells expressing a non-limiting NK72 anti-CD70 CAR against 786-O cells at an E:T ratio of 1:4. Data were collected 14 days post-transduction. [Figure 40]Figures 40A-40B show data regarding the cytotoxicity of NK cells expressing an anti-CD70 CAR and subjected to knockout of various NK cell regulatory targets. Figure 40A shows the cytotoxic effect of engineered NK cells expressing the non-limiting NK72 anti-CD70 CAR against ACHN cells expressing low levels of CD70 at an E:T ratio of 1:1. Figure 40B shows the cytotoxic effect of engineered NK cells expressing the non-limiting NK71 anti-CD70 CAR against ACHN cells at an E:T ratio of 1:2. Data were collected 14 days post-transduction. [Figure 41]Figures 41A-41O relate to gene editing protocols and assessment of expression of various editing targets, as well as expression of an anti-CD70 CAR. Figure 41A shows a non-limiting embodiment of the gene editing protocol used. Figure 41B shows an unstained control (no anti-CD70 antibody) representing background signal. Figure 41B shows a control in which CD70 expression was measured on NK cells transduced with a CAR that does not target CD70 and does not contain a CD70 subunit. This represents baseline NK cell CD70 expression. Figure 41D shows CD70 expression on NK cells subjected to gene editing to knock out CD70 expression. Figure 41E shows expression of a non-limiting NK71 anti-CD70 CAR on NK cells in which CD70 expression has been knocked out. Figure 41F shows CD70 expression on NK cells in which CD70 expression has been knocked out. Figure 41G shows expression of a non-limiting NK72 anti-CD70 CAR on NK cells in which CD70 expression has been knocked out. Figure 41H shows CD70 expression on NK cells subjected to gene editing to knock out CD70 and CISH expression. Figure 41F shows the expression of a non-limiting NK71 anti-CD70 CAR on NK cells in which CD70 and CISH expression have been knocked out. Figure 41J shows CD70 expression on NK cells subjected to gene editing to knock out CD70 and CISH expression. Figure 41GK shows the expression of a non-limiting NK72 anti-CD70 CAR on NK cells in which CD70 and CISH expression have been knocked out. Figure 41L shows CD70 expression on NK cells subjected to electroporation alone as a control. Figure 41M shows the expression of a non-limiting NK71 anti-CD70 CAR on NK cells subjected to electroporation alone as a control. Figure 41N shows CD70 expression on NK cells subjected to electroporation alone as a control. Figure 41O shows, as a control, the expression of non-limiting NK72 anti-CD70 CAR on NK cells subjected to electroporation alone. [Figure 42]Figures 42A-42C relate to cytotoxicity data for NK cells that have been subjected to gene editing and engineered to express an anti-CD70 CAR. Figure 42A shows the cytotoxicity of NK cells treated in the indicated manner against 786-O cells expressing high levels of CD70, assessed at an E:T ratio of 1:2, 7 days after transduction. Figure 42B shows the cytotoxicity of NK cells treated in the indicated manner against ACHN cells expressing low levels of CD70, assessed at an E:T ratio of 1:2, 7 days after transduction. Figure 42C shows the cytotoxicity of NK cells treated in the indicated manner against 786-O cells expressing high levels of CD70, assessed at an E:T ratio of 1:2, 7 days after transduction. [Figure 43]Figures 43A-43I relate to CD70 expression on NK cells (7 days after electroporation) using various guide RNAs. Figure 43A shows CD70 expression levels by NK cells subjected to gene editing to knock out CD70 expression using gRNA1. Figure 43B shows CD70 expression levels by NK cells subjected to gene editing to knock out CD70 expression using gRNA2. Figure 43C shows CD70 expression levels by NK cells subjected to gene editing to knock out CD70 expression using gRNA3. Figure 43D shows CD70 expression levels by NK cells subjected to gene editing to knock out CD70 expression using gRNA1 and 3. Figure 43E shows CD70 expression levels by NK cells subjected to gene editing to knock out CD70 expression using gRNA1 and 2. Figure 43F shows CD70 expression levels by NK cells subjected to gene editing to knock out CD70 expression using gRNA2 and 3. Figure 43G shows CD70 expression levels by NK cells subjected to gene editing to knock out CD70 expression using gRNA1 (for CD70) and a guide RNA to knock out CISH. Figure 43H shows CD70 expression levels by NK cells subjected to gene editing to knock out CD70 expression using gRNA1 (for CD70) and a guide RNA to knock out adenosine receptor (A2AR). Figure 43I shows control data from NK cells subjected to electroporation alone (no gene editing enzyme or guide RNA). [Figure 44]Figures 44A-44D relate to cytotoxicity assessments of NK cells subjected to various gene editing protocols and engineered to express an anti-CD70 CAR (here, the anti-CD70 CAR, NK71, is used as a non-limiting example). Figure 44A shows the cytotoxicity of the indicated NK cells against Reh tumor cells at either a 1:1 or 1:2 effector:target ratio. Figure 44B shows a summary histogram of cytotoxicity at 1:1. Figure 44C shows a summary histogram of cytotoxicity at 1:2. Figure 44D shows the cytotoxicity of the indicated constructs against Nalm-6 tumor cells. [Figure 45] Figures 45A-45B show cytotoxicity assessments of NK cells subjected to various gene editing protocols and engineered to express an anti-CD70 CAR (here, the anti-CD70 CAR, NK71, is used as a non-limiting example). Figure 45A shows the cytotoxicity of the indicated NK cells against Reh tumor cells at an E:T ratio of 1:1. Figure 45B shows the cytotoxicity of the indicated NK cells against Reh tumor cells at an E:T ratio of 1:2. [Figure 46]Figures 46A-46J show data regarding the expression of an anti-CD70 CAR in NK cells subjected to gene editing to knock out native CD70 expression (and / or CISH or adenosine receptor expression), as well as the expression of native CD70 by NK cells. Figure 46A shows data regarding anti-CD70 CAR expression and native CD70 expression levels by NK cells subjected to gene editing with gRNA1 (for CD70). Figure 46B shows data regarding anti-CD70 CAR expression and native CD70 expression levels by NK cells subjected to gene editing with gRNA2 (for CD70). Figure 46C shows data regarding anti-CD70 CAR expression and native CD70 expression levels by NK cells subjected to gene editing with gRNA3 (for CD70). Figure 46D shows data regarding anti-CD70 CAR expression and native CD70 expression levels by NK cells subjected to gene editing with gRNA1 and 3 (both for CD70). Figure 46E shows data on anti-CD70 CAR expression and native CD70 expression levels by NK cells subjected to gene editing with gRNA2 and 3 (both for CD70). Figure 46F shows data on anti-CD70 CAR expression and native CD70 expression levels by NK cells subjected to gene editing with gRNA1 and 2 (both for CD70). Figure 46G shows data on anti-CD70 CAR expression and native CD70 expression levels by NK cells subjected to gene editing with gRNA1 (for CD70) and an additional gRNA to knock out CISH. Figure 46H shows data on anti-CD70 CAR expression and native CD70 expression levels by NK cells subjected to gene editing with gRNA1 (for CD70) and an additional gRNA to knock out adenosine receptor (A2AR). Figure 46I shows control data in which NK cells were subjected to electroporation alone, but without gRNA and engineered CAR expression. Figure 46J shows control data using non-transduced NK cells. [Figure 47]Figures 47A-47F show cytotoxicity data for anti-CD70 CAR-expressing and gene-edited NK cells against tumor cells. Figure 47A shows cytotoxicity data for the indicated NK cells against 786-O cells at an E:T ratio of 1:2. Figure 47B shows cytotoxicity data for the indicated NK cells against 786-O cells at an E:T ratio of 1:4. Figure 47C shows cytotoxicity data for the indicated NK cells against 786-O cells at an E:T ratio of 1:8. Figure 47D shows cytotoxicity data for the indicated NK cells against ACHN cells at an E:T ratio of 1:2. Figure 47E shows cytotoxicity data for the indicated NK cells against ACHN cells at an E:T ratio of 1:4. Figure 47F shows cytotoxicity data for the indicated NK cells against ACHN cells at an E:T ratio of 1:8. [Figure 48] Figures 48A-48D show cytotoxicity data from various gene-edited NK cells that were also engineered to express an anti-CD70 CAR against tumor cells. Figure 48A shows cytotoxicity data from the indicated gene-edited NK cells expressing an anti-CD70 CAR at a 1:1 ratio to 786-O cells, with data collected 72 hours after co-culture with tumor cells. Figure 48B shows summary data for GFP detection, a surrogate for the number of viable tumor cells, measured as a percent of baseline GFP detection (time zero). Figure 48C shows cytotoxicity data from the indicated gene-edited NK cells expressing an anti-CD70 CAR at a 1:2 ratio to 786-O cells, with data collected 72 hours after co-culture with tumor cells. Figure 48D shows summary data for GFP detection, a surrogate for the number of viable tumor cells, measured as a percent of baseline GFP detection (time zero). [Figure 49]Figures 49A-49D show additional gene schematics and knockout data from various gene-edited NK cells that were also engineered to express an anti-CD70 CAR against tumor cells. Figure 49A shows a schematic in which NK cells are first gene-edited (e.g., using CRISPR) to knock out both CD70 and the target gene, expanded, transduced with an NK71 anti-CD70 CAR construct (a non-limiting example of a CAR according to the present disclosure), and then assayed for tumor-killing efficacy. Figure 49B shows the consistent CD70 expression percentage at day 7 following CD70 and target gene double knockout for different target gene conditions. Figure 49C shows essentially undetectable CD70 expression at day 10 following dual CD70 and target gene double knockout under different target gene conditions and consistent expression of the non-limiting NK71 CAR. Figure 49D shows PCR amplification data and amplicon indel frequency of the target locus for the knockout NK cell population. Figures 49E-49G show cytotoxicity data for NK cell genes edited for SMAD3. Figure 49E shows successful knockout of SMAD3 in NK cell populations. Figure 49F shows cytotoxicity data from NK cells expressing SMAD3 or CISH gene-edited NK71 CARs against 786-O cells at a 1:4 ratio with or without treatment with 20 ng / mL TGFb for up to 6.5 days. Figure 49G shows the percentage of 786-O cells remaining relative to the starting amount 3 days after treatment with NK cells expressing a non-limiting example of a SMAD3 or CISH gene-edited CAR, NK71, at a 1:1 ratio with or without treatment with 20 ng / mL TGFb. Figures 49H-49I show cytotoxicity data for NK cell genes edited for A2AR. Figure 49H shows cytotoxicity data from NK cells expressing A2AR or CISH gene edited, NK71 CAR against 786-O cells at a 1:4 ratio with or without treatment with 10 μM NECA for up to 6 days.Figure 49I shows the percentage of 786-O cells remaining relative to the starting dose 3 days after treatment with NK cells expressing A2AR or CISH gene-edited NK71 CAR at a 1:1 ratio, with or without treatment with 10 μM NECA. Figures 49J-49K show cytotoxicity data for NK cell genes edited for MAPKAPK3. Figure 49J shows cytotoxicity data from NK cells expressing MAPKAPK3 (MK3) or CISH gene-edited NK71 CAR against 786-O cells at a 1:2 ratio for up to 94 hours. Figure 49K shows the percentage of 786-O cells remaining relative to the starting dose 3 days after treatment with NK cells expressing MK3 or CISH gene-edited NK71 CAR at a 1:1 ratio. Figures 49L-49P show cytotoxicity data for NK cell genes edited for NKG2A. Figure 49L shows cytotoxicity data from NK cells expressing NKG2A or CISH gene-edited, NK71 CAR against 786-O cells at a 1:1 ratio for up to 72 hours. Figure 49M shows cytotoxicity data from NK cells expressing NKG2A, MK3, or CISH gene-edited, NK71 CAR against 786-O cells at a 1:1 ratio for up to 94 hours. Figure 49N shows cytotoxicity data from NK cells expressing NKG2A, MK3, or CISH gene-edited, NK71 CAR against 786-O cells at a 1:1 ratio for up to 94 hours. Figure 49O shows cytotoxicity data from NK cells expressing NKG2A or CISH gene-edited, NK71 CAR against 786-O cells at a 1:1 ratio with or without treatment with 20 ng / mL TGFb for up to 72 hours. Figure 49P shows cytotoxicity data from NKG2A or CISH gene-edited NK cells expressing NK71 CAR against 786-O cells at a 1:2 ratio with or without treatment with 10 μM NECA for up to 54 hours. Figures 49Q-49R show cytotoxicity data for NK cell genes edited for DDIT4.Figure 49Q shows cytotoxicity data from NK cells expressing DDIT4 or CISH gene-edited NK71 CAR against 786-O cells at a 1:1 ratio with or without treatment with 50 μM CoCl2 for up to 94 hours. Figure 49R shows the percentage of remaining 786-O cells relative to the starting amount 3 days after treatment with NK cells expressing DDIT4 or CISH gene-edited NK71 CAR at a 1:1 ratio with or without treatment with 50 μM CoCl2. Figures 49S-49T show cytotoxicity data for NK cell genes edited for CEACAM1. Figure 49S shows cytotoxicity data from NK cells expressing CEACAM1 or CISH gene-edited NK71 CAR against 786-O cells at a 1:2 ratio with or without treatment with 1 μg / mL CEACAM5 for up to 72 hours. Figure 49T shows the percentage of surviving 786-O cells relative to the starting amount 3 days after treatment with NK cells expressing CEACAM1 or CISH gene edited NK71 CAR at a 1:2 ratio, with or without treatment with 1 μg / mL CEACAM5. Figures 49U and 49V show survival over 49 days for NK cells expressing NK71 CAR gene edited for CD70 and various indicated gene targets. [Figure 50]Figure 50A shows exemplary heavy chain variable region (VH) and light chain variable region (VL) peptide and nucleic acid sequences for selected anti-CD70 scFvs disclosed herein. The sequences disclosed herein can be used in any of the embodiments disclosed herein. Figure 50B shows exemplary heavy chain variable region and light chain variable region complementarity-determining regions (CDRs) for selected anti-CD70 scFvs disclosed herein. In some embodiments, other combinations of CDRs can be used to prepare other anti-CD70 scFvs or other binding domains. The CDRS disclosed herein can be used in any of the embodiments disclosed herein. Figure 50C shows a schematic for selecting a CAR comprising an anti-CD70 binding domain based on tonic signaling and immune cell activation. Figure 50D shows data related to the extent of tonic signaling in a Jurkat lineage. Figure 50E shows CAR activation relative to tonic signaling, further ordered by overall CAR expression, in Jurkat cells expressing the disclosed anti-CD70 CARs. The circled dots indicate selected, non-limiting embodiments of anti-CD70 CAR constructs that result in significant on-target activation versus sustained signaling. Figure 50F shows a non-limiting list of 10 anti-CD70 CAR constructs selected for desirable activation and limited / minimal sustained signaling effects in Jurkat cells. These constructs were tested in additional assays disclosed herein. [Figure 51] Figures 51A-51B show the expression levels of tested anti-CD70 CARs in donor NK cell populations gene-edited to knock out CD70. Figure 51A shows flow cytometry plots detecting CAR expression (with anti-FLAG antibody conjugated with allophycocyanin (APC)) and loss of CD70 expression (with anti-CD70 antibody conjugated with phycoerythrin (PE)). Figure 51B shows quantification of anti-CD70 CAR and CD70 expression in the NK cell populations in Figure 51A. NK8 refers to a control construct expressing GFP instead of CAR / mbIL15. [Figure 52] Figures 52A-52D show the expression levels and preliminary cytotoxicity assays of tested anti-CD70 CARs in different donor NK cell populations edited to knock out CD70. Figure 52A shows flow cytometry plots detecting CAR expression (by anti-FLAG antibody conjugated with APC) and loss of CD70 expression (by anti-CD70 antibody conjugated with PE). Figure 52B shows quantification of anti-CD70 CAR and CD70 expression in the NK cell population of Figure 52A. Figure 52C shows the raw mean fluorescence intensity (MFI) used to quantify CAR expression. Figure 52D shows the cytotoxicity assay of the anti-CD70 CAR NK cell population against 786-O tumor cells at different effector:target (E:T) ratios and the EC50 calculated from the assay. [Figure 53] Figures 53A-53F show cytotoxicity data of tested anti-CD70 CARs on CD70 knockout NK cells. Figure 53A shows cytotoxicity data of NK cells tested against 786-O cells at a 1:2 ratio for up to 7 days. Figure 53B shows the remaining 786-O cells at 73.5 hours after 1:2 NK cell co-culture, as measured by 786-O GFP fluorescence. Figure 53C shows the cytotoxicity data of the tested NK cells in Figure 53A, but extended to 10 days. On day 7, the cultures were re-challenged with additional tumor cells. Figure 53D shows the cytotoxicity data of NK cells tested against 786-O cells at a 1:4 ratio for up to 7 days. Figure 53E shows the remaining 786-O cells at 73.5 hours after 1:4 NK cell co-culture, as measured by 786-O GFP fluorescence. Figure 53F shows the cytotoxicity data of NK cells tested against 786-O cells at a ratio of 1:8 for up to 7 days. [Figure 54]Figures 54A-54D show cytotoxicity data of anti-CD70 CARs tested on CD70 knockout NK cells against either 786-O cells or ACHN cells. Figure 54A shows cytotoxicity data for NK cells tested against 786-O cells at a 1:2 ratio for up to 5 days. 786-O cells express GFP. Figure 54B shows cytotoxicity data for NK cells tested against ACHN cells at a 1:2 ratio for up to 5 days. ACHN cells are stained with NucRed. Figure 54C shows cytotoxicity data for NK cells tested against 786-O cells at a 1:4 ratio for up to 5 days. Figure 54D shows cytotoxicity data for NK cells tested against ACHN cells at a 1:4 ratio for up to 5 days. [Figure 55]Figures 55A-55M show cytotoxicity data of tested anti-CD70 CARs on CD70 knockout NK cells from one donor. Figure 55A shows flow cytometry plots detecting CAR expression (by APC anti-FLAG) and loss of CD70 expression (by PE anti-CD70). Figure 55B shows preliminary cytotoxicity data for tested NK cells against 786-O cells at different E:T ratios at 4 hours of coculture. Figure 55C shows quantification of expressed anti-CD70 CAR, loss of CD70 expression, and the EC50 calculated from the assay in Figure 55B. Figure 55D shows cytotoxicity data for tested NK cells against 786-O cells at a 1:2 ratio for up to 64 hours. Figure 55E shows cytotoxicity data for tested NK cells against 786-O cells at a 1:2 ratio for up to 6 days. Figure 55F shows cytotoxicity data of NK cells tested against 786-O cells at a 1:2 ratio for up to 7 days. Figure 55G shows cytotoxicity data of NK cells tested as in Figure 55F, but extended to 11 days and re-challenged with additional tumor cells on day 7. Figure 55H shows remaining 786-O cells 51 hours after 1:2 NK cell co-culture as measured by 786-O GFP fluorescence. Figure 55I shows remaining 786-O cells 66 hours after 1:2 NK cell co-culture as measured by 786-O GFP fluorescence. Figure 55J shows cytotoxicity data of NK cells tested against 786-O cells at a 1:4 ratio for up to 64 hours. Figure 55K shows cytotoxicity data of NK cells tested against 786-O cells at a 1:4 ratio for up to 6 days. Figure 55L shows cytotoxicity data of NK cells tested against 786-O cells at a 1:4 ratio for up to 7 days. Figure 55M shows cytotoxicity data of NK cells tested against 786-O cells at a 1:4 ratio for up to 11 days. On day 7, cultures were re-challenged with additional tumor cells. [Figure 56]Figures 56A-56J show cytotoxicity data of tested anti-CD70 CARs on CD70 knockout NK cells from another donor. Figure 56A shows flow cytometry plots detecting CAR expression (by APC anti-FLAG) and loss of CD70 expression (by PE anti-CD70). Figure 56B shows preliminary cytotoxicity data for tested NK cells against 786-O cells at different E:T ratios at 4 hours of coculture. Figure 56C shows quantification of expressed anti-CD70 CAR, loss of CD70 expression, and calculated EC50 from the assay in Figure 56B. Figure 56D shows cytotoxicity data for tested NK cells against 786-O cells at a 1:2 ratio for up to 5.75 days. Figure 56E shows cytotoxicity data for tested NK cells, as seen in Figure 56H, but extended to 10 days and re-challenged with additional tumor cells on day 6. Figure 56F shows the cytotoxicity data of tested NK cells against 786-O cells at a 1:4 ratio for up to 42 hours. Figure 56G shows the cytotoxicity data of tested NK cells against 786-O cells at a 1:4 ratio for up to 5 days. Figure 56H shows the cytotoxicity data of tested NK cells against 768-O cells at a 1:4 ratio for up to 5.75 days. Figure 56I shows the cytotoxicity data of tested NK cells against 786-O cells at a 1:8 ratio for up to 42 hours. Figure 56J shows the cytotoxicity data of tested NK cells against 786-O cells at a 1:8 ratio for up to 5 days. [Figure 57]Figures 57A-57H show cytotoxicity data of tested anti-CD70 CARs in CD70 knockout NK cells from one donor against ACHN cells. Figure 57A shows cytotoxicity data of tested NK cells against ACHN cells at a 1:2 ratio for up to 5 days. At approximately 3.75 days, cultures were re-challenged with additional tumor cells. Figure 57B shows the cytotoxicity data of Figure 57A, but cell numbers are normalized to day 0. Figure 57C shows the cytotoxicity data of tested NK cells in Figure 57B over a longer period after re-challenge. Figure 57D shows the remaining ACHN cells after 51 hours of NK cell 1:2 co-culture, as measured by ACHN fluorescence. Figure 57E shows the remaining ACHN cells after 66 hours of NK cell 1:2 co-culture, as measured by ACHN fluorescence. Figure 57F shows cytotoxicity data of the tested NK cells against ACHN cells at a 1:4 ratio for up to 5 days. Cultures were re-challenged with additional tumor cells approximately 3.75 days later. Figure 57G shows the cytotoxicity data of Figure 57F, but cell numbers are normalized to day 0. Figure 57H shows the cytotoxicity data of the tested NK cells of Figure 57G over a longer period after re-challenge. [Figure 58]Figures 58A-58H show cytotoxicity data of tested anti-CD70 CARs in CD70 knockout NK cells from another donor against ACHN cells. Figure 58A shows cytotoxicity data of tested NK cells against ACHN cells at a 1:4 ratio for up to 5 days. After approximately 3.75 days, cultures were re-challenged with additional tumor cells. Figure 58B shows the cytotoxicity data of Figure 58A, but cell numbers are normalized to day 0. Figure 58C shows cytotoxicity data of tested NK cells in Figure 58B over a longer period after re-challenge. Figure 58D shows remaining ACHN cells after 51 hours of 1:4 NK cell co-culture as measured by ACHN fluorescence. Figure 58E shows remaining ACHN cells after 66 hours of 1:4 NK cell co-culture as measured by ACHN fluorescence. Figure 58F shows cytotoxicity data of tested NK cells against ACHN cells at a 1:8 ratio for up to 5 days. Cultures were re-challenged with additional tumor cells approximately 3.75 days later. Figure 58G shows the cytotoxicity data of Figure 58D, but cell numbers are normalized to day 0. Figure 58H shows the cytotoxicity data of the tested NK cells of Figure 58G over a longer period after re-challenge. [Figure 59] Figures 59A-59D show expression levels and preliminary cytotoxicity data for additional anti-CD70 CARs in NK cell genes edited to knock out CD70 in one donor. Figure 59A shows flow cytometry plots detecting CAR expression (with APC anti-FLAG) and loss of CD70 expression (with PE anti-CD70). Figure 59B shows quantification of anti-CD70 CAR and CD70 expression in the NK cell population in Figure 59A. Figure 59C shows the MFI used to quantify CAR expression. Figure 59D shows a cytotoxicity assay of the anti-CD70 CAR NK cell population against 786-O tumor cells at different effector:target ratios and the EC50 calculated from this assay. [Figure 60]Figures 60A-60O show cytotoxicity data of tested anti-CD70 CARs in donor-derived CD70 knockout NK cells against either 786-O or ACHN tumor cells. Figure 60A shows cytotoxicity data of tested NK cells against 786-O cells at a 1:2 ratio for up to 51 hours. Figure 60B shows cytotoxicity data of tested NK cells in Figure 60A, extended to 90 hours and re-challenged with additional tumor cells at 70 hours. Figure 60C shows remaining 786-O cells after 51 hours of NK cell 1:2 co-culture as measured by 786-O fluorescence. Figure 60D shows remaining 786-O cells after 66 hours of NK cell 1:2 co-culture as measured by 786-O fluorescence. Figure 60E shows cytotoxicity data of tested NK cells against ACHN cells at a 1:2 ratio for up to 51 hours. Figure 60F shows cytotoxicity data for the tested NK cells of Figure 60E extended to 90 hours and re-challenged with additional tumor cells at 70 hours. Figure 60G shows the remaining ACHN cells after 51 hours of 1:2 NK cell co-culture as measured by ACHN fluorescence. Figure 60H shows the remaining ACHN cells after 66 hours of 1:2 NK cell co-culture as measured by ACHN fluorescence. Figure 60I shows cytotoxicity data for the tested NK cells against 786-O cells at a 1:4 ratio for up to 51 hours. Figure 60J shows the cytotoxicity data for the tested NK cell culture of Figure 60I extended to 90 hours and re-challenged with additional tumor cells at 70 hours. Figure 60K shows the remaining 786-O cells after 51 hours of 1:4 NK cell co-culture as measured by 786-O fluorescence. Figure 60L shows the remaining 786-O cells after 66 hours of 1:4 NK cell co-culture as measured by 786-O fluorescence. Figure 60M shows the cytotoxicity data of tested NK cells against ACHN cells at a 1:4 ratio for up to 51 hours. Figure 60N shows the cytotoxicity data of the tested NK cell cultures of Figure 60M, but extended to 90 hours and re-challenged with additional tumor cells at 70 hours. Figure 60O shows the remaining ACHN cells after 51 hours of 1:4 NK cell co-culture as measured by ACHN fluorescence. Figure 60P shows the remaining ACHN cells after 66 hours of 1:4 NK cell co-culture as measured by ACHN fluorescence. [Figure 61] Figures 61A-61N show expression levels and cytotoxicity data for additional anti-CD70 CARs in NK cell gene edited to knockout CD70 in another donor. Figure 61A shows flow cytometry plots demonstrating knockout of CD70 in a donor (designated donor 512). Figure 61B shows flow cytometry plots detecting CAR expression (by APC anti-FLAG) and loss of CD70 expression (by PE anti-CD70). Figure 61C shows quantification of anti-CD70 CAR and CD70 expression in the NK cell population in Figure 61B. Figure 61D shows the raw MFI used to quantify CAR expression in Figure 61B. Figure 61E shows the % abundance of the tested anti-CD70 CARs after 1 week of culture. Figure 61F shows the raw MFI of the tested anti-CD70 CARs after 1 week of culture. Figure 61G shows the % abundance of the tested anti-CD70 CARs after 2 weeks of culture. Figure 61H shows the raw MFI of the tested anti-CD70 CAR after 2 weeks of culture. Figure 611 shows the % abundance of the tested ant-CD70 CAR after 3 weeks of culture. Figure 61J shows the raw MFI of the tested anti-CD70 CAR after 3 weeks of culture. Figure 61K shows the cytotoxicity data of the tested NK cells against 786-O cells at an E:T ratio of 1:2 on day 3 of the total 14-day culture. Figure 61L shows the cytotoxicity data of the tested NK cells against 786-O cells at an E:T ratio of 1:4 on day 3 of the total 14-day culture. Figure 61M shows the cytotoxicity data of the tested NK cells against ACHN cells at a ratio of 1:2 on day 3 of the total 14-day culture. Figure 61N shows the cytotoxicity data of the tested NK cells against ACHN cells at a ratio of 1:4 on day 3 of the total 14-day culture. [Figure 62] Figures 62A-62B show NK cell survival over a 5-week period (week 0-5) following transduction with the tested anti-CD70 CARs. Figure 62A shows NK cell survival from one donor (designated donor 451). Figure 62B shows NK cell survival from another donor (designated donor 512). [Figure 63]Figures 63A-63B show cytotoxicity data for tested NK cell genes expressing an anti-CD70 CAR, as well as genes edited to knock out CD70 and optionally CISH. Figure 63A shows cytotoxicity data for tested NK cells against 786-O cells at a 1:8 ratio for up to 64 hours. Figure 63B shows cytotoxicity data for tested NK cells against ACHN cells at a 1:8 ratio for up to 64 hours. [Figure 64] Figures 64A-64J show additional data regarding the screening of various CD70 CAR constructs expressed by NK cells and the characterization of CD70 gene knockouts. Figure 64A shows data regarding the indicated CD70 CAR constructs and their ability to bind CD70 trimers (native CD70 conformation) when expressed by NK cells, measured one week after transduction (one week after the phenotypic analysis of gene editing following CD70 expression shown in Figures 61A-61B). Figure 64B shows similar data from another donor. Figures 64C and 64D show summary data from flow cytometry binding data, expressed as the percentage of the NK cell population binding to CD70 trimers or as the mean fluorescence intensity (MFI) detected for two respective donors. Figures 64E and 64F show graphical summary data regarding the relative expression of each of the indicated CAR constructs by NK cells, as measured by MFI. Figures 64G and 64H show flow cytometry plots demonstrating knockout of CD70 expression in NK cells from two donors, and Figures 641 and 64J show the percent of NK cells expressing CD70 as a percentage and summary data by MFI for both donors. [Figure 65]Figures 65A-65J show further data regarding the screening of various CD70 CAR constructs expressed by NK cells and the characterization of CD70 gene knockout measured two weeks after transduction of NK cells with the CAR. Figure 65A shows data regarding the indicated CD70 CAR constructs and their ability to bind native CD70 trimers when expressed by NK cells. Figure 65B shows similar data from another donor. Figures 65C and 65D show summary data from flow cytometry binding data, expressed as the percentage of the NK cell population binding to CD70 trimers or as the mean fluorescence intensity (MFI) detected for two respective donors. Figures 65E and 65F show graphical summary data regarding the relative expression of each of the indicated CAR constructs by NK cells, as measured by MFI. Figures 65G and 65H show flow cytometry plots demonstrating the maintained knockout of CD70 expression in NK cells from each of two donors. Figures 65I and 65J show the percent of NK cells expressing CD70 as a percentage and summarized data by MFI for both donors. [Figure 66]Figures 66A-66I show further data regarding the screening of various CD70 CAR constructs expressed by NK cells and the characterization of CD70 gene knockout measured 3 weeks after transduction of NK cells with the CAR. Figure 66A shows data regarding the indicated CD70 CAR constructs and their ability to bind native CD70 trimers when expressed by NK cells. Figure 66B shows similar data from another donor. Figure 66C shows summary data from flow cytometry binding data, expressed as the percentage of the NK cell population binding to CD70 trimers or as the mean fluorescence intensity (MFI) detected for two respective donors. Figures 66D and 66E show graphical summary data regarding the relative expression of each of the indicated CAR constructs by NK cells, as measured by MFI. Figures 66F and 66G show flow cytometry plots demonstrating the maintained knockout of CD70 expression in NK cells from each of two donors. Figures 66H and 66I show summary data for the percent of NK cells expressing CD70 as a percentage and by MFI, as well as viability for both donors. [Figure 67]Figures 67A-67J show further data regarding the screening of various CD70 CAR constructs expressed by NK cells and the characterization of CD70 gene knockout measured 4 weeks after transduction of NK cells with the CAR. Figure 67A shows data regarding the indicated CD70 CAR constructs and their ability to bind native CD70 trimers when expressed by NK cells. Figure 67B shows similar data from another donor. Figures 67C and 67D show summary data from flow cytometry binding data, expressed as the percentage of the NK cell population binding to CD70 trimers or as the mean fluorescence intensity (MFI) detected for two respective donors. Figures 67E and 67F show graphical summary data regarding the relative expression of each of the indicated CAR constructs by NK cells, as measured by MFI. Figures 67G and 67H show flow cytometry plots demonstrating the maintained knockout of CD70 expression in NK cells from each of two donors. Figures 67I and 67J show the percent of NK cells expressing CD70 as a percentage and summarized data by MFI for both donors. [Figure 68]Figures 68A-68I show further data regarding the screening of various CD70 CAR constructs expressed by NK cells and the characterization of CD70 gene knockout measured 5 weeks after transduction of NK cells with the CAR. Figure 68A shows data regarding the indicated CD70 CAR constructs and their ability to bind native CD70 trimers when expressed by NK cells. Figure 68B shows similar data from another donor. Figures 68C and 68D show summary data from flow cytometry binding data, expressed as the percentage of the NK cell population binding to CD70 trimers or as the mean fluorescence intensity (MFI) detected for two respective donors. Figures 68E and 68F show graphical summary data regarding the relative expression of each of the indicated CAR constructs by NK cells, as measured by MFI. Figures 68G and 68H show flow cytometry plots demonstrating the maintained knockout of CD70 expression in NK cells from each of two donors. Figure 68I shows the percent of NK cells expressing CD70 as a percentage and summarized data by MFI for both donors. [Figure 69]Figures 69A-69H show further data regarding the screening of various CD70 CAR constructs expressed by NK cells and the characterization of CD70 gene knockout measured 7 weeks after transduction of NK cells with the CAR. Figure 69A shows data regarding the indicated CD70 CAR constructs and their ability to bind native CD70 trimers when expressed by NK cells, and Figure 69B shows corresponding data from another donor. Figure 69C shows summary data from flow cytometry binding data, expressed as the percentage of the NK cell population binding to CD70 trimers or as the mean fluorescence intensity (MFI) detected for two respective donors. Figures 69D and 69E show graphical summary data regarding the relative expression of each of the indicated CAR constructs by NK cells, as measured by MFI. Figures 69F and 69G show flow cytometry plots demonstrating the maintained knockout of CD70 expression in NK cells from each of two donors. Figure 69H shows the percent of NK cells expressing CD70 as a percentage and summarized data by MFI for both donors. [Figure 70] Figures 70A-70B show data regarding the expression of select, non-limiting anti-CD70 CARs by NK cells over time. Figure 70A tracks the expression of three non-limiting CARs on NK cells from a first donor, and Figure 70B tracks the expression of the same CARs on NK cells from a second donor. [Figure 71]Figures 71A-71T show data on cytokines released by NK cells from one of two donors expressing various CD70 CAR constructs (and edited to knock out CD70) when co-cultured with either ACHN or 786-O tumor cells at various effector:target ratios. Figures 71A and 71B show levels of interferon-gamma release when cells from the first donor are co-cultured with 786-O cells (71A) or ACHN cells (71B) at an E:T ratio of 1:2; data are collected on days 14 and 28 after initiation of the process to produce gene-edited and transduced cells (e.g., D0 of the non-limiting example process shown in Figure 49A). Figures 71C and 71D show corresponding data from the second donor. Figures 71E and 71F show the levels of GMCSF release when cells from a first donor were co-cultured with 786-O cells (71E) or ACHN cells (71F) at an E:T ratio of 1:2, with data collected 14 and 28 days after initiation of the cell production process. Figures 71G and 71H show corresponding data from a second donor. Figures 711 and 71J show the levels of TNF-alpha release when cells from a first donor were co-cultured with 786-O cells (711) or ACHN cells (71J) at an E:T ratio of 1:2, with data collected 14 and 28 days after initiation of the cell production process. Figures 71K and 71L show corresponding data from a second donor. Figures 71M and 71N show the levels of perforin release when cells from a first donor were co-cultured with 786-O cells (71M) or ACHN cells (71N) at an E:T ratio of 1:2, with data collected 14 and 28 days after initiation of the cell production process. Figures 71O and 71P show corresponding data from a second donor. Figures 71Q and 71R show the levels of granzyme B release when cells from a first donor were co-cultured with 786-O cells (71Q) or ACHN cells (71R) at an E:T ratio of 1:2, with data collected 14 and 28 days after initiation of the cell production process. Figures 71S and 71T show corresponding data from a second donor. [Figure 72]Figures 72A-72M show data on the persistence (both CAR expression and cell viability) and cytotoxicity of NK cells edited to express a CD70 CAR and knock out one or both of CD70 and CISH. Figure 72A shows expression data for the indicated CAR over an 8-week period after transduction, as measured by the percentage of the population expressing the CAR. Figure 72B shows similar data, as measured by MFI. Figure 72C shows data on NK cell survival over an 8-week in vitro period. Figure 72D shows data on the cytotoxicity of NK cells expressing the indicated CARs against 786-O cells at an E:T of 1:8, and Figure 72E is a histogram showing the final number of green objects per well (indicating the remaining tumor cell population) for each of the constructs from Figure 72D. Figures 72F and 72G are histograms showing tumor cell counts at the end of co-culture of donor NK cells expressing the indicated CARs and the indicated edits with ACHN cells (72F) or 786-O cells (72G) at an E:T ratio of 1:4. Figures 72H and 72I show corresponding data at an E:T ratio of 1:8. Figures 72J and 72K show cytotoxicity curves in a re-challenge experimental setup, in which NK cells expressing the indicated CARs edited for CD70 knockout and edited for CISH knockout (or unedited) are re-challenged with ACHN (72J) or 786-O (72K) cells at an E:T ratio of 1:4. Figures 72L and 72M show corresponding data using an E:T ratio of 1:8. [Figure 73]Figures 73A-73H show data related to the cytotoxicity of NK cells expressing selected CARs against tumor cells in in vitro assays before and after re-challenge (assays beginning 21 days after the start of the cell production process). Figure 73A shows data regarding the presence of 786-O tumor cells using an E:T ratio of 1:4 72 hours before re-challenge. Figure 73B shows data regarding the presence of 786-O tumor cells using an E:T ratio of 1:8 72 hours before re-challenge. Figure 73C shows data regarding the presence of 786-O tumor cells using an E:T ratio of 1:4 6 days after re-challenge of NK cells with additional 786-O cells. Figure 73D shows data regarding the presence of 786-O tumor cells using an E:T ratio of 1:8 6 days after re-challenge of NK cells with additional 786-O cells. Figure 73E shows data regarding the presence of ACHN tumor cells using an E:T ratio of 1:4 72 hours before re-challenge. Figure 73F shows data on the presence of ACHN tumor cells 72 hours before rechallenge using an E:T ratio of 1:8. Figure 73G shows data on the presence of ACHN tumor cells 6 days after rechallenge of NK cells with additional ACHN cells using an E:T ratio of 1:4. Figure 73H shows data on the presence of ACHN tumor cells 6 days after rechallenge of NK cells with additional ACHN cells using an E:T ratio of 1:8. [Figure 74]Figures 74A-74H show data regarding the cytotoxicity of select CAR-expressing NK cells against tumor cells in in vitro assays before and after re-challenge (assays beginning 28 days after the start of the cell production process). Figure 74A shows data regarding the presence of 786-O tumor cells using an E:T ratio of 1:4 72 hours prior to re-challenge. Figure 74B shows data regarding the presence of 786-O tumor cells using an E:T ratio of 1:8 72 hours prior to re-challenge. Figure 74C shows data regarding the presence of 786-O tumor cells using an E:T ratio of 1:4 6 days after re-challenge of NK cells with additional 786-O cells. Figure 74D shows data regarding the presence of 786-O tumor cells using an E:T ratio of 1:8 6 days after re-challenge of NK cells with additional 786-O cells. Figure 74E shows data regarding the presence of ACHN tumor cells using an E:T ratio of 1:4 72 hours prior to re-challenge. Figure 74F shows data on the presence of ACHN tumor cells using a 1:8 E:T ratio 72 hours prior to rechallenge. Figure 74G shows data on the presence of ACHN tumor cells using a 1:4 E:T ratio 6 days after rechallenge of NK cells with additional ACHN cells. Figure 74H shows data on the presence of ACHN tumor cells using a 1:8 E:T ratio 6 days after rechallenge of NK cells with additional ACHN cells. [Figure 75] Figures 75A-75F show data regarding the frequency and evaluation of CISH KO. Figure 75A shows the detection of indel frequency associated with editing in CISH. Figure 75B shows the detection of indel frequency associated with editing in CD70. Figure 75C shows a schematic of the CISH signaling pathway. Figure 75D shows Western blot data evaluating the expression of phosphorylated Stat 5, a downstream signaling molecule of CISH. Figure 75E shows quantitative data normalized to electroporation control. Figure 75F shows data normalized to electroporation control with a value of 1. DETAILED DESCRIPTION OF THE INVENTION

[0060] Some embodiments of the methods and compositions provided herein relate to engineered immune cells and combinations thereof for use in immunotherapy. In some embodiments, the engineered cells are engineered in multiple ways, for example, to express a cytotoxicity-inducing receptor complex. As used herein, the term "cytotoxicity receptor complex" shall be given its ordinary meaning and (unless otherwise specified) shall refer to a chimeric antigen receptor (CAR), a chimeric receptor (in the case of an NKG2D chimeric receptor, also referred to as an activating chimeric receptor). In some embodiments, the cells are further engineered to achieve modified cellular responsiveness to non-tumor tissue and / or other therapeutic cells. In some embodiments, natural killer (NK) cells are also engineered to express a cytotoxicity-inducing receptor complex (e.g., a chimeric antigen receptor or a chimeric receptor), for example, to target tumor cells expressing CD70. In some embodiments, NK cells are gene-edited to reduce and / or eliminate specific markers / proteins that would otherwise inhibit or limit the therapeutic efficacy of CAR-expressing NK cells. In some embodiments, certain markers / proteins have expression that is upregulated or otherwise induced by one or more processes performed to engineer and / or expand NK cells. For example, in some embodiments, the process of expanding NK cells in culture substantially increases CD70 expression by the NK cells. In embodiments in which a CD70 CAR is engineered to be expressed by the expanded NK cells, the CAR actually targets not only the CD70-expressing tumor, but also the other engineered and expanded NK cells (based on the increased CD70 expression obtained from the cell culture). Thus, for example, in some embodiments, therapeutic NK cells are engineered to express a CAR that targets CD70, and are also gene-edited to knock out CD70 expression on the NK cells themselves, if present, which causes targeting of the tumor and, in turn, the therapeutic NK cells by the CAR-expressing NK cells. This would otherwise result in a self-limiting therapeutic effect that allows tumor expansion and cancer progression.

[0061] The term "anti-cancer effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in cancer cell number, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, and / or amelioration of various physiological symptoms associated with a cancerous condition.

[0062] cell type Some embodiments of the methods and compositions provided herein refer to cells, such as immune cells. For example, immune cells, such as NK cells or T cells, may be engineered to contain a chimeric receptor, such as a CD70-directed chimeric receptor, or may be engineered to contain a nucleic acid encoding said chimeric receptor, as described herein. Further embodiments relate to engineering a second set of cells to express another cytotoxic receptor complex, such as the NKG2D chimeric receptor complex disclosed herein. Still additional embodiments relate to further genetic engineering of cells (e.g., donor NK cells) to reduce, disrupt, minimize, and / or eliminate expression of one or more markers / proteins by the NK cells, resulting in increased potency and / or persistence of the engineered NK cells.

[0063] Traditional anticancer therapies have relied on surgical approaches, radiation therapy, chemotherapy, or a combination of these methods. As research has led to a better understanding of some of the mechanisms of certain cancers, this knowledge has been used to develop targeted cancer therapies. Targeted therapy is a cancer treatment that uses specific drugs to target specific genes or proteins found in cancer cells or cells that support cancer growth (such as blood vessel cells) to suppress or prevent cancer cell growth. More recently, genetic engineering has made it possible to develop approaches that harness specific aspects of the immune system to fight cancer. In some cases, a patient's own immune cells are modified to specifically eradicate that patient's type of cancer. As described in more detail below, various types of immune cells can be used, such as T cells, natural killer (NK) cells, or a combination thereof.

[0064] To facilitate cancer immunotherapy, polynucleotides, polypeptides, and vectors are provided herein that encode chimeric antigen receptors (CARs) comprising a target-binding moiety (e.g., an extracellular binding portion of a ligand, or a tumor marker-directed chimeric receptor expressed by cancer cells) and a cytotoxic signaling complex. For example, some embodiments include polynucleotides, polypeptides, or vectors that encode chimeric antigen receptors directed against, among others, tumor markers, such as CD70, CD19, CD123, Her2, mesothelin, claudin 6, BCMA, and EGFR, to facilitate immune cell targeting to cancer and exert cytotoxic effects in cancer cells. Also provided are engineered immune cells (e.g., NK cells and / or T cells) that express such CARs. Also provided herein in some embodiments are polynucleotides, polypeptides, and vectors encoding constructs comprising two or more subdomains, e.g., a first CD70-targeting subdomain comprising an anti-CD70 binding domain disclosed herein, and an extracellular domain comprising an additional binding moiety, e.g., a C-type lectin-like receptor and cytotoxic signaling complex, or a second subdomain comprising another anti-CD70 binding domain. Also provided are engineered immune cells (e.g., NK cells and / or T cells) expressing such bispecific constructs. Also provided herein are methods for treating cancer and other uses of such cells for cancer immunotherapy.

[0065] Also provided herein are polynucleotides, polypeptides, and vectors encoding chimeric receptors comprising a target-binding moiety (e.g., an extracellular binding portion of a ligand expressed by cancer cells) and a cytotoxic signaling complex to facilitate cancer immunotherapy. For example, some embodiments include polynucleotides, polypeptides, or vectors encoding activating chimeric receptors comprising an NKG2D extracellular domain directed against, among other things, tumor markers, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, to facilitate immune cell targeting to cancer and exert a cytotoxic effect on cancer cells. Also provided are engineered immune cells (e.g., NK cells and / or T cells) expressing such chimeric receptors. Also provided herein, in some embodiments, are polynucleotides, polypeptides, and vectors encoding constructs comprising two or more subdomains, e.g., an extracellular domain comprising a first and a second ligand-binding receptor and a cytotoxic signaling complex. Also provided are engineered immune cells (e.g., NK cells and / or T cells) that express such bispecific constructs (in some embodiments, the first and second ligand-binding domains target the same ligand). Methods of treating cancer and other uses of such cells for cancer immunotherapy are also provided herein.

[0066] Engineered cells for immunotherapy In some embodiments, immune system cells are engineered to have enhanced cytotoxic effects against target cells, such as tumor cells. For example, immune system cells can be engineered to contain a tumor-targeting chimeric receptor and / or a tumor-targeting CAR, as described herein. In some embodiments, white blood cells or leukocytes are used because their primary function is to defend the body against abnormal cell proliferation and infection. There are various types of white blood cells that play specific roles in the human immune system and are therefore a preferred starting point for engineering the cells disclosed herein. Leukocytes include granulocytes and agranulocytes (the presence or absence of granules in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils, and mast cells. Agranulocytes include lymphocytes and monocytes. The following cells, or other cells, such as those described herein, can be engineered to contain a chimeric antigen receptor, e.g., a CD70-targeting CAR, or a nucleic acid encoding a CAR. In some embodiments, the cells may be engineered to co-express a membrane-bound interleukin-15 (mbIL15) domain. As discussed in more detail below, in some embodiments, the therapeutic cells are further genetically modified to increase cytotoxicity and / or cellular persistence. In some embodiments, the genetic modification increases the ability of the cells to resist signals emanating from the tumor microenvironment that cause reduced efficacy or shortened lifespan of the therapeutic cells.

[0067] Monocytes for immunotherapy Monocytes are a subtype of white blood cell. They can differentiate into macrophages and myeloid dendritic cells. Monocytes are associated with the adaptive immune system and are responsible for the key functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of ingesting cellular material, or whole cells, followed by digestion and destruction of the ingested cellular material. In some embodiments, monocytes are used in conjunction with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions described herein relate to monocytes comprising tumor-targeting CARs or nucleic acids encoding tumor-targeting CARs. Some embodiments of the methods and compositions disclosed herein relate to monocytes engineered to express CARs that target tumor markers, such as CD70, CD19, CD123, Her2, mesothelin, claudin 6, BCMA, and EGFR, among others, and optionally induce a membrane-bound interleukin-15 (mbIL15) domain. Some embodiments of the methods and compositions disclosed herein relate to monocytes engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally the membrane-bound interleukin-15 (mbIL15) domain.

[0068] Lymphocytes for immunotherapy Lymphocytes are another primary subtype of white blood cells and 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 some embodiments disclosed herein, some 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 from different donors). Some embodiments of the methods and compositions disclosed herein relate to lymphocytes engineered to express a CAR that targets, inter alia, tumor markers, e.g., CD70, CD19, CD123, Her2, mesothelin, claudin 6, BCMA, EGFR, and optionally includes a membrane-bound interleukin-15 (mbIL15) domain. Some embodiments of the methods and compositions disclosed herein relate to lymphocytes engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally the membrane-bound interleukin-15 (mbIL15) domain.

[0069] T Cells for Immunotherapy T cells can be distinguished from other lymphocyte subtypes (e.g., B cells or NK cells) based on the presence of T cell receptors on the cell surface. T cells are divided into various subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. In some embodiments, T cells of a specific subtype are engineered. In some embodiments, a mixed pool of T cell subtypes is engineered. In some embodiments, there is no specific selection of T cell types engineered to express the cytotoxic receptor complexes disclosed herein. In some embodiments, specific techniques, such as the use of cytokine stimulation, are used to increase the expansion / collection of T cells with specific marker profiles. For example, in some embodiments, activation of specific human T cells, e.g., CD4+ T cells, CD8+ T cells, is achieved by the use of CD3 and / or CD28 as stimulatory molecules. In some embodiments, methods are provided for treating or preventing cancer or infectious diseases, comprising administering a therapeutically effective amount of T cells expressing a cytotoxic receptor complex and / or a homing moiety as described herein. In some embodiments, the engineered T cells are autologous, while in some embodiments, the T cells are allogeneic. Some embodiments of the methods and compositions disclosed herein relate to T cells engineered to express a CAR that targets, among other things, a tumor marker, e.g., CD70, CD19, CD123, Her2, mesothelin, claudin 6, BCMA, EGFR, and optionally includes a membrane-bound interleukin-15 (mbIL15) domain. Some embodiments of the methods and compositions disclosed herein relate to T cells engineered to express an activating chimeric receptor that targets a ligand on tumor cells, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally a membrane-bound interleukin-15 (mbIL15) costimulatory domain.

[0070] NK cells for immunotherapy In some embodiments, methods are provided for treating or preventing cancer or infectious diseases, comprising administering a therapeutically effective amount of natural killer (NK) cells expressing a cytotoxic receptor complex and / or a homing moiety as described herein. In some embodiments, the engineered NK cells are autologous cells, while in some embodiments, the NK cells are allogeneic cells. In some embodiments, NK cells are preferred due to their relatively high natural cytotoxic potential. In some embodiments, it is unexpectedly beneficial that the engineered cells disclosed herein can further upregulate the cytotoxic activity of NK cells, resulting in more effective activity against target cells (e.g., tumor or other disease cells). Some embodiments of the methods and compositions described herein relate to NK cells engineered to express a CAR that targets, inter alia, tumor markers, e.g., CD70, CD19, CD123, Her2, mesothelin, claudin 6, BCMA, EGFR, and optionally includes a membrane-bound interleukin-15 (mbIL15) domain. Some embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express activating chimeric receptors targeting ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally, a membrane-bound interleukin-15 (mbIL15) domain. In some embodiments, immortalized NK cells are used and subjected to gene editing and / or engineering as disclosed herein. In some embodiments, the NK cells are derived from the cell line NK-92. NK-92 cells are derived from NK cells, but lack the major inhibitory receptors displayed by normal NK cells while retaining most of the activating receptors. Some embodiments of NK-92 cells are described herein in connection with NK-92 cells engineered to silence certain additional inhibitory receptors, such as SMAD3, allowing for upregulation of interferon-γ (IFNγ), granzyme B, and / or perforin production.Further information regarding the NK-92 cell line is disclosed in International Publication No. 1998 / 49268 and U.S. Patent Application Publication No. 2002 / 0068044, which are incorporated herein by reference in their entireties. In some embodiments, NK-92 cells are used in combination with one or more of the other cell types disclosed herein. For example, in one embodiment, NK-92 cells are used in combination with the NK cells disclosed herein. In additional embodiments, NK-92 cells are used in combination with the T cells disclosed herein.

[0071] In some embodiments, genetic manipulation of NK cells is used to further increase the potency and / or persistence of NK cells. For example, in some embodiments, the expression of various markers / proteins is reduced, substantially reduced, or knocked out (eliminated) via gene editing techniques. Depending on the embodiment, this may involve gene editing to reduce the expression of one or more of the following: cytokine-inducible SH2-containing protein encoded by the CISH gene, transforming growth factor beta receptor (e.g., TGFBR2), natural killer group 2, member A (NKG2A) receptor, Cbl proto-oncogene B protein encoded by the CBLB gene, tripartite motif-containing protein 29 protein encoded by the TRIM29 gene, suppressor of cytokine signaling 2 protein encoded by the SOCS2 gene, mothers against decapentaplegic homolog 3 (SMAD3) protein encoded by the SMAD3 gene, MAP kinase-activated protein kinase 3 (MAPKAPK3) protein encoded by the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) gene, and / or DNA damage-induced transcript 4 (DDIT4) protein encoded by the DDIT4 gene. In some embodiments, reduced expression is achieved through targeted introduction of DNA breaks and subsequent DNA repair mechanisms. In some embodiments, double-strand breaks in DNA are repaired by non-homologous end joining (NHEJ), and enzymes are used to directly connect DNA ends to each other to repair the break.However, in some embodiments, double-strand breaks are repaired by homology-directed repair (HDR), which is advantageously more accurate, thereby enabling sequence-specific breakage and repair.HDR uses homologous sequences as templates for regenerating the missing DNA sequence at the break point, such as a vector with desired genetic elements (e.g., insertion elements that destroy the coding sequence of target proteins, such as CD70 and / or CISH) in the sequence that is homologous to the adjacent sequence of double-strand break.This results in the desired change (e.g., insertion) that is inserted into the site of DSB.

[0072] In some embodiments, gene editing is achieved by one or more of a variety of engineered nucleases. In some embodiments, restriction enzymes are used, especially when double-strand breaks are desired in multiple regions. In some embodiments, bioengineered nucleases are used. Depending on the embodiment, one of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and / or clustered regularly interspaced short palindromic repeats (CRISPR / Cas9) systems is used to specifically edit genes encoding one or more target proteins, such as CD70 and / or CISH.

[0073] Meganucleases are characterized by their ability to recognize and cleave large DNA sequences (14-40 base pairs). In some embodiments, meganucleases from the LAGLIDADG family are used and subjected to mutagenesis and screening to generate meganuclease variants that recognize unique sequence(s), such as specific sites in genes encoding target proteins of interest. In some embodiments, two or more meganucleases, or functional fragments thereof, are fused to create hybrid enzymes that recognize desired target sequences within genes encoding target proteins of interest, such as CD70 and / or CISH.

[0074] In contrast to meganucleases, ZFNs and TALENs function based on a nonspecific DNA cleavage catalytic domain linked to a specific DNA sequence-recognizing peptide, such as a zinc finger or transcription activator-like effector (TALE). Advantageously, ZFNs and TALENs thus enable sequence-independent DNA cleavage and have a high degree of sequence specificity in target recognition. Zinc finger motifs naturally function in transcription factors to recognize specific DNA sequences for transcription. The C-terminal portion of each finger is responsible for specific recognition of the DNA sequence. While the sequences recognized by ZFNs are relatively short (e.g., about 3 base pairs), in some embodiments, combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more zinc fingers with characterized recognition sites are used, thereby enabling targeting of specific sequences, such as portions of genes encoding target proteins typically expressed by NK cells, such as CD70 and / or CISH. The combined ZFNs are then fused to the catalytic domain(s) of an endonuclease, such as FokI (optionally a FokI heterodimer), to induce targeted DNA cleavage. Additional information regarding the use of ZFNs to edit target genes of interest, such as CD70 or CISH, can be found in U.S. Patent No. 9,597,357, which is incorporated herein by reference.

[0075] Transcription activator-like effector nucleases (TALENs) are specific DNA-binding proteins characterized by an array of 33- or 34-amino acid repeats. Similar to ZFNs, TALENs fuse the DNA cleavage domain of a nuclease to a TALE domain, allowing for sequence-independent double-stranded DNA breaks and highly precise target site recognition. TALENs can create double-stranded breaks at target sites, which are repaired by error-prone non-homologous end joining (NHEJ), resulting in gene disruption by introducing small insertions or deletions. TALENs are advantageously used in some embodiments, at least in part due to their higher specificity in DNA binding, reduced off-target effects, and ease of constructing DNA-binding domains.

[0076] CRISPR (clustered regularly interspaced short palindromic repeats) is a genetic element used by bacteria to defend against viruses. The repeats are short sequences derived from viral genomes and integrated into the bacterial genome. Cas (CRISPR-associated proteins) process these sequences and cleave matching viral DNA sequences. By introducing a plasmid containing a Cas gene into a eukaryotic cell and specifically assembling CRISPR, the eukaryotic genome can be cleaved at any desired location. Additional information regarding CRISPR can be found in U.S. Patent Publication No. 2014 / 0068797, which is incorporated herein by reference. In some embodiments, CRISPR is used to manipulate genes encoding one or more TCRs of a T cell and / or genes encoding one or more immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor is selected from one or more of CTLA4 and PD1. In some embodiments, native CD70 expression by NK cells is disrupted or substantially eliminated by targeting the CD70-encoding gene using the CRISPR / Cas system. In some embodiments, one or more additional target proteins normally expressed by NK cells are disrupted or substantially eliminated by targeting the corresponding encoding genes with a CRISPR / Cas system.Depending on the embodiment, one or more of the following proteins are targeted using the CRISPR / Cas system: cytokine-inducible SH2-containing protein encoded by the CISH gene, transforming growth factor beta receptor (e.g., TGFBR2), natural killer group 2, member A (NKG2A) receptor, Cbl proto-oncogene B protein encoded by the CBLB gene, tripartite motif-containing protein 29 protein encoded by the TRIM29 gene, suppressor of cytokine signaling 2 protein encoded by the SOCS2 gene, SMAD3 protein encoded by the SMAD3 gene, MAPKAPK3 protein encoded by the MAPKAPK3 gene, CEACAM1 protein encoded by the CEACAM1 gene, and / or DDIT4 protein encoded by the DDIT4 gene. Depending on the embodiment, Class 1 or Class 2 Cas is used. In some embodiments, 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, IIID, IV, IVA, IVB, and combinations thereof. In some embodiments, the Cas is selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, and combinations thereof. In some embodiments, Class 2 Cas is used, and the Cas type is selected from the following types: II, IIA, IIB, IIC, V, VI, and combinations thereof. In some embodiments, the Cas is selected from the group consisting of Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a (formerly known as C2c2), Cas13b, Cas13c, CasX, CasY, and combinations thereof. In some embodiments, a class 2 CasX is used, wherein the CasX can form a complex with a guide nucleic acid, and the complex can bind to a target DNA, wherein the target DNA comprises a non-target strand and a target strand.In some embodiments, class 2 CasY is used, which is capable of binding and modifying a target nucleic acid and / or a polypeptide associated with the target nucleic acid.

[0077] Hematopoietic stem cells for cancer immunotherapy In some embodiments, hematopoietic stem cells (HSCs) are used in the immunotherapy methods disclosed herein. In some embodiments, the cells are engineered to express a homing moiety and / or a cytotoxic receptor complex. HSCs are used in some embodiments to take advantage of their ability to engraft for long-term blood cell production, which can provide a sustained source of targeted anti-cancer effector cells, for example, to combat cancer remission. In some embodiments, this continuous production helps offset anergy or depletion of other cell types, for example, by the tumor microenvironment. In some embodiments, allogeneic HSCs are used, while in some embodiments, autologous HSCs are used. In some embodiments, HSCs are used in combination with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions described herein relate to stem cells, such as hematopoietic stem cells, engineered to express a CAR that targets a tumor marker, e.g., CD70, CD19, CD123, Her2, mesothelin, claudin 6, BCMA, EGFR, among others, and optionally includes a membrane-bound interleukin 15 (mbIL15) domain. Some 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 tumor cells, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally includes a membrane-bound interleukin 15 (mbIL15) domain.

[0078] induced pluripotent stem cells In some embodiments, induced pluripotent stem cells (iPSCs) are used in the immunotherapy methods disclosed herein. iPSCs are used to exploit their ability to differentiate and induce non-pluripotent cells, including, but not limited to, CD34 cells, hemogenic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells, containing one or several genetic modifications at selected sites, via differentiation of iPSCs or less differentiated cells containing the same genetic modifications at selected sites. In some embodiments, iPSCs are used to generate iPSC-derived NK or T cells. In some embodiments, the cells are engineered to express a homing moiety and / or a cytotoxic receptor complex. In some embodiments, iPSCs are used in combination with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions described herein relate to stem cells, such as induced pluripotent stem cells, engineered to express a CAR that targets a tumor marker, e.g., CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, EGFR, any of the others disclosed herein, and optionally a membrane-bound interleukin-15 (mbIL15) costimulatory domain. Some 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 tumor cells, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally a membrane-bound interleukin-15 (mbIL15) costimulatory domain.

[0079] Genetic manipulation of immune cells As discussed above, various cell types can be utilized for cellular immunotherapy. Furthermore, as described in more detail below and shown in the Examples, genetic modifications can be made to these cells to increase one or more aspects of their efficacy (e.g., cytotoxicity) and / or durability (e.g., active lifespan). As discussed herein, in some embodiments, NK cells are used for immunotherapy. In some embodiments provided herein, gene editing of NK cells confers various beneficial characteristics to the cells, such as, for example, increased proliferation, increased cytotoxicity, and / or increased persistence. In some embodiments provided herein, gene editing of NK cells can advantageously confer the edited NK cells the ability to resist and / or overcome various inhibitory signals generated in the tumor microenvironment. Tumors are known to produce various signaling molecules intended to reduce the anti-tumor effects of immune cells. As discussed in more detail below, in some embodiments, gene editing of NK cells limits this tumor microenvironment suppressive effect in NK cells, T cells, a combination of NK and T cells, or any of the edited / engineered immune cells provided herein.

[0080] As discussed below, in some embodiments, gene editing is employed to reduce or knock out the expression of a target protein, for example, by disrupting the underlying gene encoding the protein. In some embodiments, gene editing can reduce the expression of the target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99%, or more (including any amount in between). In some embodiments, the gene is completely knocked out so that the expression of the target protein is undetectable. In some embodiments, gene editing is used to "knock in" or alternatively increase the expression of the target protein. In some embodiments, target protein expression may be 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 in between those listed).

[0081] According to further embodiments, other regulators of one or more aspects of NK cell (or T cell) function are modulated via gene editing. Various cytokines provide immune cells with negative or positive signals (similar to TGF-beta, described above). As a non-limiting example, IL15, as disclosed herein, is a positive regulator of NK cells and can increase one or more of NK cell homing, NK cell migration, NK cell expansion / proliferation, NK cell cytotoxicity, and / or NK cell persistence. To suppress NK cells under normal physiological circumstances, cytokine-inducible SH2-containing protein (CIS, encoded by the CISH gene) acts as a key negative regulator of IL-15 signaling in NK cells. As discussed herein, this is because IL15 biology affects multiple aspects of NK cell functionality, including, but not limited to, proliferation / expansion, activation, cytotoxicity, persistence, homing, migration, etc., among others. Thus, according to some embodiments, editing CISH increases NK cell functionality across multiple functions, resulting in more effective and long-lasting NK cell therapy. In some embodiments, CIS inhibitors are used in conjunction with the administration of engineered NK cells.In some embodiments, CIS expression is knocked down or knocked out through gene editing of CISH gene, for example, by using CRISPR-Cas editing.In other embodiments, small interfering RNA, antisense RNA, TALEN or zinc finger are used.In some embodiments, CIS expression in T cell is knocked down through gene editing.

[0082] In some embodiments, CISH gene editing confers an increased ability for NK cells to home to target sites. In some embodiments, CISH gene editing confers an increased ability for NK cells to migrate, e.g., within a tissue, e.g., in response to a chemoattractant or away from a repellent. In some embodiments, CISH gene editing confers an increased ability for NK cells to be activated and thus exert, e.g., an anti-tumor effect. In some embodiments, CISH gene editing confers an increased proliferative capacity for NK cells, and in some embodiments, allows for the generation of robust NK cell numbers from a donor blood sample. Furthermore, in such embodiments, NK cells edited for CISH and engineered to express a CAR are more easily, robustly, and consistently expanded in culture. In some embodiments, CISH gene editing confers an increased cytotoxicity for NK cells. In some embodiments, CISH editing synergistically increases the cytotoxic effect of engineered NK cells and / or engineered T cells expressing a CAR.

[0083] In some embodiments, CISH gene editing activates or inhibits a wide range of pathways. CIS proteins are negative regulators of IL15 signaling, for example, by inhibiting the JAK-STAT signaling pathway. These pathways typically result in the transcription of IL15-responsive genes (including CISH). In some embodiments, knockdown of CISH disinhibits JAK-STAT (e.g., JAK1-STAT5) signaling and increases the transcription of IL15-responsive genes. In some embodiments, knockout of CISH results in increased signaling through the mammalian target of rapamycin (mTOR), with a corresponding increase in the expression of genes related to cellular metabolism and respiration. In some embodiments, knockout of CISH results in increased IL15-induced expression of IL-2Rα (CD25), but not IL-15Rα or IL-2 / 15Rβ, increased NK cell membrane binding of IL15 and / or IL2, increased phosphorylation of STAT-3 and / or STAT-5, and increased expression of anti-apoptotic proteins such as Bcl-2. In some embodiments, CISH knockout results in IL15-induced upregulation of selected genes related to mitochondrial function (e.g., electron transport chain and cellular respiration) and the cell cycle. Thus, in some embodiments, knockout of CISH by gene editing increases NK cell cytotoxicity and / or persistence, at least in part through metabolic reprogramming. In some embodiments, negative regulators of cellular metabolism, such as TXNIP, are downregulated in response to CISH knockout. In some embodiments, promoters for cell survival and proliferation, including BIRC5 (survivin), TOP2A, CKS2, and RACGAP1, are upregulated after CISH knockout, while antiproliferative or proapoptotic proteins, such as TGFB1, ATM, and PTCH1, are downregulated.In some embodiments, the CISH knockout alters the state of signaling (e.g., activates or inactivates) via or through one or more of CXCL-10, IL2, TNF, IFNg, IL13, IL4, Jnk, PRF1, STAT5, PRKCQ, IL2 receptor beta, SOCS2, MYD88, STAT3, STAT1, TBX21, LCK, JAK3, IL& receptor, ABL1, IL9, STAT5A, STAT5B, Tcf7, PRDM1, and / or EOMES.

[0084] In some embodiments, gene editing of immune cells can also provide unexpected increases in the expansion, persistence, and / or cytotoxicity of the edited immune cells. As disclosed herein, engineered cells (e.g., cells expressing a CAR) can also be edited, the combination providing robust cells for immunotherapy. In some embodiments, editing allows for unexpectedly improved NK cell expansion, persistence, and / or cytotoxicity. In some embodiments, knocking out CISH expression in NK cells removes a potent negative regulator of IL15-mediated signaling in NK cells, disinhibiting NK cells and allowing one or more of increased NK cell homing, NK cell migration, NK cell activation, expansion, cytotoxicity, and / or persistence. Furthermore, in some embodiments, editing can increase NK cell and / or T cell function in an otherwise suppressive tumor microenvironment. In some embodiments, CISH gene editing results in increased NK cell expansion, persistence, and / or cytotoxicity without the need for exogenously provided Notch ligand.

[0085] As a non-limiting example, TGF-beta is one such cytokine released by tumor cells that leads to immunosuppression within the tumor microenvironment. Immunosuppression reduces the ability of immune cells, even engineered CAR-immune cells, to destroy tumor cells, thereby allowing tumor progression. In some embodiments, immune checkpoint inhibitors are disrupted via gene editing, as discussed in detail below. In some embodiments, blockers of immunosuppressive cytokines in the tumor microenvironment are used, including blockers or competitive inhibitors of their release, which reduce the ability of signaling molecules to bind to and inhibit immune cells. Such signaling molecules include, but are not limited to, TGF-beta, IL10, arginase, inducible NOS, reactive NOS, Arg1, indoleamine 2,3-dioxygenase (IDO), and PGE2. However, in further embodiments, immune cells, such as NK cells, are provided in which the ability of the NK cells (or other cells) to respond to certain immunosuppressive signaling molecules is disrupted and / or eliminated. For example, in some embodiments, NK cells or T cells are gene-edited to have reduced sensitivity to TGF-beta. TGF-beta is an inhibitor of NK cell function, at least at the level of proliferation and cytotoxicity. See, for example, Figure 8A, which schematically illustrates some of the inhibitory pathways by which TGF-beta reduces NK cell activity and / or proliferation. Thus, according to some embodiments, expression of TGF-beta receptor is knocked down or knocked out via gene editing so that edited NK cells are resistant to the immunosuppressive effects of TGF-beta in the tumor microenvironment. In some embodiments, the TGFB2 receptor is knocked down or knocked out via gene editing, for example, by using CRISPR-Cas editing. In other embodiments, small interfering RNA, antisense RNA, TALEN, or zinc finger is used. Other isoforms of TGF-beta receptor (e.g., TGF-beta 1 and / or TGF-beta 3) are edited in some embodiments. In some embodiments, the TGF-beta receptor in T cells is knocked down via gene editing.

[0086] Extracellular domain (tumor-binding factor) Some embodiments of the compositions and methods described herein relate to chimeric antigen receptors (chimeric antigen receptors) comprising an extracellular domain that includes a tumor-binding domain (also referred to as an antigen-binding protein or antigen-binding domain), as described herein. The tumor-binding domain targets, for example, CD70, CD19, CD123, Her2, mesothelin, claudin 6, BCMA, or EGFR, among others, depending on the embodiment. Some embodiments of the compositions and methods described herein relate to chimeric receptors (chimeric receptors) comprising an extracellular domain that includes a ligand-binding domain that binds to a ligand expressed by tumor cells, as described herein (also referred to as an activating chimeric receptor). The ligand-binding domain targets, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), depending on the embodiment.

[0087] In some embodiments, the antigen-binding domain is derived from or comprises a wild-type or non-wild-type sequence of an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single-domain antibody (SDAB), vH or vL domain, camelid VHH domain, or a non-immunoglobulin scaffold, such as a DARPIN, affibody, affilin, adnectin, affitin, repebody, fynomer, alphabody, avimer, atrimer, centilin, pronectin, anticalin, Kunitz domain, armadillo repeat protein, autoantigen, receptor, or ligand. In some embodiments, the tumor-binding domain comprises more than one antigen-binding domain.

[0088] antigen-binding proteins In some embodiments, antigen-binding proteins are provided. As used herein, the term "antigen-binding protein" shall be given its ordinary meaning and shall also refer to antigen-binding fragments that bind to an antigen and, where appropriate, proteins that include a scaffold or framework portion that allows the antigen-binding fragment to adopt a conformation that promotes binding of the antigen-binding protein to the antigen. In some embodiments, the antigen is a cancer antigen (e.g., CD70) or a fragment thereof. In some embodiments, the antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen. In some embodiments, the antigen-binding fragment comprises all three CDRs from the heavy chain or all three CDRs from the light chain of the antibody that binds the antigen. In further embodiments, the antigen-binding fragment comprises all six CDRs from the antibody that binds the antigen (three from the heavy chain and three from the light chain). In some embodiments, the antigen-binding fragment comprises one, two, three, four, five, or six CDRs from the antibody that binds the antigen, and in some embodiments, the CDRs may be any combination of heavy and / or light chain CDRs. In some embodiments, the antigen-binding fragment is an antibody fragment.

[0089] Non-limiting examples of antigen-binding proteins include antibodies, antibody fragments (e.g., antigen-binding fragments of antibodies), antibody derivatives, and antibody analogs. Further specific examples include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., the VH domain of a camelid heavy chain antibody; VHH fragments), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments. These molecules can be derived from any mammalian source, such as human, mouse, rat, rabbit, pig, dog, or camel. Antibody fragments can compete with intact (e.g., native) antibodies for target antigen binding, and fragments can be synthesized de novo using modification of intact antibodies (e.g., enzymatic or chemical cleavage) or recombinant DNA technology or peptide synthesis. Antigen-binding proteins can include, for example, alternative protein frameworks or artificial scaffolds with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing, for example, mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, as well as fully synthetic scaffolds containing, for example, biocompatible polymers. Additionally, peptide antibody mimetics ("PAMs") can be used, as well as scaffolds based on antibody mimetics that utilize fibronectin components as the scaffold.

[0090] In some embodiments, the antigen-binding protein comprises one or more antibody fragments incorporated into a single polypeptide chain or multiple polypeptide chains. For example, antigen-binding proteins can include, but are not limited to, diabodies, intrabodies, domain antibodies (a single VL or VH domain, or two or more VH domains connected by a peptide linker), maxibodies (two scFvs fused to an Fc region), triabodies, tetrabodies, minibodies (scFvs fused to a CH3 domain), peptibodies (one or more peptides bound to an Fc region), linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with complementary light chain polypeptides), small modular immunopharmaceuticals, and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0091] In some embodiments, the antigen-binding protein has the structure of an immunoglobulin. As used herein, the term "immunoglobulin" is given its ordinary meaning and refers to a tetrameric molecule, each tetramer comprising two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0092] Within light and heavy chains, the variable (V) and constant (C) regions are joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 or more amino acids. The variable regions of each light / heavy chain pair form the antibody binding site, such that an intact immunoglobulin has two binding sites.

[0093] Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity-determining regions or CDRs. From the N-terminus to the C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0094] Human light chains are classified as kappa and lambda light chains. An antibody "light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (K) and lambda (λ) light chains refer to the two major antibody light chain isotypes. A light chain can comprise, from the amino terminus to the carboxyl terminus, a polypeptide comprising a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL).

[0095] Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. An antibody "heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations and typically determines the class to which the antibody belongs. A heavy chain can comprise, from amino to carboxyl terminus, a polypeptide comprising a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and, optionally, immunoglobulin heavy chain constant domain 4 (CH4).

[0096] The IgG class is further divided into subclasses, i.e., IgG1, IgG2, IgG3, and IgG4. The IgA class is further divided into subclasses, i.e., IgA1 and IgA2. IgM has subclasses, including, but not limited to, IgM1 and IgM2. The heavy chains of IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be derived from any immunoglobulin isotype, including subtypes. Antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL and CH1 domains (e.g., between the light and heavy chains) and between the hinge regions of the antibody heavy chains.

[0097] In some embodiments, the antigen-binding protein is an antibody. The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be monoclonal, polyclonal, multi- or single-chain, or intact immunoglobulins and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. Antibodies can be "humanized," "chimeric," or non-human. Antibodies can include intact immunoglobulins of any isotype, including, for example, chimeric, humanized, human, and bispecific antibodies. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains. Antibody sequences can be derived solely from a single species or can be "chimeric," i.e., different portions of the antibody can be derived from two different species, as further described below. Unless otherwise indicated, the term "antibody" also includes antibodies comprising two substantially full-length heavy chains and two substantially full-length light chains, provided that the antibody retains the same or similar binding and / or function as an antibody composed of two full-length light and heavy chains. For example, antibodies with substitutions, insertions, or deletions of 1, 2, 3, 4, or 5 amino acid residues at the N-terminus and / or C-terminus of the heavy and / or light chains are included in the definition, provided that the antibody retains the same or similar binding and / or function as an antibody comprising two full-length heavy chains and two full-length light chains. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and synthetic antibodies. In some embodiments, monoclonal and polyclonal antibodies are provided. As used herein, the term "polyclonal antibody" shall be given its ordinary meaning and shall refer to an antibody population that typically varies widely in composition and binding specificity. As used herein, the term "monoclonal antibody" ("mAb") shall be given its ordinary meaning and shall refer to one or more of a population of antibodies having identical sequence. A monoclonal antibody binds to an antigen at a specific epitope on the antigen.

[0098] In some embodiments, the antigen-binding protein is an antibody fragment or antigen-binding fragment. The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CHI domains, linear antibodies, single-domain antibodies such as sdAb (either vL or vH), camelid vHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (see, e.g., U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). Antibody fragments can include Fab, Fab', F(ab')2, and / or Fv fragments that contain at least one CDR of an immunoglobulin sufficient to confer specific antigen binding to a cancer antigen (e.g., CD19). Antibody fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.

[0099] In some embodiments, Fab fragments are provided. Fab fragments are monovalent fragments containing the VL, VH, CL, and CH1 domains; F(ab')2 fragments are bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments contain the VH and CH1 domains; Fv fragments contain the VL and VH domains of a single antibody arm; and dAb fragments contain the VH domain, VL domain, or antigen-binding fragments of the VH or VL domain. In some embodiments, these antibody fragments can be incorporated into single-domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs. In some embodiments, the antibody comprises at least one CDR as described herein.

[0100] Also provided herein, in some embodiments, are single-chain variable fragments. As used herein, the term "single-chain variable fragment" ("scFv") shall be given its ordinary meaning and shall refer to a fusion protein in which a VL and VH domain are connected via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, the linker being long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site. For clarity, unless otherwise indicated, a "single-chain variable fragment" is not an antibody or antibody fragment as defined herein. Diabodies are bivalent antibodies comprising two polypeptide chains, each of which comprises a VH and a VL domain connected by a linker configured to reduce or prevent pairing between the two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain. According to some embodiments, when the two polypeptide chains of a diabody are identical, the diabody resulting from their pairing has two identical antigen-binding sites. Polypeptide chains with different sequences can be used to generate diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, respectively, which can be the same or different.

[0101] In some embodiments, an antigen-binding protein comprises one or more CDRs. As used herein, the term "CDR" shall be given its ordinary meaning and shall also refer to the complementarity-determining regions (also called "minimal recognition units" or "hypervariable regions") within an antibody variable sequence. CDRs enable an antigen-binding protein to specifically bind to a particular antigen of interest. There are three heavy chain variable region CDRs (CDR-H1, CDR-H2, and CDR-H3) and three light chain variable region CDRs (CDR-L1, CDR-L2, and CDR-L3). The CDRs in each of the two chains are typically aligned by framework regions to form a structure that specifically binds to a particular epitope or domain on the target protein. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically have these elements in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. For the heavy chain variable region, the order is typically, from N-terminus to C-terminus, FW-H1, CDR-H1, FW-H2, CDR-H2, FW-H3, CDR-H3, and FW-H4. For the light chain variable region, the order is typically, from N-terminus to C-terminus, FW-L1, CDR-L1, FW-L2, CDR-L2, FW-L3, CDR-L3, and FW-L4. A numbering system has been devised to number the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using this system.Other numbering systems for amino acids in immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001). The binding domains disclosed herein can utilize CDRs defined according to any of these systems. For any given embodiment containing more than one CDR, the CDRs can be defined according to any of the Kabat, Chothia, extended, IMGT, Paratome, AbM, and / or conformational definitions, or any combination of the above. Any CDR, either separately or within the context of a variable domain, can be interpreted under any of these numbering systems as appropriate by one of skill in the art. One or more CDRs can be incorporated covalently or noncovalently into a molecule to make it an antigen-binding protein.

[0102] In some embodiments, the antigen binding proteins provided herein comprise one or more CDR(s) as part of a larger polypeptide chain. In some embodiments, the antigen binding protein covalently links one or more CDR(s) to another polypeptide chain. In some embodiments, the antigen binding protein incorporates one or more CDRs non-covalently. In some embodiments, the antigen binding protein may comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In some embodiments, the biocompatible framework structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support, or framework, or scaffold, capable of presenting one or more sequences of amino acids that bind to an antigen (e.g., a CDR, a variable region, etc.) at a localized surface area. Such a structure may be a naturally occurring polypeptide or polypeptide "fold" (structural motif), or may have one or more modifications, such as amino acid additions, deletions, and / or substitutions, relative to a naturally occurring polypeptide or fold. Depending on the embodiment, the scaffold can be derived from polypeptides from a variety of different species (or more than one species), such as humans, non-human primates or other mammals, other vertebrates, invertebrates, plants, bacteria or viruses.

[0103] The term "consensus sequence," as used herein with respect to sequences, refers to a generalized sequence that represents all different combinations of allowed amino acids at each position in a group of sequences. Consensus sequences can provide insight into the conserved regions of related sequences, where the units (e.g., amino acids or nucleotides) are identical in most or all of the sequences, and the regions that show differences between sequences.In the case of antibodies, the consensus sequence of CDRs can indicate the amino acids that are important or not essential for antigen binding.It is envisioned that consensus sequences can be prepared for any sequence provided herein, and the resulting various sequences derived from consensus sequences can be verified to have the same effect as the template sequence.

[0104] In some embodiments, an antibody or its binding fragment comprises a combination of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and one or more of these CDRs are defined by a consensus sequence.The consensus sequence provided herein is derived from the alignment of the CDRs provided herein.However, it is envisioned that alternative alignments can be performed (for example, using global or local alignment, or using different algorithms such as Hidden Markov Models, seed-guided trees, Needleman-Wunsch algorithm, or Smith-Waterman algorithm), and such alternative consensus sequences can be derived.

[0105] In some embodiments, CDR-H1 is defined by the formula X1TFX4X5X6X7X8X9 (SEQ ID NO: 1202), where X1 is G or Y; X4 is R or T; X5 is D, E, N or S; X6 is N or Y; X7 is A, D, E, G or Y; X8 is I, L or M; and X9 is H, N or S. In some embodiments, CDR-H1 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, CDR-H1 comprises a sequence with 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0106] In some embodiments, the CDR-H2 has the formula GX2X3X4X5X6X7GX9X 10 X 11 X is defined by YA (SEQ ID NO: 1203), wherein X2 is G, I, V, or W; X3 is I or M; X4 is I, N, or S; X5 is A or P; X6 is I, N, S, or Y; X7 is F, G, N, or S; X9 is A, D, G, H, N, S, or T; 10is A or T;X 11 is G, I, N, or S. In some embodiments, CDR-H2 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, CDR-H2 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0107] In some embodiments, the CDR-H3 has the formula CAX3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X is defined by X, K, or R; X is D, E, G, S, or Y; X is F, H, I, M, P, R, S, W, or Y; X is G, S, or V; X is no amino acid, A, D, G, or V; X is no amino acid, A, G, N, W, or Y; X is no amino acid, A, P, T, or Y; 10 is no amino acid, or is A, E, G, H, R, or Y; X 11 is no amino acid, or is A, D, G, H, or S; X 12 is no amino acid, or is D, F, G, or W; X 13 is no amino acid, or is A, D, E, G, V, or Y; X 14 is no amino acid, or is F, M, or Y; X 15 is no amino acid or Y; X 16 is no amino acid or Y; X 17 is the absence of an amino acid or is not G; X 18 is the absence of an amino acid or M; X 19 is D or G; X 20is I, L, V, or Y. In some embodiments, the CDR-H3 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to this consensus sequence. In some embodiments, the CDR-H3 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0108] In some embodiments, CDR-L1 has the formula X1ASQX5X6X7X8X9LX 11 (SEQ ID NO: 1205), wherein X1 is Q or R; X5 is D, G, S or T; X6 is I or V; X7 is G, R or S; X8 is N, R or S; X9 is F, W or Y; 11 is A or N. In some embodiments, CDR-L1 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, CDR-L1 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0109] In some embodiments, CDR-L2 is defined by the formula X1X2SX4X5X6X7 (SEQ ID NO: 1206), where X1 is A, D, or G; X2 is A or T; X4 is D, N, S, or T; X5 is L or R; X6 is A, E, or Q; and X7 is A, N, S, or T. In some embodiments, CDR-L2 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, CDR-L2 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0110] In some embodiments, the CDR-L3 has the formula CQQX4X5X6X7X8X9X 10 X 11 (SEQ ID NO: 1207), wherein X4 is A, S, or Y; X5 is D, H, I, or Y; X6 is N, S, or T; X7 is A, F, P, S, or T; X8 is L or P; X9 is L, S, T, V, W, or Y; 10 is missing, F, or T. In some embodiments, the CDR-L3 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, the CDR-L3 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0111] In some embodiments, CDR-H1 is of the formula X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X is defined by (SEQ ID NO: 1208), wherein X1 is F, G, N, or Y; X2 is I, R, S, T, or V; X3 is F or L; X4 is A, D, I, N, R, S, or T; X5 is A, D, E, G, N, R, S, or T; X6 is no amino acid, H, S, or Y; X7 is no amino acid, A, D, G, T, or V; X8 is no amino acid, D, F, I, or M; X9 is H, N, Q, S, or Y; 10 is no amino acid or is A, E, F, G, H, L, S, or Y; X 11 is no amino acid or is I, L, M, T, or V; X 12is no amino acid, H, or Y. In some embodiments, CDR-H1 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, CDR-H1 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0112] In some embodiments, the CDR-H2 is of the formula X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X is defined by (SEQ ID NO: 1209), wherein X1 is A, G, or S; X2 is A, G, I, M, R, S, T, V, or W; X3 is no amino acid, F, I, M, or V; X4 is D, I, N, S, or T; X5 is A, K, P, S, or T; X6 is D, G, H, I, M, N, R, S, T, or Y; X7 is A, D, F, G, N, S, or T; X8 is A or G; X9 is A, D, G, H, I, K, N, R, S, T, V, or Y; 10 is A, E, N, P, S, or T; X 11 is A, D, G, H, I, K, L, N, Q, S, T or Y; X 12 is F, N or Y; X 13 is A or Y; X 14 is no amino acid, an A, or a V. In some embodiments, CDR-H2 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, CDR-H2 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0113] In some embodiments, the CDR-H3 is of the formula X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X1 is defined by no amino acid or C; X2 is no amino acid, A or C; X3 is no amino acid, A, C, K or V; X4 is no amino acid, A, D, G, K, M, R, S or W; X5 is no amino acid, A, D, E, G, H, T or V; X6 is no amino acid, C, D, E, F, G, H, L, M, N, P, Q, R, S, T, V or Y; X7 is no amino acid, A, D, E, G, I, L, M, N, Q, R, S, V or Y; X8 is no amino acid, A, F, I, L, P, R, T, V, W or Y; X9 is no amino acid, D, E or Y; 10 is no amino acid, or is G, S, V, or Y; X 11 is no amino acid, or is E, G, I, or S; X 12 is no amino acid or G; X 13 is the absence, L, or T amino acid; X 14 is either absent or is D, L, or T; X 15 is no amino acid, or is A, C, D, G, H, or P; X 16 is no amino acid, or is A, C, F, G, L, M, or Y; X 17 is no amino acid or is A, C, D, E, G, K, N, R, S, T, or V; X 18 is no amino acid or is A, C, D, E, G, I, L, N, P, R, S, T, V, W, or Y; X 19is no amino acid or is A, D, E, F, G, H, K, L, N, Q, R, S, T, W, or Y; X 20 is no amino acid or is A, C, D, E, G, I, M, P, Q, S, T, V, W, or Y; X 21 is no amino acid or is A, D, E, F, G, H, L, Q, S, V, W, or Y; X 22 is the absence of an amino acid or is A, D, E, F, G, H, I, L, M, N, P, Q, S, T, W, or Y; X 23 is no amino acid or is A, D, E, G, H, L, P, S, T, V, W, or Y; X 24 is no amino acid or is A, D, E, F, G, I, L, Q, S, T, V, W, or Y; X 25 is the absence of an amino acid or is A, F, I, L, M, S, V, or Y; X 26 is no amino acid, or is D, G, L, or V; X 27 is I, L, N, P, V, or Y. In some embodiments, the CDR-H3 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, the CDR-H3 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0114] In some embodiments, CDR-L1 is of the formula X1X2SX4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X is defined by (SEQ ID NO: 1211), wherein X1 is K, Q, or R; X2 is A, S, or T; X4 is E, H, Q, S, or T; X5 is no amino acid or S; X6 is no amino acid, L, or V; X7 is no amino acid or L; X8 is no amino acid, H, or Y; X9 is no amino acid or S; 10is no amino acid or S; X 11 is D, E, G, N, R, S or T; X 12 is G, I, N, or V; X 13 is D, G, K, N, R, S, T or Y; X 14 is D, G, H, I, K, N, R, S, or T; X 15 is D, F, G, N, S, W or Y; X 16 is L or V; X 17 is A, D, G, H, or N. In some embodiments, CDR-L1 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, CDR-L1 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0115] In some embodiments, CDR-L2 is defined by the formula X1X2X3X4X5X6X7 (SEQ ID NO: 1212), where Xi is A, D, E, G, H, L, Q, S, W, or Y; X2 is A, G, T, or V; X3 is S or T; X4 is D, N, S, T, or Y; X5 is L or R; X6 is A, D, E, H, or Q; and X7 is A, G, I, N, R, S, or T. In some embodiments, CDR-L2 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, CDR-L2 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0116] In some embodiments, the CDR-L3 is of the formula X1X2X3X4X5X6X7X8X9X 10 X 11X is defined by (SEQ ID NO: 1213), wherein X1 is C or L; X2 is L, M, Q or S; X3 is K, Q or T; X4 is A, D, G, N, S, T or Y; X5 is A, D, F, H, I, L, N, R, T or Y; X6 is A, D, E, G, H, I, N, Q, R, S or T; X7 is A, F, G, I, P, S, T, W or Y; X8 is L, P or T; X9 is A, F, I, L, M, P, S, T, V, W or Y; 10 is the absence of an amino acid or is A, F, H, R, S, or T; X 11 is no amino acid or F. In some embodiments, the CDR-L3 comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, the CDR-L3 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.

[0117] Depending on the embodiment, the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains, hi some such embodiments, these framework structures are based on fibronectin, ankyrin, lipocalin, neocarzinostatin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain, and / or tendamistat domain.

[0118] In some embodiments, antigen-binding proteins having more than one binding site are also provided. In some embodiments, the binding sites are identical to each other, while in some embodiments, the binding sites are different from each other. For example, antibodies typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two different binding sites. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes, which may be present on the same or different protein targets. In some embodiments, this is particularly advantageous because bispecific chimeric antigen receptors can confer the engineered cells the ability to target multiple tumor markers. For example, bispecific antibodies can bind to CD70 and additional tumor markers, such as CD123, CD19, Her2, mesothelin, claudin 6, BCMA, EGFR, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, among others, or any other marker disclosed herein or recognized in the art as a tumor-specific or tumor-associated antigen.

[0119] As used herein, the term "chimeric antibody" shall be given its ordinary meaning and shall also refer to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In some embodiments, one or more CDRs are derived from an anti-cancer antigen antibody (e.g., CD70, CD19, CD123, Her2, mesothelin, PD-L1, claudin 6, BCMA, EGFR, etc.). In some embodiments, all of the CDRs are derived from an anti-cancer antigen antibody (e.g., an anti-CD70 antibody). In some embodiments, CDRs from more than one anti-cancer antigen antibody are mixed and matched in a chimeric antibody. For example, a chimeric antibody may contain CDR1 from the light chain of a first anti-cancer antigen antibody, CDR2 and CDR3 from the light chain of a second anti-cancer antigen antibody, and CDRs from the heavy chain of a third anti-cancer antigen antibody. Furthermore, the framework regions of the antigen-binding proteins disclosed herein can be derived from one of the same anti-cancer antigen (e.g., CD70, CD123, CD19, Her2, mesothelin, claudin 6, BCMA, EGFR, etc.) antibodies, one or more different antibodies, such as a human antibody, or a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and / or light chain is identical to, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to, homologous to, or derived from an antibody from another species or belonging to another antibody class or subclass. Also provided herein are fragments of such antibodies that exhibit desired biological activity. In some embodiments, the CARs disclosed herein comprise an anti-CD70 binding domain. In some embodiments, the anti-CD70 binding domain is an scFv. In some embodiments, the CARs disclosed herein comprise an scFv as a binder to a tumor antigen. In some embodiments, the scFv is encoded by a polynucleotide comprising a sequence having at least about 85%, about 90%, about 95%, or more sequence identity to one or more of SEQ ID NOs: 36-120, 221-229, 1038-1111, 1112-1185.In some embodiments, the scFv comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or more sequence identity to one or more of SEQ ID NOs: 230-312, 890-963, and / or 964-1037.

[0120] Natural killer group domains that bind to tumor ligands In some embodiments, engineered immune cells, such as NK cells, are utilized for their ability to recognize and destroy tumor cells. For example, engineered NK cells may contain a CD70-directed chimeric antigen receptor or a nucleic acid encoding such a chimeric antigen receptor (or a CAR directed against one or more of, for example, CD123, CD19, Her2, mesothelin, claudin 6, BCMA, EGFR, etc.). NK cells express both inhibitory and activating receptors on their cell surface. Inhibitory receptors bind to self-molecules expressed on the surface of healthy cells (thus preventing an immune response against "self" cells), while activating receptors bind to ligands expressed on abnormal cells, such as tumor cells. When the balance between inhibitory and activating receptor activation is in favor of activating receptors, NK cell activation occurs, resulting in the lysis of target (e.g., tumor) cells.

[0121] Natural killer group 2 member D (NKG2D) is an NK cell-activating receptor that recognizes various ligands expressed on cells. Surface expression of various NKG2D ligands is generally low on healthy cells but is upregulated, for example, by malignant transformation. Non-limiting examples of ligands recognized by NKG2D include, but are not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, as well as other molecules expressed on target cells that control the cytolytic or cytotoxic function of NK cells. In some embodiments, T cells are engineered to express an extracellular domain to bind to one or more tumor ligands and activate the T cells. For example, in some embodiments, T cells are engineered to express the NKG2D receptor as a binding / activating moiety. In some embodiments, the engineered cells disclosed herein are engineered to express another member of the NKG2 family, e.g., NKG2A, NKG2C, and / or NKG2E. In some embodiments, a combination of such receptors is engineered. Additionally, in some embodiments, other receptors are expressed, such as killer cell immunoglobulin-like receptors (KIRs).

[0122] In some embodiments, the cells are engineered to express a cytotoxicity receptor complex comprising full-length NKG2D as an extracellular component for recognizing a ligand on the surface of tumor cells (e.g., hepatocytes). In one embodiment, the full-length NKG2D has the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the full-length NKG2D, or a functional fragment thereof, is human NKG2D. Further information regarding chimeric receptors for use in the methods and compositions of the present disclosure is described in PCT Patent Publication No. 2018 / 183385, which is incorporated herein by reference in its entirety.

[0123] In some embodiments, cells are engineered to express a cytotoxicity receptor complex comprising a functional fragment of NKG2D as an extracellular component for recognizing a ligand on the surface of tumor cells or other diseased cells. In one embodiment, the functional fragment of NKG2D has the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the fragment of NKG2D has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to full-length wild-type NKG2D. In some embodiments, the fragment can have one or more additional mutations from SEQ ID NO: 25 but retain ligand-binding function, or in some embodiments, have enhanced ligand-binding function. In some embodiments, the functional fragment of NKG2D comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the NKG2D fragment is provided as a dimer, trimer, or other concatemeric format; such embodiments provide enhanced ligand-binding activity. In some embodiments, the sequence encoding the NKG2D fragment may be fully or partially codon-optimized. In one embodiment, the sequence encoding the codon-optimized NKG2D fragment comprises the sequence of SEQ ID NO: 28. According to some embodiments, advantageously, the functional fragment lacks its native transmembrane or intracellular domain, but retains its ability to bind to NKG2D's ligand and its ability to transmit an activation signal upon ligand binding. A further advantage of such fragments is that DAP10 expression is not required to localize NKG2D to the cell membrane. Thus, in some embodiments, the cytotoxicity receptor complex encoded by the polypeptide disclosed herein does not include DAP10.In some embodiments, immune cells such as NK or T cells (e.g., non-alloreactive T cells engineered according to embodiments disclosed herein) are engineered to express one or more chimeric receptors targeting, for example, CD70, CD19, CD123, Her2, mesothelin, claudin 6, BCMA, EGFR, and NKG2D ligands, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. Such cells, in some embodiments, also co-express mbIL15.

[0124] In some embodiments, the cytotoxic receptor complex is configured to dimerize. Dimerization can include homodimerization or heterodimerization, depending on the embodiment. In some embodiments, dimerization results in improved ligand recognition by the cytotoxic receptor complex (and thus the NK cell expressing the receptor), resulting in a reduction (or absence) of adverse toxic effects. In some embodiments, the cytotoxic receptor complex employs an internal dimer, or repeats of one or more component subunits. For example, in some embodiments, the cytotoxic receptor complex may include a first NKG2D extracellular domain linked to a second NKG2D extracellular domain, and a transmembrane / signaling region (or a separate transmembrane region along with a separate signaling region).

[0125] In some embodiments, the various domains / subdomains are separated by a linker, for example, a GS3 linker (SEQ ID NOS: 15 and 16, nucleotide and protein, respectively) (or a GSn linker) is used. Other linkers for use in accordance with various embodiments disclosed herein include, but are not limited to, those encoded by SEQ ID NOS: 17, 19, 21, or 23. In some embodiments, other linkers comprise the peptide sequence of one of SEQ ID NOS: 18, 20, 22, and 24. This provides the possibility to separate the various component parts of the receptor complex along with a polynucleotide that can increase the expression, stability, and / or functionality of the receptor complex.

[0126] Cytotoxic signaling complex Some embodiments of the compositions and methods described herein relate to chimeric antigen receptors (e.g., CARs directed against CD70) comprising a cytotoxic signaling complex, or chimeric receptors directed against NKG2D ligands such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. As disclosed herein, according to some embodiments, the provided cytotoxic receptor complexes comprise one or more transmembrane and / or intracellular domains that initiate a cytotoxic signaling cascade upon extracellular domain(s) that bind to a ligand on the surface of a target cell.

[0127] In some embodiments, the cytotoxic signaling complex comprises at least one transmembrane domain, at least one costimulatory domain, and / or at least one signaling domain. In some embodiments, more than one component moiety comprises a given domain—e.g., a costimulatory domain can comprise two subdomains. Furthermore, in some embodiments, a domain can serve multiple functions; for example, a transmembrane domain can serve to provide a signaling function.

[0128] Transmembrane domain Some embodiments of the compositions and methods described herein relate to chimeric receptors (e.g., tumor antigen-directed CARs and / or ligand-directed chimeric receptors) that include a transmembrane domain. Some embodiments include a transmembrane domain from NKG2D or another transmembrane protein. In some embodiments in which a transmembrane domain is employed, the portion of the transmembrane protein employed retains at least a portion of its normal transmembrane domain.

[0129] However, in some embodiments, the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein normally expressed on both T cells and NK cells. In some embodiments, the transmembrane domain comprises CD8α. In some embodiments, the transmembrane domain is referred to as a "hinge." In some embodiments, the "hinge" of CD8α has the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the CD8α hinge is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a CD8α having the sequence of SEQ ID NO: 1. In some embodiments, the "hinge" of CD8α comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, CD8α can be truncated or modified to have 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: 2.

[0130] In some embodiments, the transmembrane domain comprises a CD8α transmembrane region. In some embodiments, the CD8α transmembrane domain has the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the CD8α hinge is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a CD8α having the sequence of SEQ ID NO: 3. In some embodiments, the CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD8α hinge is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a CD8α having the sequence of SEQ ID NO: 4.

[0131] In some embodiments, together, the CD8 hinge / transmembrane complex is encoded by the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the CD8 hinge / transmembrane complex is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a CD8 hinge / transmembrane complex having the sequence of SEQ ID NO: 13. In some embodiments, the CD8 hinge / transmembrane complex comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CD8 hinge / transmembrane complex hinge is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a CD8 hinge / transmembrane complex having the sequence of SEQ ID NO: 14.

[0132] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain or a fragment thereof. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the CD28 transmembrane domain complex hinge is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a CD28 transmembrane domain having the sequence of SEQ ID NO: 30.

[0133] Costimulatory domain Some embodiments of the compositions and methods described herein relate to chimeric receptors (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors) that include a costimulatory domain. Furthermore, in some embodiments, various transmembrane and signaling domains (and transmembrane / signaling domain combinations) can be provided, along with additional coactivating molecules. These can be, for example, specific molecules that further enhance the activity of immune cells. Cytokines can be used in some embodiments. For example, specific interleukins such as IL-2 and / or IL-15 are used, as non-limiting examples. In some embodiments, therapeutic immune cells are engineered to express secreted forms of such molecules. In further embodiments, such costimulatory domains are engineered to be membrane-bound, acting as autocrine stimulatory molecules (or even as paracrine stimulators for neighboring cells).

[0134] In some embodiments, the NK cells disclosed herein are engineered to express interleukin 15 (IL15, IL-15). In some embodiments, IL15 is expressed from a separate cassette on a construct comprising any one of the CARs disclosed herein. In some embodiments, IL15 is expressed on the same cassette as any one of the CARs disclosed herein, optionally separated by a cleavage site, e.g., a proteolytic cleavage site or a T2A, P2A, E2A, or F2A autocleaving peptide cleavage site. In some embodiments, IL15 is membrane-bound IL15 (mbIL15). In some embodiments, mbIL15 comprises a native IL15 sequence, such as a human native IL15 sequence, and at least one transmembrane domain. In some embodiments, the native IL15 sequence is encoded by a sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 11. In some embodiments, the native IL15 sequence comprises a peptide sequence having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the at least one transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, mbIL15 may comprise additional components such as a leader sequence and / or a hinge sequence. In some embodiments, the leader sequence is a CD8 leader sequence. In some embodiments, the hinge sequence is a CD8 hinge sequence.

[0135] In some embodiments, the tumor antigen-directed CAR and / or tumor ligand-directed chimeric receptor is encoded by a polynucleotide encoding one or more cytoplasmic protease cleavage sites. Such sites can be recognized and cleaved by a cytoplasmic protease, resulting in the separation (and separate expression) of the various component parts of the receptor encoded by the polynucleotide. In some embodiments, the tumor antigen-directed CAR and / or tumor ligand-directed chimeric receptor is encoded by a polynucleotide encoding one or more self-cleaving peptides, such as a T2A cleavage site, a P2A cleavage site, an E2A cleavage site, and / or an F2A cleavage site. As a result, depending on the embodiment, various components of the engineered cytotoxic receptor complex can be delivered to NK cells or T cells by a single vector or multiple vectors. Thus, as shown schematically in the figures, the construct can be encoded by a single polynucleotide but also contain cleavage sites, so that downstream elements of the construct are expressed by the cell as separate proteins (as in some embodiments with IL-15). In some embodiments, a T2A cleavage site is used. In some embodiments, the T2A cleavage site has the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the T2A cleavage site may be truncated or modified to have 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: 9. In some embodiments, the T2A cleavage site comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the T2A cleavage site is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a T2A cleavage site having the sequence of SEQ ID NO: 10.

[0136] In some embodiments, NK cells are engineered to express membrane-bound interleukin-15 (mbIL15). In such embodiments, mbIL15 expression on NK cells increases the cytotoxic effect of the engineered NK cells by increasing their proliferation and / or lifespan. In some embodiments, mbIL15 is encoded by the same polynucleotide as the CAR. In some embodiments, mbIL15 is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 11 and a sequence encoding a transmembrane domain. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 12 operably linked to the amino acid sequence of the transmembrane domain. In some embodiments, mbIL15 has the nucleic acid sequence of SEQ ID NO: 1188. In some embodiments, mbIL15 can be truncated or modified to have 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: 1188. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 1189. In some embodiments, the mbIL15 is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to mbIL15 having the sequence of SEQ ID NO: 1189. Membrane-bound IL15 sequences are found in PCT Publication Nos. 2018 / 183385 and 2020 / 056045, each of which is expressly incorporated herein by reference in its entirety, and relate to membrane-bound IL15 sequences.

[0137] Signaling domains Some embodiments of the compositions and methods described herein relate to chimeric receptors (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors) comprising a signaling domain. For example, immune cells engineered according to some embodiments disclosed herein may comprise at least one subunit of the CD3 T cell receptor complex (or a fragment thereof). In some embodiments, the signaling domain comprises a CD3 zeta subunit. In some embodiments, CD3 zeta is encoded by the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, CD3 zeta can be truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to CD3 zeta having the sequence of SEQ ID NO: 7. In some embodiments, the CD3 zeta domain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CD3 zeta domain is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a CD3 zeta domain having the sequence of SEQ ID NO:8.

[0138] In some embodiments, unexpectedly increased signaling is achieved through the use of multiple signaling domains whose activities act synergistically. For example, in some embodiments, the signaling domain further comprises an OX40 domain. In some embodiments, the OX40 domain is an intracellular signaling domain. In some embodiments, the OX40 intracellular signaling domain has the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the OX40 intracellular signaling domain can be truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to OX40 having the sequence of SEQ ID NO:5. In some embodiments, the OX40 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the OX40 intracellular signaling domain is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the OX40 intracellular signaling domain having the sequence of SEQ ID NO:6. In some embodiments, OX40 is used as the only transmembrane / signaling domain in the construct, although in some embodiments, OX40 can be used in conjunction with one or more other domains. For example, in some embodiments, a combination of OX40 and CD3 zeta is used. As a further example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used.

[0139] In some embodiments, the signaling domain comprises a 4-1BB domain. In some embodiments, the 4-1BB domain is an intracellular signaling domain. In some embodiments, the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the 4-1BB intracellular signaling domain is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 29. In some embodiments, 4-1BB is used as the only transmembrane / signaling domain in the construct, although in some embodiments, 4-1BB may be used in conjunction with one or more other domains. For example, in some embodiments, a combination of 4-1BB and CD3 zeta is used. By way of further example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used.

[0140] In some embodiments, the signaling domain comprises a CD28 domain. In some embodiments, the CD28 domain is an intracellular signaling domain. In some embodiments, the CD28 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the CD28 intracellular signaling domain is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CD28 intracellular signaling domain having the sequence of SEQ ID NO: 31. In some embodiments, CD28 is used as the only transmembrane / signaling domain in the construct, although in some embodiments, CD28 may be used in conjunction with one or more other domains. For example, in some embodiments, a combination of CD28 and CD3 zeta is used. By way of further example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used.

[0141] Cytotoxicity receptor complex construct Some embodiments of the compositions and methods described herein relate to chimeric antigen receptors, such as CD19-directed chimeric receptors and activating chimeric receptors (ACRs) that target NKG2D ligands. The expression of these cytotoxic receptor complexes in immune cells, such as genetically modified non-alloreactive T cells and / or NK cells, allows for the targeting and destruction of specific target cells, such as cancerous cells. Non-limiting examples of such cytotoxic receptor complexes are discussed in more detail below.

[0142] Chimeric antigen receptor cytotoxicity receptor complex construct In some embodiments, various cytotoxic receptor complexes (also referred to as cytotoxic receptors) are provided herein, along with the general structure of chimeric antigen receptors. Figures 1-7 show non-limiting schematic diagrams of constructs containing tumor-binding moieties that bind to tumor antigens or tumor-associated antigens expressed on the surface of cancer cells and activate engineered cells expressing chimeric antigen receptors. Figure 7 shows a schematic diagram of a chimeric receptor complex with an NKG2D-activating chimeric receptor as a non-limiting example (see NKG2D ACRa and ACRb). Figure 6 shows a schematic diagram of a bispecific CD70 CAR / chimeric receptor complex, as well as two non-limiting constructs, NK71 and NK72, that target CD70.

[0143] As shown in the figures, some embodiments of CARs comprise an anti-tumor binding agent, a CD8a hinge domain, an Ig4 SH domain (or hinge), a CD8a transmembrane domain, a CD28 transmembrane domain, an OX40 domain, a 4-1BB domain, a CD28 domain, a CD3ζ ITAM domain or subdomain, a CD3 zeta domain, an NKp80 domain, a CD16 IC domain, a 2A cleavage site, and / or a membrane-bound IL-15 domain (although in some embodiments, soluble IL-15 is used). In some embodiments, the binding and activation functions are engineered to be performed by separate domains. Some embodiments relate to complexes with more than one tumor-binding agent moiety or other binding agent / activation moiety. In some embodiments, the binding agent / activation moiety targets a marker other than CD70, e.g., a cancer target described herein, e.g., CD19, CD123, CLDN6, BCMA, HER2, mesothelin, PD-L1, or EGFR. In some embodiments, constructs that target NKG2D ligands on tumor cells are provided that can be used with the CARs disclosed herein. In some embodiments, the general structure of a chimeric antigen receptor construct includes a hinge and / or transmembrane domain. In some embodiments, these can be achieved by a single domain, or in some embodiments, multiple subdomains can be used. The receptor complex further includes a signaling domain that transmits a signal after the homing moiety binds to the target cell, ultimately resulting in a cytotoxic effect in the target cell. In some embodiments, the complex further includes a costimulatory domain, which in some embodiments acts synergistically to enhance the function of the signaling domain. Expression of these complexes in immune cells, such as NK cells and / or T cells, enables the targeting and destruction of specific target cells, such as cancerous cells expressing a predetermined tumor marker. Some of these receptor complexes include an extracellular domain containing an anti-CD70 moiety or CD70-binding moiety that binds to CD70 on the surface of target cells and activates the engineered cells. The CD3 zeta ITAM subdomain can act in concert as a signaling domain.The IL-15 domain, e.g., the mbIL-15 domain, can act as a costimulatory domain. The IL-15 domain, e.g., the mbIL-15 domain, can make immune cells (e.g., NK or T cells) that express it particularly effective against target tumor cells. It is recognized that, in some embodiments, the IL-15 domain, such as the mbIL-15 domain, can be encoded on a separate construct. Furthermore, each component can be encoded on one or more separate components.

[0144] In some embodiments, anti-CD70 binding domains are disclosed herein. In some embodiments, the anti-CD70 binding domains are scFvs. These anti-CD70 binding domains are specific for and / or preferentially bind to CD70. The anti-CD70 binding domains disclosed herein can be incorporated into any one of the chimeric antigen receptor constructs disclosed herein. The anti-CD70 binding domains disclosed herein can also be expressed by cells separately or within an anti-CD70 CAR.

[0145] In some embodiments, the anti-CD70 binding domain comprises a polynucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of either SEQ ID NO: 36 and / or SEQ ID NO: 37, or to a range defined by any two of the foregoing percentages.

[0146] In some embodiments, the anti-CD70 binding domain comprises a heavy chain variable region and a light chain variable region, in which the heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3. In some embodiments, CDR-H1 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 428-501; CDR-H2 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 502-575; and CDR-H3 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 576-649. CDR-L1 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 668-741; CDR-L2 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 742-815; and CDR-L3 comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 816-889.

[0147] In some embodiments of the anti-CD70 binding domain, the heavy chain variable region comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any sequence selected from SEQ ID NOs: 890-963. In some embodiments, the light chain variable region comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any sequence selected from SEQ ID NOs: 964-1037. In some embodiments of the anti-CD70 binding domain, 1) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 890, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 964; 2) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 891, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 965; 3) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 892, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 965. 3) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 893, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 967; 4) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 893, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 967; 5) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 894, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 968; 6) the heavy chain variable region comprises CDR-H1, CDR-H2 within SEQ ID NO: 895. , CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 969; 7) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 896, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 970; 8) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 897, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 971; 9) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 898 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 972; 10) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 899 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 973; 11) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 900 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 974;12) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 901, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 975; 13) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 902, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 976; 14) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 903, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 977. 15) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 904, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 978; 16) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 905, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 979; 17) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 906. 18) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 907, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 981; 19) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 908, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 982; 20) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 909, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 981. 21) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 910, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 984; 22) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 911, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 985;23) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 912, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 986; 24) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 913, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 987; 25) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 914, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 988. 26) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 915, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 989; 27) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 916, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 990; 28) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 917. 19) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 918, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 992; 20) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 919, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 993; 21) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 920, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 921; 31) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 994; 32) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 921, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 995; 33) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 922, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 996;34) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 923, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 997; 35) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 924, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 998; 36) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 925, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 999 37) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 926, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1000; 38) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 927, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1001; 39) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 928, 40) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 929, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1003; 41) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 930, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1004; 42) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 931. 41) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 932, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1005; 42) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 932, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1006; 43) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 932, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1006; 44) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 933, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1007;45) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 934, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1008; 46) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 935, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1009; 47) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 936, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1010. 48) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 937, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1011; 49) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 938, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1012; 50) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 939. 51) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 940, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1014; 52) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 941, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1015; 53) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 942 53) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 943, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1017; 54) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 943, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1017; 55) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 944, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1018;56) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 945, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1019; 57) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 946, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1020; 58) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 947. 59) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 948, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1022; 60) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 949, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1023; 61) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 950, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1024; 62) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 951, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1025; 63) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 952, and the light chain variable region comprises 64) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 953, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1027; 65) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 954, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1028; 66) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 955, 65) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 956, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1030; 66) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 957, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1031; 67) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 956, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1030; 68) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 957, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1031; 69) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 95 70) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 959 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1033; 71) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 960 and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1034;72) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 961, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1035; 73) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 962, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1036; or 74) the heavy chain variable region comprises CDR-H1, CDR-H2, CDR-H3 within SEQ ID NO: 963, and the light chain variable region comprises CDR-L1, CDR-L2, CDR-L3 within SEQ ID NO: 1037.

[0148] In some embodiments of the anti-CD70 binding domain, 1) the heavy chain variable region comprises SEQ ID NO:890 and the light chain variable region comprises SEQ ID NO:964; 2) the heavy chain variable region comprises SEQ ID NO:891 and the light chain variable region comprises SEQ ID NO:965; 3) the heavy chain variable region comprises SEQ ID NO:892 and the light chain variable region comprises SEQ ID NO:966; 4) the heavy chain variable region comprises SEQ ID NO:893 and the light chain variable region comprises SEQ ID NO:967; 5) the heavy chain variable region comprises SEQ ID NO:894 and the light chain variable region comprises SEQ ID NO:968; 6) the heavy chain variable region comprises SEQ ID NO:895 and the light chain variable region comprises SEQ ID NO:969. 7) the heavy chain variable region comprises SEQ ID NO: 896 and the light chain variable region comprises SEQ ID NO: 970; 8) the heavy chain variable region comprises SEQ ID NO: 897 and the light chain variable region comprises SEQ ID NO: 971; 9) the heavy chain variable region comprises SEQ ID NO: 898 and the light chain variable region comprises SEQ ID NO: 972; 10) the heavy chain variable region comprises SEQ ID NO: 899 and the light chain variable region comprises SEQ ID NO: 973; 11) the heavy chain variable region comprises SEQ ID NO: 900 and the light chain variable region comprises SEQ ID NO: 974; 12) the heavy chain variable region comprises SEQ ID NO: 901 and the light chain variable region comprises SEQ ID NO: 975; 13) the heavy chain variable region comprises SEQ ID NO: 902 and the light chain variable region comprises SEQ ID NO:976; 14) the heavy chain variable region comprises SEQ ID NO:903 and the light chain variable region comprises SEQ ID NO:977; 15) the heavy chain variable region comprises SEQ ID NO:904 and the light chain variable region comprises SEQ ID NO:978; 16) the heavy chain variable region comprises SEQ ID NO:905 and the light chain variable region comprises SEQ ID NO:979; 17) the heavy chain variable region comprises SEQ ID NO:906 and the light chain variable region comprises SEQ ID NO:980; 18) the heavy chain variable region comprises SEQ ID NO:907 and the light chain variable region comprises SEQ ID NO:981; 19) the heavy chain variable region comprises SEQ ID NO:908 and the light chain variable region comprises SEQ ID NO:909 20) the heavy chain variable region comprises SEQ ID NO:909 and the light chain variable region comprises SEQ ID NO:983; 21) the heavy chain variable region comprises SEQ ID NO:910 and the light chain variable region comprises SEQ ID NO:984; 22) the heavy chain variable region comprises SEQ ID NO:911 and the light chain variable region comprises SEQ ID NO:985; 23) the heavy chain variable region comprises SEQ ID NO:912 and the light chain variable region comprises SEQ ID NO:986; 24) the heavy chain variable region comprises SEQ ID NO:913 and the light chain variable region comprises SEQ ID NO:987; 25) the heavy chain variable region comprises SEQ ID NO:914 and the light chain variable region comprises SEQ ID NO:988;26) the heavy chain variable region comprises SEQ ID NO: 915 and the light chain variable region comprises SEQ ID NO: 989; 27) the heavy chain variable region comprises SEQ ID NO: 916 and the light chain variable region comprises SEQ ID NO: 990; 28) the heavy chain variable region comprises SEQ ID NO: 917 and the light chain variable region comprises SEQ ID NO: 991; 29) the heavy chain variable region comprises SEQ ID NO: 918 and the light chain variable region comprises SEQ ID NO: 992; 30) the heavy chain variable region comprises SEQ ID NO: 919 and the light chain variable region comprises SEQ ID NO: 993; 31) the heavy chain variable region comprises SEQ ID NO: 920 and the light chain variable region comprises SEQ ID NO: 994; 32) the heavy chain variable region comprises SEQ ID NO: 31) the heavy chain variable region comprises SEQ ID NO: 921 and the light chain variable region comprises SEQ ID NO: 995; 32) the heavy chain variable region comprises SEQ ID NO: 922 and the light chain variable region comprises SEQ ID NO: 996; 33) the heavy chain variable region comprises SEQ ID NO: 922 and the light chain variable region comprises SEQ ID NO: 996; 34) the heavy chain variable region comprises SEQ ID NO: 923 and the light chain variable region comprises SEQ ID NO: 997; 35) the heavy chain variable region comprises SEQ ID NO: 924 and the light chain variable region comprises SEQ ID NO: 998; 36) the heavy chain variable region comprises SEQ ID NO: 925 and the light chain variable region comprises SEQ ID NO: 999; 37) the heavy chain variable region comprises SEQ ID NO: 926 and the light chain variable region comprises SEQ ID NO: 1000; 38) the heavy chain variable region comprises SEQ ID NO: 927 and the light chain variable region comprises SEQ ID NO: 929 39) the heavy chain variable region comprises SEQ ID NO:928 and the light chain variable region comprises SEQ ID NO:1002; 40) the heavy chain variable region comprises SEQ ID NO:929 and the light chain variable region comprises SEQ ID NO:1003; 41) the heavy chain variable region comprises SEQ ID NO:930 and the light chain variable region comprises SEQ ID NO:1004; 42) the heavy chain variable region comprises SEQ ID NO:931 and the light chain variable region comprises SEQ ID NO:1005; 43) the heavy chain variable region comprises SEQ ID NO:932 and the light chain variable region comprises SEQ ID NO:1006; 44) the heavy chain variable region comprises SEQ ID NO:933 and the light chain variable region comprises SEQ ID NO: 1007; 45) the heavy chain variable region comprises SEQ ID NO:934 and the light chain variable region comprises SEQ ID NO:1008; 46) the heavy chain variable region comprises SEQ ID NO:935 and the light chain variable region comprises SEQ ID NO:1009; 47) the heavy chain variable region comprises SEQ ID NO:936 and the light chain variable region comprises SEQ ID NO:1010; 48) the heavy chain variable region comprises SEQ ID NO:937 and the light chain variable region comprises SEQ ID NO:1011; 49) the heavy chain variable region comprises SEQ ID NO:938 and the light chain variable region comprises SEQ ID NO:1012; 50) the heavy chain variable region comprises SEQ ID NO:939 and the light chain variable region comprises SEQ ID NO:1013;51) the heavy chain variable region comprises SEQ ID NO:940 and the light chain variable region comprises SEQ ID NO:1014; 52) the heavy chain variable region comprises SEQ ID NO:941 and the light chain variable region comprises SEQ ID NO:1015; 53) the heavy chain variable region comprises SEQ ID NO:942 and the light chain variable region comprises SEQ ID NO:1016; 54) the heavy chain variable region comprises SEQ ID NO:943 and the light chain variable region comprises SEQ ID NO:1017; 55) the heavy chain variable region comprises SEQ ID NO:944 and the light chain variable region comprises SEQ ID NO:1018; 56) the heavy chain variable region comprises SEQ ID NO:945 and the light chain variable region comprises SEQ ID NO:1019; 57) the heavy chain variable region comprises SEQ ID NO: 946 and the light chain variable region comprises SEQ ID NO: 1020; 58) the heavy chain variable region comprises SEQ ID NO: 947 and the light chain variable region comprises SEQ ID NO: 1021; 59) the heavy chain variable region comprises SEQ ID NO: 948 and the light chain variable region comprises SEQ ID NO: 1022; 60) the heavy chain variable region comprises SEQ ID NO: 949 and the light chain variable region comprises SEQ ID NO: 1023; 61) the heavy chain variable region comprises SEQ ID NO: 950 and the light chain variable region comprises SEQ ID NO: 1024; 62) the heavy chain variable region comprises SEQ ID NO: 951 and the light chain variable region comprises SEQ ID NO: 1025; 3) the heavy chain variable region comprises SEQ ID NO:952 and the light chain variable region comprises SEQ ID NO:1026; 64) the heavy chain variable region comprises SEQ ID NO:953 and the light chain variable region comprises SEQ ID NO:1027; 65) the heavy chain variable region comprises SEQ ID NO:954 and the light chain variable region comprises SEQ ID NO:1028; 66) the heavy chain variable region comprises SEQ ID NO:955 and the light chain variable region comprises SEQ ID NO:1029; 67) the heavy chain variable region comprises SEQ ID NO:956 and the light chain variable region comprises SEQ ID NO:1030; 68) the heavy chain variable region comprises SEQ ID NO:957 and the light chain variable region comprises SEQ ID NO:1031; 69 70) the heavy chain variable region comprises SEQ ID NO:959 and the light chain variable region comprises SEQ ID NO:1033; 71) the heavy chain variable region comprises SEQ ID NO:960 and the light chain variable region comprises SEQ ID NO:1034; 72) the heavy chain variable region comprises SEQ ID NO:961 and the light chain variable region comprises SEQ ID NO:1035; 73) the heavy chain variable region comprises SEQ ID NO:962 and the light chain variable region comprises SEQ ID NO:1036; or 74) the heavy chain variable region comprises SEQ ID NO:963 and the light chain variable region comprises SEQ ID NO:1037.

[0149] In some embodiments of the anti-CD70 binding domain, the heavy chain variable region and / or the light chain variable region comprise a framework. In some embodiments, the heavy chain variable region comprises FW-H1, FW-H2, FW-H3, and FW-H4. In some embodiments, the heavy chain variable region comprises, from N-terminus to C-terminus, FW-H1, CDR-H1, FW-H2, CDR-H2, FW-H3, CDR-H3, and FW-H4. In some embodiments, the light chain variable region comprises FW-L1, FW-L2, FW-L3, and FW-L4. In some embodiments, the light chain variable region comprises, from N-terminus to C-terminus, FW-L1, CDR-L1, FW-L2, CDR-L2, FW-L3, CDR-L3, FW-L4. In some embodiments, FW-H1 comprises a sequence having at least 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 399-402, FW-H2 comprises a sequence having at least 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 403-406, FW-H3 comprises a sequence having at least 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 407-422, and FW-H4 comprises a sequence having at least 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 423-427. FW-L1 comprises a sequence having at least 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 650 to 653, FW-L2 comprises a sequence having at least 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 654 to 657, FW-L3 comprises a sequence having at least 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 658 to 661, and FW-L4 comprises a sequence having at least 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 662 to 667.

[0150] In some embodiments of the anti-CD70 binding domain, the heavy chain variable domain is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any sequence selected from SEQ ID NOs: 1038-1111.

[0151] In some embodiments of the anti-CD70 binding domain, the light chain variable domain is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any sequence selected from SEQ ID NOs: 1112-1185.

[0152] In some embodiments, the anti-CD70 binding domain is an antibody, a Fab' fragment, a F(ab')2 fragment, or an scFv.

[0153] In some embodiments, the anti-CD70 binding domain is encoded by a polynucleotide sequence comprising a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical, or to a range defined by any two of the foregoing percentages, to one or more of SEQ ID NOs: 38-120, 221-229, 1038-1111, and / or 1112-1185. In some embodiments, the anti-CD70 binding domain comprises an amino acid sequence that has at least about 85%, about 90%, about 95%, or more sequence identity to one or more of SEQ ID NOs: 230-312, 890-963, 964-1037.

[0154] CARs are also disclosed herein. In some embodiments, the CAR is an anti-CD70 CAR. In some embodiments, the CAR comprises any one or more of the anti-CD70 binding domains disclosed herein.

[0155] In some embodiments, the CAR further comprises an OX40 subdomain and a CD3 zeta subdomain. In some embodiments, the OX40 subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO:5. In some embodiments, the OX40 subdomain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% sequence identity to SEQ ID NO:6. In some embodiments, the CD3 zeta subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO:7. In some embodiments, the CD3 zeta subdomain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% sequence identity to SEQ ID NO:8. In some embodiments, mbIL15 is encoded by a sequence having at least 95% sequence identity to SEQ ID NO:1188. In some embodiments, one or more of SEQ ID NOs: 36-120, 221-229, 1038-1111, and / or 1112-1185, the polynucleotide encoding the OX40 subdomain, the polynucleotide encoding the CD3 zeta subdomain, and the polynucleotide encoding mbIL15 are arranged in a 5' to 3' direction within the polynucleotide.

[0156] In some embodiments, an anti-CD70 CAR is provided that is encoded by a polynucleotide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to one or more of SEQ ID NOs: 138-220, or a portion thereof (e.g., the portion excluding the mbIL15 sequence and / or the self-cleaving peptide sequence), or to a range defined by any two of the foregoing percentages. In some embodiments, the CAR comprises amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to one or more of SEQ ID NOs: 313-395, or a portion thereof (e.g., the portion excluding the mbIL15 sequence and / or the self-cleaving peptide sequence), or to a range defined by any two of the foregoing percentages.

[0157] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8 hinge-CD8™ / 4-1BB / CD3 zeta chimeric antigen receptor complex is provided (see Figure 1, CAR1a). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB domain, and a CD3 zeta domain, as described herein. In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0158] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8 hinge-CD8™ / 4-1BB / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex is provided (see Figure 1, CAR1b). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB domain, a CD3 zeta domain, a 2A cleavage site, and a mIL-15 domain, as described herein. In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0159] In one embodiment, a polynucleotide encoding a tumor-binding factor / CD8 hinge-CD8™ / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 1, CAR1c). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain, as described herein. In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence sharing 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 with a sequence resulting from a combination of one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0160] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge-CD8™ / OX40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex is provided (see Figure 1, CAR 1d). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, an OX40 domain, a CD3 zeta domain, a 2A cleavage site, and an mIL-15 domain, as described herein. In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs: described herein. It is recognized that certain sequence variations, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites). In some embodiments, the anti-CD70 binding domain comprises a polynucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of either SEQ ID NO:36 and / or SEQ ID NO:37, or within a range defined by any two of the foregoing percentages.In some embodiments, the anti-CD70 binding domain comprises a polynucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of SEQ ID NOs: 38-120, 221-229, 1038-1111, and / or 1112-1185, or within a range defined by any two of the foregoing percentages. In some embodiments, an anti-CD70 CAR is provided that is encoded by a polynucleotide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to one or more of SEQ ID NOs: 138-220, or a portion thereof (e.g., the portion excluding the mbIL15 sequence and / or the self-cleaving peptide sequence), or to a range defined by any two of the foregoing percentages. In some embodiments, the CAR comprises amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to one or more of SEQ ID NOs: 313-395, or a portion thereof (e.g., the portion excluding the mbIL15 sequence and / or the self-cleaving peptide sequence), or to a range defined by any two of the foregoing percentages.

[0161] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge-CD28TM / CD28 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 1, CAR1e). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD28 transmembrane domain, a CD28 domain, and a CD3 zeta domain, as described herein. In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence sharing 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 with a sequence resulting from a combination of one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0162] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge-CD28™ / CD28 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex is provided (see Figure 1, CAR1f). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD28 transmembrane domain, a CD28 domain, a CD3 zeta domain, a 2A cleavage site, and a mIL-15 domain, as described herein. In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0163] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8a™ / ICOS / CD3 zeta chimeric antigen receptor complex is provided (see Figure 1, CAR1g). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, an inducible costimulatory factor (ICOS) signaling domain, and a CD3 zeta domain. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 1, CAR1h). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0164] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8a™ / CD28 / 4-1BB / CD3 zeta chimeric antigen receptor complex is provided (see Figure 1, CAR1i). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a CD28 signaling domain, a 4-1BB signaling domain, and a CD3 zeta domain. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 1, CAR1j). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0165] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / NKG2D™ / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 2, CAR2a). The polynucleotide comprises or consists of a tumor-binding factor (such as a light chain variable region of an scFv), a CD8a hinge, an NKG2D transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 2, CAR2b). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0166] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8a™ / OX40 / CD3 zeta / 2A / EGFRt chimeric antigen receptor complex is provided (see Figure 2, CAR2e). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, a CD3 zeta domain, a 2A cleavage site (side), and a truncated form of epidermal growth factor receptor (EGFRt). In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 2, CAR2f). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0167] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / NKG2DTM / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 2, CAR2g). The polynucleotide comprises or consists of a tumor-binding factor (such as a heavy chain variable region of an scFv), a CD8a hinge, an NKG2D transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 2, CAR2h). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0168] In some embodiments, a polynucleotide encoding a tumor-binding factor / Ig4SH-CD28TM / CD28 / CD3zeta chimeric antigen receptor complex is provided (see Figure 2, CAR2i). The polynucleotide comprises or consists of a tumor-binding factor, an Ig4 SH domain, a CD28 transmembrane domain, a CD28 domain, and a CD3zeta domain, as described herein. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 2, CAR2j). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0169] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8a™ / CD27 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 3, CAR3a). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a CD27 signaling domain, and a CD3 zeta domain. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 3, CAR3b). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0170] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8a™ / CD70 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 3, CAR3c). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a CD70 signaling domain, and a CD3 zeta domain. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 3, CAR3d). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0171] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8a™ / CD161 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 3, CAR3e). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a CD161 signaling domain, and a CD3 zeta domain. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 3, CAR3f). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0172] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8a™ / CD40L / CD3 zeta chimeric antigen receptor complex is provided (see Figure 3, CAR3g). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a CD40L signaling domain, and a CD3 zeta domain. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 3, CAR3h). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0173] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8a™ / CD44 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 3, CAR3i). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a CD44 signaling domain, and a CD3 zeta domain. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 3, CAR3j). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0174] In some embodiments, a polynucleotide encoding a tumor-binding factor / Ig4SH-CD8TM / 4-1BB / CD3zeta chimeric antigen receptor complex is provided (see Figure 4, CAR4a). The polynucleotide comprises or consists of a tumor-binding factor, an Ig4 SH domain, a CD8a transmembrane domain, a 4-1BB domain, and a CD3zeta domain, as described herein. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 4, CAR4b). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0175] In some embodiments, a polynucleotide encoding a tumor-binding factor / Ig4SH-CD8TM / OX40 / CD3zeta chimeric antigen receptor complex is provided (see Figure 4, CAR4c). The polynucleotide comprises or consists of a tumor-binding factor, an Ig4 SH domain, a CD8a transmembrane domain, an OX40 domain, and a CD3zeta domain, as described herein. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 4, CAR4d). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares 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 with a sequence resulting from combining one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creation of restriction sites).

[0176] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge-CD3αTM / CD28 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 4, CAR4e). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD3α transmembrane domain, a CD28 domain, and a CD3 zeta domain, as described herein. In some embodiments, the polynucleotide further encodes mbIL15 (see Figure 4, CAR4f). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those incl...

Claims

1. an anti-CD70 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises CDR-H1, CDR-H2, and CDR-H3 contained within the VH amino acid sequence set forth in SEQ ID NO: 956; and the VL comprises CDR-L1, CDR-L2, and CDR-L3 contained within the VL amino acid sequence set forth in SEQ ID NO: 1030; An anti-CD70 antibody or antigen-binding fragment thereof.

2. VH comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 956, and VL comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1030; The anti-CD70 antibody or antigen-binding fragment thereof according to claim 1.

3. VH comprises the amino acid sequence of SEQ ID NO: 956, and VL comprises the amino acid sequence of SEQ ID NO: 1030; The anti-CD70 antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. The anti-CD70 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-CD70 antibody or antigen-binding fragment thereof is a Fab' fragment.

5. The anti-CD70 antibody or antigen-binding fragment thereof may be F(ab') ２ The anti-CD70 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is a fragment thereof.

6. The anti-CD70 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-CD70 antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv).

7. A chimeric antigen receptor (CAR) comprising an antigen-binding fragment of the anti-CD70 antibody of any one of claims 1 to 6, a transmembrane domain, and a cytotoxic signaling complex.

8. The CAR of claim 7, wherein the cytotoxic signaling complex comprises an OX40 subdomain and a CD3 zeta subdomain.

9. The CAR of claim 7 or 8, wherein the transmembrane domain comprises a CD8α transmembrane region.

10. A cell comprising the anti-CD70 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the CAR according to any one of claims 7 to 9.

11. The cell of claim 10 , wherein the cell is an immune cell.

12. The cell of claim 10 or 11, wherein the cell is a natural killer (NK) cell.

13. The cell of claim 10 or 11, wherein the cell is a T cell.

14. 14. The cell of any one of claims 10-13, wherein the cell has been gene edited with an endogenous CD70 gene such that the cell expresses reduced levels of CD70 protein compared to a cell in which the endogenous CD70 gene has not been edited.

15. 15. The cell of any one of claims 10-14, wherein the cell has been gene-edited to express reduced levels of CISH, adenosine receptor, A2A adenosine receptor, A2B adenosine receptor, A3 adenosine receptor, A1 adenosine receptor, A2AR, TGFBR, B2M, CIITA, NKG2A, CBLB, TRIM29, SOCS2, SMAD3, MAPKAPK3, CEACAM1 or DDIT4, or any combination thereof, compared to an unedited cell.

16. 16. The cell of any one of claims 10-15, wherein the cell has been gene-edited to express reduced levels of CISH compared to an unedited cell.

17. The cell of any one of claims 10 to 16, wherein the cell is engineered to express membrane-bound interleukin 15 (mbIL15).

18. A genetically engineered natural killer (NK) cell population, the population comprising a plurality of NK cells expanded in culture; The plurality of NK cells are engineered to express a chimeric antigen receptor (CAR) comprising an antigen-binding fragment of an anti-CD70 antibody, a transmembrane domain, and a cytotoxic signaling complex; The antigen-binding fragment of the anti-CD70 antibody comprises a heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3 contained within the VH amino acid sequence set forth in SEQ ID NO: 956, and a light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3 contained within the VL amino acid sequence set forth in SEQ ID NO: 1030; the cytotoxic signaling complex comprises an OX-40 subdomain and a CD3 zeta subdomain; NK cells are engineered to express membrane-bound IL-15 (mbIL15) and The NK cells have been expanded in culture and have been gene-edited with an endogenous CD70 gene to express reduced levels of CD70 protein compared to NK cells in which the endogenous CD70 gene has not been edited. Genetically engineered NK cell populations.

19. NK cells have been gene-edited with an endogenous CISH gene to express reduced levels of cytokine-inducible SH2-containing (CIS) proteins compared to NK cells in which the endogenous CISH gene has not been edited; and The genetically engineered NK cells exhibit one or more of increased expansion capacity, increased cytotoxicity against target cells, and increased persistence compared to NK cells expressing natural levels of CIS protein.

19. The genetically engineered NK cell population of claim 18.

20. 20. The genetically engineered NK cell population of claim 18 or 19, wherein the VH comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 956, and the VL comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1030.

21. 21. The genetically engineered NK cell population of any one of claims 18 to 20, wherein the VH comprises the amino acid sequence of SEQ ID NO: 956 and the VL comprises the amino acid sequence of SEQ ID NO: 1030.

22. 22. The genetically engineered NK cell population of any one of claims 18 to 21, wherein the transmembrane domain comprises the CD8α transmembrane region.

23. 23. The genetically engineered NK cell population of any one of claims 18-22, wherein the NK cells have been further gene-edited to express reduced levels of Cbl proto-oncogene B protein (Cblb), transforming growth factor beta receptor (TGFBR), beta-2 microglobulin (B2M), class II major histocompatibility complex transactivator (CIITA), natural killer group 2, member A (NKG2A) receptor, tripartite motif-containing protein 29 (TRIM29), or suppressor of cytokine signaling 2 (SOCS2) compared to unedited NK cells.

24. 24. The genetically engineered NK cell population of any one of claims 18-23, wherein the NK cells have been further gene-edited to express reduced levels of Cbl proto-oncogene B protein (Cblb) compared to non-edited NK cells.

25. 25. The genetically engineered NK cell population of any one of claims 18 to 24, wherein the gene editing is performed using the CRISPR-Cas system, zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs).

26. The genetically engineered NK cell population of any one of claims 18 to 25, wherein the gene editing is performed using a CRISPR-Cas system, wherein the Cas is Cas9.

27. 27. The genetically engineered NK cell population of any one of claims 19 to 26, wherein the CD70 gene is edited using one or more guide RNAs comprising the sequence of SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO: 123, or the CISH gene is edited using one or more guide RNAs comprising the sequence of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO:

134.

28. A genetically engineered immune cell population comprising a plurality of immune cells, The plurality of immune cells are engineered to express a chimeric antigen receptor (CAR) comprising an antigen-binding fragment of an anti-CD70 antibody, a transmembrane domain, and a cytotoxic signaling complex; The antigen-binding fragment of the anti-CD70 antibody comprises a heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3 contained within the VH amino acid sequence set forth in SEQ ID NO: 956, and a light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3 contained within the VL amino acid sequence set forth in SEQ ID NO: 1030; The immune cells are gene-edited to express reduced levels of CD70 compared to non-edited immune cells, and the reduced CD70 expression is engineered via editing of the endogenous CD70 gene. Immune cell populations.

29. 29. The genetically engineered immune cell population of claim 28, wherein the plurality of immune cells are natural killer (NK) cells.

30. 30. The genetically engineered immune cell population of claim 28 or 29, wherein the cytotoxic signaling complex comprises an OX40 subdomain and a CD3 zeta subdomain.

31. 31. The genetically engineered immune cell population of any one of claims 28 to 30, wherein the NK cells are engineered to express membrane-bound IL-15 (mbIL15).

32. 32. A pharmaceutical composition for treating a subject having cancer, comprising the anti-CD70 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the CAR according to any one of claims 7 to 9, the cell according to any one of claims 10 to 17, the genetically engineered NK cell population according to any one of claims 18 to 27, or the genetically engineered immune cell population according to any one of claims 28 to 31, wherein the cancer is a CD70-expressing cancer.

33. 33. The pharmaceutical composition of claim 32, wherein the cancer is lung cancer, renal cell carcinoma, or melanoma.

34. 33. The pharmaceutical composition of claim 32, wherein the cancer is leukemia.

35. The pharmaceutical composition of any one of claims 32 to 34, wherein the cells are allogeneic cells to the subject.

36. The pharmaceutical composition of any one of claims 32 to 34, wherein the cells are autologous to the subject.

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