Combinations of engineered natural killer cells and engineered t cells for immunotherapy

Abstract

To provide methods and compositions comprising genetically engineered cells for cancer immunotherapy, particularly combinations of genetically engineered immune cell types.SOLUTION: Some embodiments of methods and compositions disclosed herein relate to immune cells that are engineered to express chimeric antigen receptors and / or genetically modified to enhance one or more aspects of the efficacy of the immune cells in cellular immunotherapy. Some embodiments relate to genetic modifications which reduce potential side effects of cellular immunotherapy. In some embodiments, combinations of cells are used to achieve both rapid and long-term tumor reduction with reduced or eliminated potential for graft versus host effects.SELECTED DRAWING: Figure 21-2

Description

Technical Field

[0001] Related Cases This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 857,167, filed on June 4, 2019, and U.S. Provisional Patent Application No. 62 / 943,697, filed on December 4, 2019, 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, particularly combinations of genetically engineered immune cell types, for cancer immunotherapy. In some embodiments, the disclosure relates to cells engineered to express chimeric antigen receptors. In some embodiments, further engineering is performed to increase efficacy and / or reduce potential side effects when the cells are used in cancer immunotherapy.

Background Art

[0003] As more knowledge is gained about various cancers and the properties of cancerous cells that can be used to specifically distinguish cancerous cells from healthy cells, therapeutic agents that utilize prominent features of cancerous cells are under development. Immunotherapy using engineered immune cells is one approach to treating cancer.

[0004] Incorporation by reference of information in an ASCII text file This application incorporates by reference the sequence listing contained in the following ASCII text file, which is being filed concurrently: File name: NKT043WO_ST25.txt; created on June 1, 2020, and having a size of 327 KB.

Summary of the Invention

Means for Solving the Problems

[0005] Immunotherapy offers new technological advances in the treatment of diseases, where immune cells are engineered to express specific targeting and / or effector molecules that specifically recognize and respond to diseased or damaged cells. This represents a promising advance, at least in part, due to the potential to specifically target diseased or damaged cells, as opposed to more traditional approaches such as chemotherapy that affect all cells, and the desired outcome is for sufficient healthy cells to survive for the patient to survive. One immunotherapy approach is the recombinant expression of chimeric receptors in immune cells to achieve targeted recognition and destruction of abnormal cells of interest.

[0006] In some embodiments, the cells for immunotherapy are genetically modified to increase one or more properties of the cells that result in more effective treatment. In some embodiments, one or more of the expandability, cytotoxicity, and / or persistence of the genetically modified immune cells is increased. In some embodiments, the immune cells are also engineered to express a cytotoxic receptor that targets tumors. As described herein, in some embodiments, there is provided a genetically engineered NK cell population for cancer immunotherapy that includes a plurality of natural killer (NK) cells, wherein the plurality of NK cells are engineered to express a cytotoxic receptor that includes an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex, and the NK cells are gene edited to express a reduced level of cytokine-inducible SH2-containing (CIS) protein encoded by the CISH gene as compared to non-engineered NK cells, and the reduced CIS expression is engineered via editing of the CISH gene, and the genetically engineered NK cells exhibit increased expansion ability, increased cytotoxicity against target cells, and increased persistence as compared to NK cells expressing natural levels of CIS. In some embodiments, the cytotoxic signaling complex includes an OX-40 subdomain and a CD3 zeta subdomain. In some embodiments, the NK cells are engineered to express membrane-bound IL-15. In some embodiments, T cells are engineered and used instead of or in addition to NK cells. In some embodiments, NKT cells are not included in the engineered immune cell population. In some embodiments, the immune cell population comprises, consists of, or consists essentially of engineered NK cells.

[0007] In some embodiments, the extracellular ligand-binding domain comprises a receptor having specificity for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the cytotoxic receptor expressed by NK cells comprises or consists essentially of (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3z co-stimulatory subdomain. In some embodiments, the cytotoxic receptor is encoded by a polynucleotide having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 145. In some embodiments, the cytotoxic receptor has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 174.

[0008] In some embodiments, the cytotoxic receptor expressed by the NK cell comprises or consists essentially of a chimeric antigen receptor (CAR) comprising: (i) a tumor-binding domain comprising an anti-CD19 antibody fragment; (ii) a CD8 transmembrane domain; and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3z co-stimulatory subdomain. In some embodiments, the anti-CD19 antibody comprises a variable heavy chain (VH) domain of a single-chain variable fragment (scFv) and a variable light chain (VL) domain of the scFv, the VH domain comprising the amino acid sequence of SEQ ID NO: 120 and the encoded VL domain comprising the amino acid sequence of SEQ ID NO: 118. In some embodiments, the CAR expressed by the T cell has at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, the anti-CD19 antibody fragment is designed (e.g., engineered) to reduce the potential antigenicity of the encoded protein and / or to increase one or more properties of the encoded protein (e.g., target recognition and / or binding properties). Thus, according to some embodiments, the anti-CD19 antibody fragment does not contain a specific sequence. For example, according to some embodiments, the anti-CD19 antibody fragment is not encoded by SEQ ID NO: 116 and does not include the VL region of SEQ ID NO: 105 or 107 or the VH region of SEQ ID NO: 104 or 106. In some embodiments, the anti-CD19 antibody fragment does not include one or more CDRs selected from SEQ ID NOs: 108-115.

[0009] In some embodiments, the expression of CIS is substantially reduced compared to unmanipulated NK cells. According to certain embodiments provided herein, gene editing can reduce the expression of a target protein such as CIS (or others disclosed herein) by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99%, or more (including any amount between the recited ones). In some embodiments, the gene is completely knocked out so that the expression of the target protein cannot be detected. Thus, in some embodiments, immune cells (e.g., NK cells) do not express detectable levels of the CIS protein.

[0010] In some embodiments, the NK cells are further genetically engineered to express reduced levels of transforming growth factor beta receptor (TGFBR) compared to unmanipulated NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of TGFBR. In some embodiments, the NK cells are further gene edited to express reduced levels of beta-2 microglobulin (B2M) compared to unmanipulated NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of the B2M surface protein. In some embodiments, the NK cells are further gene edited to express reduced levels of CIITA (class II major histocompatibility complex transactivator) compared to unmanipulated NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of CIITA. In some embodiments, the NK cells are further gene edited to express reduced levels of natural killer group 2, member A (NKG2A) receptor compared to unmanipulated NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of NKG2A. In some embodiments, the NK cells are further gene edited to express reduced levels of Cbl proto-oncogene B protein, encoded by the CBLB gene, compared to unmanipulated NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of Cbl proto-oncogene B protein. In some embodiments, the NK cells are further gene edited to express reduced levels of tripartite motif-containing protein 29 protein, encoded by the TRIM29 gene, compared to unmanipulated NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of the TRIM29 protein.In some embodiments, the NK cells are further genetically edited to express a reduced level of suppressor of cytokine signaling 2 protein encoded by the SOCS2 gene, as compared to unmanipulated NK cells. In some embodiments, at least 50% of the NK cell population does not express detectable levels of SOCS2 protein. Depending on the embodiment, any combination of the above target proteins / genes can be edited to a desired level such that the protein is not expressed at detectable levels, including in combination with CIS. In some embodiments, some amount of detectable protein may remain, but the function of the protein is disrupted, substantially disrupted, eliminated, or substantially eliminated. In some embodiments, even if some functionality remains, the positive effects conferred on the engineered immune cells (e.g., NK cells or T cells) remain and help increase one or more anti-cancer aspects of the cells.

[0011] In some embodiments, the NK cells are further genetically edited to disrupt the 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.

[0012] In some embodiments, gene editing is used to "knock in" or alternatively increase the expression of a target protein. In some embodiments, the expression of the target protein can 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 between the recited ones). For example, in some embodiments, NK cells are further gene edited to express CD47. In some embodiments, NK cells are further genetically engineered to express HLA-E. The knocked-in gene can be knocked in in combination with either the knocked-out or alternatively disrupted gene.

[0013] In some embodiments, the genetically engineered NK cell population further comprises a genetically engineered T cell population. In some embodiments, the T cell population is at least partially, if not substantially, allogeneic non-reactive. In some embodiments, the allogeneic non-reactive T cells comprise at least one gene-edited subunit of the T cell receptor (TCR) such that the allogeneic non-reactive T cells do not exhibit an allogeneic reactive effect against the recipient subject's cells. In some embodiments, the T cell population is engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker, where the tumor marker is one or more of CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, EGFR. In some embodiments, combinations of two or more of these tumor markers can be targeted. In some embodiments, the CAR expressed by the T cells has specificity for CD19. In some embodiments, the CAR expressed by the T cells has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, the CAR targets CD19. In some embodiments, the CAR is designed (e.g., engineered) to reduce the potential antigenicity of the encoded protein and / or increase one or more properties of the encoded protein (e.g., target recognition and / or binding properties). Thus, according to some embodiments, the anti-CD19 CAR does not contain a particular sequence. For example, according to some embodiments, the anti-CD19 CAR is not composed of SEQ ID NO: 116, SEQ ID NO: 105, 107, 104, or 106. In some embodiments, the anti-CD19 antibody fragment does not contain one or more CDRs selected from SEQ ID NOs: 108-115.

[0014] In some embodiments, the TCR subunit of the engineered T cell is TCRα. In some embodiments, modification of the TCR of the T cell results in at least 80%, 85%, or 90% of the T cell population no longer expressing detectable levels of TCR. Similar to the engineered NK cells disclosed herein, in some embodiments, the T cells are further gene edited to reduce the expression of one or more of CIS, TGFBR, B2M, CIITA, TRIM29, and SOCS2 or to express CD47 or HLA-E compared to unengineered T cells. In some embodiments, the T cells are further gene edited to disrupt the expression of at least one immune checkpoint protein by the T cells, and the at least one immune checkpoint protein is selected from CTLA4, PD-1, and lymphocyte activation gene (LAG-3).

[0015] Depending on the embodiment, gene editing of NK cells and / or T cells to reduce expression, and / or gene editing to induce expression, is performed using 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, and combinations thereof. In some embodiments, the Cas is Cas9. In 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, gene editing of NK cells and / or T cells to reduce expression, and / or gene editing to induce expression, is performed using zinc finger nucleases (ZFNs). In some embodiments, gene editing of NK cells and / or T cells to reduce expression, and / or gene editing to induce expression, is performed using transcription activator-like effector nucleases (TALENs).

[0016] In some embodiments, the genetically engineered NK cells and / or genetically engineered T cells have an OX40 subdomain encoded by a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 5. In some embodiments, the genetically engineered NK cells and / or genetically engineered T cells have a CD3 zeta subdomain encoded by a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 7. In some embodiments, the genetically engineered NK cells and / or genetically engineered T cells have mbIL15 encoded by a sequence having at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 11.

[0017] Also provided herein are methods of treating cancer in a subject, including administering to the subject a genetically engineered NK cell population (and / or a genetically engineered T cell population) as disclosed herein. Also provided herein is the use of a genetically engineered NK cell population (and / or a genetically engineered T cell population) as disclosed herein in the treatment of cancer. Also provided herein is the use of a genetically engineered NK cell population (and / or a genetically engineered T cell population) as disclosed herein in the manufacture of a medicament for the treatment of cancer.

[0018] Methods of treating cancer are also provided herein. In some embodiments, provided are methods of treating cancer in a subject, including administering to the subject a genetically engineered immune cell population, wherein (i) the genetically engineered immune cell population comprises a plurality of NK cells that are engineered to express a cytotoxic receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex, and the NK cells are gene-edited to express a reduced level of cytokine-inducible SH2-containing (CIS) protein encoded by the CISH gene, such that the reduced CIS expression is engineered via gene 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 as compared to non-engineered NK cells; and optionally (ii) a plurality of T cells.

[0019] In some embodiments, the cytotoxic signaling complex comprises an OX-40 subdomain and a CD3 zeta subdomain. In some embodiments, the NK cells are also engineered to express membrane-bound IL-15.

[0020] In some embodiments, when included, the plurality of T cells are substantially non-alloreactive. Advantageously, in some embodiments, the non-alloreactive T cells include at least one modification to a subunit of the T cell receptor (TCR) such that the non-alloreactive T cells do not exhibit an alloreactive effect against the cells of the recipient subject. In some embodiments, the T cells are also engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker that can be selected from CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, EGFR, and combinations thereof.

[0021] In some embodiments, the cytotoxic receptor expressed by the NK cell comprises (i) an NKG2D ligand binding domain, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3z co-stimulatory subdomain. In some embodiments, the cytotoxic receptor is encoded by a polynucleotide having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 145. In some embodiments, the cytotoxic receptor has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 174. In some embodiments, the cytotoxic receptor expressed by the NK cell has specificity for CD19. In some embodiments, the cytotoxic receptor expressed by the NK cell has at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, the CAR expressed by the T cell has specificity for CD19. In some embodiments, the CAR expressed by the T cell (and NK cell) comprises (i) a tumor-binding domain comprising an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3z co-stimulatory subdomain. In some embodiments, the polynucleotide encoding the CAR also encodes a membrane-bound IL15. In some embodiments, the anti-CD19 antibody fragment comprises a variable heavy (VH) domain of a single-chain variable fragment (scFv) and a variable light (VL) domain of the scFv. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 120 and the VL domain comprises the amino acid sequence of SEQ ID NO: 118.

[0022] In some embodiments, the NK cell and / or T cell is further genetically edited to reduce the expression of one or more of CIS, TGFBR, B2M, CIITA, TRIM29, and SOCS2, or to express CD47 or HLA-E, as compared to an unmanipulated T cell.

[0023] In some embodiments, the NK cells and / or T cells are further gene edited to disrupt the expression of at least one immune checkpoint protein by the cell, and the at least one immune checkpoint protein is selected from CTLA4, PD-1, and lymphocyte activation gene (LAG-3), NKG2A receptor, KIR2DL-1, KIR2DL-2, KIR2DL-3, KIR2DS-1 and / or KIR2DA-2.

[0024] In some embodiments, the OX40 subdomain is encoded by a sequence having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 5. In some embodiments, the CD3 zeta subdomain is encoded by a sequence having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 7. In some embodiments, mbIL15 is encoded by a sequence having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 11.

[0025] Depending on the embodiments of the methods disclosed herein that are applicable, gene editing of NK cells and / or T cells to reduce expression, and / or gene editing to induce expression, is performed using the CRISPR-Cas system. In some embodiments, the CRISPR-Cas system comprises a Cas selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, and combinations thereof. In some embodiments, the Cas is Cas9. In some embodiments, the CRISPR-Cas system 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, gene editing of NK cells and / or T cells to reduce expression, and / or gene editing to induce expression, is performed using zinc finger nucleases (ZFNs). In some embodiments, gene editing of NK cells and / or T cells to reduce expression, and / or gene editing to induce expression, is performed using transcription activator-like effector nucleases (TALENs).

[0026] Furthermore, provided herein is a mixed population of engineered cells for cancer immunotherapy comprising a plurality of NK cells, wherein the plurality of NK cells are engineered to express a cytotoxic receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex, and the NK cells are genetically edited to express, by the cell, a reduced level of cytokine-inducible SH2-containing (CIS) protein encoded by the CISH gene as compared to unengineered NK cells, and the reduced CIS expression is engineered via genetic editing of the CISH gene, and the genetically engineered NK cells exhibit increased expansion ability, increased cytotoxicity against target cells, and increased persistence as compared to NK cells expressing natural levels of CIS, and the plurality of T cells are substantially allogeneic non-reactive via at least one modification to a subunit of the T cell receptor (TCR), and the T cell population is engineered to express a chimeric antigen receptor (CAR) having specificity for one or more tumor markers selected from CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, and EGFR. In some embodiments, the cytotoxic signaling complex of the cytotoxic receptor and / or the CAR comprises an OX-40 subdomain and a CD3 zeta subdomain. In some embodiments, the NK cells and / or the T cells are engineered to express membrane-bound IL-15. In some embodiments, the cytotoxic receptor expressed by the NK cells has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 174. In some embodiments, the cytotoxic receptor expressed by the NK cells has at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 178. In some embodiments, the CAR expressed by the T cells has at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 178.

[0027] This specification provides, in some embodiments, a genetically modified population of immune cells for cancer immunotherapy, genetically modified to reduce the expression of cytokine-inducible SH2-containing protein encoded by the CISH gene by the immune cells, genetically modified to reduce the expression of transforming growth factor beta receptor by the immune cells, genetically modified to reduce the expression of natural killer group 2, member A (NKG2A) by the immune cells, genetically modified to reduce the expression of Cbl proto-oncogene B protein encoded by the CBLB gene by the immune cells, genetically modified to reduce the expression of tripartite motif-containing protein 29 protein encoded by the TRIM29 gene by the immune cells, and / or genetically modified to reduce the expression of suppressor of cytokine signaling 2 protein encoded by the SOCS2 gene by the immune cells, and genetically engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker present on the target tumor cells. In some embodiments, the population comprises, consists of, or consists essentially of natural killer cells. In some embodiments, the population further comprises T cells. In some embodiments, the CAR has specificity for CD19. In some embodiments, the CAR comprises one or more humanized CDR sequences. In some embodiments, the CAR has specificity for an NKG2D ligand. In some embodiments, the genetic modification of the cells 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, and combinations thereof. In some embodiments, the Cas is Cas9.In some embodiments, the modification is to CISH, and the CRISPR-Cas system is guided by one or more guide RNAs selected from those comprising the sequences of SEQ ID NO: 153, 154, 155, 156, or 157; the modification is to TGFBR2, and the CRISPR-Cas system is guided by one or more guide RNAs selected from those comprising the sequences of SEQ ID NO: 147, 148, 149, 150, 151, or 152; the modification is to NKG2A, and the CRISPR-Cas system is guided by one or more guide RNAs selected from those comprising the sequences of SEQ ID NO: 158, 159, or 160; the modification is to CBLB, and the CRISPR-Cas system is guided by one or more guide RNAs selected from those comprising the sequences of SEQ ID NO: 164, 165, or 166; the modification is to TRIM29, and the CRISPR-Cas system is guided by one or more guide RNAs selected from those comprising the sequences of SEQ ID NO: 167, 168, or 169; and / or the modification is to SOCS2, and the CRISPR-Cas system is guided by one or more guide RNAs selected from those comprising the sequences of SEQ ID NO: 171, 172, or 173.

[0028] In some embodiments, the gene modification(s) is / are performed using zinc finger nucleases (ZFNs). In some embodiments, the gene modification(s) is / are performed using transcription activator-like effector nucleases (TALENs).

[0029] In some embodiments, the genetically modified immune cells exhibit increased cytotoxicity, increased survival rate, and / or increased anti-tumor cytokine release profile compared to unmodified immune cells. In some embodiments, the genetically modified immune cells are further genetically modified to reduce alloreactivity to the cells when administered to a subject who is not the donor of the cells.

[0030] Also provided herein is a mixed population for cancer immunotherapy, comprising a T cell population that is substantially non-alloreactive through at least one modification to a subunit of a T cell receptor (TCR) selected from TCRα, TCRβ, TCRγ, and TCRδ, whereby the TCR does not recognize differences in the major histocompatibility gene complex among the T cells of a recipient subject to whom the mixed population of immune cells is administered, the T cell population is engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker, the tumor marker is selected from the group consisting of CD19, CD123, CD70, mesothelin, claudin 6, BCMA, PD-L1, EGFR, and combinations thereof, the natural killer (NK) cell population is engineered to express a chimeric receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex, and the extracellular ligand-binding domain has specificity for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the modified TCR subunit is TCRα.

[0031] In some embodiments, the T cells and / or NK cells are modified to express reduced levels of MHC I and / or MHC II molecules, thereby inducing a reduced immune response from the immune system of a recipient subject in which the NK cells and T cells are allogeneic. In some embodiments, the MHC I and / or MHC II molecules are beta-microglobulin and / or CIITA (class II major histocompatibility complex transactivator). In some embodiments, the T cells and / or NK cells further comprise a modification that disrupts the expression of at least one immune checkpoint protein by the T cells and / or NK cells. Depending on the embodiment, 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.

[0032] In some embodiments, the NK cells and / or T cells are further modified to reduce or substantially eliminate the expression and / or function of CIS. In some embodiments, the NK cells are further engineered to express membrane-bound IL-15.

[0033] In some embodiments, the CAR expressed by the T cell comprises: (i) a tumor-binding domain comprising an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3ζ co-stimulatory subdomain. In some embodiments, the T cell also expresses membrane-bound IL15. In some embodiments, mbIL15 is encoded by the same polynucleotide encoding the CAR. In some embodiments, the anti-CD19 antibody comprises a variable heavy (VH) domain of a single-chain variable fragment (scFv) and a variable light (VL) domain of the scFv. In some such embodiments, the VH domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 120. In some embodiments, the encoded VL domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 118. In some embodiments, the OX40 subdomain is encoded by a sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 5. In some embodiments, the CD3 zeta subdomain is encoded by a sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 7. In some embodiments, mbIL15 is encoded by a sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 11. In some embodiments, the CAR expressed by the T cell has at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 178. In some embodiments, the chimeric receptor expressed by the NK cell comprises: (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3ζ co-stimulatory subdomain. In some embodiments, the NK cell is further engineered to express membrane-bound IL15 (which may be encoded by the same polynucleotide encoding the chimeric receptor). In some embodiments, the chimeric receptor is encoded by a polynucleotide having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 145.In some embodiments, the chimeric receptor has at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 174.

[0034] In some embodiments, modification of the TCR results in at least 80% of the T cell population no longer expressing detectable levels of TCR, but at least 70% of the T cell population expressing detectable levels of CAR. In some embodiments, the T cells and / or NK cells are further modified to reduce the expression of one or more of the B2M surface protein, cytokine-inducible SH2-containing protein (CIS) encoded by the CISH gene, transforming growth factor beta receptor, natural killer group 2, member A (NKG2A) receptor, Cbl proto-oncogene B protein encoded by the CBLB gene, tripartite motif-containing protein 29 protein encoded by the TRIM29 gene, suppressor of cytokine signaling 2 protein encoded by the SOCS2 gene by T cells and / or NK cells. In some embodiments, gene editing can reduce the expression of any of these target proteins 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 between those listed). In some embodiments, the gene is completely knocked out so that the expression of the target protein cannot be detected. In some embodiments, the target protein expression can 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 between those listed). For example, in some embodiments, the T cells and / or NK cells are further gene edited to express CD47. In some embodiments, the NK cells are further genetically engineered to express HLA-E. Any gene to be knocked in can be knocked in in combination with either the gene that is knocked out or the gene that is disrupted instead.

[0035] In some embodiments, the modification(s) to the TCR, or further modification of NK cells or T cells, 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, and combinations thereof. In some embodiments, the Cas is Cas9. In 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 modification(s) to the TCR, or further modification of NK cells or T cells, is performed using a zinc finger nuclease (ZFN). In some embodiments, the modification(s) to the TCR, or further modification of NK cells or T cells, is performed using a transcription activator-like effector nuclease (TALEN).

[0036] Also provided herein is a mixed population of immune cells for cancer immunotherapy, the T cell population being substantially allogeneic reactive due to at least one modification to a subunit of the T cell receptor (TCR), whereby the allogeneic reactive T cells do not exhibit an allogeneic reactive effect against the cells of the recipient subject, and the T cell population is engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker selected from CD19, CD123, CD70, mesothelin, claudin 6, BCMA, PD-L1, EGFR, and combinations thereof, and a natural killer (NK) cell population, the NK cell population being engineered to express a chimeric receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex, the extracellular ligand-binding domain having specificity for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0037] Also provided herein is a method of treating cancer in a subject without inducing graft-versus-host disease, comprising administering to the subject a mixed population of immune cells according to the present disclosure. Also provided herein is the use of a mixed population of immune cells according to the present disclosure in the treatment of cancer. Also provided herein is the use of a mixed population of immune cells according to the present disclosure in the manufacture of a medicament for treating cancer.

[0038] In some embodiments, a method for treating cancer in a subject is provided, comprising administering to the subject at least a first dose of a mixed population of immune cells, the mixed population of cells comprising a substantially allogeneic non-reactive T cell population engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker selected from CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, EGFR, and combinations thereof, and a natural killer (NK) cell population engineered to express a chimeric receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex, the extracellular ligand binding domain having specificity for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0039] In some embodiments, the alloreactive T cells comprise at least one modification to a subunit of the T cell receptor (TCR) such that the alloreactive T cells do not exhibit an alloreactive effect against the recipient subject's cells. In some embodiments, the CAR expressed by the T cells has specificity for CD19. In some embodiments, the CAR expressed by the T cells comprises (i) a tumor-binding domain comprising an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3z co-stimulatory subdomain. In some embodiments, the polynucleotide encoding the CAR also encodes a membrane-bound IL15. In some embodiments, the anti-CD19 antibody comprises a variable heavy (VH) domain of a single-chain variable fragment (scFv) and a variable light (VL) domain of the scFv. In some embodiments, the VH domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 120, and the VL domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 118. In some embodiments, the CAR expressed by the T cells has at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, the chimeric receptor expressed by the NK cells comprises (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3z co-stimulatory subdomain. In some embodiments, the polynucleotide encoding the chimeric receptor also encodes a membrane-bound IL15. In some embodiments, the chimeric receptor is encoded by a polynucleotide having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 145. In some embodiments, the chimeric receptor has at least 95%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 174. In some embodiments, the OX40 subdomain of the CAR and / or chimeric receptor is encoded by a sequence having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 5.In some embodiments, the CD3 zeta subdomain of the CAR and / or chimeric receptor is encoded by a sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 7. In some embodiments, the mbIL15 expressed by T cells and / or NK cells is encoded by a sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 11.

[0040] In some embodiments, a mixed population of immune cells for cancer immunotherapy is provided, the mixed population comprising a T cell population expressing a CAR having specificity for a tumor antigen, the T cell population being genetically modified such that the T cells are substantially allogeneic non-reactive, and an NK cell population expressing a CAR having specificity for the same tumor antigen. In some embodiments, a mixed population of immune cells for cancer immunotherapy is provided, the mixed population comprising a T cell population expressing a CAR having specificity for a tumor antigen, the T cell population being genetically modified such that the T cells are substantially allogeneic non-reactive, and an NK cell population expressing a CAR having specificity for an additional tumor antigen. In some embodiments, a mixed population of immune cells for cancer immunotherapy is provided, the mixed population comprising a T cell population that is substantially allogeneic non-reactive and an NK cell population expressing a chimeric receptor that targets a tumor ligand.

[0041] In some embodiments, the allogeneic non-reactive T cells comprise at least one modification to a subunit of the T cell receptor (TCR) such that the TCR recognizes an antigen without recognizing differences in the major histocompatibility complex between T cells of a subject to whom the mixed population of immune cells is administered. In some embodiments, the allogeneic non-reactive T cell population is engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker (e.g., a tumor associated antigen or tumor antigen). Depending on the embodiment, the CAR can be engineered to target one or more of CD19, CD123, CD70, Her2, mesothelin, claudin 6 (but not other claudins), BCMA, PD-L1, EGFR.

[0042] In some embodiments, the NK cell population is engineered to express a chimeric receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and a cytotoxic signaling complex, wherein the extracellular ligand-binding domain has specificity for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the NK cells may also be engineered to express a CAR, which may be engineered to target one or more of CD19, CD123, CD70, Her2, mesothelin, claudin 6 (but not other claudins), BCMA, PD-L1, EGFR (or any other antigen such that both T cells and NK cells target the same antigen of interest).

[0043] In some embodiments, the T cells comprise mutations that disrupt the expression of at least one immune checkpoint protein by the T cells. For example, the T cells can be mutated with respect to immune checkpoint proteins selected from CTLA4, PD-1, and combinations thereof. In some embodiments, the blockade of B7-1 / B7-2 to CTLA4 is also used to reduce T cells that are maintained in an inactive state. Thus, in some embodiments, the T cells are modified to express CTLA4 that they mismatched or mutated, but in some embodiments, exogenous substances are used, for example, to bind to antigen-presenting cells and / or to inhibit the ability to interact with CTLA4 of B7-1 / B7-2 on antigen-presenting cells instead. Similarly, in some embodiments, NK cells can be modified to disrupt the expression of at least one checkpoint inhibitor. In some embodiments, for example, CDTLA4 or PD-1 is modified, for example, mutated, to reduce its ability to reduce the NK cell cytotoxic response of such checkpoint inhibitors. In some embodiments, lymphocyte activation gene 3 (LAG-3, CD223) is disrupted in NK cells (and / or T cells). In some embodiments, the inhibitory NKG2A receptor is mutated, knocked out, or inhibited, for example, by an antibody. Monalizumab is used, in some embodiments, as a non-limiting example, to disrupt inhibitory signaling by the NKG2A receptor. In some embodiments, one or more killer inhibitory receptors (KIRs) on NK cells are disrupted and / or blocked (e.g., via genetic modification). For example, in some embodiments, one or more of KIR2DL-1, KIR2DL-2, KIR2DL-3, KIR2DS-1, and / or KIR2DA-2 are disrupted or blocked, thereby preventing their binding to HLA-C MHC molecules.Furthermore, in some embodiments, TIM3 is modified, mutated (e.g., via gene editing), or alternatively functionally disrupted (e.g., blocked by an antibody), whereby its normal function of suppressing the response of immune cells upon ligand binding is disrupted. In some such embodiments, disruption of TIM3 expression or function (e.g., by CRISPr or other methods disclosed herein), optionally in combination with disruption of one or more immune checkpoint modulators, results in administered T cells and / or NK cells having increased antitumor activity. Tim-3 is involved in galectin-9 secretion, and the latter functions to impair the anticancer activity of cytotoxic lymphoid cells including natural killer (NK) cells. TIM3 is also expressed in a soluble form and inhibits the secretion of interleukin-2 (IL-2). Thus, in some embodiments, disruption of TIM3, expression, secretion, or pathway functionality provides an increase in T cell and / or NK cell activity.

[0044] In some embodiments, TIGIT (also referred to as VSTM3) is modified, mutated (e.g., via gene editing), or alternatively functionally disrupted (e.g., blocked by an antibody), whereby its normal function of suppressing the response of immune cells upon ligand binding is disrupted. CD155 is a ligand for TIGIT. In some embodiments, TIGIT expression is decreased or knocked out. In some embodiments, TIGIT is blocked by an inactivating ligand or its activity is decreased via a competitive inhibitor of CD155 (which does not activate TIGIT). TIGIT contains an inhibitory ITIM motif and in some embodiments is excised, for example, via gene editing using CRISPr or other methods disclosed herein. In such embodiments, the function of TIGIT is decreased, enabling an increase in T cell and / or NK cell activity.

[0045] In some embodiments, the adenosine receptor A1 is modified, mutated (e.g., via gene editing), or alternatively functionally disrupted (e.g., blocked by an antibody), thereby disrupting its normal function of suppressing the response of immune cells upon ligand binding. Adenosine signaling is involved in tumor immunity as a result of its function as an immunosuppressive metabolite. Thus, in some embodiments, adenosine receptor A1 expression is decreased or knocked out. In some embodiments, the adenosine receptor A1 is blocked by an inactivating ligand, or its activity is decreased via a competitive inhibitor of adenosine (which does not activate the adenosine signaling pathway). In some embodiments, the adenosine receptor is modified, e.g., by gene editing using CRISPr or other methods disclosed herein, to decrease its function or expression and enable an increase in T cell and / or NK cell activity.

[0046] In some embodiments, the modified TCR subunit is selected from TCRα, TCRβ, TCRγ, and TCRδ. In some embodiments, the modified TCR subunit is TCRα.

[0047] In some embodiments, the modification to the TCR is performed using the CRISPR-Cas system. In some embodiments, disruption of the expression of at least one immune checkpoint protein by T cells or NK cells is performed using the CRISPR-Cas system. For example, Cas can be selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, and combinations thereof. In some embodiments, Cas is Cas9. In 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.

[0048] In some embodiments, the modification to the TCR is performed using zinc finger nucleases (ZFNs). In some embodiments, disruption of the expression of at least one immune checkpoint protein by T cells or NK cells is performed using zinc finger nucleases (ZFNs). In some embodiments, the modification to the TCR is performed using transcription activator-like effector nucleases (TALENs). In some embodiments, disruption of the expression of at least one immune checkpoint protein by T cells or NK cells is performed using transcription activator-like effector nucleases (TALENs). Combinations of ZFNs and TALENs (and optionally CRISPR-Cas) are used in some embodiments to modify either or both NK cells and T cells.

[0049] According to some embodiments, either NK cells, allogeneic non-reactive T cells, or both are further engineered to express membrane-bound IL-15.

[0050] Advantageously, the mixed cell population is useful in the methods provided herein, and cancer in a subject can be treated without inducing graft-versus-host disease. In some embodiments, the method comprises administering to a subject a mixed population of alloreactive T cells expressing a CAR and engineered NK cells expressing a chimeric receptor. Also provided is the use of a mixed population of alloreactive T cells expressing a CAR and engineered NK cells expressing a chimeric receptor in the treatment of cancer and / or in the manufacture of a medicament for the treatment of cancer. In still further embodiments, the NK cells and T cells are allogeneic with respect to the subject receiving them. In some embodiments, such combinations involve NK cells and T cells having specificity for the same target antigen. For example, in some embodiments, both the NK cells and the T cells (e.g., alloreactive T cells) are allogeneic with respect to the subject receiving them and are engineered to express a CAR that targets the same antigen, e.g., CD19. In some embodiments, the NK cells and T cells are configured to both target target cells expressing another marker, e.g., CD123, CD70, Her2, mesothelin, claudin 6 (but not other claudins), BCMA, PD-L1, EGFR (or any other antigen such that both the T cells and NK cells target the same antigen of interest).

[0051] In some embodiments, modification of the TCR results in at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the T cell population no longer expressing detectable levels of the TCR, while at the same time at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of the T cell population expressing detectable levels of the CAR. Thus, these cells are primarily alloreactive and are activated with an anti-tumor directed CAR. Further, in some embodiments, useful for limiting the immune response from allogeneic T cells, at least 50% of the engineered T cells express detectable levels of the CAR and do not express detectable levels of the TCR surface protein or the B2M surface protein.

[0052] In some embodiments, the NK cells are genetically modified to reduce the immune response that can occur in an allogeneic host against non-self NK cells. In some embodiments, the NK cells are engineered to exhibit reduced expression of one or more MCH class I and / or one or more MHC class II molecules. In some embodiments, the expression of beta-microglobulin is substantially, significantly, or completely reduced in at least a portion of the NK cells that express (or are modified to express) a CAR having specificity for a tumor antigen such as CD19 (or any other antigen disclosed herein). In some embodiments, the expression of CIITA (class II major histocompatibility complex transactivator) is substantially, significantly, or completely reduced in at least a portion of the NK cells that express (or are modified to express) a CAR having specificity for a tumor antigen such as CD19 (or any other antigen disclosed herein). In some embodiments, such genetically modified NK cells are generated using the CRISPr-Cas system, TALEN, zinc fingers, RNAi, or other gene editing techniques. As discussed herein, in some embodiments, NK cells with reduced alloreactivity are used in combination with non-alloreactive T cells. In some embodiments, the NK cells are modified to express CD47 in the modified NK cells, which helps to avoid detection by the recipient's endogenous innate immune cells. In some embodiments, the T cells are modified in a similar manner. In some embodiments, both the NK cells and the T cells are modified to express CD47, which helps with the persistence of NK and / or T cells in the recipient and thus increases the anti-tumor effect. In some embodiments, the NK cells are modified to express HLA-G in the modified NK cells, which helps to avoid detection by the recipient's endogenous innate immune cells. In some embodiments, the T cells are modified in a similar manner.In some embodiments, both NK cells and T cells are modified to express HLA-G, which serves to enhance the persistence of NK and / or T cells in the recipient and thus increase the anti-tumor effect. In some embodiments, T cells and NK cells that have reduced alloreactivity and are engineered to express CARs against the same antigen are used to treat cancer in allogeneic patients.

[0053] In some embodiments, a genetically modified population of immune cells for cancer immunotherapy is provided, genetically modified to reduce the expression of the transforming growth factor beta receptor by the immune cells, and genetically engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker present on a target tumor cell. In further embodiments, a genetically modified population of immune cells for cancer immunotherapy is provided, genetically modified to reduce the expression of the natural killer group 2, member A (NKG2A) receptor by the immune cells, and genetically engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker present on a target tumor cell. In further embodiments, a genetically modified population of immune cells for cancer immunotherapy is provided, genetically modified to reduce the expression of the cytokine-inducible SH2-containing protein encoded by the CISH gene by the immune cells, and genetically engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker present on a target tumor cell. CISH is an inhibitory checkpoint in NK cell-mediated cytotoxicity. In further embodiments, a genetically modified population of immune cells for cancer immunotherapy is provided, genetically modified to reduce the expression of the Cbl proto-oncogene B protein encoded by the CBLB gene by the immune cells, and genetically engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker present on a target tumor cell. CBLB is an E3 ubiquitin ligase and a negative regulator of NK cell activation. In further embodiments, a genetically modified population of immune cells for cancer immunotherapy is provided, genetically modified to reduce the expression of the tripartite motif-containing protein 29 protein encoded by the TRIM29 gene by the immune cells, and genetically engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker present on a target tumor cell. TRIM29 is an E3 ubiquitin ligase and a negative regulator of NK cell function after activation.In a further embodiment, a genetically modified population of immune cells for cancer immunotherapy is provided, genetically modified to reduce the expression of a suppressor of cytokine signaling 2 protein encoded by the SOCS2 gene by the immune cells, and genetically engineered to express a chimeric antigen receptor (CAR) having specificity for a tumor marker present on the target tumor cells. SOCS2 is a negative regulator of NK cell function. In some embodiments, the genetically modified population of immune cells comprises NK cells, T cells, or a combination thereof. In some embodiments, additional immune cells such as gamma delta T cells, NK T cells, etc. are also included. In some embodiments, the CAR has specificity for CD19. In some such embodiments, the CAR comprises one or more humanized CDR sequences. In a further embodiment, the CAR has specificity for CD123. In some embodiments, the genetically modified cells are engineered to express more than one CAR having specificity for more than one target. Optionally, a mixed population of T cells and NK cells is used, and the T cells and NK cells can each express at least one CAR that may or may not have specificity for the same cancer marker, depending on the embodiment. In some embodiments, the cells express a CAR having specificity for an NKG2D ligand.

[0054] As discussed above, in some embodiments, the cells are edited using a CRISPr-based approach. In some embodiments, the modification is to TGFBR2, and the CRISPR-Cas system is guided by one or more guide RNAs selected from the sequences of SEQ ID NO: 147, 148, 149, 150, 151, or 152, or sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequences of SEQ ID NO: 147, 148, 149, 150, 151, or 152. In some embodiments, the modification is to NKG2A, and the CRISPR-Cas system is guided by one or more guide RNAs selected from the sequences of SEQ ID NO: 158, 159, or 160, or sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequences of SEQ ID NO: 158, 159, or 160. In some embodiments, the modification is to CISH, and the CRISPR-Cas system is guided by one or more guide RNAs selected from the sequences of SEQ ID NO: 153, 154, 155, 156, or 157, or sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequences of SEQ ID NO: 153, 154, 155, 156, or 157. In some embodiments, the modification is to CBLB, and the CRISPR-Cas system is guided by one or more guide RNAs selected from the sequences of SEQ ID NO: 164, 165, or 166, or sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequences of SEQ ID NO: 164, 165, or 166.In some embodiments, the modification is to TRIM29, and the CRISPR-Cas system is guided by one or more guide RNAs selected from those comprising the sequence of SEQ ID NO: 167, 168, or 169, or a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to a sequence comprising the sequence of SEQ ID NO: 167, 168, or 169. In some embodiments, the modification is to SOCS2, and the CRISPR-Cas system is guided by one or more guide RNAs selected from those comprising the sequence of SEQ ID NO: 171, 172, or 173, or a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to a sequence comprising the sequence of SEQ ID NO: 171, 172, or 173. In some embodiments, the guide RNA is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length.

[0055] In some embodiments, a method of generating engineered T cells suitable for allogeneic transplantation is provided, the method comprising introducing into T cells a vector comprising an RNA-guided nuclease, a gRNA targeting a T cell receptor gene, and a donor template comprising a nucleic acid encoding a CAR, wherein the CAR comprises (i) a tumor-binding domain comprising an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3z co-stimulatory subdomain, and (iv) a membrane-bound IL15, and the nucleic acid encoding the CAR is flanked by left and right homology arms adjacent to the T cell receptor locus, and (b) expanding the engineered T cells in culture.

[0056] Also provided is a further method for engineered T cells suitable for allogeneic transplantation, the method comprising delivering to T cells an RNA-guided nuclease and a gRNA targeting a T cell receptor gene to disrupt the expression of at least one subunit of the TCR, delivering to the T cells a nucleic acid vector comprising a nucleic acid encoding a CAR, wherein the CAR comprises (i) a tumor-binding domain comprising an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3z co-stimulatory subdomain, and (iv) a membrane-bound IL15, and expanding the engineered T cells in culture.

[0057] Furthermore, a method is provided, for example, a method of generating engineered T cells suitable for allogeneic transplantation, the method comprising delivering to T cells a nuclease capable of inducing a targeted double-stranded DNA break in a target region of a T cell receptor gene to disrupt the expression of at least one subunit of the TCR, delivering to the T cells a vector comprising a nucleic acid encoding a CAR, the CAR comprising (i) a tumor-binding domain comprising an antibody fragment that recognizes one or more of CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, and EGFR, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 co-stimulatory subdomain and a CD3z co-stimulatory subdomain, and (iv) a membrane-bound IL15, and expanding the engineered T cells in culture. In some embodiments, the method further comprises modifying the T cells by inactivating at least a first gene encoding an immune checkpoint protein. In some embodiments, the immune checkpoint gene is selected from the group consisting of PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, and 2B4.

[0058] A method of treating cancer is provided, the method comprising generating T cells suitable for allogeneic transplantation according to the embodiments disclosed herein, the T cells being donor-derived and transduced into a population of NK cells expanded from the same donor to express an activating chimeric receptor comprising an extracellular ligand-binding domain having specificity for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, generating a population of engineered NK cells, optionally further expanding the T cells and / or the population of engineered NK cells, combining the T cells suitable for allogeneic transplantation with the population of engineered NK cells, and administering the combined NK cell and T cell population to a subject that is allogeneic with respect to the donor.

[0059] A method of treating cancer is provided, the method comprising generating T cells suitable for allogeneic transplantation according to embodiments disclosed herein, the T cells being donor-derived and modified to express a chimeric antigen receptor (CAR) having specificity for CD19, CD123, CD70, Her2, mesothelin, claudin 6 (but not other claudins), BCMA, PD-L1, or EGFR, transducing an expanded NK cell population from the same donor to express a CAR having specificity for CD19, CD123, CD70, Her2, mesothelin, claudin 6 (but not other claudins), BCMA, PD-L1, or EGFR, generating an engineered NK cell population, optionally further expanding the T cells and / or the engineered NK cell population, combining the T cells suitable for allogeneic transplantation with the engineered NK cell population, and administering the combined NK cell and T cell population to a subject that is allogeneic with respect to the donor.

[0060] A further method for treating a cancer subject is also provided, the method comprising generating T cells suitable for allogeneic transplantation according to embodiments disclosed herein, the T cells being derived from a first donor and transducing an expanded NK cell population from a second donor to express an activating chimeric receptor comprising an extracellular ligand-binding domain having specificity for a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, generating an engineered NK cell population, optionally further expanding the T cells and / or the engineered NK cell population, combining the T cells suitable for allogeneic transplantation with the engineered NK cell population, and administering the combined NK cell and T cell population to a subject that is allogeneic with respect to the first and second donors.

[0061] In some embodiments, provided herein are immune cells that express a CD19-directed chimeric receptor, and even an immune cell population, wherein the chimeric receptor comprises an extracellular anti-CD19 binding portion, a hinge and / or transmembrane domain, and an intracellular signaling domain. Also provided herein are a CD19-directed chimeric antigen receptor, an extracellular anti-CD19 binding portion, a hinge and / or transmembrane domain, and a polynucleotide encoding a chimeric antigen receptor comprising an intracellular signaling domain (and a vector for transfecting cells therewith).

[0062] Also provided herein are, in some embodiments, a polynucleotide encoding a CD19-directed chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular anti-CD19 binding portion, the anti-CD19 binding portion comprises an scFv, a hinge, the hinge is a CD8 alpha hinge, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprises a CD3 zeta ITAM.

[0063] Also provided herein are, in some embodiments, a polynucleotide encoding a CD19-directed chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular anti-CD19 binding portion, the anti-CD19 binding portion comprises a variable heavy chain of an scFv or a variable light chain of an scFv, a hinge is a CD8 alpha hinge, a transmembrane domain, the transmembrane domain comprises a CD8 alpha transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprises a CD3 zeta ITAM.

[0064] In some embodiments, the transmembrane domain comprises a CD8 alpha transmembrane domain. In some embodiments, the transmembrane domain comprises an NKG2D transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0065] In some embodiments, the intracellular signaling domain comprises or further comprises a CD28 signaling domain. In some embodiments, the intracellular signaling domain comprises or further comprises a 4-1BB signaling domain. In some embodiments, the intracellular signaling domain comprises or further comprises an OX40 domain. In some embodiments, the intracellular signaling domain comprises or further comprises a 4-1BB signaling domain. In some embodiments, the intracellular signaling domain comprises or further comprises a domain selected from ICOS, CD70, CD161, CD40L, CD44, and combinations thereof.

[0066] In some embodiments, the polynucleotide also encodes a truncated epidermal growth factor receptor (EGFRt). In some embodiments, EGFRt is expressed intracellularly as a soluble factor. In some embodiments, EGFRt is expressed in a membrane-bound form. In some embodiments, the polynucleotide also encodes a membrane-bound interleukin-15 (mbIL15). Also provided herein are engineered immune cells (e.g., NK or T cells, or mixtures thereof) that express a CD19-directed chimeric antigen receptor encoded by a polynucleotide disclosed herein. Further provided is a method of treating cancer in a subject, comprising administering to the subject an engineered immune cell that expresses a chimeric antigen receptor disclosed herein. In some embodiments, provided is the use of a polynucleotide disclosed herein in the treatment of cancer and / or in the manufacture of a medicament for the treatment of cancer.

[0067] In some embodiments, the anti-CD19 binding portion comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the VH domain has at least 95% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the VL domain has at least 95% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the anti-CD19 binding portion is derived from the VH and / or VL sequences of SEQ ID NO: 33 or 32. For example, in some embodiments, the VH and VL sequences of SEQ ID NO: 33 and / or 32 are the subject of a humanization campaign and are thus more readily expressed and / or less immunogenic when administered to a human subject. In some embodiments, the anti-CD19 binding portion comprises a scFv that targets CD19, and the scFv comprises a heavy chain variable region that comprises the sequence of SEQ ID NO: 35 or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 35. In some embodiments, the anti-CD19 binding portion comprises a scFv that targets CD19, and the scFv comprises a light chain variable region that comprises the sequence of SEQ ID NO: 36 or a sequence that is at least 95% identical to the sequence of SEQ ID NO: 36. In some embodiments, the anti-CD19 binding portion comprises a light chain CDR (LC CDR1, LC CDR2, and LC CDR3, respectively) that comprises first, second, and third complementarity determining regions, and / or a heavy chain CDR (HC CDR1, HC CDR2, and HC CDR3, respectively) that comprises first, second, and third complementarity determining regions. Depending on the embodiment, various combinations of LC CDRs and HC CDRs are used. For example, in one embodiment, the anti-CD19 binding portion comprises LC CDR1, LC CDR3, HC CD2, and HC CDR3. In some embodiments, other combinations are used. In some embodiments, LC CDR1 comprises the sequence of SEQ ID NO: 37, or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 37. In some embodiments, LC CDR2 comprises the sequence of SEQ ID NO: 38, or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 38. In some embodiments, LC CDR3 comprises the sequence of SEQ ID NO: 39, or a sequence that is at least about 95% homologous to the sequence of SEQ ID NO: 39.In some embodiments, HC CDR1 comprises the sequence of SEQ ID NO: 40, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 40. In some embodiments, HC CDR2 comprises the sequence of SEQ ID NO: 41, 42, or 43, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 41, 42, or 43. In some embodiments, HC CDR3 comprises the sequence of SEQ ID NO: 44, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 44.

[0068] In some embodiments, an anti-CD19 binding portion comprising a variable light chain region (VL) and a variable heavy chain region (HL) is also provided, the VL region comprising first, second and third complementarity determining regions (VL CDR1, VL CDR2, and VL CDR3, respectively), and the VH region comprising first, second and third complementarity determining regions (VH CDR1, VH CDR2, and VH CDR3, respectively). In some embodiments, the VL region comprises the sequence of SEQ ID NO: 45, 46, 47, or 48, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 45, 46, 47, or 48. In some embodiments, the VH region comprises the sequence of SEQ ID NO: 49, 50, 51 or 52, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 49, 50, 51 or 52.

[0069] In some embodiments, an anti-CD19 binding portion is also provided that includes light chain CDRs (LC CDR1, LC CDR2, and LC CDR3, respectively) that include first, second, and third complementarity determining regions. In some embodiments, the anti-CD19 binding portion further includes heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3, respectively) that include first, second, and third complementarity determining regions. In some embodiments, LC CDR1 includes the sequence of SEQ ID NO: 53, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 53. In some embodiments, LC CDR2 includes the sequence of SEQ ID NO: 54, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 54. In some embodiments, LC CDR3 includes the sequence of SEQ ID NO: 55, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 55. In some embodiments, HC CDR1 includes the sequence of SEQ ID NO: 56, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 56. In some embodiments, HC CDR2 includes the sequence of SEQ ID NO: 57, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 57. In some embodiments, HC CDR3 includes the sequence of SEQ ID NO: 58, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 58.

[0070] In some embodiments, the intracellular signaling domain of the chimeric receptor includes an OX40 subdomain. In some embodiments, the intracellular signaling domain further includes a CD3 zeta subdomain. In some embodiments, the OX40 subdomain includes the amino acid sequence of SEQ ID NO: 6 (or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 6), and the CD3 zeta subdomain includes the amino acid sequence of SEQ ID NO: 8 (or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 8).

[0071] In some embodiments, the hinge domain includes a CD8a hinge domain. In some embodiments, the CD8a hinge domain includes the amino acid sequence of SEQ ID NO: 2 (or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 2).

[0072] In some embodiments, the immune cells also express membrane-bound interleukin-15 (mbIL15). In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 12, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 12.

[0073] In some embodiments, the chimeric receptor further comprises the extracellular domain of the NKG2D receptor. In some embodiments, the immune cells express a second chimeric receptor comprising the extracellular domain of the NKG2D receptor, a transmembrane domain, a cytotoxic signaling complex, and optionally mbIL15. In some embodiments, the extracellular domain of the NKG2D receptor comprises a functional fragment of NKG2D comprising the amino acid sequence of SEQ ID NO: 26, or a sequence that is at least about 95% identical to the sequence of SEQ ID NO: 26. In various embodiments, the chimeric antigen receptors and / or immune cells engineered to express the chimeric receptors disclosed herein are NK cells. In some embodiments, T cells are used. In some embodiments, a combination of NK and T cells (and / or other immune cells) is used.

[0074] In some embodiments, provided herein are methods of treating cancer in a subject, comprising administering to the subject engineered immune cells that target CD19 disclosed herein. Also provided herein is the use of immune cells that target CD19 disclosed herein for the treatment of cancer. Similarly, provided herein is the use of immune cells that target CD19 disclosed herein in the preparation of a medicament for the treatment of cancer. In some embodiments, the cancer being treated is acute lymphoblastic leukemia.

[0075] Some embodiments of the methods and compositions described herein relate to immune cells. In some embodiments, the immune cells express a CD19-directed chimeric receptor comprising an extracellular anti-CD19 moiety, a hinge and / or transmembrane domain, and / or an intracellular signaling domain. In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the immune cells are T cells.

[0076] In some embodiments, the hinge domain comprises a CD8a hinge domain. In some embodiments, the hinge domain comprises an Ig4 SH domain.

[0077] In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD3 transmembrane domain.

[0078] In some embodiments, the signaling domain comprises an OX40 signaling domain. In some embodiments, the signaling domain comprises a 4-1BB signaling domain. In some embodiments, the signaling domain comprises a CD28 signaling domain. In some embodiments, the signaling domain comprises an NKp80 signaling domain. In some embodiments, the signaling domain comprises a CD16 IC signaling domain. In some embodiments, the signaling domain comprises a CD3 zeta or CD3ζ ITAM signaling domain. In some embodiments, the signaling domain comprises an mbIL-15 signaling domain. In some embodiments, the signaling domain comprises a 2A cleavage domain. In some embodiments, the mIL-15 signaling domain is separated from the remainder or another portion of the CD19-directed chimeric receptor by a 2A cleavage domain.

[0079] One embodiment relates to a method comprising administering the immune cells described herein to a subject in need thereof. In some embodiments, the subject has cancer. In some embodiments, the administration treats, inhibits, or prevents the progression of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0104] 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 "cytotoxic receptor complex" is given its ordinary meaning and refers to (unless otherwise indicated) chimeric antigen receptors (CARs), chimeric receptors (also referred to as activating chimeric receptors in the case of NKG2D chimeric receptors). In some embodiments, the cells are further engineered to achieve modification of the reactivity of the cells towards non-tumor tissues. Some embodiments relate to the modification of T cells via various genetic engineering methods such that the resulting T cells have reduced and / or eliminated alloreactivity. Such alloreactivity-free T cells can also be engineered to express a chimeric antigen receptor (CAR) that enables the alloreactivity-free T cells to confer a cytotoxic effect on tumor cells. In some embodiments, natural killer (NK) cells are also engineered to express an urban-inducing receptor complex (e.g., a chimeric antigen receptor or a chimeric receptor). In some embodiments, combinations of these engineered immune cell types are used in immunotherapy, resulting in both rapid (NK cell-based) and sustained (T cell-based) anti-tumor effects, both of which advantageously have little or no graft-versus-host disease. Some embodiments include methods of using the compositions or cells in immunotherapy.

[0105] The term "anticancer effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in mean lifespan, a decrease in cancer cell proliferation, a decrease in cancer cell survival, and / or alleviation of various physiological symptoms associated with the cancer condition.

[0106] Cell type Some embodiments of the methods and compositions provided herein relate to cells such as immune cells, for example, immune cells such as T cells can be engineered to contain chimeric receptors such as CD19-directed chimeric receptors, or can be engineered to contain nucleic acids encoding such chimeric receptors 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 further embodiments relate to further genetic engineering of T cells (e.g., donor T cells) to reduce, destroy, minimize and / or eliminate the ability of donor T cells that are alloreactive against recipient cells (graft-versus-host disease).

[0107] Traditional cancer therapies relied on surgical approaches, radiation therapy, chemotherapy, or combinations of these methods. As research has led to a greater understanding of some of the mechanisms of certain cancers, this knowledge has been utilized in the development of targeted cancer therapies. Targeted therapies are cancer treatments that use specific drugs to target specific genes or proteins found in cancer cells, or cells that support cancer growth (such as blood vessel cells), to inhibit or prevent the growth of cancer cells. More recently, genetic engineering has made it possible to develop approaches that utilize specific aspects of the immune system to fight cancer. In some cases, a patient's own immune cells are modified to specifically eradicate that patient's cancer type. As described in more detail below, various types of immune cells can be used, such as T cells, natural killer (NK cells), or combinations thereof.

[0108] To facilitate cancer immunotherapy, provided herein are polynucleotides, polypeptides, and vectors encoding chimeric antigen receptors (CARs) comprising a target-binding moiety (e.g., an extracellular binder of a ligand, or a tumor marker-directed chimeric receptor expressed by a cancer cell) and a cytotoxic signaling complex. For example, some embodiments include polynucleotides, polypeptides, or vectors encoding chimeric antigen receptors having specificity for, inter alia, tumor markers such as CD19, CD123, CD70, Her2, mesothelin, Claudin 6, BCMA, EGFR, to promote targeting of immune cells to cancer and exert a cytotoxic effect on cancer cells. Also provided are engineered immune cells (e.g., T cells or NK cells) expressing such CARs. Also provided herein are, in some embodiments, polynucleotides, polypeptides, and vectors encoding constructs comprising an extracellular domain comprising two or more subdomains, e.g., a first CD19-targeting subdomain comprising a CD19-binding moiety disclosed herein, and a second subdomain comprising a C-type lectin-like receptor and a cytotoxic signaling complex. Also provided are engineered immune cells (e.g., T cells or NK cells) expressing such bispecific constructs. Also provided herein are methods of treating cancer and other uses of such cells for cancer immunotherapy.

[0109] Also provided herein are polynucleotides, polypeptides, and vectors encoding chimeric receptors comprising a target binding moiety (e.g., an extracellular binder of a ligand expressed by cancer cells) and a cytotoxic signaling complex, for example, to facilitate cancer immunotherapy. For example, some embodiments include polynucleotides, polypeptides, or vectors encoding an activating chimeric receptor comprising an NKG2D extracellular domain having specificity for tumor markers such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, to promote targeting of immune cells to cancer and exert a cytotoxic effect on cancer cells. Also provided are engineered immune cells (e.g., T cells or NK cells) that express such chimeric receptors. Also provided herein are, in some embodiments, polynucleotides, polypeptides, and vectors encoding constructs comprising an extracellular domain comprising two or more subdomains, e.g., a first and second ligand-binding receptor and a cytotoxic signaling complex. Also provided are engineered immune cells (e.g., T cells or NK cells) that express such bispecific constructs (in some embodiments, the first and second ligand-binding domains target the same ligand). Also provided herein are methods of treating cancer and other uses of such cells for cancer immunotherapy.

[0110] Engineered cells for immunotherapy In some embodiments, cells of the immune system are engineered to have an increased cytotoxic effect against target cells such as tumor cells. For example, cells of the immune system can be engineered to include a tumor-directed chimeric receptor and / or a tumor-directed CAR as described herein. In some embodiments, white blood cells or leukocytes are used because their native function is to defend the body against abnormal cell growth and infections. There are various types of white blood cells that play specific roles in the human immune system and are thus a preferred starting point for the engineering of the cells disclosed herein. White blood cells include granulocytes and agranulocytes (the presence or absence of granules in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils, and mast cells. Agranulocytes include lymphocytes and monocytes. Cells such as the following cells or other cells described herein can be engineered to include a chimeric receptor, such as an NKG2D chimeric receptor, and / or a CAR, such as a CD19-directed CAR, or a nucleic acid encoding a chimeric receptor or CAR. In some embodiments, the cells may be engineered to co-express a membrane-bound interleukin 15 (mbIL15) co-stimulatory domain. As will be discussed in more detail below, in some embodiments, cells, particularly T cells, are further genetically modified to reduce and / or eliminate alloreactivity of the cells.

[0111] Monocytes for Immunotherapy Monocytes are a subtype of white blood cells. Monocytes can differentiate into macrophages and myeloid dendritic cells. Monocytes are associated with the adaptive immune system and are responsible for the main functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is a process in which, after taking in cellular material or an entire cell, the ingested cellular material is digested and destroyed. In some embodiments, monocytes are used in association with one or more further engineered cells, as disclosed herein. Some embodiments of the methods and compositions described herein relate to monocytes comprising a tumor-directed CAR or a nucleic acid encoding a tumor-directed CAR. Some embodiments of the methods and compositions disclosed herein particularly relate to monocytes engineered to express a CAR that targets a tumor marker, such as CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, EGFR, and the membrane-bound interleukin 15 (mbIL15) co-stimulatory domain. Some embodiments of the methods and compositions disclosed herein relate to monocytes engineered to express an activating chimeric receptor that targets a ligand on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally the membrane-bound interleukin 15 (mbIL15) co-stimulatory domain.

[0112] 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). B cells are engineered according to some of the embodiments disclosed herein, although some embodiments also relate to engineered T cells or engineered NK cells (a mixture of T cells and NK cells may be used in some embodiments, either from the same donor or different donors). Some embodiments of the methods and compositions disclosed herein relate, inter alia, to lymphocytes engineered to express a chimeric antigen receptor (CAR) that targets a tumor marker, such as CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, EGFR, and the membrane-bound interleukin 15 (mbIL15) co-stimulatory domain. Some embodiments of the methods and compositions disclosed herein relate to lymphocytes engineered to express an activating chimeric receptor that targets a ligand on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally the membrane-bound interleukin 15 (mbIL15) co-stimulatory domain.

[0113] 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 their 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, specific subtypes of T cells 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 / enrichment of T cells with a specific marker profile. For example, in some embodiments, the 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 an infectious disease, including administering a therapeutically effective amount of T cells engineered to express a cytotoxic receptor complex and / or a homing moiety as described herein. In some embodiments, the engineered T cells are autologous cells, while in some embodiments, the T cells are allogeneic cells. Some embodiments of the methods and compositions disclosed herein relate, inter alia, to T cells engineered to express a chimeric antigen receptor (CAR) targeting a tumor marker, e.g., CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, EGFR, and the membrane-bound interleukin 15 (mbIL15) co-stimulatory domain. Some embodiments of the methods and compositions disclosed herein relate to T cells engineered to express an activating chimeric receptor targeting a ligand on tumor cells, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally the membrane-bound interleukin 15 (mbIL15) co-stimulatory domain.

[0114] NK Cells for Immunotherapy In some embodiments, provided are methods of treating or preventing cancer or an infection, comprising administering a therapeutically effective amount of natural killer (NK) cells that express 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 because of their relatively high natural cytotoxic potential. In some embodiments, the engineered cells disclosed herein can further upregulate the cytotoxic activity of NK cells, which unexpectedly provides a more effective activity against target cells (e.g., tumors or other diseased cells). Some embodiments of the methods and compositions described herein relate, inter alia, to NK cells engineered to express a chimeric antigen receptor (CAR) that targets a tumor marker, such as CD19, CD123, CD70, Her2, mesothelin, Claudin 6, BCMA, EGFR, and optionally a membrane-bound interleukin 15 (mbIL15) co-stimulatory domain. Some embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express an activating chimeric receptor that targets a ligand on tumor cells, such as, among others, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, and optionally a membrane-bound interleukin 15 (mbIL15) co-stimulatory domain.

[0115] Hematopoietic stem cells for cancer immunotherapy In some embodiments, hematopoietic stem cells (HSCs) are used in the methods of immunotherapy 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 hematopoiesis, and can provide, for example, a continuous source of targeted anti-cancer effector cells to fight cancer remission. In some embodiments, this continuous production helps counteract, for example, the anergy or depletion of other cell types 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, inter alia, to stem cells, such as hematopoietic stem cells engineered to express a chimeric antigen receptor (CAR) that targets tumor markers, such as CD19, CD123, CD70, Her2, mesothelin, Claudin 6, BCMA, EGFR, and optionally a membrane-bound interleukin 15 (mbIL15) co-stimulatory domain. Some embodiments of the methods and compositions disclosed herein relate to hematopoietic stem cells engineered to express an activating chimeric receptor that targets ligands on tumor cells, such as, inter alia, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, and optionally a membrane-bound interleukin 15 (mbIL15) co-stimulatory domain.

[0116] Genetic engineering of immune cells As discussed above, various cell types can be utilized for adoptive cell therapy. Further, as described in more detail below and shown in the Examples, genetic modification can be performed on these cells to increase one or more aspects of their efficacy (e.g., cytotoxicity) and / or persistence (e.g., active lifespan). As discussed herein, in some embodiments, NK cells are used for immunotherapy. In some embodiments provided herein, genetic editing of NK cells can advantageously confer on 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, genetic editing of NK cells limits this tumor microenvironment inhibitory effect in NK cells, T cells, combinations of NK and T cells, or any edited / engineered immune cells provided herein. As discussed below, in some embodiments, genetic 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, genetic 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 between the recited values). In some embodiments, the gene is completely knocked out such that the expression of the target protein cannot be detected. In some embodiments, genetic editing is used to "knock in" or alternatively increase the expression of the target protein. In some embodiments, the expression of the target protein can increase by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between the recited values).

[0117] As a non-limiting example, TGF-beta is one such cytokine released by tumor cells that results in immunosuppression in 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, as discussed in detail below, immune checkpoint inhibitors are disrupted via gene editing. In some embodiments, blockers of immunosuppressive cytokines in the tumor microenvironment are used, including blockers or competitive inhibitors of their release that bind to and reduce the ability of signaling molecules to 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 PGE 2However, in further embodiments, immune cells such as NK cells are provided in which the ability of NK cells (or other cells) to respond to a given immunosuppressive signaling molecule is disrupted and / or eliminated. For example, in some embodiments, NK cells or T cells are gene edited to be less sensitive to TGF-beta. TGF-beta is an inhibitor of NK cell function, at least with respect to levels of proliferation and cytotoxicity. See, for example, FIG. 8A which schematically shows some of the inhibitory pathways by which TGF-beta decreases NK cell activity and / or proliferation. Thus, according to some embodiments, the expression of the TGF-beta receptor is knocked down or knocked out via gene editing such that the engineered NKs 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 use of CRISPR-Cas editing. In other embodiments, small interfering RNAs, antisense RNAs, TALENs or zinc fingers are used. Other isoforms of the TGF-beta receptor (e.g., TGF-beta1 and / or TGF-beta3) are edited in some embodiments. In some embodiments, the TGF-beta receptor in T cells is knocked down via gene editing.

[0118] According to further embodiments, other regulators of one or more aspects of NK cell (or T cell) function are regulated via gene editing. Various cytokines give negative or positive signals to immune cells (similar to the above-mentioned TGF-beta). As a non-limiting example, IL15 is a positive regulator of NK cells as disclosed herein 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 conditions, cytokine-inducible SH2-containing protein (CIS, encoded by the CISH gene) acts as an important negative regulator of IL-15 signaling in NK cells. As considered 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. Thus, according to some embodiments, editing of CISH increases NK cell functionality across multiple functions, resulting in a more effective and long-lasting NK cell therapy. In some embodiments, an inhibitor of CIS is used in combination with the administration of engineered NK cells. In some embodiments, CIS expression is knocked down or knocked out via gene editing of the CISH gene, for example, by the use of CRISPR-Cas editing. In other embodiments, small interfering RNA, antisense RNA, TALEN or zinc finger is used. In some embodiments, CIS expression in T cells is knocked down via gene editing.

[0119] In some embodiments, CISH gene editing confers upon NK cells an increased ability to home to target sites. In some embodiments, CISH gene editing confers upon NK cells an increased ability to migrate, for example, within a tissue, in response to, for example, a chemoattractant, or away from a repellent. In some embodiments, CISH gene editing activates NK cells and thus confers upon them an increased ability to exert, for example, an anti-tumor effect. In some embodiments, CISH gene editing confers upon NK cells an increased proliferative capacity and, in some embodiments, enables the generation of robust numbers of NK cells from donor blood samples. Further, in such embodiments, NK cells edited for CISH and engineered to express a CAR expand more readily, robustly, and consistently in culture. In some embodiments, CISH gene editing confers upon NK cells an increased cytotoxicity. In some embodiments, editing of CISH synergistically increases the cytotoxic effects of engineered NK cells expressing a CAR and / or engineered T cells.

[0120] In some embodiments, CISH gene editing activates or inhibits broad pathways. The CIS protein is a negative regulator 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 derepresses 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 mammalian target of rapamycin (mTOR), corresponding to increased expression of genes related to cell metabolism and respiration. In some embodiments, knockout of CISH results in increased expression of IL15-induced 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 regulation of selected genes related to mitochondrial function (e.g., electron transport chain and cell respiration) and the cell cycle. Thus, in some embodiments, knockout of CISH by gene editing increases the cytotoxicity and / or persistence of NK cells, at least in part, through metabolic reprogramming. In some embodiments, negative regulators of cell 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 anti-proliferative or pro-apoptotic proteins such as TGFB1, ATM, and PTCH1 are downregulated.In some embodiments, CISH knockout alters (e.g., activates or inactivates) the signaling state through or via 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.

[0121] In some embodiments, gene editing of immune cells can also provide an unexpected increase 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, and the combination provides robust cells for immunotherapy. In some embodiments, the editing enables unexpectedly improved expansion, persistence, and / or cytotoxicity of NK cells. In some embodiments, knockout of CISH expression in NK cells removes a potent negative regulator of IL15-mediated signaling in NK cells, de-represses NK cells, and enables one or more of increased NK cell homing, NK cell migration, NK cell activation, expansion, cytotoxicity, and / or persistence. Further, in some embodiments, the 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.

[0122] As discussed above, T cells engineered to express a CAR or chimeric receptor are employed in some embodiments. Also, as noted above, T cells express a T cell receptor (TCR) on their surface. As disclosed herein, in some embodiments, autologous immune cells are returned to the original donor of the cells. In such embodiments, immune cells such as NK cells or T cells may be obtained from a patient, expanded, genetically modified (e.g., using a CAR or chimeric receptor), and / or further expanded and reintroduced into the patient. As disclosed herein, in some embodiments, allogeneic immune cells are transferred to a subject who is not the original donor of the cells. In such embodiments, immune cells such as NK cells or T cells may be obtained from a donor, expanded, genetically modified (e.g., using a CAR or chimeric receptor), and / or further expanded and administered to the subject.

[0123] Allogeneic immunotherapy has several hurdles to overcome. In immunocompetent hosts, administered allogeneic cells are rapidly rejected, known as host-versus-graft rejection (HvG). This substantially limits the effectiveness of the administered cells, particularly their persistence. In immunocompromised hosts, allogeneic cells can engraft. However, when the administered cells contain T cells (some embodiments disclosed herein use a mixed population of NK cells and T cells), the endogenous T cell receptor (TCR) specificity recognizes host tissue as foreign and causes graft-versus-host disease (GvHD). GvHD can cause significant tissue damage in the host (cell recipient). Some embodiments disclosed herein address both of these hurdles, thereby enabling effective and safe allogeneic immunotherapy. In some embodiments, gene editing can advantageously serve to reduce and / or avoid graft-versus-host disease (GvHD). A non-limiting embodiment of such an approach using a mixed population of NK cells and T cells is schematically illustrated in FIG. 8C, where NK cells are engineered to express a CAR and T cells are not only engineered to express a CAR but also edited to be non-alloreactive. FIG. 8D schematically shows the mechanism by which graft-versus-host disease occurs. Both allogeneic T cells and allogeneic NK cells are engineered to express a CAR that targets the tumor and are introduced into the host. However, the T cells still have a native T cell receptor (TCR). This TCR can recognize the HLA type of host cells as "non-self" and can exert cytotoxicity against host cells. FIG. 8E shows a non-limiting embodiment of how graft-versus-host disease can be reduced or alternatively avoided by gene editing of T cells. Briefly, this approach can perform gene editing to knock out the native TCR on T cells, as will be discussed in more detail below. When the TCR is deficient, allogeneic T cells cannot detect the "non-self" HLA of host cells and are thus not induced to exert cytotoxicity against host cells.Thus, in some embodiments, T cells are subjected to gene editing to reduce the functionality of native T cells and / or to reduce or eliminate the expression of native T cells. In some embodiments, CRISPR is used to knockout the TCR. These and other embodiments are considered below.

[0124] The T cell receptor (TCR) is a cell surface receptor that is involved in the activation of T cells in response to antigen presentation. The TCR consists of two different protein chains (which are heterodimers). The majority of human T cells have a TCR consisting of an alpha (α) chain and a beta (β) chain (encoded by separate genes). A small percentage of T cells have a TCR consisting of a gamma chain and a delta chain (γ / δ chain) (the cells are known as gamma-delta T cells).

[0125] Rather than recognizing intact antigens (like immunoglobulins), T cells are activated by processed peptide fragments in association with MHC molecules. This is known as MHC restriction. When the TCR recognizes differences between donor MHC and recipient MHC, that recognition stimulates T cell proliferation and the potential development of GVHD. In some embodiments, the gene encoding any of TCRα, TCRβ, TCRγ, and / or TCRδ is disrupted or alternatively modified to reduce the tendency of donor T cells to recognize differences between donor MHC and host MHC, thereby reducing the recognition of allogeneic antigens and GVHD.

[0126] T cell-mediated immunity involves a balance between co-stimulatory and inhibitory signals that help fine-tune the immune response. Inhibitory signals, also known as immune checkpoints, allow for the avoidance of autoimmunity (e.g., self-tolerance) and also limit immune-mediated damage. Immune checkpoint protein expression is often altered by tumors, increasing immune resistance in tumor cells and limiting the effectiveness of immunotherapy. CTLA4 downregulates the amplitude of T cell activation. In contrast, PD1 restricts T cell effector functions in peripheral tissues during inflammatory responses and also restricts autoimmunity. Immune checkpoint blockade, in some embodiments, helps overcome barriers to the activation of functional cellular immunity. In some embodiments, agonistic antibodies specific for inhibitory ligands on T cells, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4; also known as CD152) and programmed cell death protein 1 (PD1 or PDCD1, also known as CD279), are used to enhance immunotherapy.

[0127] In some embodiments, genetically modified T cells that are non-alloreactive and highly active are provided. In some embodiments, the T cells are further modified such that specific immune checkpoint genes are inactivated, and thus, immune checkpoint proteins are not expressed by the T cells. In some embodiments, this is done in the absence of manipulation or disruption of the CD3z signaling domain (e.g., the TCR can still initiate T cell signaling).

[0128] In some embodiments, genetic inactivation of TCR alpha and / or TCR beta, in combination with inactivation of immune checkpoint genes in T lymphocytes from allogeneic donors, significantly reduces the risk of GVHD. In some embodiments, this is done by removing at least a portion of one or more substituted protein chains (alpha, beta, gamma, and / or delta) involved in recognition of MHC differences between donor and recipient cells. In some embodiments, this is done while still allowing T cell proliferation and activity.

[0129] In some embodiments in which allogeneic cells are administered, the recipient subject may receive some other adjuvant treatment to support or alternatively enhance the function of the administered immune cells. In some embodiments, the subject may be pre-treated (e.g., using radiation or chemotherapy). In some embodiments, the adjuvant treatment includes administration of lymphocyte growth factors (such as IL-2).

[0130] Furthermore, in some embodiments, editing can improve the persistence of administered cells (either NK cells, T cells, or any others) by, for example, masking the cells from the host immune response. In some cases, the recipient's immune cells attack donor cells, particularly donor cells from allogeneic donors, which is known as host-versus-graft disease (HvG). FIG. 8F shows a schematic of HvG where host T cells, together with the native / functional TCR, recognize HLA on donor T and / or donor NK cells as non-self. In such cases, when the host T cell TCR binds to allogeneic cell HLA, the allogeneic cells are removed and the persistence of the donor-engineered NK / T cells is reduced. With respect to HvG, to prevent the rejection of administered allogeneic T cells, the subject receiving the cells needs to have their immune system suppressed. In some embodiments, glucocorticoids are used, including but not limited to, beclomethasone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone, triamcinolone. Activation of the glucocorticoid receptor in the recipient's own T cells changes the expression of genes involved in the immune response, leading to a decrease in the level of cytokine production, which leads to T cell anergy and interference with T cell activation (in the recipient). Other embodiments relate to the administration of antibodies that can deplete specific types of recipient immune cells. One such target is CD52, which is expressed at high levels on T and B lymphocytes, at lower levels on monocytes, but not on granulocytes and bone marrow progenitor cells. Treatment or pretreatment of the recipient with alemtuzumab, a humanized monoclonal antibody directed against CD52, has been shown to induce rapid depletion of circulating lymphocytes and monocytes, which, considering the reduction of recipient immune cells, reduces the likelihood of HvG. Immunosuppressive drugs may limit the effectiveness of administered allogeneic engineered T cells. Thus, as disclosed herein, some embodiments relate to genetically engineered allogeneic donor cells that are resistant to immunosuppressive therapy.In some embodiments, immune cells such as NK cells and / or T cells are engineered (in addition to being engineered to express a CAR) to have their persistence extended by avoiding a cytotoxic response from host immune cells. In some embodiments, gene editing to remove one or more HLA molecules from allogeneic NK and / or T cells reduces clearance by host T cells. In some embodiments, allogeneic NK and / or T cells are engineered to knockout one or more of beta-2 microglobulin (an HLA class I molecule) and CIITA (an HLA class II molecule). Figure 8G schematically illustrates this approach.

[0131] In some embodiments of the mixed allogeneic cell therapy, the engineered cell populations actually target each other, for example, when the therapeutic cells are edited to remove HLA molecules to avoid HvG. For example, such editing of CAR T cells can render the engineered allogeneic CAR T cells vulnerable to cytotoxic attack by CAR NK cells and elimination by host NK cells. This is generally caused by the lack of the "self" inhibitory signal presented by KIR molecules. Figure 8H schematically shows this process. In some embodiments, gene editing can be used to knock in the expression of one or more "masking" molecules that mask allogeneic cells from the host immune system and fratricide by other administered engineered cells. Figure 8I schematically shows this approach. In some embodiments, a protein can be expressed on the surface of allogeneic cells to inhibit targeting by NK (both engineered and host NK), and advantageously, extend the persistence of both allogeneic CAR-T and CAR-NK. In some embodiments, gene editing is used to knock in CD47, the expression of which effectively functions as a "don't eat me" signal. In some embodiments, gene editing is used to knock in the expression of HLA-E. HLA-E binds to both the inhibitory and activating receptors NKG2A and NKG2C present on the surface of NK cells, respectively. However, NKG2A is more highly expressed in most human NK cells, and thus, in some embodiments, the expression of HLA-E on engineered cells results in an inhibitory effect of NK cells (both host and donor) on HLA-E edited (or naturally expressed) cells. Further, in some embodiments, one or more viral HLA homologs are knocked in such that they are expressed by the engineered NK and / or T cells, thus conferring on the cells the ability of the virus to evade the host immune system. In some embodiments, these approaches advantageously extend the persistence of both allogeneic CAR-T and CAR-NK.

[0132] In some embodiments, any gene editing (either knockout or knock-in) of a target gene (e.g., CISH, TGFBR, TCR, B2M, CIISH, CD47, HLA-E, or any other target gene disclosed herein) is achieved via targeted introduction of DNA cleavage 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 the 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 cleavage and repair. HDR uses a homologous sequence as a template for the regeneration of the missing DNA sequence at the cleavage point, such as a vector having a desired genetic factor (e.g., an insertion factor that disrupts the coding sequence of TCR) within a sequence that is homologous to the adjacent sequence of the double-strand break. This results in the desired change (e.g., insertion) being inserted at the site of the DSB.

[0133] In some embodiments, gene editing is achieved by one or more of various engineered nucleases. In some embodiments, restriction enzymes are used, particularly when double-strand breaks are desired in multiple regions. In some embodiments, bioengineered nucleases are used. One or more of Zinc finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), Meganucleases, and / or Clustered regularly interspaced short palindromic repeat (CRISPR / Cas9) systems are used to specifically edit the gene encoding one or more TCR subunits.

[0134] 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 mutants that recognize unique sequences (s) such as specific sites in TCR or CISH, or any other target gene disclosed herein. The target site of TCR can be readily identified. Further information on the target site within the region of TCR can be found in U.S. Patent Application Publication No. 2018 / 0325955 and U.S. Patent Application Publication No. 2015 / 0017136, each of which is incorporated herein by reference in its entirety. In some embodiments, two or more meganucleases or their functional fragments are fused to create a hybrid enzyme that recognizes a desired target sequence within a target gene (e.g., CISH).

[0135] In contrast to meganucleases, ZFNs and TALENs have a function based on a nonspecific DNA cleavage catalytic domain linked to a specific DNA sequence that recognizes peptides such as zinc fingers or transcription activator-like effectors (TALEs). Advantageously, ZFNs and TALENs thus enable sequence-independent DNA cleavage and have a high degree of sequence specificity in target recognition. The zinc finger motif functions naturally in transcription factors to recognize specific DNA sequences for transcription. The C-terminal portion of each finger is involved in the specific recognition of the DNA sequence. The sequences recognized by ZFNs are relatively short (e.g., about 3 base pairs), but in some embodiments, combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more zinc fingers, for which the recognition sites are characterized, are used, thereby enabling the targeting of specific sequences such as part of a TCR (or immune checkpoint inhibitor). Next, the combined ZFNs are 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 for editing TCRs and / or immune checkpoint inhibitors can be found in U.S. Patent No. 9,597,357, which is incorporated herein by reference.

[0136] 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 are a fusion of a nuclease DNA cleavage domain to a TALE domain, enabling the introduction of sequence-independent double-strand DNA cleavage and allowing for very accurate target site recognition. TALENs can cause double-strand breaks at the target site, which are repaired by error-prone non-homologous end joining (NHEJ), resulting in gene disruption by introducing small insertions or deletions. Advantageously, TALENs are used in some embodiments, at least in part, due to their higher specificity in DNA binding, reduced off-target effects, and ease of construction of the DNA binding domain.

[0137] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a genetic element used by bacteria as a defense against viruses. These 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 the Cas gene into eukaryotic cells and specifically constructing 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 (s) encoding target genes that are knocked out or knocked in, for example, in CISH, TGFBR2, TCR, B2M, CIITA, CD47, HLA-E, etc. In some embodiments, CRISPR is used to edit one or more of the TCRs of T cells 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, CRISPR is used to cleave one or more of TCRα, TCRβ, TCRγ, and TCRδ. In some embodiments, the TCR is cleaved without affecting the function of the CD3z signaling domain of the TCR. Depending on the embodiment and which target genes are to be edited, 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, 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 II, IIA, IIB, IIC, V, VI, and combinations thereof. In some embodiments, Cas is selected from the group consisting of Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a (formerly known as C2c2), Cas13b, Cas13c, and combinations thereof.

[0138] In some embodiments, as discussed above, editing of CISH advantageously confers increased expansion, cytotoxicity and / or persistence to the edited cells, particularly edited NK cells. Further, in some embodiments, modification of the TCR includes modification of TCRα, but does not affect signaling through the CD3 complex and enables T cell proliferation. In one embodiment, TCRα is inactivated by the expression of pre-Tα in the cell, thus restoring a functional CD3 complex in the absence of a functional alpha / beta TCR. As disclosed herein, allogeneic non-reactive modified T cells also express a CAR and are engineered to direct allogeneic non-reactive T cell specificity towards tumor markers, but are independent of MHC. Combinations of editing are used in some embodiments, as non-limiting examples, such as, for example, combined TCR and CISH knockout, or CISH knockout and CD47 knock-in.

[0139] Extracellular domain (tumor binding factor) Some embodiments of the compositions and methods described herein relate to chimeric antigen receptors that include an extracellular domain that includes a tumor-binding domain (also referred to as an antigen-binding protein or antigen-binding domain). The tumor-binding domain targets, inter alia, CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, EGFR, depending on the embodiment. Some embodiments of the compositions and methods described herein relate to chimeric receptors (also referred to as activating chimeric receptors) that include an extracellular domain that includes a ligand-binding domain that binds to a ligand expressed by a tumor cell. The ligand-binding domain targets, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, inter alia, depending on the embodiment.

[0140] In some embodiments, the antigen-binding domain is 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, an affibody, an affilin, an adnectin, an avimer, a repebody, a fibronomer, an alphabody, an abimer, an atrimer, a centyrin, a pronectin, an anticalin, a knotted domain, an armadillo repeat protein, a wild-type or non-wild-type sequence derived from or including an autoantigen, a receptor, or a ligand. In some embodiments, the tumor-binding domain includes more than one antigen-binding domain. In embodiments, the antigen-binding domain is operably linked directly or via any linker to the NH2 terminus of a TCR domain (e.g., the constant chain of TCR-alpha, TCR-beta1, TCR-beta2, preTCR-alpha, pre-TCR-alpha-Del48, TCR-gamma, or TCR-delta).

[0141] Antigen-binding protein In some embodiments, an antigen-binding protein is provided. As used herein, the term "antigen-binding protein" is given its ordinary meaning and also refers to an antigen-binding fragment that binds to an antigen and, optionally, a protein that includes a scaffold or framework portion that enables the antigen-binding fragment to adopt a three-dimensional structure that facilitates binding of the antigen-binding protein to the antigen. In some embodiments, the antigen is a cancer antigen (e.g., CD19) or a fragment thereof. In some embodiments, the antigen-binding fragment includes at least one CDR derived from an antibody that binds to the antigen. In some embodiments, the antigen-binding fragment includes all three CDRs derived from the heavy chain of an antibody that binds to the antigen or all three CDRs derived from the light chain of an antibody that binds to the antigen. In still further embodiments, the antigen-binding fragment includes all six CDRs derived from an antibody that binds to the antigen (three from the heavy chain and three from the light chain). In some embodiments, the antigen-binding fragment includes one, two, three, four, five, or six CDRs derived from an antibody that binds to the antigen, and in some embodiments, the CDRs can be any combination of heavy chain and / or light chain CDRs. The antigen-binding fragment in some embodiments is an antibody fragment.

[0142] Non-limiting examples of antigen-binding proteins include antibodies, antibody fragments (e.g., antigen-binding fragments of antibodies), antibody derivatives, and antibody mimetics. Further specific examples include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., VH domains of camelid heavy-chain antibodies; 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, or pig, dog, or camel. Antibody fragments can compete with intact (e.g., native) antibodies for binding to a target antigen, and the 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 scaffolds or artificial scaffolds having transplanted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, as well as fully synthetic scaffolds containing, for example, biocompatible polymers. Further, peptide antibody mimetics (“PAMs”), as well as scaffolds based on antibody mimetics that utilize fibronectin components as scaffolds, can be used.

[0143] 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, the antigen-binding protein can include, but is not limited to, diabodies; intracellular antibodies; domain antibodies (a single VL or VH domain, or two or more VH domains connected by a peptide linker); maxibodies (two scFvs fused to the Fc region); tribodies; tetrabodies; minibodies (scFv fused to the CH3 domain); peptibodies (one or more peptides bound to the 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).

[0144] 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 (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 - 110 or more amino acids that is primarily involved in antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is primarily involved in effector functions.

[0145] Within the light and heavy chains, the variable region (V) and the constant region (C) are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 or more amino acids. The variable regions of each light chain / heavy chain pair form an antibody binding site such that a native immunoglobulin has two binding sites.

[0146] Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also known as complementarity-determining regions or CDRs. From the N-terminus to the C-terminus, both the light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0147] Human light chains are classified into kappa and lambda light chains. The antibody "light chain" refers to the smaller of the two types of polypeptide chains that exist in their native three-dimensional structure within the antibody molecule. Kappa (K) and lambda (λ) light chains refer to the two major antibody light chain isotypes. A light chain can contain a polypeptide that includes a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus to the carboxyl terminus.

[0148] Heavy chains are classified into mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. The antibody "heavy chain" refers to the larger of the two types of polypeptide chains that exist in the antibody molecule in its native three-dimensional structure and usually determines the class to which the antibody belongs. A heavy chain can contain a polypeptide that includes a single immunoglobulin heavy chain variable region (VH), an immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3), and, optionally, an immunoglobulin heavy chain constant domain 4 (CH4) from the amino terminus to the carboxyl terminus.

[0149] The IgG class is further divided into subclasses, namely, IgG1, IgG2, IgG3, and IgG4. The IgA class is further divided into subclasses, namely 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), and the heavy chains of IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). Immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. Antibody chains are linked to each other via inter-polypeptide 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.

[0150] In some embodiments, the antigen-binding protein is an antibody. As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be monoclonal, or polyclonal, multivalent 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 include at least two full-length heavy chains and two full-length light chains. Antibody sequences can be derived from only 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 that include 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 chains and heavy chains. For example, antibodies having 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 chain and / or light chain are included in the definition, provided that the antibody retains the same or similar binding and / or function as an antibody that includes 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" is given its ordinary meaning and is intended to refer to a population of antibodies that typically vary widely in composition and binding specificity. As used herein, the term "monoclonal antibody" ("mAb") is given its ordinary meaning and is intended to refer to one or more of a population of antibodies having the same sequence. Monoclonal antibodies bind to an antigen at a specific epitope on the antigen.

[0151] In some embodiments, the antigen-binding protein is an antibody fragment or antigen-binding fragment. The term "antibody fragment" refers to at least one portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen. 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, bispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region, and isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides 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 techniques or by enzymatic or chemical cleavage of intact antibodies.

[0152] In some embodiments, Fab fragments are provided. A Fab fragment is a monovalent fragment having VL, VH, CL, and CH1 domains; an F(ab’)2 fragment is a divalent fragment having two Fab fragments linked by a disulfide bridge in the hinge region; an Fd fragment has VH and CH1 domains; an Fv fragment has the VL and VH domains of a single arm of an antibody; and a dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain. In some embodiments, these antibody fragments can be incorporated into single-domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs. In some embodiments, an antibody comprises at least one CDR as described herein.

[0153] In some embodiments, single-chain variable fragments are also provided herein. As used herein, the term "single-chain variable fragment" ("scFv") is given its ordinary meaning and refers to a fusion protein in which the VL and VH regions are connected via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, and the linker is of sufficient length for the protein chain to fold back on itself to form a monovalent antigen-binding site. For clarity, unless otherwise indicated, "single-chain variable fragment" is not an antibody or antibody fragment as defined herein. A diabody is a bivalent antibody comprising two polypeptide chains, each polypeptide chain comprising a VH and a VL domain connected by a linker configured to reduce or prevent pairing between the two domains on the same chain, such that each domain is able to pair with a complementary domain on a different 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. Diabodies having two different antigen-binding sites can be made using polypeptide chains having different sequences. Similarly, a tribody and a tetrabody are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, which can be the same or different.

[0154] In some embodiments, the antigen-binding protein comprises one or more CDRs. As used herein, the term "CDR" is given its ordinary meaning and is also intended to refer to complementarity-determining regions (also referred to as "minimal recognition units" or "hypervariable regions") within antibody variable sequences. CDRs enable the antigen-binding protein to specifically bind to a particular antigen of interest. There are three heavy-chain variable region CDRs (CDRH1, CDRH2, and CDRH3) and three light-chain variable region CDRs (CDRL1, CDRL2, and CDRL3). The CDRs in each of the two chains are typically aligned by framework regions and 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 the naturally occurring light-chain and heavy-chain variable regions typically conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to number the amino acids occupying 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). One or more CDRs can be incorporated into a molecule, either covalently or non-covalently, to make it an antigen-binding protein.

[0155] In some embodiments, the antigen-binding proteins provided herein include one or more CDRs (plural available) as part of a larger polypeptide chain. In some embodiments, the antigen-binding protein covalently attaches one or more CDRs (plural available) to another polypeptide chain. In some embodiments, one or more CDRs are non-covalently incorporated into the antigen-binding protein. In some embodiments, the antigen-binding protein may include at least one of the CDRs described herein incorporated into a biocompatible framework structure. In some embodiments, the biocompatible framework structure is a structurally stable structural support, or framework, or scaffold that can present one or more sequences of amino acids that bind to an antigen (e.g., CDR, variable region, etc.) in a localized surface region, or includes a polypeptide or a portion thereof sufficient to form a scaffold. Such a structure can be a naturally occurring polypeptide or polypeptide "fold" (structural motif), or can have one or more modifications such as addition, deletion, and / or substitution of amino acids relative to a naturally occurring polypeptide or fold. Depending on the embodiment, the scaffold can be derived from polypeptides of various different species (or more than one species) such as human, non-human primate or other mammals, other vertebrates, invertebrates, plants, bacteria or viruses.

[0156] Depending on the embodiment, the biocompatible framework structure is based on a protein scaffold or backbone other than an immunoglobulin domain. In 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.

[0157] 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, an antibody typically has two identical binding sites, while a "bispecific" or "bifunctional" antibody has two different binding sites. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes that may be present on the same or different protein targets. In some embodiments, this is particularly advantageous because a bispecific chimeric antigen receptor can endow engineered cells with the ability to target multiple tumor markers. For example, CD19 and additional tumor markers, particularly, for example, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, EGFR, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, or any other marker disclosed herein or recognized in the art as a tumor-specific antigen or tumor-associated antigen, may be bound by a bispecific antibody.

[0158] As used herein, the term "chimeric antibody" shall be given its ordinary meaning and shall also refer to an antibody that includes 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 (e.g., CD19, CD123, CD70, Her2, mesothelin, PD-L1, claudin 6, BCMA, EGFR, etc.) antibody. In some embodiments, all of the CDRs are derived from an anti-cancer antigen antibody (e.g., anti-CD19 antibody). In some embodiments, CDRs derived from more than one anti-cancer antigen antibody are mixed and adapted in the chimeric antibody. For example, a chimeric antibody can include 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. Further, the framework regions of the antigen-binding proteins disclosed herein can be derived from one of the same anti-cancer antigen (e.g., CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, EGFR, etc.) antibody, one or more different antibodies such as human antibodies, or a humanized antibody. In one example of a chimeric antibody, a portion of the heavy chain and / or light chain is identical to, homologous to, or derived from an antibody from a particular species, or an antibody 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 an antibody belonging to another antibody class or subclass. Also provided herein are fragments of such antibodies that exhibit the desired biological activity.

[0159] In some embodiments, an antigen-binding protein is provided that comprises a heavy-chain variable domain having at least 90% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the antigen-binding protein comprises a heavy-chain variable domain having at least 95% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the antigen-binding protein comprises a heavy-chain variable domain having at least 96, 97, 98, or 99% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the heavy-chain variable domain may have one or more additional mutations (e.g., for the purpose of humanization) in the VH domain amino acid sequence shown in SEQ ID NO: 33, but may retain specific binding to a cancer antigen (e.g., CD19). In some embodiments, the heavy-chain variable domain may have one or more additional mutations in the VH domain amino acid sequence shown in SEQ ID NO: 33, but the specific binding to a cancer antigen (e.g., CD19) is improved.

[0160] In some embodiments, the antigen-binding protein comprises a light-chain variable domain having at least 90% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the antigen-binding protein comprises a light-chain variable domain having at least 95% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the antigen-binding protein comprises a light-chain variable domain having at least 96, 97, 98, or 99% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the light-chain variable domain may have one or more additional mutations (e.g., for the purpose of humanization) in the VL domain amino acid sequence shown in SEQ ID NO: 32, but may retain specific binding to a cancer antigen (e.g., CD19). In some embodiments, the light-chain variable domain may have one or more additional mutations in the VL domain amino acid sequence shown in SEQ ID NO: 32, but the specific binding to a cancer antigen (e.g., CD19) is improved.

[0161] In some embodiments, the antigen-binding protein comprises a heavy-chain variable domain having at least 90% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33, and a light-chain variable domain having at least 90% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the antigen-binding protein comprises a heavy-chain variable domain having at least 95% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33, and a light-chain variable domain having at least 95% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the antigen-binding protein comprises a heavy-chain variable domain having at least 96, 97, 98, or 99% identity with the VH domain amino acid sequence shown in SEQ ID NO: 33, and a light-chain variable domain having at least 96, 97, 98, or 99% identity with the VL domain amino acid sequence shown in SEQ ID NO: 32.

[0162] In some embodiments, the antigen-binding protein comprises a heavy-chain variable domain having the VH domain amino acid sequence shown in SEQ ID NO: 33, and a light-chain variable domain having the VL domain amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the light-chain variable domain comprises an amino acid 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 the light-chain variable domain of SEQ ID NO: 32. In some embodiments, the light-chain variable domain comprises an amino acid 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 the heavy-chain variable domain according to SEQ ID NO: 33.

[0163] In some embodiments, the light chain variable domain comprises an amino acid sequence encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to polynucleotide SEQ ID NO: 32. In some embodiments, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of the polynucleotide encoding the light chain variable domain according to the sequence of SEQ ID NO: 32. In some embodiments, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a complement of the polynucleotide encoding the light chain variable domain according to the sequence of SEQ ID NO: 32.

[0164] In some embodiments, the heavy chain variable domain comprises an amino acid 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 the heavy chain variable domain according to the sequence of SEQ ID NO: 33. In some embodiments, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of the polynucleotide encoding the heavy chain variable domain according to the sequence of SEQ ID NO: 33. In some embodiments, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a complement of the polynucleotide encoding the heavy chain variable domain according to the sequence of SEQ ID NO: 33.

[0165] In some embodiments, additional anti-CD19 binding constructs are provided. For example, in some embodiments, an scFv targeting CD19 is provided that includes a heavy chain variable region comprising the sequence of SEQ ID NO: 35. In some embodiments, the antigen-binding protein includes a heavy chain variable domain having at least 95% identity to the HCV domain amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the antigen-binding protein includes a heavy chain variable domain having at least 96, 97, 98, or 99% identity to the HCV domain amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the heavy chain variable domain may have one or more additional mutations (e.g., for the purpose of humanization) in the HCV domain amino acid sequence shown in SEQ ID NO: 35, but may retain specific binding to a cancer antigen (e.g., CD19). In some embodiments, the heavy chain variable domain may have one or more additional mutations in the HCV domain amino acid sequence shown in SEQ ID NO: 35, but the specific binding to a cancer antigen (e.g., CD19) is improved.

[0166] Furthermore, in some embodiments, the scFv targeting CD19 includes a light chain variable region comprising the sequence of SEQ ID NO: 36. In some embodiments, the antigen-binding protein includes a light chain variable domain having at least 95% identity to the LCV domain amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the antigen-binding protein includes a light chain variable domain having at least 96, 97, 98, or 99% identity to the LCV domain amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the light chain variable domain may have one or more additional mutations (e.g., for the purpose of humanization) in the LCV domain amino acid sequence shown in SEQ ID NO: 36, but may retain specific binding to a cancer antigen (e.g., CD19). In some embodiments, the light chain variable domain may have one or more additional mutations in the LCV domain amino acid sequence shown in SEQ ID NO: 36, but the specific binding to a cancer antigen (e.g., CD19) is improved.

[0167] In some embodiments, an anti-CD19 binding portion is also provided that includes light chain CDRs (LC CDR1, LC CDR2, and LC CDR3, respectively) that include a first, second, and third complementarity determining region. In some embodiments, the anti-CD19 binding portion further includes heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3, respectively) that include a first, second, and third complementarity determining region. In some embodiments, LC CDR1 includes the sequence of SEQ ID NO: 37. In some embodiments, LC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 37. In some embodiments, LC CDR2 includes the sequence of SEQ ID NO: 38. In some embodiments, LC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 38. In some embodiments, LC CDR3 includes the sequence of SEQ ID NO: 39. In some embodiments, LC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 39. In some embodiments, HC CDR1 includes the sequence of SEQ ID NO: 40. In some embodiments, HC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 40. In some embodiments, HC CDR2 includes the sequence of SEQ ID NO: 41, 42, or 43. In some embodiments, HC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 41, 42, or 43. In some embodiments, HC CDR3 includes the sequence of SEQ ID NO: 44. In some embodiments, HC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 44.

[0168] In some embodiments, an anti-CD19 binding portion comprising a light chain variable region (VL) and a heavy chain variable region (HL) is also provided, where the VL region comprises first, second, and third complementarity determining regions (VL CDR1, VL CDR2, and VL CDR3, respectively), and the VH region comprises first, second, and third complementarity determining regions (VH CDR1, VH CDR2, and VH CDR3, respectively). In some embodiments, the VL region comprises the sequence of SEQ ID NO: 45, 46, 47, or 48. In some embodiments, the VL region comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 45, 46, 47, or 48. In some embodiments, the VH region comprises the sequence of SEQ ID NO: 49, 50, 51, or 52. In some embodiments, the VH region comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 49, 50, 51, or 52.

[0169] In some embodiments, an anti-CD19 binding portion is also provided that includes light chain CDRs (LC CDR1, LC CDR2, and LC CDR3, respectively) that include first, second, and third complementarity determining regions. In some embodiments, the anti-CD19 binding portion further includes heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3, respectively) that include first, second, and third complementarity determining regions. In some embodiments, LC CDR1 includes the sequence of SEQ ID NO: 53. In some embodiments, LC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 53. In some embodiments, LC CDR2 includes the sequence of SEQ ID NO: 54. In some embodiments, LC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 54. In some embodiments, LC CDR3 includes the sequence of SEQ ID NO: 55. In some embodiments, LC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 55. In some embodiments, HC CDR1 includes the sequence of SEQ ID NO: 56. In some embodiments, HC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 56. In some embodiments, HC CDR2 includes the sequence of SEQ ID NO: 57. In some embodiments, HC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 57. In some embodiments, HC CDR3 includes the sequence of SEQ ID NO: 58. In some embodiments, HC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 58.

[0170] In some embodiments, the antigen-binding protein comprises a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 104. In some embodiments, the antigen-binding protein comprises a heavy-chain variable region having at least 90% identity with the VH domain amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, the antigen-binding protein comprises a heavy-chain variable domain having at least 95% sequence identity with the VH domain amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, the antigen-binding protein comprises a heavy-chain variable domain having at least 96, 97, 98, or 99% sequence identity with the VH domain amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, the heavy-chain variable domain may have one or more additional mutations (e.g., for the purpose of humanization) in the VH domain amino acid sequence set forth in SEQ ID NO: 104, but may retain specific binding to a cancer antigen (e.g., CD19). In some embodiments, the heavy-chain variable domain may have one or more additional mutations in the VH domain amino acid sequence set forth in SEQ ID NO: 104, but the specific binding to a cancer antigen (e.g., CD19) is improved.

[0171] In some embodiments, the antigen-binding protein comprises a variable light chain region comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, the antigen-binding protein comprises a variable light chain region having at least 90% sequence identity with the VL domain amino acid sequence shown in SEQ ID NO: 105. In some embodiments, the antigen-binding protein comprises a variable light chain domain having at least 95% sequence identity with the VL domain amino acid sequence shown in SEQ ID NO: 105. In some embodiments, the antigen-binding protein comprises a variable light chain domain having at least 96, 97, 98, or 99% sequence identity with the VL domain amino acid sequence shown in SEQ ID NO: 105. In some embodiments, the variable light chain domain may have one or more additional mutations (e.g., for the purpose of humanization) in the VL domain amino acid sequence shown in SEQ ID NO: 105, but may retain specific binding to a cancer antigen (e.g., CD19). In some embodiments, the variable light chain domain may have one or more additional mutations in the VL domain amino acid sequence shown in SEQ ID NO: 105, but the specific binding to a cancer antigen (e.g., CD19) is improved.

[0172] In some embodiments, the antigen-binding protein comprises a variable heavy chain domain having the VH domain amino acid sequence shown in SEQ ID NO: 104 and a variable light chain domain having the VL domain amino acid sequence shown in SEQ ID NO: 105. In some embodiments, the variable light chain domain comprises an amino acid 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 the variable light chain domain of SEQ ID NO: 105. In some embodiments, the variable heavy chain domain comprises an amino acid 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 the variable heavy chain domain according to SEQ ID NO: 104.

[0173] In some embodiments, the antigen-binding protein comprises a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 106. In some embodiments, the antigen-binding protein comprises a heavy-chain variable region having at least 90% sequence identity with the VH amino acid sequence shown in SEQ ID NO: 106. In some embodiments, the antigen-binding protein comprises a heavy-chain variable region having at least 95% sequence identity with the VH amino acid sequence shown in SEQ ID NO: 106. In some embodiments, the antigen-binding protein comprises a heavy-chain variable region having at least 96, 97, 98, or 99% identity with the VH amino acid sequence shown in SEQ ID NO: 106. In some embodiments, the heavy-chain variable region may have one or more additional mutations (e.g., for the purpose of humanization) in the VH amino acid sequence shown in SEQ ID NO: 106, but may retain specific binding to a cancer antigen (e.g., CD19). In some embodiments, the heavy-chain variable region may have one or more additional mutations in the VH amino acid sequence shown in SEQ ID NO: 106, but the specific binding to a cancer antigen (e.g., CD19) is improved.

[0174] In some embodiments, the antigen-binding protein comprises a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 107. In some embodiments, the antigen-binding protein comprises a light-chain variable region having at least 90% sequence identity with the VL amino acid sequence shown in SEQ ID NO: 107. In some embodiments, the antigen-binding protein comprises a light-chain variable region having at least 95% sequence identity with the VL amino acid sequence shown in SEQ ID NO: 107. In some embodiments, the antigen-binding protein comprises a light-chain variable region having at least 96, 97, 98, or 99% identity with the VL amino acid sequence shown in SEQ ID NO: 107. In some embodiments, the light-chain variable region may have one or more additional mutations (e.g., for the purpose of humanization) in the VL amino acid sequence shown in SEQ ID NO: 107, but may retain specific binding to a cancer antigen (e.g., CD19). In some embodiments, the light-chain variable region may have one or more additional mutations in the VL amino acid sequence shown in SEQ ID NO: 107, but the specific binding to a cancer antigen (e.g., CD19) is improved.

[0175] In some embodiments, an anti-CD19 binding portion is also provided that includes light chain CDRs (LC CDR1, LC CDR2, and LC CDR3, respectively) that include first, second, and third complementarity determining regions. In some embodiments, the anti-CD19 binding portion further includes heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3, respectively) that include first, second, and third complementarity determining regions. In some embodiments, LC CDR1 includes the sequence of SEQ ID NO: 108. In some embodiments, LC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity with the sequence of SEQ ID NO: 108. In some embodiments, LC CDR2 includes the sequence of SEQ ID NO: 109. In some embodiments, LC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity with the sequence of SEQ ID NO: 109. In some embodiments, LC CDR3 includes the sequence of SEQ ID NO: 110. In some embodiments, LC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity with the sequence of SEQ ID NO: 110. In some embodiments, HC CDR1 includes the sequence of SEQ ID NO: 111. In some embodiments, HC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity with the sequence of SEQ ID NO: 111. In some embodiments, HC CDR2 includes the sequence of SEQ ID NO: 112, 113, or 114. In some embodiments, HC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity with the sequence of SEQ ID NO: 112, 113, or 114. In some embodiments, HC CDR3 includes the sequence of SEQ ID NO: 115. In some embodiments, HC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity with the sequence of SEQ ID NO: 115. In some embodiments, the anti-CD19 binding portion includes SEQ ID NO: 116 or is a sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity with the sequence of SEQ ID NO: 116.

[0176] In some embodiments, the antigen-binding protein comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 117, 118, or 119. In some embodiments, the antigen-binding protein comprises a variable light chain region having at least 90% identity with the VL amino acid sequence set forth in SEQ ID NO: 117, 118, or 119. In some embodiments, the antigen-binding protein comprises a variable light chain comprising the VL amino acid sequence set forth in SEQ ID NO: 117, 118, or 119 and having at least 95% identity therewith. In some embodiments, the antigen-binding protein comprises a variable light chain comprising the VL amino acid sequence set forth in SEQ ID NO: 117, 118, or 119 and having at least 96, 97, 98, or 99% identity therewith. In some embodiments, the variable light chain may have one or more additional mutations (e.g., for the purpose of humanization) in the VL amino acid sequence set forth in SEQ ID NO: 117, 118, or 119, but retains specific binding to a cancer antigen (e.g., CD19). In some embodiments, the variable light chain may have one or more additional mutations in the VL amino acid sequence set forth in SEQ ID NO: 117, 118, or 119, but the specific binding to a cancer antigen (e.g., CD19) is improved.

[0177] In some embodiments, the antigen-binding protein comprises a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 120, 121, 122, or 123. In some embodiments, the antigen-binding protein comprises a heavy-chain variable region having at least 90% identity with the VH amino acid sequence set forth in SEQ ID NO: 120, 121, 122, or 123. In some embodiments, the antigen-binding protein comprises a heavy-chain variable region having at least 95% identity with the VH amino acid sequence set forth in SEQ ID NO: 120, 121, 122, or 123. In some embodiments, the antigen-binding protein comprises a heavy-chain variable region having at least 96, 97, 98, or 99% identity with the VH amino acid sequence set forth in SEQ ID NO: 120, 121, 122, or 123. In some embodiments, the heavy-chain variable region may have one or more additional mutations (e.g., for the purpose of humanization) in the VH amino acid sequence set forth in SEQ ID NO: 120, 121, 122, or 123, but retains specific binding to a cancer antigen (e.g., CD19). In some embodiments, the heavy-chain variable region may have one or more additional mutations in the VH amino acid sequence set forth in SEQ ID NO: 120, 121, 122, or 123, but has improved specific binding to a cancer antigen (e.g., CD19).

[0178] In some embodiments, an anti-CD19 binding portion is also provided that includes light chain CDRs (LC CDR1, LC CDR2, and LC CDR3, respectively) that include first, second, and third complementarity determining regions. In some embodiments, the anti-CD19 binding portion further includes heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3, respectively) that include first, second, and third complementarity determining regions. In some embodiments, LC CDR1 includes the sequence of SEQ ID NO: 124, 127, or 130. In some embodiments, LC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 124, 127, or 130. In some embodiments, LC CDR2 includes the sequence of SEQ ID NO: 125, 128, or 131. In some embodiments, LC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 125, 128, or 131. In some embodiments, LC CDR3 includes the sequence of SEQ ID NO: 126, 129, or 132. In some embodiments, LC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 126, 129, or 132. In some embodiments, HC CDR1 includes the sequence of SEQ ID NO: 133, 136, 139, or 142. In some embodiments, HC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 133, 136, 139, or 142. In some embodiments, HC CDR2 includes the sequence of SEQ ID NO: 134, 137, 140, or 143. In some embodiments, HC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 134, 137, 140, or 143. In some embodiments, HC CDR3 includes the sequence of SEQ ID NO: 135, 138, 141, or 144.In some embodiments, the HC CDR3 comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity with the sequence of SEQ ID NO: 135, 138, 141, or 144.

[0179] Additional anti-CD19 binding portions are known in the art and include, for example, those disclosed in U.S. Patent No. 8,399,645, U.S. Patent Application Publication No. 2018 / 0153977, U.S. Patent Application Publication No. 2014 / 0271635, U.S. Patent Application Publication No. 2018 / 0251514, and U.S. Patent Application Publication No. 2018 / 0312588, each of which is incorporated herein by reference in its entirety.

[0180] Some embodiments relate to CARs that have specificity for Claudin 6 and show little or no binding to Claudin 3, 4, or 9 (or other Claudins). In some embodiments, the antigen-binding protein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 88. In some embodiments, the antigen-binding protein comprises a heavy chain variable region having at least 90% identity with the VH amino acid sequence shown in SEQ ID NO: 88. In some embodiments, the antigen-binding protein comprises a heavy chain variable region having at least 95% identity with the VH amino acid sequence shown in SEQ ID NO: 88. In some embodiments, the antigen-binding protein comprises a heavy chain variable region having at least 96, 97, 98, or 99% identity with the VH amino acid sequence shown in SEQ ID NO: 88. In some embodiments, the heavy chain variable region may have one or more additional mutations (e.g., for the purpose of humanization) in the VH amino acid sequence shown in SEQ ID NO: 88, but may retain specific binding to a cancer antigen (e.g., CLDN6). In some embodiments, the heavy chain variable region may have one or more additional mutations in the VH amino acid sequence shown in SEQ ID NO: 88, but with improved specific binding to a cancer antigen (e.g., CLDN6).

[0181] In some embodiments, the antigen-binding protein comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 89, 90, or 91. In some embodiments, the antigen-binding protein comprises a variable light chain having at least 90% identity with the VL amino acid sequence set forth in SEQ ID NO: 89, 90, or 91. In some embodiments, the antigen-binding protein comprises a variable light chain having at least 95% identity with the VL amino acid sequence set forth in SEQ ID NO: 89, 90, or 91. In some embodiments, the antigen-binding protein comprises a variable light chain having at least 96, 97, 98, or 99% identity with the VL amino acid sequence set forth in SEQ ID NO: 89, 90, or 91. In some embodiments, the variable light chain may have one or more additional mutations (e.g., for the purpose of humanization) in the VL amino acid sequence set forth in SEQ ID NO: 89, 90, or 91, but retains specific binding to a cancer antigen (e.g., CLDN6). In some embodiments, the variable light chain may have one or more additional mutations in the VL amino acid sequence set forth in SEQ ID NO: 89, 90, or 91, but has improved specific binding to a cancer antigen (e.g., CLDN6).

[0182] In some embodiments, an anti-CLDN6 binding portion is also provided that includes light chain CDRs (LC CDR1, LC CDR2, and LC CDR3, respectively) that include a first, second, and third complementarity determining region. In some embodiments, the anti-CD19 binding portion further includes heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3, respectively) that include a first, second, and third complementarity determining region. In some embodiments, LC CDR1 includes the sequence of SEQ ID NO: 95, 98, or 101. In some embodiments, LC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 95, 98, or 101. In some embodiments, LC CDR2 includes the sequence of SEQ ID NO: 96, 99, or 102. In some embodiments, LC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 96, 99, or 102. In some embodiments, LC CDR3 includes the sequence of SEQ ID NO: 97, 100, or 103. In some embodiments, LC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 97, 100, or 103. In some embodiments, HC CDR1 includes the sequence of SEQ ID NO: 92. In some embodiments, HC CDR1 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 92. In some embodiments, HC CDR2 includes the sequence of SEQ ID NO: 93. In some embodiments, HC CDR2 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 93. In some embodiments, HC CDR3 includes the sequence of SEQ ID NO: 94. In some embodiments, HC CDR3 includes an amino acid sequence having at least about 85%, about 90%, about 95%, or about 98% sequence identity to the sequence of SEQ ID NO: 94. In some embodiments, the antigen-binding protein does not bind to claudins other than CLDN6.

[0183] Natural killer group domain that binds 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 can include a CD19-directed chimeric antigen receptor, or a nucleic acid encoding said chimeric antigen receptor (or a CAR having specificity for one or more of, e.g., CD123, CD70, 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. NK cell activation occurs when the balance between inhibitory receptor activation and activating receptor activation favors the activating receptor, and the target (e.g., tumor) cells are lysed.

[0184] Natural killer group 2 member D (NKG2D) is a NK cell activation receptor that recognizes various ligands expressed on cells. The surface expression of various NKG2D ligands is generally low in 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 functions of NK cells. In some embodiments, T cells are engineered to express an extracellular domain in order 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 factor / activation moiety. In some embodiments, the engineered cells disclosed herein are engineered to express another member of the NKG2 family, such as NKG2A, NKG2C, and / or NKG2E. In some embodiments, combinations of such receptors are engineered. Further, in some embodiments, other receptors such as killer cell immunoglobulin-like receptors (KIR) are expressed.

[0185] In some embodiments, cells are engineered to express a cytotoxic receptor complex that includes full-length NKG2D as an extracellular component for recognizing ligands 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.

[0186] In some embodiments, the cells are engineered to express a cytotoxic receptor complex that includes a functional fragment of NKG2D as an extracellular component for recognizing ligands 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 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the full-length wild-type NKG2D. In some embodiments, the fragment can have one or more additional mutations from SEQ ID NO: 25, but retains the ligand-binding function or, in some embodiments, has an increased ligand-binding function. In some embodiments, the functional fragment of NKG2D includes the amino acid sequence of SEQ ID NO: 26. In some embodiments, the NKG2D fragment is provided as a dimer, trimer, or other concatameric form, and such embodiments provide increased ligand-binding activity. In some embodiments, the sequence encoding the NKG2D fragment may be fully or partially optimized codons. In one embodiment, the sequence encoding the codon-optimized NKG2D fragment includes 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 ligands and its ability to transduce activation signals upon ligand binding. A further advantage of such fragments is that it is not necessary to express DAP10 to localize NKG2D to the cell membrane. Thus, in some embodiments, the cytotoxic receptor complex encoded by the polypeptides disclosed herein does not include DAP10. In some embodiments, immune cells such as NK or T cells (e.g., alloreactive T cells engineered according to the embodiments disclosed herein) are engineered to express one or more chimeric receptors that target, for example, CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, EGFR, and NKG2D ligands such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6.In some embodiments, such cells also co-express mbIL15.

[0187] In some embodiments, the cytotoxic receptor complex is configured to dimerize. The dimerization can include a homodimer or a heterodimer, depending on the embodiment. In some embodiments, the dimerization results in improved ligand recognition by the cytotoxic receptor complex (and thus the NK cells expressing the receptor) and results in a reduction (or absence) of deleterious 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 can include a first NKG2D extracellular domain bound to a second NKG2D extracellular domain, and a transmembrane / signal transduction region (or a separate transmembrane region together with a separate signal transduction region).

[0188] In some embodiments, the various domains / subdomains are separated by linkers, such as the GS3 linker (SEQ ID NOs: 15 and 16, nucleotide and protein, respectively) (or GSn linker) used. Other linkers used according to the various embodiments disclosed herein include, but are not limited to, those encoded by SEQ ID NOs: 17, 19, 21, or 23. This provides the possibility of separating the various component parts of the receptor complex along a polynucleotide that can increase the expression, stability, and / or functionality of the receptor complex.

[0189] Cytotoxic signaling complex Some embodiments of the compositions and methods described herein relate to chimeric antigen receptors that include a cytotoxic signaling complex (e.g., CARs having specificity for CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, or EGFR, among others), or chimeric receptors having specificity for 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 complex includes one or more transmembrane domains and / or intracellular domains that initiate a cytotoxic signaling cascade on an extracellular domain(s) that binds to a ligand on the surface of a target cell.

[0190] In some embodiments, the cytotoxic signaling complex includes at least one transmembrane domain, at least one co-stimulatory domain, and / or at least one signaling domain. In some embodiments, more than one component part constitutes a given domain - for example, the co-stimulatory domain can include two sub-domains. Further, in some embodiments, a domain can perform multiple functions, for example, a transmembrane domain can serve to provide a signaling function.

[0191] Transmembrane domain Some embodiments of the compositions and methods described herein relate to chimeric receptors that include a transmembrane domain (e.g., tumor antigen-directed CARs and / or ligand-directed chimeric receptors). Some embodiments include transmembrane domains from NKG2D or another transmembrane protein. In some embodiments where a transmembrane domain is employed, the portion of the transmembrane protein employed retains at least a part of its normal transmembrane domain.

[0192] However, in some embodiments, the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein that is 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 cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to 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 cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to the sequence of SEQ ID NO: 2.

[0193] In some embodiments, the transmembrane domain comprises the 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 cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to 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 cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to CD8α having the sequence of SEQ ID NO: 4.

[0194] 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 cleaved or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to the 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 cleaved or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to the CD8 hinge / transmembrane complex having the sequence of SEQ ID NO: 14.

[0195] In some embodiments, the transmembrane domain comprises the 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 cleaved or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to the CD28 transmembrane domain having the sequence of SEQ ID NO: 30.

[0196] Costimulatory domain Some embodiments of the compositions and methods described herein relate to chimeric receptors that include a co-stimulatory domain (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors). Further, in some embodiments, various transmembrane domains and signaling domains (and combinations of transmembrane / signaling domains) can be provided that can provide additional co-activating molecules. These can be, for example, certain molecules that further increase the activity of immune cells. Cytokines can be used in some embodiments. For example, by way of non-limiting example, certain interleukins such as IL-2 and / or IL-15 are used. In some embodiments, immune cells for therapy are engineered to express molecules such as secreted forms. In further embodiments, such co-stimulatory domains are engineered to be membrane-bound such that they act as autocrine stimulatory molecules (or even as paracrine stimulators to adjacent cells). In some embodiments, NK cells are engineered to express membrane-bound interleukin 15 (mbIL15). In such embodiments, mbIL15 expression on NKs increases the cytotoxic effect of the engineered NK cells by increasing NK cell proliferation and / or lifespan. In some embodiments, T cells such as the genetically engineered alloreactive T cells disclosed herein are engineered to express membrane-bound interleukin 15 (mbIL15). In such embodiments, mbIL15 expression on T cells increases the cytotoxic effect of the engineered T cells by increasing the activity and / or propagation (e.g., lifespan) of the engineered T cells. In some embodiments, mbIL15 has the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, mbIL15 can be truncated or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to the sequence of SEQ ID NO: 11. In some embodiments, mbIL15 includes the amino acid sequence of SEQ ID NO: 12.In some embodiments, mbIL15 is cleaved or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to mbIL15 having the sequence of SEQ ID NO: 12.

[0197] In some embodiments, the tumor antigen-directed CAR and / or the tumor ligand-directed chimeric receptor are encoded by a polynucleotide comprising one or more cytoplasmic protease cleavage sites, such as a T2A cleavage site, a P2A cleavage site, an E2A cleavage site, and / or an F2A cleavage site. Such sites can be recognized and cleaved by cytoplasmic proteases, resulting in the separation (and separate expression) of the various component parts of the receptor encoded by the polynucleotide. As a result, depending on the embodiment, the 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 schematically shown in the figures, the construct can be encoded by a single polynucleotide but also includes a cleavage site such that downstream elements of the construct are expressed by the cell as separate proteins (as in some embodiments having 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 can be cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous 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 cleaved or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to the T2A cleavage site having the sequence of SEQ ID NO: 10.

[0198] Signal transduction domain Some embodiments of the compositions and methods described herein relate to chimeric receptors that include a signaling domain (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors). For example, immune cells engineered according to some embodiments disclosed herein may include at least one subunit of the CD3 T cell receptor complex (or a fragment thereof). In some embodiments, the signaling domain includes the 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 may be cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to CD3 zeta having the sequence of SEQ ID NO: 7. In some embodiments, the CD3 zeta domain includes the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CD3 zeta domain is cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to the CD3 zeta domain having the sequence of SEQ ID NO: 8.

[0199] In some embodiments, unexpected increased signaling is achieved by 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 cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to an 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 cleaved or modified to be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to an 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, but in some embodiments, OX40 can be used together 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 CD zeta is used.

[0200] 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 cleaved or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to the 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 29. In some embodiments, 4-1BB is used as the sole transmembrane / signaling domain in the construct, but in some embodiments, 4-1BB can be used with one or more other domains. For example, in some embodiments, a combination of 4-1BB 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.

[0201] 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 cleaved or modified and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homologous to the CD28 intracellular signaling domain having the sequence of SEQ ID NO: 31. In some embodiments, CD28 is used as the sole transmembrane / signaling domain in the construct, but in some embodiments, CD28 can be used with one or more other domains. For example, in some embodiments, a combination of CD28 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.

[0202] Cytotoxic 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 ligands of NKG2D. Expression of these cytotoxic receptor complexes in immune cells such as genetically modified alloreactive T cells and / or NK cells enables 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.

[0203] Chimeric antigen receptor cytotoxic 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 schematically show non-limiting schematic diagrams of constructs comprising a tumor-binding moiety that binds to a tumor antigen or tumor-associated antigen that is expressed on the surface of cancer cells and activates engineered cells expressing the chimeric antigen receptor. Figure 6 shows a schematic diagram of a chimeric receptor complex having an NKG2D activating chimeric receptor as a non-limiting example (see NKG2D ACRa and ACRb). Figure 6 shows a schematic diagram of a bispecific CAR / chimeric receptor complex having an NKG2D activating chimeric receptor as a non-limiting example (see bispecific CAR / ACRa and CAR / ACRb).

[0204] As shown in the figures, some embodiments of the chimeric receptor include an anti-tumor binding factor, 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 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 factor portion or other binding factor / activation portion. In some embodiments, the binding factor / activation portion targets other markers than CD19, such as the cancer targets described herein. For example, FIGS. 6 and 7 show schematic diagrams of non-limiting examples of CAR constructs targeting different antigens such as CD123, CLDN6, BCMA, HER2, CD70, mesothelin, PD-L1, and EGFR. In some embodiments, the general structure of the 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 converts signals after binding of the homing portion to the target cell and ultimately results in a cytotoxic effect in the target cell. In some embodiments, the complex further includes a co-stimulatory domain, and in some embodiments, they act synergistically to increase the function of the signaling domain. Expression of these complexes in immune cells such as T cells and / or NK cells enables targeting and destruction of specific target cells such as cancerous cells that express a given tumor marker. Some of such receptor complexes include an extracellular domain that binds to CD19 on the surface of the target cell and an anti-CD19 portion or a CD19-binding portion that activates the engineered cell. 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 co-stimulatory domain. The IL-15 domain, e.g., the mbIL-15 domain, can make the immune cells expressing it (e.g., NK or T cells) particularly effective against target tumor cells. The IL-15 domain such as the mbIL-15 domain is approved to be encoded in a separate construct according to some embodiments. Further, each component can be encoded in one or more separate components. In some embodiments, the cytotoxic receptor or the CD19-directed receptor comprises an amino acid sequence that is identical to the sequence of SEQ ID NO: 34 in at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or more, or within a range defined by any two of the foregoing percentages.

[0205] Depending on the embodiment, CD19 can be targeted using various binding agents. In some embodiments, peptide binding agents are used, while in some embodiments, antibodies or fragments thereof are used. In some embodiments that employ antibodies, the antibody sequences are optimized from their native forms, humanized, or alternatively engineered or mutated such that one or more of the stability, affinity, avidity, or other characteristics of the antibody or fragment are increased. In some embodiments, antibodies specific for CD19 are provided. In some embodiments, scFvs specific for CD19 are provided. In some embodiments, the antibody or scFv specific for CD19 comprises a heavy chain variable portion comprising the amino acid sequence of SEQ ID NO: 104 or 106. In some embodiments, the heavy chain variable portion comprises an amino acid 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 SEQ ID NO: 104 or 106. In some embodiments, the heavy chain variable portion comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a polynucleotide that is complementary to the polynucleotide encoding the heavy chain variable portion of SEQ ID NO: 104 or 106. In some embodiments, the heavy chain variable domain is an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide that is complementary to the polynucleotide encoding the heavy chain variable portion of SEQ ID NO: 104 or 106.

[0206] In some embodiments, the antibody or scFv specific for CD19 comprises a light chain variable portion comprising the amino acid sequence of either SEQ ID NO: 105 or 107. In some embodiments, the light chain variable portion comprises an amino acid sequence encoded by a nucleotide 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 SEQ ID NO: 105 or 107. In some embodiments, the light chain variable portion comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a polynucleotide that is complementary to a polynucleotide encoding the light chain variable portion of SEQ ID NO: 105 or 107. In some embodiments, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide that is complementary to a polynucleotide encoding the light chain variable domain of SEQ ID NO: 105 or 107.

[0207] In some embodiments, the anti-CD19 antibody or scFv comprises one, two, or three heavy chain complementarity determining regions (CDRs) and one, two, or three light chain CDRs. In some embodiments, the first heavy chain CDR has the amino acid sequence of SEQ ID NO: 111. In some embodiments, the first heavy chain CDR comprises an amino acid 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 SEQ ID NO: 111. In some embodiments, the second heavy chain CDR has the amino acid sequence of SEQ ID NO: 112, 113, or 114. In some embodiments, the second heavy chain CDR comprises an amino acid 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 SEQ ID NO: 112, 113, or 114. In some embodiments, the third heavy chain CDR has the amino acid sequence of SEQ ID NO: 115. In some embodiments, the third heavy chain CDR comprises an amino acid 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 SEQ ID NO: 115.

[0208] In some embodiments, the first light chain CDR has the amino acid sequence of SEQ ID NO: 108. In some embodiments, the first light chain CDR comprises an amino acid 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 SEQ ID NO: 108. In some embodiments, the second light chain CDR has the amino acid sequence of SEQ ID NO: 109. In some embodiments, the second light chain CDR comprises an amino acid 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 SEQ ID NO: 109. In some embodiments, the third light chain CDR has the amino acid sequence of SEQ ID NO: 110. In some embodiments, the third light chain CDR comprises an amino acid 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 SEQ ID NO: 110.

[0209] In some embodiments, an anti-CD19 CAR comprising the amino acid sequence of SEQ ID NO: 116 is provided. In some embodiments, an anti-CD19 CAR is provided that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or more identical to the sequence of SEQ ID NO: 116.

[0210] In one embodiment, a polynucleotide encoding a tumor-binding factor / CD8 hinge-CD8TM / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 1, CAR1c). The polynucleotide comprises or consists of, as described herein, a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0211] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge-CD8TM / OX40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex is provided (see Figure 1, CAR 1d). The polynucleotide comprises or consists of, as described herein, 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. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are recognized as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0212] In some embodiments, a polynucleotide encoding a tumor-binding factor / Ig4SH-CD8TM / 4-1BB / CD3 zeta chimeric antigen receptor complex is provided (see FIG. 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 CD3 zeta domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0213] In some embodiments, a polynucleotide encoding a tumor-binding factor / Ig4SH-CD8TM / 4-1BB / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex is provided (see Figure 4, CAR4b). The polynucleotide, as described herein, comprises or consists of a tumor-binding factor, an Ig4 SH domain, a CD8a transmembrane domain, a 4-1BB domain, a CD3 zeta domain, a 2A cleavage site, and an mIL-15 domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0214] In some embodiments, polynucleotides encoding a tumor-binding factor / CD8 hinge-CD28TM / CD28 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 1, CAR1e). The polynucleotides include or consist 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, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0215] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge-CD28TM / CD28 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex is provided (see FIG. 1, CAR1f). The polynucleotide comprises or consists of, as described herein, a tumor-binding factor, a CD8a hinge, a CD28 transmembrane domain, a CD28 domain, a CD3 zeta domain, a 2A cleavage site, and an mIL-15 domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0216] In some embodiments, a polynucleotide encoding a tumor-binding factor / Ig4SH-CD28TM / CD28 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 2, CAR2i). The polynucleotide comprises or consists of, as described herein, a tumor-binding factor, an Ig4 SH domain, a CD28 transmembrane domain, a CD28 domain, and a CD3 zeta domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0217] In some embodiments, a polynucleotide encoding a tumor-binding factor / Ig4SH-CD28TM / CD28 / CD3ζ / 2A / mIL-15 chimeric antigen receptor complex is provided (see Figure 2, CAR2j). The polynucleotide comprises or consists of, as described herein, a tumor-binding factor, an Ig4 SH domain, a CD28 transmembrane domain, a CD28 domain, a CD3ζ domain, a 2A cleavage site, and an mIL-15 domain. In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are approved as being those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0218] In some embodiments, a polynucleotide encoding a tumor-binding factor / Ig4SH-CD8TM / OX40 / CD3 zeta 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 CD3 zeta domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0219] In some embodiments, a polynucleotide encoding a tumor-binding factor / Ig4SH-CD8TM / OX40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex is provided (see Figure 4, CAR4d). The polynucleotide comprises or consists of a tumor-binding factor, an Ig4 SH domain, 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, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that follows one or more of the SEQ ID NOs described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence 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 a combination of one or more of the SEQ ID NOs described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0220] 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, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0221] In some embodiments, polynucleotides encoding a tumor-binding factor / CD8 hinge-CD3αTM / CD28 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 4, CAR4f). The polynucleotide comprises or consists of, as described herein, a tumor-binding factor, a CD8a hinge, a CD3α transmembrane domain, a CD28 domain, a CD3 zeta domain, a 2A cleavage site, and an mIL-15 domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that follows one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are approved as being those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0222] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge-CD28TM / CD28 / 4-1BB / CD3 zeta chimeric antigen receptor complex is provided (see Figure 4, CAR 4g). The polynucleotide comprises or consists of, as described herein, a tumor-binding factor, a CD8a hinge, a CD28 transmembrane domain, a CD28 domain, a 4-1BB domain, and a CD3 zeta domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Specific sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being those that may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0223] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge-CD28TM / CD28 / 4-1BB / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex is provided (see Figure 4, CAR 4h). The polynucleotide, as described herein, comprises or consists of a tumor-binding factor, a CD8a hinge, a CD28 transmembrane domain, a CD28 domain, a 4-1BB domain, a CD3 zeta domain, a 2A cleavage site, and an mIL-15 domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that follows one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are approved as being those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0224] In some embodiments, a polynucleotide encoding a tumor binding factor / CD8 alpha hinge / CD8 alpha TM / 4-1BB / CD3 zeta chimeric antigen receptor complex is provided (see Figure 5, CAR5a). The polynucleotide comprises or consists of, as described herein, an anti-CD19 moiety, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB domain, and a CD3 zeta domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Specific sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0225] In some embodiments, a polynucleotide encoding a tumor binding factor / CD8 alpha hinge / CD8 alpha TM / 4-1BB / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex is provided (see Figure 5, CAR 5b). The polynucleotide comprises or consists of a tumor binding factor, CD8a hinge, CD8a transmembrane domain, 4-1BB domain, CD3 zeta domain, 2A cleavage site, and mIL-15 domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers included herein as examples of components, such as those described herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being able to occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0226] In some embodiments, a polynucleotide encoding a tumor binding factor / CD8 alpha hinge / CD3 TM / 4-1BB / CD3 zeta chimeric antigen receptor complex is provided (see Figure 5, CAR5c). The polynucleotide comprises or consists of a tumor binding factor, CD8a hinge, CD3 transmembrane domain, 4-1BB domain, and CD3 zeta domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence that follows one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0227] In some embodiments, polynucleotides encoding a tumor-binding factor / CD8 alpha hinge / CD3 TM / 4-1BB / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 5, CAR5d). The polynucleotide comprises or consists of, as described herein, a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB domain, a CD3 zeta domain, a 2A cleavage site, and an mIL-15 domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the SEQ ID NOs included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the SEQ ID NOs described herein. Specific sequence variations, extensions, and / or truncations of the disclosed sequences are recognized as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0228] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 alpha hinge / CD3 TM / 4-1BB / NKp80 chimeric antigen receptor complex is provided (see Figure 5, CAR5e). The polynucleotide comprises or consists of, as described herein, a tumor-binding factor, a CD8a hinge, a CD3 transmembrane domain, a 4-1BB domain, and an NKp80 domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are recognized as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0229] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 alpha hinge / CD3 TM / 4-1BB / NKp80 / 2A / mIL-15 chimeric antigen receptor complex is provided (see FIG. 5, CAR5f). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB domain, an NKp80 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 the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Specific sequence variations, extensions, and / or truncations of the disclosed sequences are recognized as those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0230] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 alpha hinge / CD3 transmembrane domain / CD16 intracellular domain / 4-1BB chimeric antigen receptor complex is provided (see Figure 5, CAR5g). The polynucleotide comprises or consists of, as described herein, a tumor-binding factor, a CD8a hinge, a CD3 transmembrane domain, a CD16 intracellular domain, and a 4-1BB domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0231] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 alpha hinge / CD3 TM / CD16 / 4-1BB / 2A / mIL-15 chimeric antigen receptor complex is provided (see Figure 5, CAR5h). The polynucleotide, as described herein, comprises or consists of a tumor-binding factor, a CD8a hinge, a CD8a transmembrane domain, a CD16 intracellular domain, a 4-1BB domain, a 2A cleavage site, and an mIL-15 domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are recognized as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for the creation of restriction sites).

[0232] In some embodiments, polynucleotides encoding a tumor-binding factor / NKG2D extracellular domain / CD8 hinge-CD8TM / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 5, bispecific CAR / ACRa). The polynucleotide, as described herein, comprises or consists of a tumor-binding factor, an NKG2D extracellular domain (either full length or a fragment), a CD8a hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0233] In some embodiments, polynucleotides encoding a tumor binding factor / NKG2D EC domain / CD8 hinge-CD8TM / OX40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex are provided (see Figure 5, bispecific CAR / ACRb). The polynucleotide comprises or consists of, as described herein, a tumor binding factor, an NKG2D extracellular domain (either full length or a fragment), a CD8a hinge, a CD8a transmembrane domain, an OX40 domain, a CD3 zeta domain, a 2A cleavage site, and an mIL-15 domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0234] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8TM / 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 a non-limiting embodiment, an anti-CD19 / CD8 hinge / CD8TM / 4-1BB / CD3 zeta chimeric antigen receptor complex is provided herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 85. In some embodiments, the nucleic acid sequence encoding the CAR1a chimeric antigen receptor 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 SEQ ID NO: 85. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the CAR1a chimeric antigen receptor comprises an amino acid 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 SEQ ID NO: 86. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are approved as may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites). In some embodiments, a CAR1a construct further comprising mbIL15 is provided as disclosed herein (see, e.g., Figure 1 CAR1b).

[0235] In some embodiments, polynucleotides encoding a tumor binding factor / CD8 hinge / CD8TM / OX40 / CD3 zeta chimeric antigen receptor complex are 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. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 1, CAR1d). As a non-limiting embodiment, provided herein is an anti-CD19 / CD8 hinge / CD8TM / OX40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, an OX40 domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 59. In some embodiments, the nucleic acid sequence encoding the CAR1d chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence to SEQ ID NO: 59. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the NK19 chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence to SEQ ID NO: 60. In some embodiments, the CD19 scFv does not contain a Flag tag. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences as a result of, for example, the ease or efficiency of cloning (e.g., for creating restriction sites).

[0236] 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 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 1, CAR1d). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD28TM / CD28 / CD3 zeta / 2A / mIL15 chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD28 transmembrane domain, a CD28 signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 61. In some embodiments, the nucleic acid sequence encoding the CAR1d chimeric antigen receptor 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 SEQ ID NO: 61. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the CAR1d chimeric antigen receptor comprises an amino acid 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 SEQ ID NO: 62. In some embodiments, the CD19 scFv does not contain a Flag tag. 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 creating restriction sites).

[0237] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8aTM / 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 chimeric antigen receptor further comprises mbIL15 (see Figure 1, CAR1h). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD8aTM / ICOS / CD3 zeta / 2A / mIL15 chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, an inducible costimulatory factor (ICOS) signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 63. In some embodiments, the nucleic acid sequence encoding the CAR1h chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 63. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, the CAR1h chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 64. In some embodiments, the CAR1h scFv does not contain a Flag tag. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences can occur when combining sequences as a result of, for example, the ease or efficiency of cloning (e.g., for creating restriction sites).

[0238] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8aTM / 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 chimeric antigen receptor further comprises mbIL15 (see Figure 3A, NK19-4b). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD8aTM / CD28 / 4-1BB / CD3 zeta / 2A / mIL-15. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, a CD28 signaling domain, a 4-1BB signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 65. In some embodiments, the nucleic acid sequence encoding the CAR1h chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence to SEQ ID NO: 65. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, the CAR1h chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence to SEQ ID NO: 66. In some embodiments, the CAR1h scFv does not contain a Flag tag. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are approved as being those that may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0239] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / NKG2D TM / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 2, CAR2a). The polynucleotide comprises or consists of a tumor-binding factor, a CD8a hinge, an NKG2D transmembrane domain, an OX40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 2, CAR2b). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / NKG2D TM / OX40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, an NKG2D transmembrane domain, an OX40 signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 67. In some embodiments, the nucleic acid sequence encoding the CAR2b chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 67. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the CAR2b chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 68. In some embodiments, the CD19 scFv does not contain a Flag tag. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0240] In some embodiments, a polynucleotide encoding a tumor binding factor / CD8 hinge / CD8aTM / CD40 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 2 CAR2c). The polynucleotide comprises or consists of a tumor binding factor, a CD8a hinge, a CD8a transmembrane domain, a CD40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 2, CAR2d). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD8aTM / CD40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv variable heavy chain, a CD8a hinge, a CD8a transmembrane domain, a CD40 signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 69. In some embodiments, the nucleic acid sequence encoding the CAR2d chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 69. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the CAR2d chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 70. In some embodiments, the CD19 scFv does not contain a Flag tag. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences can occur when combining sequences as a result of, for example, the ease or efficiency of cloning (e.g., for creating restriction sites).

[0241] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8aTM / 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, and a truncated form of epidermal growth factor receptor (EGFRt). In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 2, CAR2f). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD8aTM / OX40 / CD3 zeta / 2A / mIL-15 / 2A / EGFRt chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, an OX40 signaling domain, a CD3 zeta domain, a 2A cleavage site, a truncated form of epidermal growth factor receptor (EGFRt), an additional 2A cleavage site, and an mbIL-15 domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 71. In some embodiments, the nucleic acid sequence encoding the CAR2f chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 71. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the CAR2f chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 72. In some embodiments, the CD19 scFv does not contain a Flag tag.Certain array changes, extensions, and / or truncations of the disclosed arrays are approved as being possible when combining arrays, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0242] In some embodiments, a polynucleotide encoding a tumor binding factor / CD8 hinge / CD8aTM / CD40 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 2, CAR2g). The polynucleotide comprises or consists of a tumor binding factor, a CD8a hinge, a CD8a transmembrane domain, a CD40 signaling domain, and a CD3 zeta domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15 (see Figure 2, CAR2h). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD8aTM / CD40 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv variable light chain, a CD8a hinge, a CD8a transmembrane domain, a CD40 signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 73. In some embodiments, the nucleic acid sequence encoding the CAR2h chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 73. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 74. In some embodiments, the CAR2h chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 74. In some embodiments, the CD19 scFv does not contain a Flag tag. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as may occur when combining sequences as a result of, for example, the ease or efficiency of cloning (e.g., for creating restriction sites).

[0243] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8aTM / 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 chimeric antigen receptor further comprises mbIL15 (see Figure 3, CAR3b). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD8aTM / CD27 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, a CD27 signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 75. In some embodiments, the nucleic acid sequence encoding the CAR3b chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 75. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the CAR3b chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 76. In some embodiments, the CD19 scFv does not contain a Flag tag. 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 creating restriction sites).

[0244] In some embodiments, a polynucleotide encoding a tumor-binding factor / CD8 hinge / CD8aTM / 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 chimeric antigen receptor further comprises mbIL15 (see Figure 3, CAR3d). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD8aTM / CD70 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, a CD70 signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 77. In some embodiments, the nucleic acid sequence encoding the CAR3d chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 77. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the CAR3d chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 78. In some embodiments, the CD19 scFv does not contain a Flag tag. 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 creating restriction sites).

[0245] In some embodiments, a polynucleotide encoding a tumor binding factor / CD8 hinge / CD8aTM / 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 chimeric antigen receptor further comprises mbIL15 (see Figure 3, CAR3f). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD8aTM / CD161 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, a CD161 signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 79. In some embodiments, the nucleic acid sequence encoding the CAR3f chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence to SEQ ID NO: 79. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, the CAR3f chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence to SEQ ID NO: 80. In some embodiments, the CD19 scFv does not contain a Flag tag. 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).

[0246] In some embodiments, polynucleotides encoding a tumor binding factor / CD8 hinge / CD8aTM / CD40L / CD3 zeta chimeric antigen receptor complex are 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 chimeric antigen receptor further comprises mbIL15 (see Figure 3, CAR3h). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD8aTM / CD40L / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, a CD40L signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 81. In some embodiments, the nucleic acid sequence encoding the CAR3h chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence to SEQ ID NO: 81. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the CAR3h chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology and / or functional equivalence to SEQ ID NO: 82. In some embodiments, the CD19 scFv does not contain a Flag tag. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites).

[0247] In some embodiments, a polynucleotide encoding a tumor binding factor / CD8 hinge / CD8aTM / 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 chimeric antigen receptor further comprises mbIL15 (see Figure 3, CAR3j). As a non-limiting embodiment, provided herein is an anti-CD19 moiety / CD8 hinge / CD8aTM / CD44 / CD3 zeta / 2A / mIL-15 chimeric antigen receptor complex. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, a CD44 signaling domain, a CD3 zeta domain, a 2A cleavage site, and an mbIL-15 domain as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO: 83. In some embodiments, the nucleic acid sequence encoding the CAR3j chimeric antigen receptor comprises a sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 83. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the CAR3j chimeric antigen receptor comprises an amino acid sequence having at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence to SEQ ID NO: 84. In some embodiments, the CD19 scFv does not contain a Flag tag. It is recognized that certain sequence variations, extensions, and / or truncations of the disclosed sequences may occur when combining sequences as a result of, for example, ease or efficiency of cloning (e.g., for creating restriction sites).

[0248] In some embodiments, a polynucleotide encoding an anti-CD123 / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see FIG. 6, CD123 CARa). The polynucleotide comprises or consists of an anti-CD123 portion, a CD8 alpha hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the SEQ ID NOs included herein as examples of the components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the SEQ ID NOs described herein. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are contemplated as resulting from, for example, the ease or efficiency of cloning (e.g., for the creation of restriction sites) when combining the sequences. In some embodiments, a CD123 CAR construct further comprising mbIL15 disclosed herein is provided (see, e.g., FIG. 6, CD123 CARb).

[0249] In some embodiments, a polynucleotide encoding an anti-CLDN6 / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 6, CLDN6 CARa). The polynucleotide comprises or consists of an anti-CLDN6 binding portion, a CD8 alpha hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as those that 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, a CLDN6 CAR construct further comprising mbIL15 disclosed herein is provided (see, for example, Figure 6, CLDN6 CARb).

[0250] Depending on the embodiment, various binding agents can be used to target CLDN6. In some embodiments, peptide binding agents are used, while in some embodiments, antibodies or fragments thereof are used. In some embodiments that employ antibodies, the antibody sequences are optimized from their native forms, humanized, or alternatively engineered or mutated such that one or more of the stability, affinity, avidity, or other characteristics of the antibody or fragment are increased. In some embodiments, antibodies specific for CLDN6 are provided. In some embodiments, scFvs specific for CLDN6 are provided. In some embodiments, the antibody or scFv specific for CLDN6 comprises a heavy chain variable portion comprising the amino acid sequence of SEQ ID NO: 88. In some embodiments, the heavy chain variable portion comprises an amino acid 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 SEQ ID NO: 88. In some embodiments, the heavy chain variable portion comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a polynucleotide that is complementary to the polynucleotide encoding the heavy chain variable portion of SEQ ID NO: 88. In some embodiments, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide that is complementary to the polynucleotide encoding the heavy chain variable portion of SEQ ID NO: 88.

[0251] In some embodiments, an antibody or scFv specific for CLDN6 comprises a light chain variable region comprising the amino acid sequence of any of SEQ ID NOs: 89, 90, or 91. In some embodiments, the light chain variable region comprises an amino acid sequence encoded by a nucleotide 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 SEQ ID NOs: 89, 90, or 91. In some embodiments, the light chain variable region comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a polynucleotide that is complementary to the polynucleotide encoding the light chain variable region of SEQ ID NOs: 89, 90, or 91. In some embodiments, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide that is complementary to the polynucleotide encoding the light chain variable domain of SEQ ID NOs: 89, 90, or 91.

[0252] In some embodiments, an anti-CLDN6 antibody or scFv comprises one, two, or three heavy chain complementarity determining regions (CDRs) and one, two, or three light chain CDRs. In some embodiments, the first heavy chain CDR has the amino acid sequence of SEQ ID NO: 92. In some embodiments, the first heavy chain CDR comprises an amino acid 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 SEQ ID NO: 92. In some embodiments, the second heavy chain CDR has the amino acid sequence of SEQ ID NO: 93. In some embodiments, the second heavy chain CDR comprises an amino acid 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 SEQ ID NO: 93. In some embodiments, the third heavy chain CDR has the amino acid sequence of SEQ ID NO: 94. In some embodiments, the third heavy chain CDR comprises an amino acid 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 SEQ ID NO: 94.

[0253] In some embodiments, the first light chain CDR has the amino acid sequence of SEQ ID NO: 95, 98, or 101. In some embodiments, the first light chain CDR comprises an amino acid 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 SEQ ID NO: 95, 98, or 101. In some embodiments, the second light chain CDR has the amino acid sequence of SEQ ID NO: 96, 99, or 102. In some embodiments, the second light chain CDR comprises an amino acid 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 SEQ ID NO: 96, 99, or 102. In some embodiments, the third light chain CDR has the amino acid sequence of SEQ ID NO: 97, 100, or 103. In some embodiments, the third light chain CDR comprises an amino acid 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 SEQ ID NO: 97, 100, or 103.

[0254] Advantageously, in some embodiments, the CLDN6 CAR is highly specific for CLDN6 and does not substantially bind to any of CLDN3, 4, or 9.

[0255] In some embodiments, a polynucleotide encoding an anti-BCMA / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see FIG. 6, BCMA CARa). The polynucleotide comprises or consists of an anti-BCMA binding portion, a CD8α hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are contemplated as may occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites). In some embodiments, a BCMA CAR construct further comprising mbIL15 disclosed herein is provided (see, e.g., FIG. 6, BCMA CARb).

[0256] In some embodiments, a polynucleotide encoding an anti-HER2 / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see FIG. 6, HER2 CARa). The polynucleotide comprises or consists of an anti-HER2 binding portion, a CD8α hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are approved as being those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites). In some embodiments, a HER2 CAR construct further comprising mbIL15 disclosed herein is provided (see, e.g., FIG. 6, HER2 CARb).

[0257] In some embodiments, polynucleotides encoding NKG2D / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta activating chimeric receptor complexes are provided (see FIG. 6, NKG2D ACRa). The polynucleotides, as described herein, include or consist of fragments of the NKG2D receptor that can bind to the ligand of the NKG2D receptor, the CD8α hinge, the CD8a transmembrane domain, the OX40 domain, and the CD3 zeta domain. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 145. In yet another embodiment, this chimeric receptor is encoded by the amino acid sequence of SEQ ID NO: 174. In some embodiments, the sequence of the chimeric receptor can be different from SEQ ID NO: 145, but depending on the embodiment, retains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology with SEQ ID NO: 145. In some embodiments, the chimeric receptor can be different from SEQ ID NO: 145, but the chimeric receptor retains or, in some embodiments, increases NK cell activation and / or cytotoxic function. Further, in some embodiments, this construct may be one that can be co-expressed with mbIL15 (see FIG. 7, NKG2D ACRb). Further information regarding chimeric receptors for use in the methods and compositions of the present disclosure can be found in PCT Patent Publication No. 2018 / 183385, which is hereby incorporated by reference in its entirety.

[0258] In some embodiments, a polynucleotide encoding an anti-CD70 / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see FIG. 7, CD70 CARa). The polynucleotide comprises or consists of an anti-CD70 binding portion, a CD8α 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 the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are approved as being possible when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites). In some embodiments, a CD70 CAR construct further comprising mbIL15 disclosed herein is provided (see, e.g., FIG. 7, CD70 CARb).

[0259] In some embodiments, a polynucleotide encoding an anti - mesothelin / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see Figure 7, mesothelin CARa). The polynucleotide comprises or consists of an anti - mesothelin binding portion, a CD8α hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence variations, extensions, and / or truncations of the disclosed sequences are approved as being those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites). In some embodiments, a mesothelin CAR construct further comprising mbIL15 disclosed herein is provided (see, for example, Figure 7, mesothelin CARb).

[0260] In some embodiments, polynucleotides encoding an anti-PD-L1 / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta chimeric antigen receptor complex are provided (see Figure 7, PD-L1 CARa). The polynucleotide comprises or consists of an anti-PD-L1 binding portion, a CD8α 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 the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. Certain sequence changes, extensions, and / or truncations of the disclosed sequences are approved as being those that can occur when combining sequences, for example, as a result of the ease or efficiency of cloning (e.g., for creating restriction sites). In some embodiments, a PD-L1 CAR construct further comprising mbIL15 disclosed herein is provided (see, for example, Figure 7, PD-L1 CARb).

[0261] In some embodiments, a polynucleotide encoding an anti-EGFR / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta chimeric antigen receptor complex is provided (see FIG. 7, EGFR CARa). The polynucleotide comprises or consists of an anti-EGFR binding portion, a CD8α hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain, as described herein. In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more of the sequence numbers included herein as examples of components. In some embodiments, the encoding nucleic acid sequence or amino acid sequence 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 a combination of one or more of the sequence numbers described herein. It should be understood 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 creating restriction sites). In some embodiments, an EGFR CAR construct further comprising mbIL15 disclosed herein is provided (see, e.g., FIG. 7, EGFR CARb).

[0262] In some embodiments, an expression vector such as the MSCV-IRES-GFP plasmid, a non-limiting example of which is provided in SEQ ID NO: 87, is used to express any of the chimeric antigen receptors provided herein.

[0263] Treatment methods Some embodiments relate to methods of treating, alleviating, inhibiting, or preventing cancer using cells or immune cells comprising chimeric antigen receptors and / or activating chimeric receptors, as disclosed herein. In some embodiments, the method comprises treating or preventing cancer. In some embodiments, the method comprises administering a therapeutically effective amount of immune cells that express a tumor-directed chimeric antigen receptor and / or a tumor-directed chimeric receptor, as described herein. Examples of types of cancer that can be treated in this manner are described herein.

[0264] In certain embodiments, treatment of a subject using the genetically engineered cell(s) described herein achieves one, two, three, four, or more of the following effects, e.g., (i) a decrease or alleviation in the severity of the disease or symptoms associated therewith; (ii) a decrease in the duration of symptoms associated with the disease; (iii) protection against progression of the disease or symptoms associated therewith; (iv) regression of the disease or symptoms associated therewith; (v) protection against the development or onset of symptoms associated with the disease; (vi) protection against recurrence of symptoms associated with the disease; (vii) a decrease in hospitalization of the subject; (viii) a decrease in length of hospit...

Claims

1. 1. A genetically engineered population of natural killer (NK) cells for cancer immunotherapy, comprising a plurality of NK cells, The plurality of NK cells are engineered to express a cytotoxicity receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxicity signaling complex; the cytotoxic signaling complex comprises an OX-40 subdomain and a CD3 zeta subdomain; NK cells are engineered to express membrane-bound IL-15; The NK cells are gene-edited to express reduced levels of a cytokine-inducible SH2-containing (CIS) protein encoded by the CISH gene compared to non-manipulated NK cells; Reduced CIS expression is engineered through editing of the CISH gene, and The genetically engineered NK cell population, wherein 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.

2. 2. The genetically engineered NK cell population of claim 1, wherein the extracellular ligand binding domain comprises a receptor directed against a tumor marker selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

3. 2. The genetically engineered NK cell population of claim 1, wherein the cytotoxicity receptor expressed by the NK cells comprises (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 costimulatory subdomain and a CD3z costimulatory subdomain.

4. 2. The genetically engineered NK cell population of claim 1, wherein the cytotoxic receptor is encoded by a polynucleotide having at least 95% sequence identity to SEQ ID NO:

145.

5. 2. The genetically engineered NK cell population of claim 1, wherein the cytotoxic receptor has at least 95% sequence identity to SEQ ID NO:

174.

6. 2. The genetically engineered NK cell population of claim 1, wherein the cytotoxic receptor expressed by the NK cells comprises a chimeric antigen receptor (CAR) comprising: (i) a tumor-binding domain comprising an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 costimulatory subdomain and a CD3z costimulatory subdomain.

7. 7. The genetically engineered NK cell population of claim 6, wherein the anti-CD19 antibody comprises a single chain variable fragment (scFv) variable heavy (VH) domain and a scFv variable light (VL) domain, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 120 and the encoded VL domain comprises the amino acid sequence of SEQ ID NO:

118.

8. 8. The genetically engineered NK cell population of claim 7, wherein the CAR expressed by the T cells has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:

178.

9. The genetically engineered NK cell of any one of claims 1 to 8, wherein expression of CIS is substantially reduced compared to non-engineered NK cells.

10. The genetically engineered NK cell of any one of claims 1 to 8, wherein the NK cell does not express detectable levels of CIS protein.

11. 9. The genetically engineered NK cell of any one of claims 1 to 8, wherein the NK cell is further genetically engineered to express reduced levels of transforming growth factor beta receptor (TGFBR) compared to a non-engineered NK cell.

12. 9. The genetically engineered NK cell of any one of claims 1 to 8, wherein the NK cell has been further genetically edited to express reduced levels of beta-2 microgobulin (B2M) compared to a non-engineered NK cell.

13. 9. The genetically engineered NK cell of any one of claims 1 to 8, wherein the NK cell has been further genetically edited to express reduced levels of CIITA (class II major histocompatibility complex transactivator) compared to a non-engineered NK cell.

14. 9. The genetically engineered NK cell of any one of claims 1 to 8, wherein the NK cell has been further genetically edited to express reduced levels of the Natural Killer Group 2, member A (NKG2A) receptor compared to a non-engineered NK cell.

15. 9. The genetically engineered NK cell of any one of claims 1 to 8, wherein the NK cell has been further genetically edited to express reduced levels of Cbl proto-oncogene B protein, encoded by the CBLB gene, compared to a non-engineered NK cell.

16. 9. The genetically engineered NK cell of any one of claims 1 to 8, wherein the NK cell has been further genetically edited to express reduced levels of triplicate motif-containing protein 29 protein, encoded by the TRIM29 gene, compared to a non-engineered NK cell.

17. 9. The genetically engineered NK cell of any one of claims 1 to 8, wherein the NK cell has been further genetically edited to express reduced levels of suppressor of cytokine signaling 2 protein, encoded by the SOCS2 gene, compared to a non-engineered NK cell.

18. The genetically engineered NK cell of any one of claims 1 to 8, wherein the NK cell is further genetically edited to express CD47.

19. The genetically engineered NK cell of claims 1 to 8, wherein the NK cell is further genetically engineered to express HLA-E.

20. The genetically engineered NK cell of claims 1-8, wherein the NK cell has been further genetically edited to disrupt expression of at least one immune checkpoint protein by the NK cell.

21. 21. The genetically engineered NK cell of claim 20, wherein 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.

22. further comprising a genetically engineered T cell population, the T cell population is substantially non-alloreactive; The non-allo-reactive T cells comprise at least one gene-edited subunit of a T cell receptor (TCR), such that the non-allo-reactive T cells do not exhibit alloreactive effects against cells of the recipient subject; The T cell population is engineered to express a chimeric antigen receptor (CAR) directed against a tumor marker; 9. The genetically engineered NK cell population of any one of claims 1 to 8, wherein the tumor marker is selected from the group consisting of CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, EGFR, and combinations thereof.

23. 23. The genetically engineered NK cell population of claim 22, wherein the CAR expressed by the T cells has a tropism for CD19.

24. 23. The genetically engineered NK cell population of claim 22, wherein the CAR expressed by the T cells has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:

178.

25. 23. The genetically engineered NK cell population of claim 22, wherein the TCR subunit of the modified T cells is TCRα.

26. 23. The genetically engineered NK cell population of claim 22, wherein the modification to the TCR of the T cells results in at least 90% of the T cell population not expressing detectable levels of the TCR.

27. 23. The genetically engineered NK cell population of claim 22, wherein the T cells have been further gene edited to reduce expression of one or more of CIS, TGFBR, B2M, and CIITA, or to express CD47 or HLA-E, compared to non-engineered T cells.

28. 23. The genetically engineered NK cell population of claim 22, wherein the T cells have been further gene edited to reduce expression of one or more of TRIM29 and SOCS2 compared to non-engineered T cells.

29. 23. The genetically engineered NK cell population of claim 22, wherein the T cells are further genetically edited to disrupt expression of at least one immune checkpoint protein by the T cells, wherein the at least one immune checkpoint protein is selected from CTLA4, PD-1, and lymphocyte activation gene (LAG-3).

30. The genetically engineered NK cell population according to any one of claims 1 to 29, wherein gene editing for reducing expression or gene editing for inducing expression is performed using a CRISPR-Cas system.

31. 31. The genetically engineered NK cell population of claim 30, wherein the CRISPR-Cas system comprises a Cas selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, and combinations thereof.

32. 32. The genetically engineered NK cell population of claim 31 , wherein the Cas is Cas9.

33. 33. The genetically engineered NK cell population of claim 32, wherein 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.

34. 30. The genetically engineered NK cell population of any one of claims 1 to 29, wherein gene editing to reduce expression or gene editing to induce expression is performed using zinc finger nucleases (ZFNs).

35. 30. The genetically engineered NK cell population of any one of claims 1 to 29, wherein gene editing to reduce expression or gene editing to induce expression has been performed using a transcription activator-like effector nuclease (TALEN).

36. The genetically engineered NK cell population of any one of claims 1 to 35, wherein the OX40 subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO:

5.

37. 37. The genetically engineered NK cell population of any one of claims 1 to 36, wherein the CD3 zeta subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO:

7.

38. The genetically engineered NK cell population of any one of claims 1 to 37, wherein mbIL15 is encoded by a sequence having at least 95% sequence identity to SEQ ID NO:

11.

39. 40. A method of treating cancer in a subject comprising administering to the subject the genetically engineered NK cell population of any one of claims 1 to 38.

40. Use of the genetically engineered NK cell population according to any one of claims 1 to 39 in the treatment of cancer.

41. 41. Use of a mixed population of immune cells according to any one of claims 1 to 40 in the manufacture of a medicament for the treatment of cancer.

42. 1. A method of treating cancer in a subject, comprising administering to the subject: (i) a plurality of NK cells, The plurality of NK cells are engineered to express a cytotoxicity receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxicity signaling complex; the cytotoxic signaling complex comprises an OX-40 subdomain and a CD3 zeta subdomain; NK cells are engineered to express membrane-bound IL-15; The NK cells are gene-edited to express reduced levels of a cytokine-inducible SH2-containing (CIS) protein, encoded by the CISH gene, by the cells compared to non-manipulated NK cells; Reduced CIS expression is engineered via gene editing of the CISH gene, and a plurality of NK cells, wherein the genetically engineered NK cells exhibit one or more of an increased expansion capacity, an increased cytotoxicity against target cells, and an increased persistence compared to NK cells expressing native levels of CIS; and optionally (ii) a plurality of T cells, the plurality of T cells are substantially non-alloreactive; The non-alloreactive T cells contain at least one modification to a subunit of the T cell receptor (TCR), such that the non-alloreactive T cells do not exhibit alloreactive effects against cells of the recipient subject; The T cell population is engineered to express a chimeric antigen receptor (CAR) directed against a tumor marker; The tumor marker is selected from the group consisting of CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, EGFR, and combinations thereof. The method comprises administering a genetically engineered immune cell population comprising:

43. 43. The method of claim 42, wherein the cytotoxicity receptor expressed by the NK cell comprises a signaling complex comprising (i) an NKG2D ligand-binding domain, (ii) a CD8 transmembrane domain, and (iii) an OX40 costimulatory subdomain and a CD3z costimulatory subdomain.

44. 44. The method of claim 42 or 43, wherein the cytotoxic receptor is encoded by a polynucleotide having at least 95% sequence identity to SEQ ID NO:

145.

45. The method of any one of claims 42 to 44, wherein the cytotoxic receptor has at least 95% sequence identity to SEQ ID NO:

174.

46. 43. The method of claim 42, wherein the cytotoxicity receptor expressed by the NK cell is directed against CD19.

47. 47. The method of claim 46, wherein the cytotoxicity receptor expressed by the NK cell has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:

178.

48. 43. The method of claim 42, wherein the CAR expressed by the T cell has a tropism for CD19.

49. 49. The method of any one of claims 42-48, wherein the CAR expressed by the T cell comprises (i) a tumor-binding domain comprising an anti-CD19 antibody fragment, (ii) a CD8 transmembrane domain, and (iii) a signaling complex comprising an OX40 costimulatory subdomain and a CD3z costimulatory subdomain, and (iv) membrane-bound IL15.

50. 50. The method of claim 49, wherein the anti-CD19 antibody comprises a variable heavy (VH) domain of a single chain variable fragment (scFv) and a variable light (VL) domain of the scFv.

51. 51. The method of claim 50, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 120 and the VL domain comprises the amino acid sequence of SEQ ID NO:

118.

52. 52. The method of any one of claims 42-51, wherein the NK cells and / or T cells have been further gene edited to reduce expression of one or more of CIS, TGFBR, B2M, and CIITA, or to express CD47 or HLA-E, compared to non-manipulated T cells.

53. 52. The method of any one of claims 42-51, wherein the NK cells and / or T cells have been further gene-edited to reduce expression of one or more of TRIM29 and SOCS2 compared to non-manipulated NK or T cells.

54. 54. The method of any one of claims 42-53, wherein the NK cell and / or T cell is further genetically edited to disrupt expression of at least one immune checkpoint protein by the cell, wherein the at least one immune checkpoint protein is selected from CTLA4, PD-1, and lymphocyte activation gene (LAG-3), NKG2A receptor, KIR2DL-1, KIR2DL-2, KIR2DL-3, KIR2DS-1 and / or KIR2DA-2.

55. The method of any one of claims 42 to 54, wherein the OX40 subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO:

5.

56. The method of any one of claims 42 to 55, wherein the CD3 zeta subdomain is encoded by a sequence having at least 95% sequence identity to SEQ ID NO:

7.

57. The method of any one of claims 42 to 56, wherein mbIL15 is encoded by a sequence having at least 95% sequence identity with SEQ ID NO:

11.

58. The method according to any one of claims 42 to 57, wherein gene editing to reduce expression or gene editing to induce expression is performed using a CRISPR-Cas system.

59. 59. The method of claim 58, wherein the CRISPR-Cas system comprises a Cas selected from Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, Cas13a, Cas13b, Cas13c, and combinations thereof.

60. 60. The method of claim 59, wherein the Cas is Cas9.

61. 59. The method of claim 58, wherein 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.

62. The method according to any one of claims 42 to 56, wherein the gene editing to reduce expression or the gene editing to induce expression is carried out using zinc finger nucleases (ZFNs).

63. The method of any one of claims 42 to 56, wherein the gene editing to decrease or induce expression is performed using a transcription activator-like effector nuclease (TALEN).

64. A mixed population of genetically engineered immune cells for cancer immunotherapy, comprising: (i) a plurality of NK cells, The plurality of NK cells are engineered to express a cytotoxicity receptor comprising an extracellular ligand binding domain, a transmembrane domain, and a cytotoxicity signaling complex; the cytotoxic signaling complex comprises an OX-40 subdomain and a CD3 zeta subdomain; NK cells are engineered to express membrane-bound IL-15; The NK cells are gene-edited to express reduced levels of a cytokine-inducible SH2-containing (CIS) protein, encoded by the CISH gene, by the cells compared to non-manipulated NK cells; Reduced CIS expression is engineered via gene editing of the CISH gene, and a plurality of NK cells, wherein the genetically engineered NK cells exhibit one or more of an increased expansion capacity, an increased cytotoxicity against target cells, and an increased persistence compared to NK cells expressing native levels of CIS; (ii) a plurality of T cells, the plurality of T cells are substantially non-alloreactive; The non-alloreactive T cells contain at least one modification to a subunit of the T cell receptor (TCR), such that the non-alloreactive T cells do not exhibit alloreactive effects against cells of the recipient subject; The T cell population is engineered to express a chimeric antigen receptor (CAR) directed against a tumor marker; The tumor marker is selected from the group consisting of CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, PD-L1, EGFR, and combinations thereof. A mixed population of genetically engineered immune cells, including:

65. 65. The mixed population of immune cells of claim 64, wherein the cytotoxicity receptor expressed by the NK cell has at least 95% sequence identity to SEQ ID NO:

174.

66. 65. The mixed population of immune cells of claim 64, wherein the cytotoxicity receptor expressed by the NK cell has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:

178.

67. The mixed population of immune cells of claim 64, wherein the T cell-mediated CAR has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 178.

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