Protecting cell therapies from TGF-beta induced immunosuppression

Abstract

Embodiments of the disclosure encompass at least compositions and methods for improving immune effector cell therapies, wherein the improvement allows the cells to be more effective in environments characterized by the presence of TGF-β. In some aspects, provided are effector immune cells, such as Natural Killer (NK) cells, that comprise one or more engineered mutations in an endogenous basic leucine zipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 3 (BATF3), and / or DNA damage inducible transcript 3 (DDIT3) gene of the cell. Also disclosed are methods of making engineered immune effector cells, and kits comprising said cells.

Description

PROTECTING CELL THERAPIES FROM TGF-BETA INDUCEDIMMUNOSUPPRESSIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U. S. Provisional Patent ApplicationSerial No. 63 / 608,041 filed December 8, 2023, and U.S. Provisional Patent Application Serial No. 63 / 693,336 filed September 11, 2024, the contents of each of which are hereby incorporated by reference in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on September 10, 2024, is named MDAC_P1381USP2_Sequence_Listing.xml and is 233,953 bytes in size.TECHNICAL FIELD

[0003] Embodiments of the disclosure include at least the fields of cell biology, molecular biology, immunology, and medicine, including cancer medicine.BACKGROUND

[0004] In the wake of the 2017 Food and Drug Administration (FDA) approvals of chimeric antigen receptor (CAR)-T-cell therapies for the treatment of patients with lymphoma and leukemia, adoptive cellular therapies have rapidly become a focal point for stakeholders across the field of cancer immunotherapy. This treatment modality had displayed unprecedented patient responses and offers a significant curative potential for certain hematological malignancies, however, success in other modalities is still lacking.

[0005] Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) belong to a continuous disease spectrum characterized by an overproduction of immature cells of the myeloid lineage h This overproduction leads to a disruption of effective hematopoiesis, resulting in debilitating cytopenias2. Despite advances in the understanding of the pathogenesis and molecular mechanisms of these disorders, and incremental improvements in treatment regimens, most patients with MDS and AML relapse and fail to achieve cure. These and other factors underscore the urgent need for new therapeutic alternatives that will improve the clinical outcomes of these patients.

[0006] Most of the therapies currently available for patients with myeloid malignancies include chemotherapy, targeted therapies, and epigenetic modulators such as hypomethylatingagents, histone deacetylase inhibitors, and more recently menin inhibitors3'5. Immunotherapy using checkpoint molecule inhibitors and / or adoptive cell therapy using autologous immune effector cells have unfortunately shown relatively disappointing activity in patients with MDS and AML6'8. This could be due to the low mutational burden in these malignancies, the immunosuppressive tumor microenvironment in the bone marrow niche, to intrinsic dysfunction in the immune effector cells of these patients, and / or to other unknown characteristics.BRIEF SUMMARY

[0007] The present disclosure provides solutions to long-felt needs in the art of immunotherapy. Provided herein are technologies for improved adoptive cellular therapies for treatment of diseases such as MDS and AML, and more broadly, other diseases that can be characterized as having and / or being susceptible to having a TGF-P rich microenvironment.

[0008] Embodiments of the disclosure include methods and compositions associated with cell therapy, including adoptive cell therapy. Particular embodiments of the disclosure encompass methods and compositions for cancer immunotherapy, anti-pathogen immunotherapy (e.g., including at least viruses, bacteria, fungi, and parasites), and / or immunotherapy for treatment of other conditions in which TGF-P plays a role in the pathogenesis (e.g., tissue disorders, connective tissue disorders, tissue fibrosis, chronic inflammatory conditions etc.). The disclosure encompasses immune effector cell therapies that have been improved for the explicit purpose of imparting one or more characteristics to the cells that improves their efficacy (e.g., improves their cytotoxicity and / or reduces their propensity to exhaustion, etc.). In specific embodiments, immune effector cells are modified to allow them to better kill target cells, such as cancer cells. In specific embodiments, immune effector cells are engineered to have reduced expression of one or more gene products that allow the engineered cells to be effective in an immunosuppressive environment, such as an environment (e.g., microenvironment) characterized by extracellular TGF-p.

[0009] As shown herein, NK cells from MDS / AML patients had irreversible dysfunction mediated by cell-cell contact dependent release of TGF-P by myeloid blasts. The inventors have discovered that this increased environmental TGF-P (e.g., mature TGF-P) led to NK cell epigenetic reprogramming induced by TGF-P / SMAD2 / 3 signaling and driven by the core transcription factors (TF) basic leucine zipper ATF-like transcription factor (BATF; aka SFA2, B-ATF, BATF1; identified as US NIH National Library of Medicine NCBI Gene ID: 10538), basic leucine zipper ATF-like transcription factor 3 (BATF3; aka JDP1, SNFT, JUNDM1;identified as US NIH National Library of Medicine NCBI Gene ID: 55509), and / or DNA damage inducible transcript 3 (DDIT3; aka CHOP, CEBPZ, CHOPIO, CHOP-10, GADD153, CZEBPzeta; identified as US NIH National Library of Medicine NCBI Gene ID: 1649). In some embodiments, provided herein are methods of treatment comprising immune effector cell therapies (e.g., adoptive NK cell therapy) combined with strategies (e.g., genetic engineering) aimed at preventing rather than reversing the dysfunction induced by TGF-P, such as prevention through mutation (e.g., null mutation, hypomorphic mutation, etc.) of BATF, BATF3, and / or DDIT3 to reduce or abolish gene product creation.

[0010] Myeloid blasts are inherently susceptible to NK cell killing but have been shown to evade immunosurveillance, the mechanisms by which they evade immunosurveillance remain poorly understood. As provided herein, the inventors have shown that NK cells from patients with myeloid malignancies display global dysfunction with impaired killing capacity, altered metabolism, and exhausted phenotypes at the single cell transcriptomic and proteomic levels. The data provided herein shows that this dysfunction was mediated through cell-cell contact dependent release of transforming growth factor beta (TGF-P) by myeloid blasts. The presence of TGF-P in the microenvironment induced NK dysfunction. NK dysfunction could be prevented by interrupting the TGF-P pathway (e.g., pharmacologically and / or genetically), but once the dysfunction was established, it appeared to be irreversible due to profound epigenetic reprogramming. As shown herein, among other things, the inventors have identified BATF, BATF3, and / or DDIT3 as core transcription factors that can mediate this disfunction. Furthermore, among other things, the inventors have identified BATF as a core transcription factor and a key mediator of this NK cell dysfunction downstream of SMAD2 / 3 canonical TGF-P signaling. In some embodiments, engineering of NK cells to have BATF deletion enhanced NK cell function against AML both in vitro and in vivo, and protected the NK cells from the epigenetic rewiring and immunosuppressive action of TGF-p. Collectively, the findings presented herein describe a novel mechanism of NK immune evasion manifested by irreversible epigenetic rewiring and inactivation of NK cells by myeloid blasts, and provide compositions and methods for overcoming this immune evasion.

[0011] In particular embodiments, the endogenous BATF, BATF3, and / or DDIT3 gene has been modified by genetic manipulation of the genomic locus of BATF, BATF 3, and / or DDIT3. The immune effector cells having reduced or fully inhibited expression of BATF, BATF3, and / or DDIT3 gene products (e.g., transcripts, polypeptides, etc.) may or may not be modified in an additional manner by the hand of man, such as expressing one or more exogenously provided gene products. In specific embodiments, the gene product is a receptor, cytokine,chemokine, suicide gene, or combination thereof. In particular cases, the receptor is an antigen receptor, wherein the antigen may or may not be a cancer antigen, including an antigen on solid tumor cells. In specific cases, the antigen receptor is a chimeric antigen receptor (CAR) or a T- cell receptor.

[0012] In some embodiments, the present disclosure knocks out or knocks down the gene encoding BATF, BATF3, and / or DDIT3 from immune effector cells used in various cellular therapies to render them insensitive to the immunosuppressive effects of TGF-P, and hence increase their survival, proliferation, active phenotype, and / or immune function, including at least in microenvironments characterized by extracellular TGF-P (including mature / soluble and / or complexed TGF-P). Using the gene-editing CRISPR / Cas9 technology, as one example, the feasibility has been confirmed of inhibiting the immunosuppressive TGF-P signaling pathway by knocking-out the downstream BATF TF, utilizing Cas9 preloaded with chemically synthesized crFNA:tracrRNA duplex targeting the BATF gene. Data disclosed herein demonstrate that knocking-out BATF from NK cells leads to improvement in NK cell phenotypes, such as increased expression of activation markers, improved metabolic fitness, improved secretary functions, and / or improved cytotoxicity against target cells.

[0013] In some embodiments, the genetic engineering strategy targeting BATF, BATF 3, and / or DDIT3 could be combined with different forms of cellular therapies, including CAR-T cells, CAR-NK cells, T-cell receptor (TCR)-T cells, T-cell receptor (TCR)-NK cells, tumorinfiltrating lymphocytes (TILs), or a combination thereof, to potentiate them against various diseases.

[0014] In some embodiments, immune cells (e.g., immune effector cells) that are engineered may be of any kind, but in specific embodiments the immune effector cells are T cells, natural killer (NK) cells, NK T cells, macrophages, B cells, tumor-infiltrating lymphocytes, dendritic cells, mesenchymal stem cells (MSCs), a combination thereof, and so forth. In particular cases, the immune effector cells are NK cells. In particular cases, the immune effector cells are not T cells. In particular cases, the immune effector cells are cord blood-derived NK cells.

[0015] In some embodiments, any medical conditions may be treated by administration of a therapeutically effective amount of the engineered immune effector cells of the encompassed disclosure. In particular embodiments, the cells are utilized in compositions for treatment of cancer of any kind, including blood cancers and / or solid tumors.

[0016] In some embodiments, provided herein are engineered immune effector cells, wherein the cell comprises one or more engineered mutations in an endogenous basic leucinezipper ATF-like transcription factor (BATF), basic leucine zipper ATF-like transcription factor 3 (BATF3), and / or DNA damage inducible transcript 3 (DDIT3) gene of the cell. In some embodiments, one or more mutations comprise a partial or complete loss of function, and / or knock-out (KO) mutation. In some embodiments, one or more mutations reduces or inhibits transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the one or more mutated endogenous genes relative to a non-mutated locus encoding the same endogenous gene. In some embodiments, one or more mutations reduces or inhibits transcription of one or more mRNA isoforms through elimination of one or more transcription factor binding sites. In some embodiments, one or more mutations reduces or inhibits transcription of one or more mRNA isoforms through elimination of one or more enhancer regions. In some embodiments, one or more mutations is in a coding region of the one or more genes. In some embodiments, one or more mutations is in an intron of the one or more genes. In some embodiments, one or more mutations is in an enhancer and / or promoter region of the one or more genes. In some embodiments, one or more mutations is in a phosphorylated mothers against decapentaplegic homolog 2 (p-SMAD2) and / or phosphorylated mothers against decapentaplegic homolog 3 (p-SMAD3) binding site. In some embodiments, at least one of the one or more mutations result in substantially no polypeptide gene products from at least one of the one or more genes. In some embodiments, one or more mutations comprise a homozygous mutation in at least one of the BATF, BATF3, and / or DDIT3 genes. In some embodiments, one or more mutations comprise a heterozygous mutation in one or more genes.

[0017] In some embodiments, relative to a control non-engineered cell, a cell comprising one or more engineered mutations has decreased BATF regulon transcriptional activity, increased metabolic capacity, increased cytotoxicity, increased persistence, and / or increased secretory function of the engineered cell in a microenvironment and / or a tumor microenvironment (TME) characterized by presence of transforming growth factor beta (TGF- P). In some embodiments, following exposure to TGF-P, one or more mutations provides the engineered cell with improved cytotoxicity capacity relative to a non-engineered cell exposed to TGF-p. In some embodiments, following exposure to TGF-P, the one or more mutations provides the engineered cell with improved metabolic function relative to a non-engineered cell following exposure to TGF-p. In some embodiments, improved metabolic function comprises increased glycolytic capacity, increased oxidative phosphorylation capacity, and / or increased oxygen consumption rates. In some embodiments, following exposure to TGF-P, one or more mutations provides the engineered cell with reduced levels of exhaustion markers and / or exhaustion score relative to a non-engineered cell following exposure to TGF-p. In someembodiments, reduced exhaustion markers comprise LAG3, TIM3, TIGIT, KLRG1, KLRC1, and / or CISH. In some embodiments, following exposure to TGF-P, one or more mutations provides for reduced levels of p-SMAD2 and / or p-SMAD3 in the engineered cell relative to a non-engineered cell following exposure to TGF-p. In some embodiments, following exposure to TGF-P, one or more mutations provides for reduced levels of transcripts and / or polypeptides encoded by genes TBX21, HP1BP3, TIPI, BATF, ASCL2, HOMEZ, GATA3, ZNF444, CEBPD, LEF1, ZNF71, and / or ZNF319 relative to a non-engineered cell following exposure to TGF-p. In some embodiments, following exposure to TGF-P, one or more mutations provides for reduced levels of BATF regulon activity relative to a non-engineered cell following exposure to TGF-p. In some embodiments, following exposure to TGF-pthe one or more mutations provides for reduced levels of expression of one or more, five or more, or ten or more genes identified in Table 3 relative to a non-engineered cell following exposure to TGF-p.

[0018] In some embodiments, TGF-P comprises TGF-pi, TGF-P2, and / or TGF-P3. In some embodiments, TGF-P comprises active TGF-P, TGF-P complexed with TGF-pi-latency- associated peptide (LAP), and / or TGF-P complexed with latent transforming growth factor beta binding protein 1 (LTBP1). In some embodiments, TGF-P comprises, consists essentially of, or consists of active and / or soluble TGF-p. In some embodiments, TGF-P comprises active and / or soluble TGF-p.

[0019] In some embodiments, one or more mutations results in enhanced polyfunctionality of the engineered cell relative to a control non-engineered cell in response to stimulation by and / or immunosuppressive signaling from a tumor cell. In some embodiments, enhanced polyfunctionality is evidenced by an increase in cytokine release in response to stimulation by tumor cells. In some embodiments, an increase in cytokine release comprises an increase in interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), and / or the degranulation marker CD 107a, in response to stimulation by tumor cells. In some embodiments, an increase in cytokine release comprises an increase in granulocyte-macrophage colony-stimulating factor (GMCSF), soluble CD137 (sCD137), INF-g, Granzyme A, interleukin 13 (IL-13), Granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein- 1 alpha (MIP-la), macrophage inflammatory protein-1 beta (MIP-lb), TNF-a, and / or Perforin, in response to stimulation by tumor cells. In some embodiments, one or more mutations provides an enhanced activated and / or cytotoxic phenotype to the engineered cell relative to a control non-engineered cell. In some embodiments, one or more mutations provides an enhanced activated and / or cytotoxic phenotype for the engineered cell relative to a control non-engineered cell, whereinthe enhanced activated and / or cytotoxic phenotype is associated with one or more of GSEA identified pathways: Fc Epsilon signaling, Regulation of Actin Cytoskeleton, T cell Receptor Signaling Pathway, TNF- Alpha Signaling via NF-kB, MAPK Signaling Pathway, and / or Rho GTPase Cycle. In some embodiments, one or more mutations provides for enhanced upregulation of G2M, E2F, MYC, MT0RC1, oxidative phosphorylation, and / or TNFa signaling in the engineered cell relative to a control cell. In some embodiments, one or more mutations provides for an increase in open chromatin peaks associated with transcription factors ELF3, CTCFL, RUNX1, NKX2-2, and / or IRF8 in the engineered cell relative to a control cell. In some embodiments, one or more mutations provides for an increase in closed chromatin peaks associated with transcription factors BATF, FRA1, JUNB, ATF3, and / or FOS.

[0020] In some embodiments, at least one of the one or more mutations is in endogenous gene BATF. In some embodiments, a BATF mutation is a result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NOs: 10, 11, and / or 12. In some embodiments, a BATF mutation results in a decrease in BATF polypeptide levels of greater than 60%, greater than 80%, or greater than 90% relative to a non-engineered cell. In some embodiments, at least one of the one or more mutations is in endogenous gene BATF3. In some embodiments, a BATF3 mutation is a result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NO: 27. In some embodiments, a. BA TF 3 mutation results in a decrease in BATF3 polypeptide levels of greater than 60%, greater than 80%, or greater than 90% relative to a non-engineered cell. In some embodiments, at least one of the one or more mutations is in endogenous gene DDIT3. In some embodiments, a DDIT3 mutation is a result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NOs: 27 and / or 28. In some embodiments, Z.DDIT3 mutation results in a decrease in one or more DDIT3 polypeptide isoform levels of greater than 60%, greater than 80%, or greater than 90% relative to a non-engineered cell.

[0021] In some embodiments, an engineered immune cell (including engineered immune effector cells) disclosed herein comprises a T cell, natural killer (NK) cell, NK T cell, macrophage, B cell, invariant NKT cells, gamma delta T cells, MSCs, tumor-infiltrating lymphocyte, and / or dendritic cell. In some embodiments, an engineered immune cell is an NK cell. In some embodiments, an engineered immune cell is not a T cell. In some embodiments, an engineered immune cell is not an NK T cell. In some embodiments, an engineered immune cell is not a macrophage. In some embodiments, an engineered immune cell is not a B cell. In some embodiments, an engineered immune cell is not a MSC. In some embodiments, an engineered immune cell is not a tumor-infiltrating lymphocyte. In some embodiments, anengineered immune cell is not a dendritic cell. In some embodiments, an engineered immune cell is an NK cell derived from cord blood (CB), peripheral blood (PB), an NK cell line, bone marrow, a stem cell, or a mixture thereof. In some embodiments, a NK cell is derived from cord blood.

[0022] In some embodiments, an engineered immune cell comprises one or more transgenic engineered receptors. In some embodiments, one or more engineered transgenic receptors comprises an engineered antigen receptor that specifically targets an antigen. In some embodiments, one or more engineered transgenic antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR). In some embodiments, an engineered transgenic antigen receptor is a CAR. In some embodiments, an antigen is a cancer antigen. In some embodiments, an antigen is a solid tumor antigen. In some embodiments, an antigen is a blood cancer antigen. In some embodiments, an antigen is selected from the group consisting of 5T4, 8H9, avp6integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD47, CD70, CD 123, CD 138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, FBP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-l lRa, IL-13Ra2, Lambda, Lewis- Y, L1CAM, Kappa, KDR, MCSP, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TIM3, TIGIT, TROP2, TEMs, HMW-MAA, VEGFR2, and any combination thereof. In some embodiments, an antigen comprises CD47, CD70, CD 123, CD33, and / or TIM3. In some embodiments, one or more engineered receptors comprises a cytokine receptor, chemokine receptor, homing receptor, or a combination thereof. In some embodiments, an engineered immune cell is also engineered to transgenically express or overexpress one or more chemokines and / or one or more cytokines. In some embodiments, a cytokine is IL-15, IL-12, IL-21, IL-2, IL-18, IL-7, or a combination thereof. In some embodiments, a cytokine is IL-15 and / or IL-21. In some embodiments, a cell is engineered to comprise a transgenic suicide gene. In some embodiments, one or more mutations in an endogenous are the result of homologous recombination and / or non-homologous recombination. In some embodiments, one or more mutations is mediated by contact of the cell with an endonuclease. In some embodiments, an endonuclease is an RNA guided endonuclease. In some embodiments, an RNA guided endonuclease is CRISPR-Cas9. In some embodiments, an engineered immune cell comprises one or more additional mutations in one or more genes,wherein the one or more additional gene is selected from the group consisting of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, CREB1, NKG2A, SIGLEC-7, LAG3, TIM3, GISH, F0X01, TGFBR2, TIGIT, CD96, AD0RA2, NR3C1, PD1, PDL-1, PDL- 2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, GR, and CD7.

[0023] Also provided herein, in some embodiments, are methods of making engineered immune cells of the disclosure. Also provided herein, in some embodiments, are populations of engineered immune cells of the disclosure. Also provided herein, in some embodiments, are compositions comprising one or more engineered immune cells and a pharmaceutically acceptable excipient.

[0024] Also provided herein are methods of treating a disorder in a patient in need thereof, the methods comprising a step of administering a therapeutically effective amount of the cells, population of cells, and / or compositions disclosed herein to the individual. In some embodiments, a disorder is characterized by the presence of extracellular TGF-p. In some embodiments, a disorder is characterized by the presence of soluble extracellular TGF-p. In some embodiments, an extracellular TGF-P is one the dominant or is the dominant cytokine in a microenvironment created by the disorder. In some embodiments, an extracellular TGF-P is one of the most abundant, or is the most abundant cytokine in a microenvironment created by the disorder. In some embodiments, a disorder comprises cancer, a pathogenic infection, a connective tissue disorders, tissue fibrosis, and / or a chronic inflammatory condition. In some embodiments, a disorder comprises cancer. In some embodiments, engineered immune cells are autologous, allogeneic, or xenogeneic with respect to the individual. In some embodiments, engineered immune cells are allogeneic with respect to the individual. In some embodiments, a cancer comprises a solid tumor. In some embodiments, a cancer does not comprise a solid tumor. In some embodiments, a cancer is of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, and / or cervix. In some embodiments, a cancer comprises AML and / or MDS. In some embodiments, an individual is a mammal. In some embodiments, an individual is a human, dog, cat, horse, cow, sheep, pig, or rodent. In some embodiments, an individual is a human. In some embodiments, an individual is administered at least two doses of the engineered immune cells, population of cells, or compositions of the disclosure. In some embodiments, na individual is administered one or more additional cancer therapy. In some embodiments, an additional cancer therapy comprises surgery, radiation, chemotherapy, hormone therapy, immunotherapy, or a combination thereof. In some embodiments, methodsprovided herein further comprise a step of diagnosing a disease in the individual. In some embodiments, methods provided herein further comprise a step of diagnosing cancer in the individual.

[0025] Also provided herein, in some embodiments, are polynucleotides comprising a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to one or more of SEQ ID NOs: 10-12, 17, or 27-28. Also provided herein, in some embodiments, are polypeptides and / or polynucleotides encoding the same comprising a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to one or more of SEQ ID NOs: 1-103.

[0026] Also provided herein, in some embodiments, are methods of treating a disease or disorder in an individual in need thereof, wherein the methods comprise administering a cell or population of cells comprising one or more mutations in the BA IT'. BA TT'3. and / or DDIT3 genes, wherein the disorder is characterized by the production of TGF-P and / or presence of extracellular TGF-P in a microenvironment generated by the disorder.

[0027] Also provided herein, in some embodiments, are methods of shielding an immune cell from the immunosuppressive effects of TGF-P, the methods comprising creating one or more mutations in BA IT'. BA TT'3. and / or DDIT3 genes in the cell. In some embodiments, the immune cell is an NK cell.

[0028] Also provided herein, in some embodiments, are kits for comprising an engineered cell, population of engineered cells, composition, and / or means to perform a method described herein. Also provided herein, in some embodiments, are kits for creating an engineered cell, population of engineered cells, composition, and / or means to perform a method described herein. Also provided herein, in some embodiments, are kits for using an engineered cell, population of engineered cells, composition, and / or means to perform a method described herein.

[0029] Certain embodiments of the present disclosure are characterized through the following enumerated aspects.

[0030] Aspect 1 is an engineered Natural Killer (NK) cell, wherein the NK cell comprises one or more engineered mutations in an endogenous basic leucine zipper ATF-like transcription factor (BATF basic leucine zipper ATF-like transcription factor 3 (BATF3), and / or DNA damage inducible transcript 3 (DI)IT3) gene of the cell.

[0031] Aspect 2 is an engineered immune cell, wherein the immune cell comprises one or more engineered mutations in endogenous BATF and BA TT3. BATF and DDIT3, or BATF3 and DDIT3 genes of the cell.

[0032] Aspect 3 is an engineered immune cell, wherein the immune cell comprises one or more engineered mutations in endogenous BA IT'. BA TT'S. and DDIT3 genes of the cell.

[0033] Aspect 4 is the engineered cell of any one of aspects 1-3, wherein the one or more mutations comprise a partial or complete loss of function, and / or knock-out (KO) mutation.

[0034] Aspect 5 is the engineered cell of any one of aspects 1-4, wherein the one or more mutations reduces or inhibits transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the one or more mutated endogenous genes relative to a nonmutated locus encoding the same endogenous gene.

[0035] Aspect 6 is the engineered cell of any one of aspects 1-5, wherein the one or more mutations is in a coding region of the one or more genes.

[0036] Aspect 7 is the engineered cell of any one of aspects 1-6, wherein the one or more mutations is in an intron of the one or more genes.

[0037] Aspect 8 is the engineered cell of any one of aspects 1-7, wherein the one or more mutations is in an enhancer and / or promoter region of the one or more genes.

[0038] Aspect 9 is the engineered cell of any one of aspects 1-8, wherein the one or more mutations is in a phosphorylated mothers against decapentaplegic homolog 2 (p-SMAD2) and / or phosphorylated mothers against decapentaplegic homolog 3 (p-SMAD3) binding site.

[0039] Aspect 10 is the engineered cell of any one of aspects 1-9, wherein at least one of the one or more mutations result in substantially no polypeptide gene products from at least one of the one or more genes.

[0040] Aspect 11 is the engineered cell of any one of aspects 1-10, wherein the one or more mutations comprise a homozygous mutation in at least one of the BATF, BATF3, and / or DDIT3 genes.

[0041] Aspect 12 is the engineered cell of any one of aspects 1-11, wherein the one or more mutations comprise a heterozygous mutation in one or more genes.

[0042] Aspect 13 is the engineered cell of any one of aspects 1-12, wherein relative to a control non-engineered cell, the one or more mutations result in decreased BATF regulon transcriptional activity, increased metabolic capacity, increased cytotoxicity, increased persistence, and / or increased secretory function of the engineered cell in a microenvironment and / or a tumor microenvironment (TME) characterized by presence of transforming growth factor beta (TGF-P).

[0043] Aspect 14 is the engineered cell of aspect 13, wherein following exposure to TGF- P, the one or more mutations provides the engineered cell with improved cytotoxicity capacity relative to a non-engineered cell exposed to TGF-p.

[0044] Aspect 15 is the engineered cell of aspect 13 or 14, wherein following exposure to TGF-P, the one or more mutations provides the engineered cell with improved metabolic function relative to a non-engineered cell following exposure to TGF-p.

[0045] Aspect 16 is the engineered cell of aspect 15, wherein the improved metabolic function comprises increased glycolytic capacity, increased oxidative phosphorylation capacity, and / or increased oxygen consumption rates.

[0046] Aspect 17 is the engineered cell of any one of aspects 13-16, wherein following exposure to TGF-P, the one or more mutations provides the engineered cell with reduced levels of exhaustion markers and / or exhaustion score relative to a non-engineered cell following exposure to TGF-p.

[0047] Aspect 18 is the engineered cell of aspect 17, wherein the reduced exhaustion markers comprise LAG3, TIM3, TIGIT, KLRG1, KLRC1, and / or CISH.

[0048] Aspect 19 is the engineered cell of any one of aspects 13-18, wherein following exposure to TGF-P, the one or more mutations provides for reduced levels of p-SMAD2 and / or p-SMAD3 in the engineered cell relative to a non-engineered cell following exposure to TGF- P-

[0049] Aspect 20 is the engineered cell of any one of aspects 13-19, wherein following exposure to TGF-P, the one or more mutations provides for reduced levels of transcripts and / or polypeptides encoded by genes TBX21, HP1BP3, TIPI, BATF, ASCL2, HOMEZ, GATA3, ZNF444, CEBPD, LEF1, ZNF71, and / or ZNF319 relative to a non-engineered cell following exposure to TGF-p.

[0050] Aspect 21 is the engineered cell of any one of aspects 13-20, wherein following exposure to TGF-P, the one or more mutations provides for reduced levels of BATF regulon activity relative to a non-engineered cell following exposure to TGF-p.

[0051] Aspect 22 is the engineered cell of any one of aspects 13-20, wherein following exposure to TGF-P, the one or more mutations provides for reduced levels of expression of one or more, five or more, or ten or more genes identified in Table 3 relative to a non-engineered cell following exposure to TGF-p.

[0052] Aspect 23 is the engineered cell of any one of aspects 1-22, wherein following exposure to TGF-P, the one or more mutations provides for reduced levels of expression of genes LA G3, HAVCR2, TIGIT, BATF, CTLA4, TGFBR2, ENTPD1, DNAJB1, and / or HSPA1 A relative to a non-engineered cell following exposure to TGF-p.

[0053] Aspect 24 is the engineered cell of any one of aspects 13-23, wherein the TGF-P comprises TGF-pi, TGF-P2, and / or TGF-P3.

[0054] Aspect 25 is the engineered cell of any one of aspects 13-24, wherein the TGF-P comprises active TGF-P, TGF-P complexed with TGF-pi-latency-associated peptide (LAP), and / or TGF-P complexed with latent transforming growth factor beta binding protein 1 (LTBP1).

[0055] Aspect 26 is the engineered cell of any one of aspects 13-25, wherein the TGF-P comprises, consists essentially of, or consists of active and / or soluble TGF-p.

[0056] Aspect 27 is the engineered cell of any one of aspects 1-26, wherein the one or more mutations results in enhanced poly functionality of the engineered cell relative to a control nonengineered cell in response to stimulation by and / or immunosuppressive signaling from a tumor cell.

[0057] Aspect 28 is the engineered cell of aspect 27, wherein the enhanced polyfunctionality is evidenced by an increase in cytokine release in response to stimulation by tumor cells.

[0058] Aspect 29 is the engineered cell of aspect 28, wherein the increase in cytokine release comprises an increase in interferon gamma (IFN-g), tumor necrosis factor alpha (TNF- a), and / or the degranulation marker CD 107a, in response to stimulation by tumor cells.

[0059] Aspect 30 is the engineered cell of aspect 27 or 28, wherein the increase in cytokine release comprises an increase in granulocyte-macrophage colony-stimulating factor (GMCSF), soluble CD137 (sCD137), INF-g, Granzyme A, interleukin 13 (IL-13), Granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein- 1 alpha (MIP-la), macrophage inflammatory protein-1 beta (MIP-lb), TNF-a, and / or Perforin, in response to stimulation by tumor cells.

[0060] Aspect 31 is the engineered cell of any one of aspects 1-30, wherein the one or more mutations provides an enhanced activated and / or cytotoxic phenotype to the engineered cell relative to a control non-engineered cell.

[0061] Aspect 32 is the engineered cell of any one of aspects 1-31, wherein the one or more mutations provides an enhanced activated and / or cytotoxic phenotype for the engineered cell relative to a control non-engineered cell, wherein the enhanced activated and / or cytotoxic phenotype is associated with one or more of GSEA identified pathways: Fc Epsilon signaling, Regulation of Actin Cytoskeleton, T cell Receptor Signaling Pathway, TNF-Alpha Signaling via NF-kB, MAPK Signaling Pathway, and / or Rho GTPase Cycle.

[0062] Aspect 33 is the engineered cell of any one of aspects 1-32, wherein the one or more mutations provides for enhanced upregulation of G2M, E2F, MYC, MTORC1, oxidative phosphorylation, and / or TNFa signaling in the engineered cell relative to a control cell.

[0063] Aspect 34 is the engineered cell of any one of aspects 1-33, wherein the one or more mutations provides for an increase in open chromatin peaks associated with transcription factors ELF3, CTCFL, RUNX1, NKX2-2, and / or IRF8 in the engineered cell relative to a control cell.

[0064] Aspect 35 is the engineered cell of any one of aspects 1-34, wherein the one or more mutations provides for an increase in closed chromatin peaks associated with transcription factors BATF, FRA1, JUNB, ATF3, and / or FOS.

[0065] Aspect 36 is the engineered cell of any one of aspects 1-35, wherein at least one of the one or more mutations is in endogenous gene BATF.

[0066] Aspect 37 is the engineered cell of aspect 36, wherein the BATF mutation is a result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NOs: 10, 11, and / or 12.

[0067] Aspect 38 is the engineered cell of aspect 36 or 37, wherein the BATF mutation results in a decrease in BATF polypeptide levels of greater than 60%, greater than 80%, or greater than 90% relative to a non-engineered cell.

[0068] Aspect 39 is the engineered cell of any one of aspects 1-38, wherein at least one of the one or more mutations is in endogenous gene BATF3.

[0069] Aspect 40 is the engineered cell of aspect 39, wherein the BATF3 mutation is a result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NO: 27.

[0070] Aspect 41 is the engineered cell of aspect 39 or 40, wherein the BATF3 mutation results in a decrease in BATF3 polypeptide levels of greater than 60%, greater than 80%, or greater than 90% relative to a non-engineered cell.

[0071] Aspect 42 is the engineered cell of any one of aspects 1-41, wherein at least one of the one or more mutations is in endogenous gene DDIT3.

[0072] Aspect 43 is the engineered cell of aspect 42, wherein the DDIT3 mutation is a result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NOs: 27 and / or 28.

[0073] Aspect 44 is the engineered cell of aspect 42 or 43, wherein the DDIT3 mutation results in a decrease in one or more DDIT3 polypeptide isoform levels of greater than 60%, greater than 80%, or greater than 90% relative to a non-engineered cell.

[0074] Aspect 45 is the engineered cell of any one of aspects 4-44, wherein the cell is a T cell, natural killer (NK) cell, NK T cell, macrophage, B cell, invariant NKT cells, gamma delta T cells, MSCs, tumor-infiltrating lymphocyte, or dendritic cell.

[0075] Aspect 46 is the engineered cell of any one of aspects 4-45, wherein the cell is an NK cell.

[0076] Aspect 47 is the engineered cell of any one of aspects 1-46, wherein the cell is an NK cell derived from cord blood (CB), peripheral blood (PB), an NK cell line, bone marrow, a stem cell, or a mixture thereof.

[0077] Aspect 48 is the engineered NK cell of any one of aspects 45-47, wherein the NK cell is derived from cord blood.

[0078] Aspect 49 is the engineered cell of any one of aspects 1-48, wherein the cell comprises one or more transgenic engineered receptors.

[0079] Aspect 50 is the engineered cell of aspect 49, wherein the one or more engineered transgenic receptors comprises an engineered antigen receptor that specifically targets an antigen.

[0080] Aspect 51 is the engineered cell of aspect 50, wherein the one or more engineered transgenic antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

[0081] Aspect 52 is the engineered cell of aspect 51, wherein the engineered transgenic antigen receptor is a CAR.

[0082] Aspect 53 is the engineered cell of aspect 51, wherein the engineered transgenic antigen receptor is a TCR.

[0083] Aspect 54 is the engineered cell of aspect 53, wherein the TCR is an invariant TCR (iTCR).

[0084] Aspect 55 is the engineered cell of aspect 53 or 54, wherein the TCR comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 95-98.

[0085] Aspect 56 is the engineered cell of any one of aspects 51-55, wherein the engineered cell transgenically expresses a CD3 complex.

[0086] Aspect 57 is the engineered cell of aspect 56, wherein the CD3 complex comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 99-100.

[0087] Aspect 58 is the engineered cell of any one of aspects 51-57, wherein the engineered cell transgenically expresses a Fc Receptor (FcR) extracellular binding domain.

[0088] Aspect 59 is the engineered cell of aspect 58, wherein the FcR comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 101-103.

[0089] Aspect 60 is the engineered cell of any one of aspects 1-59, wherein the engineered cell is complexed with one or more antibodies targeting antigens BCMA, CD20, CD 19, EGFR, CD30, HER2, GPRC5D, CD16, CD3, CD28, c-MET, PSMA, MUC17, CD33, FLT3, STEAP1, CLDN18.2, CD123, EpCAM, CEA, GPC3, CD38, CD33, CD22, GPA33, GD2, MUC16, DLL-3, CLEC12A, FcRH5, BlyS, and / or SSTR.

[0090] Aspect 61 is the engineered cell of aspect 60, wherein the one or more antibodies comprise Elranatamab, Glofitamab, Tafasitamab, Cetuximab, Imgatuzumab, Margetuximab, Amivantamab, Blinatumomab, Obinutuzumab, IPH61 (also known as IPH6101 or SAR443579), Teclistamab, Talquetamab, Pertuzumab, Trastuzumab, Brentuximab, Mosunetuzumab, Epcoritamab, GEN3017, Loncastuximab tesirine, Belimumab, and / or Rituximab.

[0091] Aspect 62 is the engineered cell of any one of aspects 50-59, wherein the antigen is a cancer antigen.

[0092] Aspect 63 is the engineered cell of any one of aspects 50-62, wherein the antigen is a solid tumor antigen.

[0093] Aspect 64 is the engineered cell of any one of aspects 50-63, wherein the antigen is a blood cancer antigen.

[0094] Aspect 65 is the engineered cell of any one of aspects 50-64, wherein the antigen is selected from the group consisting of CD70, 5T4, 8H9, avpe integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD47, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, FBP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-l lRa, IL-13Ra2, Lambda, Lewis- Y, L1CAM, Kappa, KDR, MCSP, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TIM3, TIGIT, TROP2, TEMs, HMW-MAA, VEGFR2, and any combination thereof.

[0095] Aspect 66 is the engineered cell of any one of aspects 50-65, wherein the antigen comprises CD70, CD47, CD19, CD20, BCMA, CD5, TROP2, CD123, CD33, PRAME, NY- ESO-1, EGFRvIII, IL-13Ra2, and / or TIM3.

[0096] Aspect 67 is the engineered cell of aspect 66, wherein the target antigen comprises CD70.

[0097] Aspect 68 is the engineered cell of aspect 67, wherein the engineered transgenic antigen receptor comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 29-37, or 58-64.

[0098] Aspect 69 is the engineered cell of aspect 66, wherein the target antigen comprises CD5.

[0099] Aspect 70 is the engineered cell of aspect 69, wherein the engineered transgenic antigen receptor comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 65-79.

[0100] Aspect 71 is the engineered cell of aspect 66, wherein the target antigen comprises TROP2.

[0101] Aspect 72 is the engineered cell of aspect 71, wherein the engineered transgenic antigen receptor comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 46-57.

[0102] Aspect 73 is the engineered cell of aspect 66, wherein the target antigen comprises PRAME.

[0103] Aspect 74 is the engineered cell of aspect 73, wherein the engineered transgenic antigen receptor comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 82-87 and / or 110-112.

[0104] Aspect 75 is the engineered cell of aspect 66, wherein the target antigen comprises NY-ESO-1.

[0105] Aspect 76 is the engineered cell of aspect 75, wherein the engineered transgenic antigen receptor comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 80-81 and / or 104-109.

[0106] Aspect 77 is the engineered cell of aspect 66, wherein the target antigen comprisesEGFRvIII and IL-13Ra2.

[0107] Aspect 78 is the engineered cell of any one of aspects 50-77, wherein the one or more engineered receptors comprises a cytokine receptor, chemokine receptor, homing receptor, or a combination thereof.

[0108] Aspect 79 is the engineered cell of any one of aspects 1-78, wherein the cell is engineered to transgenically express or overexpress one or more chemokines and / or one or more cytokines.

[0109] Aspect 80 is the engineered cell of aspect 79, wherein the cytokine is IL-15, IL-12, IL-21, IL-2, IL- 18, IL-7, or a combination thereof.

[0110] Aspect 81 is the engineered cell of aspect 80, wherein the cytokine is IL-15 and / or IL-21.

[0111] Aspect 82 is the engineered cell of any one of aspects 1-81, wherein the cell is engineered to comprise a transgenic suicide gene.

[0112] Aspect 83 is the engineered cell of any one of aspects 1-82, wherein the one or more mutations in an endogenous are the result of homologous recombination and / or non- homologous recombination.

[0113] Aspect 84 is the engineered cell of any one of aspects 1-83, wherein the one or more mutations is mediated by contact of the cell with an endonuclease.

[0114] Aspect 85 is the engineered cell of aspect 84, wherein the endonuclease is an RNA guided endonuclease.

[0115] Aspect 86 is the engineered cell of aspect 85, wherein the RNA guided endonuclease is CRISPR-Cas9.

[0116] Aspect 87 is the engineered cell of any one of aspects 1-85, wherein the cell comprises one or more additional mutations in one or more genes, wherein the one or more additional gene is selected from the group consisting of TGFBR2, GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, CREB1, NKG2A, SIGLEC-7, LAG3, TIM3, GISH, F0X01, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL J OR, CD 5, GR, and CD7.

[0117] Aspect 88 is a method of making the engineered cell according to any one of aspects 1-87.

[0118] Aspect 89 is a population of cells comprising the engineered cells of any one of aspects 1-87.

[0119] Aspect 90 is a composition comprising a pharmaceutically acceptable excipient and the population of engineered cells of aspect 89.

[0120] Aspect 91 is a method of treating a disorder in a patient in need thereof, the method comprising a step of administering a therapeutically effective amount of the engineered cell, population of engineered cells, or composition of aspects 1-90 to the individual.

[0121] Aspect 92 is a method of treating a disorder in a patient in need thereof, the method comprising a step of administering a therapeutically effective amount of the engineered cell of aspects 1-3 to the individual.

[0122] Aspect 93 is the method of aspect 91 or 92, wherein the disorder is characterized by the presence of extracellular TGF-P, optionally soluble extracellular TGF-p.

[0123] Aspect 94 is the method of aspect 93, wherein the extracellular TGF-P is one the dominant or is the dominant cytokine in a microenvironment created by the disorder.

[0124] Aspect 95 is the method of any one of aspects 93-94, wherein the extracellular TGF- P is one of the most abundant, or is the most abundant cytokine in a microenvironment created by the disorder.

[0125] Aspect 96 is the method of any one of aspects 91-95, wherein the disorder comprises cancer, a pathogenic infection, a connective tissue disorders, tissue fibrosis, and / or a chronic inflammatory condition.

[0126] Aspect 97 is the method of any one of aspects 91-96, wherein the disorder comprises cancer.

[0127] Aspect 98 is the method of any one of aspects 91-97, wherein the cells are autologous, allogeneic, or xenogeneic with respect to the individual.

[0128] Aspect 99 is the method of aspect 98, wherein the cells are allogeneic with respect to the individual.

[0129] Aspect 100 is the method of any one of aspects 96-99, wherein the cancer comprises a solid tumor.

[0130] Aspect 101 is the method of any one of aspects 96-100, wherein the cancer does not comprise a solid tumor.

[0131] Aspect 102 is the method of any one of aspects 96-101, wherein the cancer is of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, and / or cervix.

[0132] Aspect 103 is the method of any one of aspects 96-103, wherein the cancer comprises AML and / or MDS.

[0133] Aspect 104 is the method of any one of aspects 96-103, wherein the cancer comprises cells expressing one or more of antigens CD70, 5T4, 8H9, avpe integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6,CD44v7 / 8, CD47, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, FBP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3),HLA-A1+MAGE1, HLA-A1+NY-ES0-1, IL-l lRa, IL-13Ra2, Lambda, Lewis- Y, L1CAM,Kappa, KDR, MCSP, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TIM3, TIGIT, TROP2,TEMs, HMW-MAA, VEGFR2, or any combination thereof.

[0134] Aspect 105 is the method of any one of aspects 96-104, wherein the cancer comprises cells expressing one or more of antigens CD70, CD 19, CD20, BCMA, CD47, CD5,TROP2, CD 123, CD33, PRAME, NY-ESO-1, EGFRvIII, IL-13Ra2, and / or TIM3.

[0135] Aspect 106 is the method of any one of aspects 96-105, wherein the cancer comprises cells expressing CD70.

[0136] Aspect 107 is the method of any one of aspects 96-105, wherein the cancer comprises cells expressing CD5.

[0137] Aspect 108 is the method of any one of aspects 96-105, wherein the cancer comprises cells expressing TROP2.

[0138] Aspect 109 is the method of any one of aspects 96-105, wherein the cancer comprises cells expressing PRAME.

[0139] Aspect 110 is the method of any one of aspects 96-105, wherein the cancer comprises cells expressing NY-ESO-1.

[0140] Aspect 111 is the method of any one of aspects 96-105, wherein the cancer comprises cells expressing EGFRvIII and IL-13Ra2.

[0141] Aspect 112 is the method of any one of aspects 91-111, wherein the individual is a mammal.

[0142] Aspect 113 is the method of aspect 112, wherein the individual is a human, dog, cat, horse, cow, sheep, pig, or rodent.

[0143] Aspect 114 is the method of aspect 112, wherein the individual is a human.

[0144] Aspect 115 is the method of any one of aspects 91-114, wherein the individual is administered at least two doses of the cells.

[0145] Aspect 116 is the method of any one of aspects 91-115, wherein the individual is administered one or more additional cancer therapy.

[0146] Aspect 117 is the method of aspect 116, wherein the additional cancer therapy comprises surgery, radiation, chemotherapy, hormone therapy, immunotherapy, or a combination thereof.

[0147] Aspect 118 is the method of any one of aspects 96-117, further comprising the step of diagnosing cancer in the individual.

[0148] Aspect 119 is a polynucleotide comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 10-12, 17, or 27-28.

[0149] Aspect 120 is a method of treating a disease or disorder in an individual in need thereof, wherein the method comprises administering a cell or population of cells comprising one or more mutations in the BA IT'. BA TT' 3. and / or DDIT3 genes, wherein the disorder is characterized by the production of TGF-P and / or presence of extracellular TGF-P in a microenvironment generated by the disorder.

[0150] Aspect 121 is a method of shielding an immune cell from the immunosuppressive effects of TGF-P, the method comprising creating one or more mutations in BA TT'. BA TT' 3. and / or DDIT3 genes in the cell.

[0151] Aspect 122 is the method of aspect 121, wherein the immune cell is an NK cell.

[0152] Aspect 123 is a kit comprising the engineered cell, population of engineered cells, composition, and / or means to perform the methods of any one of aspects 1-122.

[0153] Aspect 124 is the use of the engineered cell of any one of aspects 1-87 in the manufacture of a medicament.

[0154] Aspect 125 is the engineered cell of any one of aspects 1-87 for use in the treatment of cancer.

[0155] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Brief Summary, Detailed Description, Claims, Abstract, and Brief Description of the Drawings.

[0156] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter which form thesubject of the claims herein. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present designs. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features which are believed to be characteristic of the designs disclosed herein, both as to the organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0157] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings.

[0158] FIGs. 1A-1K, NK cells derived from patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML) displayed reduced cytokine production, killing capacity, and altered metabolism. FIG. 1A, a schematic diagram describing the experimental plan of flow cytometry-based sorting of NK cells from healthy donors, MDS and AML patients followed by functional studies including degranulation and intracellular staining for cytokines and killing assays. FIG. IB, graphs showing percentage of NK cells expressing TNF-a from healthy controls (HC, n=6), patients with MDS (low-risk (n=6) or high-risk (n=5) by Revised International Prognostic Scoring System (IPSS-R)) and patients with AML (n=6). FIG. 1C, graphs showing percentage of NK cells expressing IFN-y from HC (n=6), patients with MDS (low-risk (n=l 1) or high-risk (n=5) by IPSS-R) and patients with AML (n=5). FIG. ID, graphs showing percentage of NK cells expressing CD 107a from healthy HC (n=7), patients with MDS (low-risk (n=l 1) or high-risk (n=5) by IPSS-R) and patients with AML (n=5). FIGs. 1E-1G, IncuCyte® assay results showing the cytotoxicity of NK cells from HC (n=6), MDS (n=4) and AML patients (n=4) against various cell lines, K562 (IE), THP-1 (IF) and M0LM14 (1G) at an effector to target (E:T) ratio of 1 :5. Graphs provided show percentage of tumor cell counts normalized to time 0 hrs. FIGs. 1H-1K provide metabolic insights into HC and AML NK cells. FIG. 1H, graphs representative extracellular acidification rate (ECAR) measurements from a glycolysis stress seahorse assay comparing HC and AML NK cells. FIG. II, a summary bar graph showing the glycolysis and glycolytic capacity measurementscomparing HC (n=4) and AML (n=4) NK cells; Each seahorse experiment was performed using 4 biological donors. FIG. 1J, graphs representative oxygen consumption rate (OCR) measurements from a mitochondrial stress seahorse assay comparing HC and AML NK cells. FIG. IK, a summary bar graph showing the basal and maximal OCR comparing HC (n=4) and AML (n=4) NK cells. Each seahorse experiment was performed using 4 biological donors. Each symbol denotes an individual donor, data were shown as mean ± SEM. P values were determined by two-tailed paired or unpaired Ltests in FIGs. 1B-1D, or two-way ANOVA test (Dunnet correction to compare the different groups to HC) in FIGs. 1E-1G, or two-tailed paired t-test in FIGs. II, and IK. * denotes P<0.05, **P<0.01, *** P<0.001.

[0159] FIGs. 2A-2H, NK cells from patients with MDS and AML exhibited altered phenotype and increased exhaustion signature at the single cell transcriptomic and proteomic levels. FIG. 2A, SPADE analysis of CyTOF data revealed the phenotypes of NK cells, in healthy controls (HC, n=4), MDS patients (n=7), and AML patients (n=8), from samples that were available for the analysis. Samples were pooled and separated into the three categories: HC vs. MDS vs. AML. Clustering by SPADE analysis revealed 6 main clusters (Cluster 1-6). Frequencies of each cluster were indicated; size and color of nodes within each cluster represented numbers of clustered cells. FIG. 2B, a heatmap representing the expression levels of NK cell markers within the main sub-clusters of Clusters 1-6. Each column represented a major node in the clusters of the spade tree. The major nodes were those that are representative of the majority of cells from all corresponding conditions. The expression level for each marker was represented on a scale ranging from low to high. FIG. 2C, a dimension reduction plot using NK cells profiled by scRNA-seq from both HCs and AML patients. FIG. 2D, a heatmap showing significantly differentially expressed genes (DEGs) in NK cells between HC and AML NK cells. Each row was a gene, while each column was an NK cell. Biologically relevant DEGs were labeled on the right side of the heatmap. Gradient colors ranging from green to pink indicated gene expression (Z-scored) ranges from low to high. FIG. 2E, violin plots showing some DEGs related to NK inhibition / exhaustion in HC and AML NK cells. Significance was indicated as FDRs underneath each plot. FIG. 2F, violin plots showing module score for exhaustion markers from HC and AML NK cells in two independent scRNA- seq datasets (Abbas 2021, Van Galen 2019). Significance was calculated using the Wilcoxon test and is labeled underneath each plot. FIG. 2G, violin plots showing differentially expressed TGF-P pathway related genes in AML NK cells compared to healthy NK cells (Abbas dataset). FIG. 2H, a bar graph depicting the differentially enriched upregulated and downregulated pathways in AML NK cells when compared to HC NK cells (Abbas dataset).

[0160] FIGs. 3A-3H, Evidence of cross-talk between NK cells and myeloid blasts leading to cell-cell contact mediated dysfunction. FIG. 3A, a schematic diagram showing conditions for NK cells cultured alone or co-cultured with AML blasts for 72 hrs then purified and used for cytotoxicity assays. FIG. 3B depicts IncuCyte® cytotoxicity assay results showing the mCherry+ M0LM14 cell count over time after culture with healthy NK cells (NK alone) vs healthy NK cells previously co-cultured with AML blasts for 72 hrs (NK:AML). Data shown as mCherry+ object count normalized to time 0 hr. n=3 biological replicates. FIG. 3C is a schematic diagram showing how NK cells were co-cultured with AML blasts either in direct contact (“Direct”) or indirect contact (separated by a transwell; “Transwell”) for 72 hrs before being purified and used for functional assays. FIG. 3D, graphs showing the percent NK cells positive for secretion of TNF-a (middle), IFN-y (left), and CD107a (right) in response to K562 targets by NK cells previously cultured in direct or indirect (transwell) contact with AML blasts for 72 hrs when compared to NK cells cultured alone. n=6 biological replicates. FIG. 3E, results from IncuCyte® cytotoxicity assays showing the mCherry+ M0LM14 object counts over time normalized to time 0 hr (left panel) following multiple tumor rechallenges (black arrow indicate timing of tumor rechallenge, at 72 hrs and 168 hrs). Right panel is a bar graph displaying the normalized mCherry+ M0LM14 object counts at day 10 (240 hrs) normalized to time 0 hr. n=3 biological donors. FIG. 3F, results from IncuCyte® cytotoxicity assays showing the normalized mCherry+ THP-1 object counts over time normalized to time 0 hr (left panel) following multiple tumor rechallenges (black arrow indicate timing of tumor rechallenge, at 72 hrs and 168 hrs). Right panel is a bar graph displaying the normalized mCherry+ THP-1 object counts at day 10 (240 hrs) normalized to time 0 hr. n=3 biological donors. FIG. 3G, bar graphs showing the percent expression of exhaustion markers (TIM3, LAG3 and TIGIT) on the surface of NK cells cultured alone (n=10 donors), or in Direct (n=10 donors) or indirect (Transwell, n=7 donors) contact with AML for 72 hrs, as measured by flow cytometry. FIG. 3H, representative histograms showing the expression of LAG3, TIM3 and TIGIT on the surface NK cells cultured alone, or in Direct or indirect (Transwell) contact with AML blasts. Each symbol represented an individual data point from a biological replicate. Data were shown as mean ± SEM. P values were determined by two-tailed paired t-test in FIGs. 3D, 3G, 3E (right panel), and 3F (right panel) or two-way ANOVA test in FIGs. 3B, 3E (left panel), and 3F (left panel). * Denotes P<0.05, **P<0.01 and *** P<0.001.

[0161] FIGs. 4A-4K, Activated TGF-p / SMAD pathway in MDS / AML NK cells altered NK function, and targeting the TGF-|J pathway prevented this dysfunction. FIG. 4A, a bar graph showing mean fluorescent intensity (MFI) of phosphorylated SMAD2 / 3 (pSmad2 / 3)in NK cells from MDS (n=6) and AML (n=15) patients compared to healthy controls (n=10). NK cells treated with exogenous TGF-P 1 (10 ng / ml) were used as positive control (n=6). FIG. 4B, representative histograms showing TGF-P-LAP expression on CD34+ cells from AML patients, low IPSS-RMDS patients, and high IPSS-RMDS patients when compared to healthy control CD34+ cells. Fluorescence minus one (FMO) was used as negative control. FIG. 4C, a graph showing the percentage of TGF-P-LAP expression on CD34+ cells from AML patients (n=15), low IPSS-R MDS patients (n=5), and high IPSS-R MDS patients (n= 4) when compared to healthy control CD34+ cells (n=7). FIG. 4D, a bar graph showing the concentration of TGF-pi (pg / ml / million cells) in the supernatant of NK cells and AML blasts either cultured alone or co-cultured together in direct contact or indirectly through a transwell. n=5 biological replicates. FIG. 4E is a schematic diagram depicting the experimental plan for FIG. 4F, where HC NK cells were cultured alone or with AML cells (“NK:AML”) for 72 hrs in the presence or absence of TGF-P inhibitors, and then purified and used in IncuCyte® cytotoxicity assays. FIG. 4F, a graph of IncuCyte® assay results showing the percentage of cytotoxicity (mCherry + NIR / mCherry) of the various groups of NK cells against M0LM14 cells normalized to time 0 hr. Prior to initiating the IncuCyte® assay, NK cells were either cultured alone or co-cultured with AML (1 :2 ratio) cells for 72 hrs with or without the presence of TGF-P inhibitors (Galunisertib (“Gal”), luspatercept (“Luspa”), or Cilengitide (“Cileng”)), n=4 biological replicates. FIG. 4G is a graph of IncuCyte® rechallenge assay results showing the mCherry+ THP-1 object counts over time normalized to time 0 hr, comparing the antitumor activity of Cas9 control NK cells vs transforming growth factor beta receptor 2 (TGFBR2) knock-out (KO) NK cells. Prior to initiating the IncuCyte® assay, Cas9 control NK cells or TGFBR2 KO NK cells were either cultured alone or co-cultured with AML cells (1 :2 ratio) for 72 hrs. FIG. 4H is a schematic diagram depicting the experimental plan of the AML mouse model testing the in-vivo efficacy of adoptively transferred NK cells (Cas9 control or TGFBR2 KO) or NK cells in combination with pharmacologic inhibitors of TGF-P (Galunisertib (“+Gal”), luspatercept (“+Luspa”), or Cilengitide (“+Cileng”)). NSG mice were injected with IxlO5THP-1 tumor cells intravenously and either left untreated or treated with TGF-P inhibitors alone, or with NK cells (CTRL or TGFBR2 KO) or with NK cells in combination with TGF-P inhibitors. FIG. 41 provides bioluminescence imaging (BLI) photos of mice in the various groups described in FIG. 4H at day 1, day 23, and day 30 following THP-1 cell injection. FIG. 4J is a bar graph showing the BLI total flux [p / s] at day 30 comparing tumor burden in the various groups of mice shown in FIG. 41. FIG. 4K are bar graphs showing the percentage of AML chimerism (GFP+ / human CD45+ THP-1 cells out oftotal live cells) in the bone marrow (BM; left panel), spleen (middle panel), and peripheral blood (PB; right panel) in the various groups of mice shown in FIG. 41. Each symbol represents an individual data point from a biological replicate. Data were shown as mean ± SEM. P values were determined by unpaired t-test in FIGs. 4A, 4C, 4K, paired t-test in FIG. 4D, or two-way ANOVA test in FIGs. 4F, 4G, and 4J. * Denotes P<0.05, **P<0.01 and *** P<0.001, ns is not significant.

[0162] FIGs. 5A-5J, Direct co-culture with AML induced epigenetic regulation of important NK dysfunction cellular programs. FIG. 5A are graphs of IncuCyte® live cell imaging assays showing the percentage of mCherry+ M0LM14 cells normalized to time 0 hr as a surrogate for NK cell killing. Prior to the IncuCyte® assay, NK cells were either cultured alone (control NK cells, blue lines) or co-cultured with AML for 72hrs (AML:NK, red lines). The top panel shows the results of the cytotoxicity assay at baseline (pre-rescue) and the bottom panel shows the results of the cytotoxicity assay following the 1 week-long activation and expansion (post-rescue). FIG. 5B shows graphs of IncuCyte® live cell imaging assays showing the percentage of mCherry+ M0LM14 cells normalized to time 0 hr, as a surrogate for NK cell killing. NK cells were either derived from healthy controls (HC NK cells) or from paired AML patient samples at diagnosis during active disease and following complete remission (CR). FIG. 5C shows an UpSet plot highlighting the number of enriched accessible chromatin regions which were either unique or shared among the three experimental conditions, 1) NK cells cultured alone (Control), 2) NK cells co-cultured with AML indirectly through a Transwell (Transwell), or 3) NK cells co-cultured with AML in direct contact (Direct). FIG. 5D is a heatmap showing differential enrichment of accessible chromatin regions in all three conditions (Control n=2, Transwell n=3, Direct n=3 biological replicates). FIG. 5E is a volcano plot showing the differential expression of genes associated with / near the open chromatin regions in direct vs transwell cultures. FIG. 5F are Integrative Genomics Viewer (IGV) plots of accessible chromatin regions from all three conditions showing gain of exclusive / differential enhancers in Direct co-culture of NK and AML cells in proximity of known inhibitory receptor genes HAVCR2 and TGFBR2. FIG. 5G show Venn diagrams of integration of differential accessible regions which were computationally linked to nearest genes from ATAC-seq and differential expression genes (DEGs) from RNA-seq in Transwell and Direct co-cultures. FIG. 5H shows transcription factor (TF) consensus motif enrichment analyses highlighting presence of distinct sets of TFs at gained accessible chromatin regions in Transwell / control (CTCFL, ISL1, ETS1, ZIC1, and RUNX1 motifs) and Direct (BATF, BATF3, DDIT3, ELF3, IRF4, and TBX4 motifs) conditions. FIG. 51 are Venn diagrams of TF circuitry derived by integration ofATAC-seq and RNA-seq informing distinct self-regulatory expressed set of TFs (in innermost red circle) driving major phenotypes in NK cells in Direct (BATF, BATF3, and DDIT3) and Transwell (CTCFL, ETS1, and RUNX1) co-culture conditions. FIG 5J are IGV plots showing gain of enhancers around BATF and BATF3 genes in Direct co-culture conditions in comparison to Transwell / Control. Data were shown as mean ± SEM. P values were determined by one way ANOVA test in FIGs. 5A and 5B. *** Denotes P<0.001.

[0163] FIGs. 6A-6L, BATF mediated gene regulatory programs were a main driver of myeloid blast induced NK epigenetic dysfunction. FIG. 6A is a Venn diagram showing gained accessible chromatin peaks with BATF motifs annotated to their nearest gene and overlapped with differentially upregulated genes. FIG. 6B shows results of a pathway enrichment analysis depicting enriched pathways for epigenetically accessible genes that were overexpressed at the RNA-seq level (termed as epigenetically upregulated). FIG. 6C are Western blots showing efficient overexpression (OE) of BATF in healthy peripheral blood NK cells from 2 representative donors; empty vector (EV) transformed healthy peripheral blood NK cells were used as negative controls. FIG. 6D are Western blots showing efficient knockout of BATF (created using sgRNAs SEQ ID NOs: 10, 11, and / or 12) in healthy peripheral blood NK cells from 2 representative donors; Cas9 electroporated NK cells were used as negative control. FIG. 6E is a graph of IncuCyte® assay results showing the GFP+ M0LM14 object count over time normalized to time 0 hrs, comparing the anti-tumor activity of empty vector (EV) control NK cells vs. BATF OE NK cells following multiple tumor rechallenges. FIG. 6F is a bar graph showing the percent of BATF OE NK cells expressing exhaustion markers compared to EV control NK cells. FIG. 6G is a graph of IncuCyte® assay results showing the GFP+ M0LM14 object count over time normalized to time 0 hrs, comparing the anti-tumor activity of Cas9 control NK cells vs. BATF KO NK cells following multiple tumor rechallenges. FIG. 6H is a bar graph showing the percent of BATF KO NK cells expressing exhaustion markers compared to Cas9 control NK cells. FIG 61 are BLI images of mice injected with THP-1 and either left untreated or treated with various NK cell preparations (EV control, BATF OE, Cas9 control, or BATF KO). FIG 6J is a bar graph showing the average radiance (p / s / cm2 / sr) at day 27 in the various mice groups shown in FIG. 6H. FIG 6K are bar graphs showing the percentage of AML chimerism (GFP+ / human CD45+ THP-1 cells out of total live cells) in the bone marrow (BM; left panel), spleen (middle panel), and peripheral blood (PB; right panel) in the various groups of mice shown in FIG. 6H. FIG. 6L are IGV plots from CUT&RUN analysis showing that following treatment with TGF-P (lOng / ml) (used to mimic NK:AML direct co-culture), BATF bound to key genes related to NK cell exhaustionand dysfunction and also bound to its own gene, showing self-transcriptional regulation. IgG was used as negative control. Highlighted grey regions represent differentially open chromatin regions identified by ATAC-seq in the NK:AML direct co-culture condition. Together these results proved that BATF was binding to these regions. Each symbol represented an individual data point from a biological replicate. Data were shown as mean ± SEM. P values were determined by two-way ANOVA in FIGs. 6E and 6G, or paired t-test in FIGs. 6F and 6H, or unpaired t-tests in FIGs. 6J and 6K. * Denotes P<0.05, **P<0.01 and *** P<0.001.

[0164] FIGs. 7A-7G, TGFBR2 KO NK cells maintained potency in direct co-culture with AML through enhancer reprogramming. FIG. 7A is a Western blot showing BATF protein expression in healthy NK cells without any treatment or following treatment with recombinant TGF-pi (10 ng / ml) alone or in addition to Luspatercept (SMAD2 / 3 inhibitor, 2 pg / ml). P-actin was used as a loading control. Left panel shows representative images from 2 donors, right panel shows a summary quantification graph of BATF expression in treated NK cells (TGF-pi alone or TGF-pi + Luspatercept) normalized to mean level of untreated control NK cells (n=4 donors). FIG. 7B is a bar graph from a ChlP-qPCR experiment showing the percent input of pSMAD2 enrichment normalized to IgG at enhancer and intronic region of BATF. NC = negative control. FIG. 7C is a Venn diagram showing shared and exclusive accessible chromatin regions between NKWTand NK / G / G'2KOcells co-cultured with AML cells in Direct contact. FIG. 7D are heatmaps showing open and closed chromatin regions in NKWTand NKTGB7?2KOcells co-cultured with AML cells in Direct contact. The average intensity plots (right side) show the differences in accessibility in open or and closed in NK' / / G'2KC)cells to NKWT. FIG. 7E is a PCA plot of gained accessible peaksand NKWTcells cultured alone or in Direct or Transwell co-culture with AML cells. FIG. 7F shows TF consensus motif analyses for gained and lost accessible peaks in NK' / / G'2KC)cells in Direct coculture, indicating distinct TF regulations (ELF3, CTCFL, RUNX1, NKX2-2, IRF8 open peaks; and BATF (NNTKASTMAN (SEQ ID NO: 88)), FRA1 (NNNTKANTMANN (SEQ ID NO: 89)), JUNB (NNTKASTMAB (SEQ ID NO: 90)), ATF3 (NVTGASTCABNN (SEQ ID NO: 91), and FOS (NNVTKABTMAHN (SEQ ID NO: 92)) closed peaks). FIG. 7G provides a pathway analyses reflecting enriched pathways in gained and lost accessible peaks in NK' / / y / '2KC)Cells in Direct co-culture compared to NKWTcells in Direct co-culture. Each symbol represented an individual data point from a biological replicate. Data were shown as mean ± SEM. P values were determined by paired t-test in FIGs. 7A and 7B, and multiple unpaired t-tests in FIG. 7C. * Denotes P<0.05, **P<0.01, ns = non-significant.

[0165] FIG. 8, AML and MDS NK cells showed distinct proteomic profiles when compared to healthy NK cells. Provided are bar graphs showing the percentage expression of activation and inhibitory markers (e.g., CD16, NKp46, NKp30, T-Bet, PFN, PD1, CD39, CD57, CD38, KIR2DL1, KIR2DL3, KIR2DL5, KIR3DL1, KIR2DS1, and KIR2DS4) on the surface of healthy NK (HC) cells (left columns), NK cells from MDS patients (MDS; middle columns), and NK cells from AML patients (AML; right columns). * Denotes P<0.05, **P<0.01 and *** P<0.001, ns not significant.

[0166] FIGs. 9A-9B, AML NK cells showed a distinct transcriptomic profile when compared to healthy NK cells. Defining the NK cell subpopulation in scRNA-seq dataset of AML bone marrow samples (Abbas 2021 dataset). FIG. 9A is a UMAP showing the NK cell subpopulation among the other immune cell populations in the Abbas 2021 scRNA-seq dataset. FIG. 9B are UMAPs with specific marker gene expression overlayed. Marker genes used to define NK cells included FCGR3A, KLRD1 and NKG7. Other markers of NK cells NCAM1 and KLRB1 were also positive in that population. Marker genes for T cells were negative in that subpopulation (CD3D. CI)4. CD8A, CDS').

[0167] FIG. 10, AML NK cells showed upregulation of cytotoxicity markers when compared to healthy NK cells. Violin plots comparing the expression of cytotoxicity markers in AML NK cells compared to HC NK cells from scRNA-seq data (Abbas dataset). Significance was indicated as FDRs underneath each plot.

[0168] FIGs. 11A-11B, Cell-cell communication identified important interactions between NK cells and myeloid blasts at the scRNA-seq level. FIG. HA is a heatmap showing interactions between prioritized ligands and predicted receptors. Each row represented a ligand (on the surface of malignant cells), while each column represented a receptor (on the surface of NK cells). Interaction potential was shown as gradient colors. FIG. 1 IB is a heatmap showing regulations between prioritized ligands and altered target genes. Each row represented a ligand (on the surface of malignant cells), while each column represented their regulated target gene (on the surface of NK cells). Regulatory potential was shown as gradient colors.

[0169] FIGs. 12A-12F, NK cells co-cultured with normal CD34+ cells did not show suppressed functionality. FIG. 12A are results from IncuCyte® cytotoxicity assays showing that the impairment in NK cell activity by myeloid blasts was time and dose dependent, being more profound when NK cells were pre-exposed to AML blasts for longer incubation time (72hrs) compared to shorter incubation time (24hrs) and with higher blast:NK ratio (2: 1, high blast) compared to (0.5: 1, low blast). FIG. 12B are IncuCyte® cytotoxicity assay results showing that the impairment in NK cell activity by myeloid blasts occurred similarly in bothcord blood (CB) derived and peripheral blood (PB) derived NK cells. FIG. 12C is a schematic diagram showing the experimental plan followed for testing the effect of co-culturing NK cells with normal CD34+ cells. FIG. 12D provides results of a chromium release assay showing the percentage of cell lysis of M0LM14 cells by NK cells previously cultured alone or with CD34+ cells for 72 hrs, no significant differences were observed. FIG. 12E is a bar graph showing the percentage of NK cells expressing TNFa, IFNy, or CD107a after being previously cultured alone or with CD34+ cells for 72 hrs; no significant differences were observed. FIG. 12F is graph showing percentage of CD 107a positive NK cells exposed to AML blasts in TIM3 / LAG3 / TIGIT (+) vs (-) populations. * Denotes P<0.05 and *** P<0.001.

[0170] FIGs. 13A-13D, Cilengitide but not MMP2 / 9 inhibitors affected TGF- i production by NK cells co-cultured directly with AML blasts. FIG. 13A is a bar graph showing the TGF-P 1 concentration in pg / ml / million cells measured by ELISA in the supernatants of AML cells, NK cells alone (NK alone), or the combination co-culture (NK:AML), with or without the addition of Cilengitide (NK:AML + Cileng) or MMP2 / 9 inhibitor (NK: AML + MMP2 / 9 inhibitor (Sigma-Aldrich CAS 193807-58-8)). The results showed significantly reduced TGF-P 1 in NK:AML + Cileng relative to NK:AML conditions. FIG. 13B is a graph of IncuCyte® data showing the normalized cell counts of mCherry+ THP- 1 cultured alone or in combination with various NK cell conditions. Prior to the IncuCyte® assay, NK cells were either cultured alone (control NK) or preincubated with AML in direct contact (NK:AML direct), or indirectly through a transwell (NK:AML transwell). Additional transwell experiments included NK cells and AML (in direct contact) in one chamber ([AML+NK] upper or lower transwell) and NK cells alone in the other chamber (NK upper or lower transwell). NK cells were then purified from the chamber where they were in contact with AML (NK from (AML+NK) transwell) or from the chamber where they were left alone and tested in the cytotoxicity assay against THP-1. FIG. 13C is a graph of IncuCyte® data showing the normalized cell counts of mCherry+ THP-1 cultured alone or in combination with various NK cell conditions. Prior to the IncuCyte® assay, NK cells were either cultured alone (control NK) or pre-incubated with GARP+Tregs or with TGF-P LAP+ blasts. FIG. 13D is a bar graph showing the percentage of TGF- P LAP+ AML blasts and GARP+ Tregs from paired AML peripheral blood samples.

[0171] FIGs. 14A-14B, TGF-P inhibitors lead to reduced phosphorylation of SMAD2 / 3 and Cilengitide decreased the release of TGF- following co-culture of NK cells with AML. FIG 14A are representative histograms showing the expression of p-SMAD2 / 3 in NK cells cultured alone or with AML with or without the addition of pharmacologic inhibitorsof TGF-P (e.g., luspatercept, Galunisertib, Cilengitide). FIG. 14B is a bar graph showing p- SMAD2 / 3 mean fluorescent intensity (MFI) in NK cells cultured alone or with AML with and without the addition of pharmacologic inhibitors of TGF- P (luspatercept, Galunisertib, Cilengitide).

[0172] FIGs. 15A-15B, TGF-P inhibitors did not cause direct cytotoxicity against AML and did not affect NK cell function. FIG. 15A is a graph of IncuCyte® data showing the cell counts of mCherry+ M0LM14 cultured alone or in the presence of TGF-P inhibitors (luspatercept, Cilengitide or Galunisertib). FIG. 15B is a graph of IncuCyte® data showing the percent cytotoxicity (percentage of (mCherry + NIR overlap) / mCherry normalized to time 0 hrs) of NK cells cultured against target M0LM14 cells, with or without the addition of TGF-P inhibitors (luspatercept, Cilengitide or Galunisertib).

[0173] FIGs. 16A-16B, TGF- inhibition enhanced NK cell cytokine production and degranulation against AML. FIG. 16A are graphs comparing the percentage of cytokine production (IFNy and TNFa; left and middle panels) and degranulation (CD 107a expression; right panel) of NK cells cultured alone or with AML blasts (NK:AML), with or without the addition of Galunisertib ((+Gal) LY). FIG. 16B are graphs comparing the percentage of cytokine production (IFNy and TNFa; left and middle panels) and degranulation (CD107a expression; right panel) of NK cells cultured alone or with AML blasts (NK:AML), with or without the addition of Cilengitide (+Cileng). * denotes P<0.05, **P<0.01, and ***P<0.001.

[0174] FIGs. 17A-17E, CRISPR mediated TGFBR2 KO was efficient and protected NK cells from the suppressive action of exogenous TGF-pi. FIG. 17A are results of PCR gel electrophoresis showing efficient KO of TGFBR2 in NK cells. FIG. 17B are representative histograms showing the phosphorylation of SMAD2 / 3 (p-SMAD2 / 3) in Cas9 control NK cells and TGFBR2 KO NK cells, with or without treatment with exogenous TGF-P (10 ng / ml). FIGs. 17C-17D are graphs of chromium release assay results showing the percentage of cytotoxicity of Cas9 control NK cells and TGFBR2 KO NK cells against M0LM13 (17C) and K562 (17D) targets, with or without treatment with exogenous TGF-P 1 (10 ng / ml). FIG. 17E is a graph of IncuCyte® data showing the normalized cell counts of mCherry+ THP-1 cultured alone or in combination with various NK cell conditions. Prior to the IncuCyte® assay, NK cells were either cultured alone (control NK) or pre-incubated with WT M0LM14 (NK:AML WT) or TGFB1 KO M0LM14 (NK:AML TGFB1 KO) or GSC272 cells or with control K562 cells for 72hrs then purified for functional assessment. Arrows indicate each tumor rechallenge. * denotes P<0.05 and *** <0.001, ns not significant.

[0175] FIGs. 18A-18D, TGF- inhibitors prevented the metabolic suppression of NK cells induced by AML. FIG. 18 shows a series of ECARs that were calculated for NK cells previously cultured alone or with AML blasts for 72 hrs, with or without the addition of TGF- P inhibitors, with the NK cells being subsequently purified and treated with 2 g / L d-glucose, 1 pM oligomycin, and 50 mM 2-Deoxyglucose (2-DG). A representative graph from 3 independent experiments was shown. FIG. 18B are summary box plots of basal ECAR and glycolytic capacity of the various NK cell conditions described in FIG. 18A (n=3). FIG 18C shows a series of OCRs that were calculated for NK cells previously cultured alone or with AML blasts for 72 hrs, with or without the addition of TGF-P inhibitors, with the NK cells being subsequently purified and treated with 0.5 pM Oligomycin, 0.5 pM FCCP, and 0.5 pM Rotenone / Antimycin (Rot / AA). FIG. 18D are summary box plots of basal and maximal OCR of the various NK cell conditions described in FIG. 18C (n=3). * denotes P<0.05, and **P<0.01, ns not significant.

[0176] FIGs. 19A-19B, TGF-P inhibition enhanced NK cell anti-leukemic activity in vivo. FIG. 19A is bioluminescent imaging (BLI) showing tumor growth in NSG mice over time in mice engrafted with M0LM14 alone or M0LM14 and then treated with 107Cas9 control NK cells or TGFBR2 KO NK cells. FIG 19B is a summary graph of average radiance over time in mice groups described in FIG. 19A.*** denotes P<0.001.

[0177] FIGs. 20A-20E, NK cells derived from AML patients exhibited irreversible dysfunction. FIG. 20A are results from an IncuCyte® cytotoxicity imaging assay showing NK cell activity against K562 mCherry+ cells, with NK cells obtained from healthy control donors (n=3) or AML patient (n=4) groups, prior to NK cell functional rescue. FIG. 20B are results from an IncuCyte® cytotoxicity imaging assay showing NK cell activity against OCI-AML3 GFP+ cells, with NK cells obtained from healthy control donors (n=3) or AML patient (n=4) groups, after NK cell functional rescue. FIG. 20C is a bar graph showing fold expansion of control NK cells and NK cells pre-exposed to AML in direct contact, following 1 week (Dl- D7) of rescue with feeder cells and IL-2 (200 u / ml). FIG. 20D is a graph of IncuCyte® data showing the normalized cell counts of mCherry+ M0LM14 cells cultured alone or in combination with control NK cells or NK cells cocultured AML in direct contact (NK:AML) with or without activation with IL-12 / IL-15 / IL-18. FIG. 20E is a graph of IncuCyte® data showing the normalized cell counts of mCherry+ primary AML blasts cultured in combination with healthy control (HC) NK cells or NK cells derived from paired samples of AML patients in active disease or following complete remission (CR). *** denotes P<0.001.

[0178] FIG. 21, NK cells in direct co-culture with AML did not show a large number of DEGs when compared to NK cells in Transwell co-culture with AML. FIG. 21 shows a bar graph showing the number of DEGs (upregulated or downregulated genes) in NK cells in direct co-culture with AML when compared to NK cells in Transwell co-culture with AML.

[0179] FIGs. 22A-22C, Direct co-culture induced activation and epigenetic poising of important NK cell dysfunction transcriptional programs. FIG. 22A provides IGV plots for regions near exhaustion related markers ENTPD1 and CTLAF showing enhancer gains in NK Cells when Direct co-cultured and compared to normal or Transwell culture conditions. FIG. 22B epigenetically upregulated cellular pathways driven by gained accessible chromatins directly regulated the transcriptome in NK cells in Direct and Transwell co-culture conditions. FIG. 22C showed normalized log2 TPM BATF, BATF3 and DDIT3 mRNA transcript expression levels in transwell and direct culture NK cells.

[0180] FIGs. 23A-23E, AML derived NK cells exhibited higher BATF expression and regulon activity, and BATF expression negatively impacted survival in patients. FIGs. 23A and 23B are Violin and tSNE plots, respectively, of BATF scRNA expression analysis distribution between control and AML(TME) patient’s derived NK cells. FIG. 23C provides a regulon analysis of Healthy control and AML patients derived NK cells exhibiting differential genes activity (e.g., relative upregulation in AML NK cells of TBX21, HP18BP3, TPI1, BATF, ASCL2, HOMEZ, GATA3, ZNF444, CEBPD, LEF1, ZNZF71, and ZNF319). FIG. 23D are violin plots representing BATF regulon activity score comparison between control and AML derived NK cells. FIG. 23E are log rank test overall survival (OS) and event free survival (EFS) probability comparisons in AML patients based on BATF high and low expression from the published GSE1159 (n=293 patients), GSE12417(n=242), GSE37642 (n=561) and GSE6891 (n=536) datasets. HR-Hazard ratio.

[0181] FIGs. 24A-24G, SMAD2 / 3 epigenetically regulated BATF. FIG. 24 is a bar graph showing BATF mRNA expression in healthy NK cells by qPCR with or without recombinant TGF-pi treatment (10 ng / ml) at different time points (2 hrs, 8 hrs and 18 hrs) normalized to control at 2 hrs. FIG. 24B are IGV plots from ChlP-seq data (GSE216432) in MCF10A cell line showing gain of SMAD2 and SMAD3 binding in an intronic region and putative enhancer around BATF gene following treatment with TGF-p. FIG. 24C is a bar graph from ChlP-qPCR experiment showing the percent input of pSMAD3 enrichment normalized to IgG at the two locations on BATF gene. FIG. 24D are results from a representative IncuCyte® rechallenge assay testing BATF KO and Cas9 NK cells with or without the addition of exogenous TGF- P 1 (lOng / ml). FIG. 24D are BLI images of mice from OCI-AML3 murinemodel showing the BLI signal from various groups: untreated mice (tumor alone), mice treated with Cas9 CTRL NK cells, TGFBR2 KO NK cells or BATF KO NK cells. FIG. 24F is a bar graph showing the BLI mean radiance (p / sec / cm2 / sr) at day 30 comparing tumor burden in the various groups of mice shown in 24E. FIG. 24G are bar graphs showing the percentage of AML chimerism (GFP+ / human CD45+ AML cells out of total live cells) in the bone marrow (BM), spleen, and peripheral blood (PB) in the various groups of mice shown in FIG 24E. Each symbol represents an individual data point from a biological replicate. * Denotes P<0.05, **P<0.01 and *** P<0.001, ns not significant.

[0182] FIG. 25, Schematic overview of proposed TGF-P induced NK cell epigenetic reprogramming through the transcription factors (TF) BATF, BATF3, and / or DDIT3.

[0183] FIG. 26, BATF bound to key genes related to exhaustion. Provided are IGV plots from CUT&RUN analysis showing that following treatment with TGF-pi (10 ng / ml), BATF bound to key genes related to NK cell exhaustion and dysfunction (e.g., CTLA4, TGFBR2, ENTPD1, DNA.J L and HSPA 1A). IgG was used as negative control. Highlighted grey regions represent differentially open chromatin regions identified by ATAC-seq in the NK:AML direct co-culture condition. Together, these results proved that BATF was binding to these regions.

[0184] FIGs. 27A-27B, BATF KO enhanced the cytotoxic activity of CD70 targeting CAR-NK cells against AML. Provided are results from Incucyte live cell imaging assays showing the cytotoxicity of CD70 targeting CAR-NK cells against THP-1 (FIG. 27A) and OCI-AML3 (FIG. 27B) at an effector to target ratio of 1 :4. Graphs showing the percentage of tumor cell counts normalized to time 0 hrs. In FIG. 27A, groups are labeled as follows: THP- 1 is labeled “i”, Cas9 is labeled “ii”, TGFBR2 KO is labeled “iii”, and BATF KO is labeled “iv”. In FIG. 27B, groups are labeled as follows: OCI3 is labeled “i”, Cas9 is labeled “ii”, TGFBR2 KO is labeled “iii”, and BATF KO is labeled “iv”.FIG. 28, BATF KO CD70 targeting CAR-NK cells enhanced tumor control in a PDX mouse model of AML. Provided are bar graphs showing the percentage of AML cells (top panels) or percentage of NK cells (lower panels) out of the total live cell population from the peripheral blood, bone marrow, or spleen collected from sacrificed mice engrafted with CD70+AML PDX cell line and either left untreated (tumor only), treated with Cas9 control, or treated with BATF KO CD70-targeting CAR-NK cells.

[0185] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilledin the art without departing from the invention. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.DETAILED DESCRIPTIONI. Examples of Definitions

[0186] In keeping with long-standing patent law convention, the words “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0187] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0188] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0189] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can referto “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0190] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0191] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure.

[0192] The term “exogenous” as used herein refers to a polynucleotide (such as one encoding a gene product or part of a gene product) that is not present endogenously in a mammalian cell, such as an immune cell, or is synthetically generated outside of a mammalian cell, such as by recombinant technology.

[0193] As used herein, the term "expression" refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Accordingly, a "gene product" as used herein, refers to transcribed mRNA, pre-splicing transcribed RNA (for example, RNA which still comprises non-coding region), translated polypeptide (for example, those with or without signal peptide or other region not present in the mature protein), and protein. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from the same sample following administration of a compound.

[0194] The term "isolated" as used herein refers to molecules or biologicals or cellular materials being substantially free from other materials. In one aspect, the term "isolated" refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, such as that are present in the natural source. The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to polypeptides that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. The term "isolated" is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.

[0195] As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition, e.g., cancer. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also include reducing the intensity, effect, symptoms and / or burden of a disease or condition prior to onset or recurrence of the disease or condition.

[0196] The term “sample,” as used herein, generally refers to a biological sample. The sample may be taken from tissue or cells from an individual. In some examples, the sample may comprise, or be derived from, a tissue biopsy, blood (e.g., whole blood), blood plasma, extracellular fluid, dried blood spots, cultured cells, discarded tissue. The sample may have been isolated from the source prior to collection. Non-limiting examples include blood, cerebral spinal fluid, pleural fluid, amniotic fluid, lymph fluid, saliva, urine, stool, tears, sweat, or mucosal excretions, and other bodily fluids isolated from the primary source prior to collection. In some examples, the sample is isolated from its primary source (cells, tissue, bodily fluids such as blood, environmental samples, etc.) during sample preparation. The sample may or may not be purified or otherwise enriched from its primary source. In some cases the primary source is homogenized prior to further processing. The sample may be filtered or centrifuged to remove buffy coat, lipids, or particulate matter. The sample may also be purified or enriched for nucleic acids, or may be treated with RNases. The sample may contain tissues or cells that are intact, fragmented, or partially degraded.

[0197] The term “subject,” as used herein, generally refers to an individual having a biological sample that is undergoing processing or analysis and, in specific cases, has or is suspected of having cancer. The subject can be any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The subject canbe a patient, e.g., have or be suspected of having a disease (that may be referred to as a medical condition), such as benign or malignant neoplasias, or cancer. The subject may be undergoing or having undergone treatment. The subject may be asymptomatic. The subject may be healthy individuals but that are desirous of prevention of cancer. The term “individual” is used interchangeably with “subject”. The “subject” or "individual", as used herein, may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The individual may be receiving one or more medical compositions via the internet. The term “individual” may refer to a human or non-human animal of any age, and therefore includes both adult and juveniles (ie., children) and infants and includes in utero individuals. It is not intended that the term connote a need for medical treatment, therefore, an individual may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies.

[0198] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0199] Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) belong to a continuous disease spectrum characterized by an overproduction of immature cells of the myeloid lineage h This overproduction leads to a disruption of effective hematopoiesis, resulting in debilitating cytopenias2. Despite advances in the understanding of the pathogenesis and molecular mechanisms of these disorders, and incremental improvements in treatment regimens, most patients with MDS and AML relapse and fail to achieve cure. These and other factors underscore the urgent need for new therapeutic alternatives that will improve the clinical outcomes of these patients.

[0200] Most of the therapies currently available for patients with myeloid malignancies include chemotherapy, targeted therapies and epigenetic modulators such as hypomethylating agents, histone deacetylase inhibitors and more recently menin inhibitors3'5. Immunotherapy using checkpoint molecule inhibitors and adoptive cell therapy using autologous immune effector cells have shown disappointing activity in patients with MDS and AML6'8. This couldbe due to the low mutational burden in these malignancies, the immunosuppressive tumor microenvironment in the bone marrow niche, and / or to intrinsic dysfunction in the immune effector cells of these patients.

[0201] Natural killer (NK) cells are innate lymphocytes that play an important role in protecting against cancer. NK cells have several distinct advantages over T cells as candidates for immunotherapy9,1°. In fact, the first compelling evidence supporting the role of NK cell immunotherapy in the treatment of myeloid malignancies came from T cell depleted haploidentical stem cell transplants showing that killer immunoglobulin-like receptor (KIR) ligand mismatch resulted in better patient outcomes due to enhanced graft-versus-leukemia effectn. Moreover, myeloid blasts are inherently susceptible to NK cell-mediated killing as they express many of the ligands recognized by NK cell activating receptors12. However, malignant myeloid blasts are capable of adapting and developing defense mechanisms that allow them to evade NK cell-mediated cytotoxicity13. These mechanisms include the release of immunosuppressive factors into the microenvironment, disruption of cytokine and chemokine signaling, and receptor-ligand interactions between NK cells and tumor cells, among others14 7Moreover, other immunosuppressive cells in the AML tumor microenvironment (TME) can lead to NK cell dysfunction. An important study by Wang et al., has shown that CD4+ regulatory T cells (Tregs) that express glycoprotein-A repetitions predominant (GARP) are capable of activating transforming growth factor beta 1 (TGF-pi) from its latent form leading to decreased NK cell metabolism and anti-leukemic activity, contributing to early relapse following allogeneic stem cell transplantation.

[0202] A number of recent studies have shown that distinct epigenetic reprogramming can be associated with exhausted T cell states. Changes in expression of certain transcription factors (TFs) could lead to these altered epigenetic patterns. Basic leucin zipper ATF-like transcription factor (BATF) is an important TF that has been implicated in epigenetic rewiring of cytotoxic T cells and CAR-T cells. To date, the role of epigenetic reprogramming in driving NK cell exhaustion and impaired anti-tumor activity has not been well explored.

[0203] Herein, the inventors show that NK cells derived from patients with MDS and AML displayed a global dysfunction with impaired killing capacity, and altered metabolism associated with a distinct exhaustion signature at the single cell transcriptomic and proteomic levels. Computational reconstruction of cell-cell communication identified inhibitory interactions between AML blasts and NK cells in single cell RNA sequencing (scRNA-seq) from patients with AML. Mechanistically, the inventors have shown that myeloid blasts are highly susceptible to NK cell mediated killing, but the myeloid blasts eventually evade NK cellimmune surveillance in a manner requiring direct cell-cell contact, leading to the release of transforming growth factor beta (TGF-P) by AML blasts. This in turn results in NK cell dysfunction and functional exhaustion. As described herein, this dysfunction can be prevented by targeting the TGF-P pathway and inhibiting it prior to induction. However, once this dysfunction was established, it could not be reversed, and persisted even in an AML free environment.

[0204] The inventors describe herein how this apparently irreversible impairment is linked to epigenetic reprogramming driven by the core transcription factors BATF, BATF3, and / or DDIT3 in a TGF-P dependent manner. The data provided herein shows that following activation of the canonical TGF-P pathway, pSMAD2 / 3 binds to an enhancer of BATF, inducing its expression. BATF in turn orchestrates a gene regulatory program, binding to key genes related to exhaustion and leading to NK cell dysfunction. BATF was also validated as a key regulatory transcription factor in single cell RNA-seq data from patients with AML. Furthermore, BATF deletion protected NK cells from TGF-B immunosuppressive action and enhanced NK cell function against AML in vitro and in vivo. Collectively, the data provided herein unravel a previously unidentified novel mechanism of NK cell immune evasion in AML, and a link between TGF-B pathway and BATF, BATF3, and / or DDIT3 mediated transcriptional programs. The findings provided herein have important translational implications and provide direction / means for overcoming this and similar immune evasion mechanisms. The findings described herein support the use of allogeneic sources for adoptive NK cell therapy in combination with strategies aiming at preventing immune suppression to treat myeloid malignancies by targeting the TGF-P pathway, such as by targeting upstream receptors or downstream transcription factors (e.g., BATF, BATF3, and / or DDIT3), potentially preferentially over therapies aimed at reversing or rescuing the function of autologous NK cells.

[0205] In some embodiments, provided herein are compositions and methods comprising immune effector cells that have been engineered to have reduced or eliminated BATF, BATF3, and / or DDIT3 gene activity.

[0206] In some embodiments, hypomorphic, loss-of-function, and / or null mutations of BATF, BATF3, and / or DDIT3 can render cellular therapies insensitive to the immunosuppressive effects of TGF-P, and hence increase the effector cells functionality in TGF-P rich microenvironments. In some embodiments, TGF-P comprises TGF-pi, TGF-P2, and / or TGF-P3. In some embodiments, TGF-P consists of TGF-pi, TGF-P2, and / or TGF-P3. In some embodiments, TGF-P comprises or consists of TGF-pi. In some embodiments, TGF- P comprises or consists of TGF-P2. In some embodiments, TGF-P comprises or consists ofTGF-P3. In some embodiments, TGF-P comprises mature (e.g., soluble, active) TGF-p. In some embodiments, TGF-P comprises non-mature TGF-P that is complexed with one or more polypeptides. In some embodiments, TGF-P comprises non-mature TGF-P that is complexed with LAP.

[0207] In some embodiments, technologies described herein comprise the use of geneediting technologies (e.g., CRISPR-Cas technology), wherein the technologies are utilized to mutate (e.g., knock-out) genetic elements disclosed herein. In some embodiments, the prominent and key transcription factor BATF is mutated (e.g., knocked-out). In some embodiments, the key transcription factor BATF3 is mutated (e.g., knocked-out). In some embodiments, the key transcription factor DDIT3 is mutated (e.g., knocked-out).

[0208] In some embodiments, mutating BA TF in NK cells provides an improvement in NK cell persistence, proliferation, cytotoxicity, secretory function, and / or metabolic fitness.

[0209] In some embodiments, mutating BA TF in NK cells provides an improvement in NK cell persistence, proliferation, cytotoxicity, secretory function, and / or metabolic fitness when challenged against targets, such as cancer cells, that can be characterized as engaging in active evasion of and / or immunosuppression of cell therapies. In some embodiments, mutating BATF in NK cells provides an improvement in NK cell persistence, proliferation, cytotoxicity, secretory function, and / or metabolic fitness when challenged against targets, such as cancer cells, that can be characterized as expressing TGF-p.

[0210] In some embodiments, a targeted genetic engineering strategy is utilized to target BATF, BA TF3, and / or DDIT3. In some embodiments, genetic engineering strategies are utilized in different forms of cellular therapies, including CAR-T cells, T cells, CAR-NK cells, NK cells, T-cell receptor (TCR)-T cells, TCR-NK cells, and / or tumor-infiltrating lymphocytes (TILs). In some embodiments, such genetic engineering potentiates cell therapies against various types of cancers, including against solid tumors.

[0211] In some embodiments, disclosed herein are compositions and methods comprising genetically engineered immune effector cells and / or methods of making the same. In some embodiments, genetic engineering comprises mutating (e.g., knocking-out) of genes encoding proteins that are involved in TGF-P / SMAD2 / 3 signal transduction. In some embodiments, genetic engineering comprises mutating (e.g., knocking-out) of genes encoding proteins that are downstream of TGF-P / SMAD2 / 3 in the immunosuppressive signal transduction pathway, such as BATF, BATF 3, and / or DDIT3.

[0212] In some embodiments, the genetic engineering is performed through the use of endonuclease mediated cleavage (e.g., CRISPR-Cas technology). In some embodiments, compositions disclosed herein are utilized in methods of cancer treatment.

[0213] In certain embodiments, the mutation (e.g., knock-out) of genes implicated in TGF- P / SMAD2 / 3 immunosuppressive signaling pathway components improves an engineered immune cells effectiveness against various tumors. For example, CAR-T cells, that are FDA- approved for the treatment of leukemia, lymphoma, and myeloma, could be genetically engineered to comprise a mutation in one or more of these genes: BATF, BATF3, and / or DDIT3, in an attempt to increase their effectiveness in environments and / or against cell types characterized by the presence of TGF-p. Moreover, this genetic engineering strategy could be used in various other forms of cellular therapies such as CAR-NK cells, TCR-NK cells, TCR- T cells, TILs, etc. to potentiate their activity against various cells and / or pathogens.II. Engineered Mutations of BATF, BATF3, and / or DDIT3 Genes

[0214] Prior to expansion and genetic modification of the cells of the disclosure, a source of cells can be obtained from a subject through a variety of non-limiting methods. Immune cells of any kind, such as NK cells, can be obtained from a number of non-limiting sources, including from peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumors, or commercially available. In certain embodiments, any number of immune cell lines available and known to those skilled in the art, may be used.

[0215] In particular embodiments, immune effector cells are engineered, such as through gene editing mediated mutation, to have modified expression of endogenous BATF, BATF 3, and / or DDIT3 genes in the cell. In specific cases, the cells are engineered to have reduced levels of expression of BATF, BATF 3, and / or DDIT3, including complete inhibition of detectable expression of certain transcript variants and / or protein isoforms of BATF, BATF3, and / or DDIT3 (e.g., that may be referred to as knocked out). In some embodiments, engineered cells may or may not be expanded prior to production and / or prior to use.

[0216] In particular cases, BATF, BATF 3, and / or DDIT3 genes are disrupted, and expression of gene products from BATF, BATF3, and / or DDIT3 may be reduced in part or in full. In some embodiments, Ax BA TF, BATF3, and / or DDIT3 gene is knocked down or knocked out using processes of the disclosure. In certain embodiments, the BATF, BATF3, and / or DDIT3 are disrupted (e.g., mutated) in such a way that one or more RNA isoforms encoded bythe mutated aforementioned one or more genes are upregulated relative to a non-mutated copy of the same gene.

[0217] A skilled artisan is aware of numerous modern techniques for engineering of cells, including, including any immune cells. In some embodiments, immune cells of the disclosure are engineered to have mutations that result in reduced or fully inhibited expression of the BATF, BATF3, and / or DDIT3 genes. In particular embodiments, engineering of BATF, BATF3, and / or DDIT3 genes targeting of a polynucleotide sequence of the specific gene desired to be mutated.

[0218] In some embodiments, an engineered mutation comprises a loss-of-function mutation. In some embodiments, an engineered mutation comprises a reduction-of-function mutation. In some embodiments, an engineered mutation results in reduced or ablated gene expression. In some embodiments, an engineered mutation results in reduced or ablated gene expression that is driven by one or more specific transcription factors (e.g., p-SMAD2 / 3 / 4, BATF, BATF3, DDIT3, etc.). In some embodiments, an engineered mutation comprises a disruption of the coding portion of the BATF, BATF3, and / or DDIT3 gene. In some embodiments, an engineered mutation comprises disruption of an enhancer of the BATF, BATF3, and / or DDIT3 gene. In some embodiments, an engineered mutation comprises disruption of a promoter of the BATF, BATF3, and / or DDIT3 gene.

[0219] In some embodiments, the BATF, BATF3, and / or DDIT3 gene is mutated in a heterozygous manner. In some embodiments, the BATF, BATF3, and / or DDIT3 gene is mutated in a homozygous manner. In some embodiments, a population of immune effector cells that have engineered mutation(s) of the BATF, BATF3, and / or DDIT3 gene comprise immune cells that are homozygous for wildtype genes, are heterozygous for a wildtype gene and a mutated gene, and / or homozygous for mutated genes. In some embodiments, the BATF, BATF3, and / or DDIT3 gene is mutated in a majority of alleles in a population of immune effector cells. In some embodiments, the BATF, BATF 3, and / or DDIT3 gene is mutated in greater than or equal to, about or exactly 50%, 55%, 60%, 65%, 70%, 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%, or any range derivable therein, of alleles for the gene in the population.A. BATF

[0220] An example of basic leucine zipper ATF-like transcription factor (BATF; aka SFA2, B-ATF, BATF1) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Databaseunder Gene ID: 10538 (BATF) on chromosome 14q24.3, at NC 000014.9 Reference GRCh38.pl 4 Primary Assembly (range, 75522469 to 75546992) for the genomic sequence which is incorporated herein by reference in its entirety. A BATF mRNA transcript can be identified as NM 006399.5, and the associated protein product can be identified as NP 006390., each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a nuclear basic leucine zipper protein that belongs to the AP-l / ATF superfamily of transcription factors. The leucine zipper of this protein can mediate dimerization with members of the Jun family of proteins. This protein may be a negative regulator of AP- l / ATF transcriptional events. In some embodiments, a BATF TF consensus motifs are provided in FIG. 5G, with the sequence NTGANTMA and FIG. 7F, with the sequence NNTKASTMAN.

[0221] In certain embodiments, an engineered mutation in a BATF gene is in a BATF regulatory sequence upstream or downstream of the BATF genomic sequence (Reference GRCh38.pl4 Primary Assembly (range, 75522469 to 75546992); SEQ ID NO: 44). In certain embodiments, an engineered mutation in a BATF gene is in a sequence that is transcribed. In certain embodiments, an engineered mutation in a BATF gene is in a sequence that is transcribed but that would be spliced out of an mRNA product (e.g., in an intronic region). In certain embodiments, an engineered mutation in a. BATF gene is in a coding sequence that can be translated into a BATF polypeptide gene product. In certain embodiments, an engineered mutation in a BATF gene is in an enhancer region. In certain embodiments, an engineered mutation in a BA IT' gene is in a 5' enhancer region, intronic enhancer region, and / or 3' enhancer region. In certain embodiments, an engineered mutation in a BATF gene is in a SMAD2 / 3 binding site enhancer region. In some embodiments, a BATF gene mutation is a knock-out mutation that results in essentially no BATF gene product (e.g., BATF polypeptide and / or encoding transcript). In some embodiments, a BATF gene mutation is a mutation that results in a reduction of BATF gene product (e.g., BATF polypeptide and / or encoding transcript) by at least, exactly, or about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, relative to wild type.

[0222] In some embodiments, a BATF gene is mutated at a site comprised in any one or more of SEQ ID NOs: 1-12 or 44. In some embodiments, a BATF gene mutation is a result of exposure of the cell to a polynucleotide comprising a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 10-12.SEQ ID NO: 1 - human BATF ene 5' enhancerAAAGCGAGCGACATGTCCCTTTGGGGAGCAGTCCCTCTGCACCCCAGAGTGAGGAGGACGCA GGGGTCAGAGGTGGCTACAGGGCAGGCAGAGGAGGCACCTGTAGGGGGTGGTGGGCTGGTGG CCCAGGAGAAGTCAGGAAGGGAGCCCAGCTGGTGACAAGAGAGCCCAGAGGTGCCTGGGGCT GAGTGTGAGAGCCCGGAAGATTTCAGCCATGCCTCACAGCTCCGACAGCAGTGACTCCAGCT TCAGCCGCTCTCCTCCCCCTGGCAAACAGGTAGAGTCCTCCTTTTTCTCT ( SEQ ID NO : 1 )SEQ ID NO: 2 - human BATF gene intronic enhancer 1CTCAGAGGTTTTCTTACTCCCCTCTTTTGAACTTAGTCTAGTCCCCTGCTTCCTAGGAGGGG TGGGTTGG ( SEQ ID NO : 2 )SEQ ID NO: 3 - human BATF gene intronic regulatory region (silencer)GCCTGTAATCCCAACACTTTGGGAGCCCGAGGCGGGTGGATCACGAGGTCAGGAGATCGAGA CCATCCTGGCTAACACGGTGAAACCATGTCTCTACTAAAAATACAAAAAATTAGCCGGGCAT GGTGGCGGGTGCCCGTAGTCCCAGCTACTAGGGAGGCTGAGGCAGGAGAATGGCATGAACCC GGGAGGCGGAGCTTGCAATGAGCCGAGAT ( SEQ ID NO : 3 )SEQ ID NO: 4 - human BATF gene intronic enhancer 2GGAATCAACAAATCATGGCCTACTTTTCTCATGTAACAAGAAGTTCAAAA ( SEQ ID NO : 4 )SEQ ID NO: 5 - human BATF gene intronic enhancer 3CTTTCATTCTCTAGGCTGTGAGATGGGTGCCGAGCCTCTCGGCATCAGTCTATGTTGAAG ( SEQ ID NO : 5 )SEQ ID NO: 6 - human BATF gene intronic enhancer 4GAAATCATAGTCTTGAAAACCAAGGACCAGGCGATAGCCTTTTCACCCTG ( SEQ ID NO : 6 )SEQ ID NO: 7 - human BATF gene mRNA sequenceAAAGCGAGCGACATGTCCCTTTGGGGAGCAGTCCCTCTGCACCCCAGAGTGAGGAGGACGCA GGGGTCAGAGGTGGCTACAGGGCAGGCAGAGGAGGCACCTGTAGGGGGTGGTGGGCTGGTGG CCCAGGAGAAGTCAGGAAGGGAGCCCAGCTGGTGACAAGAGAGCCCAGAGGTGCCTGGGGCT GAGTGTGAGAGCCCGGAAGATTTCAGCCATGCCTCACAGCTCCGACAGCAGTGACTCCAGCT TCAGCCGCTCTCCTCCCCCTGGCAAACAGGACTCATCTGATGATGTGAGAAGAGTTCAGAGG AGGGAGAAAAATCGTATTGCCGCCCAGAAGAGCCGACAGAGGCAGACACAGAAGGCCGACAC CCTGCACCTGGAGAGCGAAGACCTGGAGAAACAGAACGCGGCTCTACGCAAGGAGATCAAGC AGCTCACAGAGGAACTGAAGTACTTCACGTCGGTGCTGAACAGCCACGAGCCCCTGTGCTCG GTGCTGGCCGCCAGCACGCCCTCGCCCCCCGAGGTGGTGTACAGCGCCCACGCATTCCACCA ACCTCATGTCAGCTCCCCGCGCTTCCAGCCCTGAGCTTCCGATGCGGGGAGAGCAGAGCCTC GGGAGGGGCACACAGACTGTGGCAGAGCTGCGCCCATCCCGCAGAGGCCCCTGTCCACCTGG AGACCCGGAGACAGAGGCCTGGACAAGGAGTGAACACGGGAACTGTCACGACTGGAAGGGCG TGAGGCCTCCCAGCAGTGCCGCAGCGTTTCGAGGGGCGTGTGCTGGACCCCACCACTGTGGG TTGCAGGCCCAATGCAGAAGAGTATTAAGAAAGATGCTCAAGTCCCATGGCACAGAGCAAGG CGGGCAGGGAACGGTTATTTTTCTAAATAAATGCTTTAAAAGAAA ( SEQ ID NO : 7 )SEQ ID NO: 8 - human BATF gene coding sequenceATGCCTCACAGCTCCGACAGCAGTGACTCCAGCTTCAGCCGCTCTCCTCCCCCTGGCAAACA GGACTCATCTGATGATGTGAGAAGAGTTCAGAGGAGGGAGAAAAATCGTATTGCCGCCCAGA AGAGCCGACAGAGGCAGACACAGAAGGCCGACACCCTGCACCTGGAGAGCGAAGACCTGGAG AAACAGAACGCGGC T C TACGCAAGGAGAT CAAGCAGC T CACAGAGGAAC T GAAGTAC T T GAG GTCGGTGCTGAACAGCCACGAGCCCCTGTGCTCGGTGCTGGCCGCCAGCACGCCCTCGCCCC CCGAGGTGGTGTACAGCGCCCACGCATTCCACCAACCTCATGTCAGCTCCCCGCGCTTCCAG CCCTGA ( SEQ ID NO : 8 )SEQ ID NO: 9 - human BATF gene translated polypeptide sequenceMPHSSDSSDSSFSRSPPPGKQDSSDDVRRVQRREKNRIAAQKSRQRQTQKADTLHLESEDLE KQNAALRKEIKQLTEELKYFTSVLNSHEPLCSVLAASTPSPPEWYSAHAFHQPHVSSPRFQ P ( SEQ ID NO : 9 )SEQ ID NO: 10 - human BATF gene targeting sgRNAlGCTGTCGGAGCTGTGAGGCA ( SEQ ID NO : 10 )SEQ ID NO: 11 - human BATF gene targeting sgRNAlGGACTCTACCTGTTTGCCAG ( SEQ ID NO : 11 )SEQ ID NO: 12 - human BATF gene targeting sgRNA3TTGTCCTGCCCAGGGAGCTG ( SEQ ID NO : 12 )B. BATF3

[0223] An example of a basic leucine zipper ATF-like transcription factor 3 (BATF3; aka JDP1, SNFT, JUNDM1) gene sequence can be found in the US NIH, National Library of Medicine, GENBANK® Database under Gene ID: 55509 (BATF3) on chromosome lq32.3, at NC_000001. l l (Reference GRCh38.pl 4 Primary Assembly (range 212686417 to 212699840, complement); SEQ ID NO: 45) for the genomic sequence which is incorporated herein by reference in its entirety.

[0224] A BATF3 mRNA transcript can be identified as NM 018664.3, and the associated protein product can be identified as NP 061134.1, each of which are incorporated by reference herein in their entirety. The BATF 3 gene encodes a member of the basic leucine zipper protein family. The encoded protein can function as a transcriptional repressor when heterodimerizing with JUN. The protein may play a role in repression of interleukin-2 and matrix metalloproteinase- 1 transcription. In some embodiments, a BATF3 TF consensus motif is provided in FIG. 5G, with the sequence HDACCACA.

[0225] In certain embodiments, an engineered mutation in a BATF 3 gene is in a BATF3 regulatory sequence upstream or downstream of the BATF3 genomic sequence (Reference GRCh38.pl 4 Primary Assembly (range 212686417 to 212699840, complement)). In certainembodiments, an engineered mutation in & BA TF3 gene is in a sequence that is transcribed. In certain embodiments, an engineered mutation in a BATF3 gene is in a sequence that is transcribed but that would be spliced out of an mRNA product (e.g., in an intronic region). In certain embodiments, an engineered mutation in BATF3 gene is in a coding sequence that can be translated into a BATF3 polypeptide gene product. In certain embodiments, an engineered mutation in a BATF3 gene is in an enhancer region. In certain embodiments, an engineered mutation in a BATF3 gene is in a 5' enhancer region, intronic enhancer region, and / or 3' enhancer region. In certain embodiments, an engineered mutation in a BATF3 gene is in a SMAD2 / 3 binding site enhancer region. In some embodiments, a BATF3 gene mutation is a knock-out mutation that results in essentially noBATF3 gene product (e.g., BATF3 polypeptide and / or encoding transcript). In some embodiments, a BATF3 gene mutation is a mutation that results in a reduction of BATF3 gene product (e.g., BATF3 polypeptide and / or encoding transcript) by at least, exactly, or about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, relative to wild type.

[0226] In some embodiments, a BATF3 gene is mutated at a site comprised in any one or more of SEQ ID NOs: 13-17 or 45. In some embodiments, a BATF3 gene mutation is a result of exposure of the cell to a polynucleotide comprising a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NO: 17.SEQ ID NO: 13 - human BATF3 gene enhancer sequenceCCCAGGTATGAAAATGACCAAGGCTCCTTGCTGGGCAGAGGAGTGTCCCCGGCTTCATCGGG GCAAGCAGCCGGCCACAGGGTCCGGCCGGGGAGGCACTGGCACAAAGTTCATAGGGCAGAGC AGCAGCGGGCACATCTTCTCGTGCTCCTTCAGTGCCTCTGTCAGGTGCTTCAGCTCCTCTGT CAGCTTCCCGATCTCTCTCCGCAGCATGGTGTTTTCTTGCTCCAGGCTCTCATATTCCTGGG GGAGACAGAATGGGCAAAATCATTTCTGGTGCAGCGTTCCCTTCCTCCGTCCTCCTGTGCCG CGCTGTTCACCTCTTGCCCATGAAAGCTGTCTCATTACGCATGTCCCCTCCACTGCTCAGAT ATTCAGATATTTGTCTATTTGATGAGTGTCTCCCCCCTCCCACTGGGAGAAACAATTTATTG AG G T AT AAC T T AC G T AAAAT AAAAT GTATCTATTT T AAG TAT GAT C GAG T GAG T T T T GAG AA ATGT ( SEQ ID NO : 13 )SEQ ID NO: 14 - human BATF3 gene mRNA sequenceGGGACAGCGCCCGTAGGCAGCCCCACGGGCAGGGCGCGCGGGCGGGGCGGGGCGGGCCGGGC CAGAGGAGCGCCCGGCATGTCGCAAGGGCTCCCGGCCGCCGGCAGCGTCCTGCAGAGGAGCG TCGCGGCGCCCGGGAACCAGCCGCAGCCGCAGCCGCAGCAGCAGAGCCCTGAGGATGATGAC AGGAAGGTCCGAAGGAGAGAAAAAAACCGAGTTGCTGCTCAGAGAAGTCGGAAGAAGCAGAC CCAGAAGGC T GACAAGC T CCAT GAGGAATAT GAGAGCC T GGAGCAAGAAAACACCAT GC T GC GGAGAGAGATCGGGAAGCTGACAGAGGAGCTGAAGCACCTGACAGAGGCACTGAAGGAGCAC GAGAAGATGTGCCCGCTGCTGCTCTGCCCTATGAACTTTGTGCCAGTGCCTCCCCGGCCGGACCCTGTGGCCGGCTGCTTGCCCCGATGAAGCCGGGGACACTCCTCTGCCCAGCAAGGAGCCT TGGTCATTTTCATACCTGGGAGGAAGGCTTTTCCTTCACAATTGTATACAGGGGGCACCTGT GGCCAGGCCTCCTCCTGGGAGCTCCAGGACCAGCCAGCTGTGTTCCCTGCAGACTGGGCTCA GCCCGACATCCAACAGGCGCCAAACTCACAGAGCCCTTGTGCAGATCCAGCATGGAGGCCAC CCTCAGGAGTGACTTCTCATCCACCCTGGCAGCTAGTAGGTTCTGCTGTTATGCAGAGCCAT TTCCTCTAGAATTTGGATAATAAAGATGCTTATTGTCTCTCCCTTCTCCAGTTCTGGGAATT TACAGGCACAATACACTTCCTTTTCCTGGA ( SEQ ID NO : 14 )SEQ ID NO: 15 - human BATF3 gene coding sequenceATGTCGCAAGGGCTCCCGGCCGCCGGCAGCGTCCTGCAGAGGAGCGTCGCGGCGCCCGGGAA CCAGCCGCAGCCGCAGCCGCAGCAGCAGAGCCCTGAGGATGATGACAGGAAGGTCCGAAGGA GAGAAAAAAACCGAGTTGCTGCTCAGAGAAGTCGGAAGAAGCAGACCCAGAAGGCTGACAAG CTCCATGAGGAATATGAGAGCCTGGAGCAAGAAAACACCATGCTGCGGAGAGAGATCGGGAA GCTGACAGAGGAGCTGAAGCACCTGACAGAGGCACTGAAGGAGCACGAGAAGATGTGCCCGC TGCTGCTCTGCCCTATGAACTTTGTGCCAGTGCCTCCCCGGCCGGACCCTGTGGCCGGCTGC TTGCCCCGATGA ( SEQ ID NO : 15 )SEQ ID NO: 16 - human BATF3 gene translated polypeptide sequenceMSQGLPAAGSVLQRSVAAPGNQPQPQPQQQSPEDDDRKVRRREKNRVAAQRSRKKQTQKADK LHEEYESLEQENTMLRREIGKLTEELKHLTEALKEHEKMCPLLLCPMNFVPVPPRPDPVAGC LPR ( SEQ ID NO : 16 )SEQ ID NO: 17 - human BATF3 gene targeting sgRNAlATGATGACAGGAAGGTCCGA ( SEQ ID NO : 17 )C. DDIT3

[0227] An example of DNA damage inducible transcript 3 (DDIT3; aka CHOP, CEBPZ, CHOPIO, CHOP-10, GADD153, CZEBPzeta) gene sequence can be found in the US NIH, National Library of Medicine, NCBI GENBANK® Database under Gene ID: 1649 (DDIT3) on chromosome 12ql 3.3, atNC_000012.12 Reference GRCh38.pl4 Primary Assembly (range, 57516588 to 57520517, complement) for the genomic sequence which is incorporated herein by reference in its entirety. Multiple transcripts and multiple protein isoforms exist for the DDIT3 gene, and DDIT3 mRNA transcript can be identified as NM_001195053.1, NM_00 1195054.1, NM_001195055.1, NM_001195056.1, NM_001195057.1,NM_001413641.1, NM_001413642.1, and NM_004083.6, which respectfully encode polypeptide products NP_001181982.1, NP_001181983.1, NP_001181984.1,NP_001181985.1, NP_001181986.1, NP_001400570.1, NP_001400571.1, and NP_004074.2, each of which are incorporated by reference herein in their entirety. In some embodiments, a DDIT3 TF consensus motif is provided in FIG. 5G, with the sequence BAYWTCCY.

[0228] In certain embodiments, an engineered mutation in a DDIT3 gene is in a DDIT3 regulatory sequence upstream or downstream of the DDIT3 genomic sequence (ReferenceGRCh38.pl4 Primary Assembly (range, 57516588 to 57520517, complement)). In certain embodiments, an engineered mutation in a DDIT3 gene is in a sequence that is transcribed. In certain embodiments, an engineered mutation in a DDIT3 gene is in a sequence that is transcribed but that would be spliced out of an mRNA product (e.g., in an intronic region). In certain embodiments, an engineered mutation in a.DDIT3 gene is in a coding sequence that can be translated into a DDIT3 polypeptide gene product. In certain embodiments, an engineered mutation in a DDIT3 gene is in an enhancer region. In certain embodiments, an engineered mutation in a DDIT3 gene is in a 5' enhancer region, intronic enhancer region, and / or 3' enhancer region. In certain embodiments, an engineered mutation in a DDIT3 gene is in a SMAD2 / 3 binding site and / or enhancer region. In some embodiments, 3.1)1)113 gene mutation is a knock-out mutation that results in essentially no DDIT3 gene product (e.g., DDIT3 polypeptide and / or encoding transcript). In some embodiments, a DDIT3 gene mutation is a mutation that results in a reduction of DDIT3 gene product (e.g., DDIT3 polypeptide and / or encoding transcript) by at least, exactly, or about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, relative to wild type.

[0229] In some embodiments, a DDIT3 gene is mutated at a site comprised in any one or more of SEQ ID NOs: 18-28. In some embodiments, a DDIT3 gene mutation is a result of exposure of the cell to a polynucleotide comprising a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one or more of SEQ ID NOs: 27-28.SEQ ID NO: 18 - human DDIT3 geneGAGGTCAGAGACTTAAGTCTAAGGCACTGAGCGTATCATGTTAAAGATGAGCGGGTGGCAGC GACAGAGCCAAAATCAGAGCTGGAACCTGAGGAGAGAGGCGAGTACTGATTCCCATCTACCT TTTACCCTCCCGTCTCCTCAAAGTTGGGGCGTCCGCTCTTTAGGATCGGCCTCACTCCTCCA CAGTGAAGTTAGGGACCGTCCGAGAGAGGAATGGGGAGAGTCCCTTATTCTGGGGTGGTGCT TACAAACCCCTATTGCTTCGGACGACGGCGTCTCTCCACCCCTGCCCGGAGCCGGAACACGG GCCCTGCTCTGTGCTGCTGGGCAAAGGGACCTCGGTTGCCCTTGGGAAATTCATTCTTTCCC GTAGCCAACTTCAGGCCTCATCGTTAGGCCTGTCCGCGGGGAGGCAGGTCAGCAGGACACAC CCCCGCTCTAAGACTGGGTGACCATCGCTCAGGCCGTTTCCGCCGCTTCGCCACCAGCGGGC CTTCTCCCTACCCCACCCCCAATTCTGTCTCAGTCTCAGTGCCTCTGGTGTCAGCATGGCCA CCTTGGTAGCTGGGGCTACTGGACCCTGCAGCGGATAGGGGAACCTTGAGGAGACACAAGCC TTTGGGAGGGGTGCCGATGGACAGGGAGTGGTGTGTTTTCCTTTTGCCGTAGAGGTCTCTGG GCCTCCTGCACAAGAGAGCAGCCTGGATCTCTTAAGTGTAGGAGGCCATTTGGGGTCTCCCC AGGGTATTGTCCTTCCCTCGGGATTAGTCCCTGCCTCTTTAACCCGGTCCTGTCTCCCAGCT AATCTCTGTGTAACCATTGCATCAGGCCAGCCCCGTTTGGCTCTGCAGCCTTCTGACCTGAG GCTCTACTGCTGATGAAAGCCAAGTCCCACACACTGGAAGGCAAGGGAGGGTTCCCCAGGGA GGACAGCCCTGCAGAGAAATACTTCGGGCAATATTGCATCTCTAGCCCCTAGGGATCAGCAG CTGCCACTCTGCTTCTGCCCCTTCCCTATAAGAGAGACTGGGGGGAGTTTATCCATTCATTCTTAACAAATACTTAATGAGGACCTACTGTGTGCCACACAGTTTGGGGCTCAGGGTACATCCT TGAGCAAGAGGAAAAAATCATCTCAGTGGGAGGCCTACAGTAAACAAAATATAAGTGCCACG GAGAAAGC TAAAGCAGAGAAAGGAAT GGAGAAT GT T CAGGAT GGAGGT CAGAGT GT TACAT C AGGTGGTCAGGAATTACCTTAGGTAATTCCTCCACTCAAAACCCTTCAGTGACTTCCATGAC AT GAAATAGGAAGT CAT T GGAGGGT T T GAGCAGAGGAAT GACC T GT T T TAAAAGGC T GAG T C AGGCTGCTGTATGGTGAATAGAGTTGCGGAGGGGTGGCAAGAGAAGAAATGGGAAGACCTTC T G GAG T C AGAAAG T T T C T G GAG T AAT T T AGAGAT G G T AG T GAAT T GAT C T AGAT T G GAAAC A AT GGAAT T AGAAG T G T T T AGAT T C T T C T AAGCAAAGG T T T T AAAAAC T CAT T T T T AAAGAAT GAGTTAAGGGCCGGGCATGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGACCAGAGGT GGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGTGAAATCCCATCT TTACTAAAAATACAAAAATTAGCCGGGCATGGCAGTGCATGCCTGTAATCCCAGCTACTCCG GAGGCTGAAGCAGGAGAATCGCTTGAACCCAGCAGGCGGAGGTTGCAGTGAGCCGATTGCGC CACTGCCTTCCAGCCTGGGCAAAAAGAGTGAGACCCGTCTCAGAAAAAAAGGAATGAGTTAA AATTTGCTAGTACTTTGGATTGCAGGGTGTGAGAGAAGAGGAATGAAGGATGATACCAAGGT TTTTAGCTTAAGCAACTAGAGTTGTCATCTGAGATGGGGATGACCTTGGAAGGGGAAAATCA GCAAGAGTTTGCCTTTGCACATAGTCTTAGGTGCCTATTAGACATTGAAAAAGAAATGGCAA GTAGGCAGTAGACAGCAGAGTCTGAAGTTCTGGAAGAGGTCCAGACTGGAAATGTACATTTG GAGGATGTCAGCCCTGTGGGAATGGAGTTAGGAAAATGCTATGATTTGTTCCCTTCCCTGTA GTTTAGTTTTTACCCTGGCAGATTTGAGGCCTGCTTTGGATTTAGAGAAAGCTGAGTTGGCC AG GAC TTTACTATTATG T AAC C AG GAC T AC AAAT G T C AG C AAC T AAAAAT AAAGAAAG T C AG GCCCTCTTCTGCCCTTCGAAATGGCTACAGGGACCAAGTATGCATACCCCACAAGACCAGAA GTAAGGAAGGACCAGTAGGAGGCTGGAGGTAAAAGAAAAATAAGGGCCCAGCACGGTAGCTC ATGCCTATAATCCCAGCACTTTGGGAAGCGATGGATCACAAGGTTAAGAGATGGAGACCATC CTGGCCAACATAGTGAAACCCTATCTCTGCTAAAAACACAAAAATTAGCTGGGCGTGGTGGC ACGCGCCTGTAGTCCCAGCTACTCGGGAGGCCGAGGCAGAAGAATCACTTGAACCGAGGAGG CAGAGGTTGCAGTGAGCCGAGATCGCACCACTGCACTTCAGCCTGGCAACAGAGCAAGACTT G G T C T C AAAAAAAAAAAAAGAAAGAAAAAAAGAAAAAGAAAAG T AAG TTGCCTCTCCCCCTT CCAAAAATGGCTGACATTTCTCTTTGTTGCCCACAGTGTTCAAGAAGGAAGTGTATCTTCAT AC AT C AC C AC AC C T GAAAG C AG G T AAAC T T AAC CTACCCTTTTC C AAAAAT T T T AAAC G G C A GGACAGTAAATATTTTAGATGTTAAAAGTCCTATAGTCTCTAGCGTGACTCTTCATCTCTGC CACTGTAGCACCAAAGCAGCCATAAACAATATGTAAATAAACAGATGTGGCTGTATTCCAGT ACAACTTTACCTACAAAAACAGGCATCAGACCAGCTTGCCAACTTGTGGCATAGACTGTTTG CTACATGGAGCTTGTTCCAGCCACTCCCCATTATCCTGCAGATGTGCTTTTCCAGACTGATC CAACTGCAGAGATGGCAGCTGAGTCATTGCCTTTCTCCTTCGGGACACTGTCCAGCTGGGAG CTGGAAGCCTGGTATGAGGACCTGCAAGAGGTCCTGTCTTCAGATGAAAATGGGGGTACCTA TGTTTCACCTCCTGGAAATGAAGAGGTAAGAATGTTAGCCCTAAAGCTAAAGGGGGATGTTA CCTTTCCCTTCTCAACTAATATCTATGTTCCCTTTCCTCATTTCCTTGAAGGAAGAATCAAA AATCTTCACCACTCTTGACCCTGCTTCTCTGGCTTGGCTGACTGAGGAGGAGCCAGAACCAG CAGAGGTCACAAGCACCTCCCAGAGCCCTCACTCTCCAGATTCCAGTCAGAGCTCCCTGGCT CAGGAGGAAGAGGAGGAAGACCAAGGGAGAACCAGGAAACGGAAACAGAGTGGTCATTCCCC AGCCCGGGCTGGAAAGCAGCGCATGAAGGAGAAAGAACAGGAGAATGAAAGGAAAGTGGCAC AGCTAGCTGAAGAGAATGAACGGCTCAAGCAGGAAATCGAGCGCCTGACCAGGGAAGTAGAG GCGACTCGCCGAGCTCTGATTGACCGAATGGTGAATCTGCACCAAGCATGAACAATTGGGAG CATCAGTCCCCCACTTGGGCCACACTACCCACCTTTCCCAGAAGTGGCTACTGACTACCCTC TCACTAGTGCCAATGATGTGACCCTCAATCCCACATACGCAGGGGGAAGGCTTGGAGTAGAC AAAAG GAAAG G T C T C AG C T T G TAT AT AGAGAT T G TACAT TTATTTATTACTGTCCCTATCTA TTAAAGTGACTTTCTATGAGCCAA ( SEQ ID NO : 18 )SEQ ID NO: 19 - human DDIT3 gene enhancer 1ATATTTTGTTTACTGTAGGCCTCCCACTGAGATGATTTTTTCCTCTTGCTCAAGGATGTACC CTGAGCCCCAAACTGTGTGGCACACAGTAGGTCCTCATTAAGTATTTGTTAAGAATGAATGG ATAAACTCCCCCCAGTCTCTCTTATAGGGAAGGGGCAGAAGCAGAGTGGCAGCTGCTGATCCCTAGGGGCTAGAGATGCAATATTGCCCGAAGTATTTCTCTGCAGGGCTGTCCTCCCTGGGGAACCCTCCCTTGCCTTCCAGTGTGTGGGACTTGGCTTTCATCAGCAGTAGAGCCTCAGGTCAGAAGGCTGCAGAGCCAAACGGGGCTGGCCTGATGCAATGGTTACACAGAGATTAGCTGGGAGA CAGGACCGGGTTAAAGAGGCAGGGACTAATCCCGAGGGAAGGACAATACCCTGGGGAGACCC CAAATGGCCTCCTACACTTAAGAGATCCAGGCTGCTCTCTTGTGCAGGAGGCCCAGAGACCTCTACGGCAAAAGGAAAACACACCACTCCCTGTCCATCGGCACCCCTCCCAAAGGCTTGTGTC TCCTCAAGGTTCCCCTATCCGCTGCAGGGTCCAGTAGCCCCAGCTACCAAGGTGGCCATGCT GACACCAGAGGCACTGAGACTGAGACAGAATTGGGGGTGGGGTAGGGAGAAGGCCCGCTGGTGGCGAAGCGGCGGAAACGGCCTGAGCGATGGTCACCCAGTCTTAGAGCGGGGGTGTGTCCTGCTGACCTGCCTCCCCGCGGACAGGCCTAACGATGAGGCCTGAAGTTGGCTACGGGAAAGAAT GAATTTCCCAAGGGCAACCGAGGTCCCTTTGCCCAGCAGCACAGAGCAGGGCCCGTGTTCCG GCTCCGGGCAGGGGTGGAGAGACGCCGTCGTCCGAAGCAATAGGGGTTTGTAAGCACCACCCCAGAATAAGGGACTCTCCCCATTCCTCTCTCG ( SEQ ID NO : 19 )SEQ ID NO: 20 - human DDIT3 gene enhancer 2GAATAAGGGACTCTCCCCATTCCTCTCTCGGACGGTCCCTAACTTCACTGTGGAGGAGTGAG GCCGATCCTAAAGAGCGGACGCCCCAACTTTGAGGAGACGGGAGGGTAAAAGGTAGATGGGA ATCAGT ( SEQ ID NO : 20 )SEQ ID NO: 21 - human DDIT3 gene enhancer 3GACGGTCCCTAACTTCACTGTGGAGGAGTGAGGCCGATCCTAAAGAGCGGACGCCCCAACTT TGAGGAGACGGGAGGGTAAAAGGTAGATGGGAATCAGTACTCGCCTCTCTCCTCAGGTTCCA GCTCTGATTTTGGCTCTGTCGCTGCCACCCGCTCATCTTTAACATGATACGCTCAGTGCCTTAGACTTAAGTCTCTGACCTCGGGAGCGCCTGGCTGTAATCTTTTACCATGGCCTTGCCCTCC TCTCAGGGGCGGGACCCCAAAACCTACCAATCAGAAAGTGGCACGCCGGCATTGGCCCCGCC CCGTCCCTCGCATCCGCCACTCAGGAGCCCCGCGGCACCGCCCCCGTCGCTCCCTCTCGCTAGGGGGTCGACGTAGTGTCGGACCCGGAAGTGGCTTTGGGTCACGAGGCTTCACGGAGGAGGTGGGTGAGTCATGCGCGCGCGCGGAGGGGAGTGTAGCGGGGGGGGAGGGGAGGAAAGGGGGGCGGGGATGATGCAATGTTTGGCAACCGGTGTCTGCACGCGCACGCGCAGGGGACACCGAGGGT GGTGGGAGGTGCGGAGGGTGGGGGAGAGGAGAGAGGGCGGGGCGCCAGCTCCCGGCGCCCAA CGGCCCCAACGGCTATCAGCCTTGGTCGCCCCTAGTCGGTCGTGAGCCTCTTTGCCGCTTGTCTCCCCCAGAAGCTGCTGTTCCGCCCACCTAGCTTTACCCGCCGTTTTACCCCCACTATAAGGGACTCCCATATTTCTTGGCCCCTCCCAGCTAATGGGCACATAGGCCTTTTGAACTTTTCTT TTTAAAGTTGGTATCCATCCACTGGGATTGTCGACATCTCGAGTCTTCTTAAAGAGGTCTCC TGGCCGGGCTCAGTGGCTTACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGAGAGCGGATCACTTGAGGTCAGGAGTTCGAGATCAGCCT ( SEQ ID NO : 21 )SEQ ID NO: 22 - human DDIT3 gene enhancer 4AAGGGACTCTCCCCATTCCTCTCTCGGACGGTCCCTAACTTCACTGTGGAGGAGTGAGGCCG ATCCTAAAGAGCGGACGCCCCAACTTTGAGGAGACGGGAGGGTAAAAGGTAGATGGGAATCA GTACTCGCCTCTCTCCTCAGGTTCCAGCTCTGATTTTGGCTCTGTCGCTGCCACCCGCTCATCTTTAACATGATACGCTCAGTGCCTTAGACTTAAGTCTCTGACCTCGGGAGCGCCTGGCTGTAATCTTTTACCATGGCCTTGCCCTCCTCTCAGGGGCGGGACCCCAAAACCTACCAATCAGAA AGTGGCACGCCGGCATTGGCCCCGCCCCGTCCCTCGCATCCGCCACTCAGGAGCCCCGCGGC ACCGCCCCCGTCGCTCCCTCTCGCTAGGGGGTCGACGTAGTGTCGGACCCGGAAGTGGCTTTGGGTCACGAGGCTTCACGGAGGAGGTGGGTGAGTCATGCGCGCGCGCGGAGGGGAGTGTAGC GGGGGGGGAGGGGAGGAAAGGGGGGCGGGGATGATGCAATGTTTGGCAACCGGTGTCTGCAC GCGCACGCGCAGGGGACACCGAGGGTGGTGGGAGGTGCGGAGGGTGGGGGAGAGGAGAGAGG GCGGGGCGCCAGCTCCCGGCGCCCAACGGCCCCAACGGCTATCAGCCTTGGTCGCCCCTAGT CGGTCGTGAGCCTCTTTGCCGCTTGTCTCCCCCAGAAGCTGCTGTTCCGCCCACCTAGCTTT ACCCGCCGTTTTACCCCCACTATAAGGGACTCCCATATTTCTTGGCCCCTCCCAGCTAATGG GCACATAGGCCTTTTGAACTTTTCTTTTTAAAGTTGGTATCCATCCACTGGGATTGTCGACA TCTCGAGTCTTCTTAAAGAGGTCTCCTGGCCGGGCTCAGTGGCTTACGCCTGTAATCCCAGC ACTTTGGGAGGCTGAGGAGAGCGGATCACTTGAGGTCAGGAGTTCGAGATCAGCCTGACCAA CATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGTGTGGTGATGCGCGCCT GTAATCCCAGCTACTCTGGAGGCAGAGGCAGAAGAATCGCTTGAACCCTGGAGGCGGATGTT GCAGTGACCGAGATCGCGCCACTGCACTCCAGCCTGGGTGACAGAGCGAGACTACGTTTCACCAAAAACAAAAAACAAAACAAA ( SEQ ID NO : 22 )SEQ ID NO: 23 - human DDIT3 gene coding sequence for isoform 1ATGGAGCTTGTTCCAGCCACTCCCCATTATCCTGCAGATGTGCTTTTCCAGACTGATCCAAC TGCAGAGATGGCAGCTGAGTCATTGCCTTTCTCCTTCGGGACACTGTCCAGCTGGGAGCTGG AAGCCTGGTATGAGGACCTGCAAGAGGTCCTGTCTTCAGATGAAAATGGGGGTACCTATGTT TCACCTCCTGGAAATGAAGAGGAAGAATCAAAAATCTTCACCACTCTTGACCCTGCTTCTCT GGCTTGGCTGACTGAGGAGGAGCCAGAACCAGCAGAGGTCACAAGCACCTCCCAGAGCCCTC ACTCTCCAGATTCCAGTCAGAGCTCCCTGGCTCAGGAGGAAGAGGAGGAAGACCAAGGGAGA ACCAGGAAACGGAAACAGAGTGGTCATTCCCCAGCCCGGGCTGGAAAGCAGCGCATGAAGGA GAAAGAACAGGAGAAT GAAAGGAAAGT GGCACAGC TAGC T GAAGAGAAT GAACGGC T CAAGC AGGAAATCGAGCGCCTGACCAGGGAAGTAGAGGCGACTCGCCGAGCTCTGATTGACCGAATG GTGAATCTGCACCAAGCATGA ( SEQ ID NO : 23 )SEQ ID NO: 24 - human DDIT3 gene coding sequence for isoform 2ATGGCAGCTGAGTCATTGCCTTTCTCCTTCGGGACACTGTCCAGCTGGGAGCTGGAAGCCTG GTATGAGGACCTGCAAGAGGTCCTGTCTTCAGATGAAAATGGGGGTACCTATGTTTCACCTC CTGGAAATGAAGAGGAAGAATCAAAAATCTTCACCACTCTTGACCCTGCTTCTCTGGCTTGG CTGACTGAGGAGGAGCCAGAACCAGCAGAGGTCACAAGCACCTCCCAGAGCCCTCACTCTCC AGATTCCAGTCAGAGCTCCCTGGCTCAGGAGGAAGAGGAGGAAGACCAAGGGAGAACCAGGA AACGGAAACAGAGTGGTCATTCCCCAGCCCGGGCTGGAAAGCAGCGCATGAAGGAGAAAGAA CAGGAGAAT GAAAGGAAAGT GGCACAGC TAGC T GAAGAGAAT GAACGGC T CAAGCAGGAAAT CGAGCGCCTGACCAGGGAAGTAGAGGCGACTCGCCGAGCTCTGATTGACCGAATGGTGAATC TGCACCAAGCATGA ( SEQ ID NO : 24 )SEQ ID NO: 25 - human DDIT3 gene translated isoform 1 polypeptide sequenceMELVPATPHYPADVLFQTDPTAEMAAESLPFS FGTLSSWELEAWYEDLQEVLSSDENGGTYV SPPGNEEEESKI FTTLDPASLAWLTEEEPEPAEVTSTSQSPHSPDSSQSSLAQEEEEEDQGR TRKRKQSGHSPARAGKQRMKEKEQENERKVAQLAEENERLKQE IERLTREVEATRRALIDRM VNLHQA ( SEQ ID NO : 25 )SEQ ID NO: 26 - human DDIT3 gene translated isoform 2 polypeptide sequenceMAAESLPFS FGTLSSWELEAWYEDLQEVLSSDENGGTYVSPPGNEEEESKI FTTLDPASLAW LTEEEPEPAEVTSTSQSPHSPDSSQSSLAQEEEEEDQGRTRKRKQSGHSPARAGKQRMKEKE QENERKVAQLAEENERLKQE IERLTREVEATRRALIDRMVNLHQA ( SEQ ID NO : 2 6 )SEQ ID NO: 27 - human DDIT3 gene targeting sgRNAlGGTACCTATGTTTCACCTCC ( SEQ ID NO : 27 )SEQ ID NO: 28 - human DDIT3 gene targeting sgRNA2GTCTGGAAAAGCACATCTGC ( SEQ ID NO : 28 )

[0230] In some embodiments, 5 '-3' sequences of exemplary guide RNAs for mutating the BATF, BATF3, and / or DDIT3 gene are provided as SEQ ID NOs: 10-12, 17, or 27-28. In some embodiments, these guide RNA sequences are suitable for use with CRISPR / Cas9 technology which utilizes guide RNAs (complementary to short target DNA sequences on the targeted gene) in order to perform double-stranded DNA cleavage. Guide RNAs could be positively stranded or negatively stranded but since the cleavage made using CRISPR / Cas9 technology affects both strands of the target DNA, shown here is the target sequence on the positive DNA strand of the sequence.

[0231] In some embodiments, following mutation with an endonuclease, oligonucleotide amplification techniques (e.g., PCR) can be utilized to determine mutation efficiency and / or mutation type. In some embodiments, for a PCR reaction, primers that encompass (e.g., flank) an edited region are utilized to amplify a target sequence. One of skill in the art can design appropriate primers suitable for amplification of a target loci comprised in BATF, BATF3, and / or DDIT3 genes.

[0232] Embodiments of the disclosure include methods of knocking out or down expression of endogenous BATF, BATF3, and / or DDIT3 in a cell, comprising contacting the cell at least with Cas9, or a functionally equivalent alternative, and an appropriate guide RNA that targets BATF, BATF3, and / or DDIT3. The Cas9 and / or guide RNA may be provided to the cell through expression from one or more expression vectors coding therefor. The vector may be viral (retroviral, lentiviral, adenoviral, adeno-associated viral) or non-viral (naked plasmid DNA or chemically-modified mRNA).

[0233] In specific cases, other gene(s) than BATF, BATF3, and / or DDIT3 are mutated (e.g., gene edited, e.g., knocked down or knocked out, etc.), and this may or may not occur in the same step as the BATF, BATF3, and / or DDIT3 mutation. The reduction or full inhibition of expression may or may not utilize the same mechanism of gene editing as that for BATF, BATF3, and / or DDIT3, and the reduction or full inhibition of expression of the other gene(s) may occur before, during, or after the gene editing for BATF, BATF3, and / or DDIT3. The genes that are edited in the cells may be of any kind, but in specific embodiments the genes are genes whose gene products inhibit activity and / or proliferation of the BATF, BATF 3, and / or DDIT3KO cells. In specific cases the genes that are edited in addition io BATE, BATF3, and / or DDIT3 allow the cells to work more effectively in a tumor microenvironment. In specific cases, the genes are one or more of GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, CREB1, NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, AD0RA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD38, GR, and CD7. In specific embodiments, the TGFBR2 gene is knocked out or knocked down in the cells. In specific embodiments, the CISH gene is knocked out or knocked down in the cells. In specific embodiments, the CD38 gene is knocked out or knocked down in the cells. In specific embodiments, the Glucocorticoid receptor (GR) gene is knocked out or knocked down in the cells. In specific embodiments, the CREM gene is knocked out or down in the cells. In specific embodiments, the ICER gene is knocked out or down in the cells.

[0234] In some embodiments, any gene editing in the cells is carried out using one or more DNA-binding molecules, such as an endonuclease, for example but not limited to, Cas enzymes (and variants thereof), zinc-finger nucleases, TALENs, and Meganucleases, etc. In some embodiments, any gene editing in the cells is carried out by one or more DNA-binding nucleic acids, such as alteration via an RNA-guided endonuclease (RGEN). For example, the alteration can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins; in some embodiments, Cpfl is utilized instead of Cas9. In general, "CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system), and / or other sequences and transcripts from a CRISPR locus.

[0235] The CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a noncoding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains). One or more elements of a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.

[0236] In some aspects, a Cas nuclease and gRNA (including a fusion of crRNA specific for the target sequence and fixed tracrRNA) are introduced into the cell. In general, target sitesat the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing. The target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG. In this respect, the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Typically, "target sequence" generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.

[0237] The CRISPR system can induce double stranded breaks (DSBs) at the target site, followed by disruptions or alterations as discussed herein. In other embodiments, Cas9 variants, deemed "nickases," are used to nick a single strand at the target site. Paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, to affect gene expression.

[0238] The target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of the cell, such as within an organelle of the cell. Generally, a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.

[0239] Typically, in the context of an endogenous CRISPR system, formation of the CRISPR complex (comprising the guide sequence hybridized to the target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. The tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wildtype tracr sequence), may also form part of the CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. The tracr sequence has sufficient complementarity to atracr mate sequence to hybridize and participate in formation of the CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0240] One or more vectors driving expression of one or more elements of the CRISPR system can be introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites. Components can also be delivered to cells as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site"). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell.

[0241] A vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2.

[0242] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia). In some cases, Cpfl may be used as an endonuclease instead of Cas9. The CRISPR enzyme can exert direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector can encode a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10 A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from anuclease that cleaves both strands to a nickase (cleaves a single strand). In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.

[0243] In some embodiments, an enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0244] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more.

[0245] Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0246] The CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples ofprotein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione- 5- transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluore scent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by reference.

[0247] In some embodiments, a gene editing molecule includes a DNA-binding protein such as one or more zinc finger protein (ZFP) or transcription activator-like protein (TAL), fused to an effector protein such as an endonuclease. Examples include at least ZFNs, TALEs, and TALENs.

[0248] In some embodiments, a gene editing molecule comprises one or more zinc-finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or domain within a larger protein that binds DNA in a sequencespecific manner through one or more zinc fingers, regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among the ZFPs are artificial ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers. ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers. Generally, sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (-1, 2, 3 and 6) on a zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing moleculeis non-naturally occurring, e.g., is engineered to bind to a target site of choice. In some embodiments, the DNA-targeting molecule is or comprises a zinc-finger DNA binding domain fused to a DNA cleavage domain to form a zinc-finger nuclease (ZFN). In some embodiments, fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is from the Type IIS restriction endonuclease Fok I. Fok I generally catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. Many gene-specific engineered zinc fingers are available commercially. For example, Sangamo Biosciences (Richmond, CA, USA) has developed a platform (CompoZr) for zinc-finger construction in partnership with Sigma-Aldrich (St. Louis, MO, USA), allowing investigators to bypass zinc-finger construction and validation altogether, and provides specifically targeted zinc fingers for thousands of proteins (Gaj etal., Trends in Biotechnology, 10 2013, 31(7), 397-405). In some embodiments, commercially available zinc fingers are used or are custom designed. (See, for example, Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTil-IKT, and PZD0020).

[0249] In some embodiments, a gene editing molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein, See, e.g., U.S. Patent Publication No. 2011 / 0301073, incorporated by reference in its entirety herein. A TALE DNA binding domain or TALE is a polypeptide comprising one or more TALE repeat domains / units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. Each TALE repeat unit includes 1 or 2 DNA-binding residues making up the Repeat Variable Di-residue (RVD), typically at positions 12 and / or 13 of the repeat. The natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, NN binds to G or A, and NO binds to T and noncanonical (atypical) RVDs are also known. In some embodiments, TALEs may be targeted to any gene by design of TAL arrays with specificity to the target DNA sequence. The target sequence generally begins with a thymidine. In some embodiments, the molecule is a DNA binding endonuclease, such as a TALE nuclease (TALEN). In some aspects the TALEN is a fusion protein comprising a DNA binding domain derived from a TALE and a nuclease catalyticdomain to cleave a nucleic acid target sequence. In some embodiments, the TALEN recognizes and cleaves the target sequence in the gene. In some aspects, cleavage of the DNA results in double-stranded breaks. In some aspects the breaks stimulate the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. In some aspects, repair mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation or via the so-called microhomology -mediated end joining. In some embodiments, repair via NHEJ results in small insertions or deletions and can be used to disrupt and thereby repress the gene. In some embodiments, the modification may be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells in which a cleavage- induced mutagenesis event, i.e. a mutagenesis event consecutive to an NHEJ event, has occurred can be identified and / or selected by well-known methods in the art In some embodiments, TALE repeats are assembled to specifically target a gene. (Gaj et al., 2013). A library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., 2013). Custom-designed TALE arrays are commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). Specifically, TALENs that target CD38 are commercially available (See Gencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3). Exemplary molecules are described, e.g., in U.S. Patent Publication Nos. US 2014 / 0120622, and 2013 / 0315884. In some embodiments, TALENs are introduced as trans genes encoded by one or more plasmid vectors. In some aspects, the plasmid vector can contain a selection marker which provides for identification and / or selection of cells which received said vector.III. Immune Effector Cells

[0250] In some embodiments, the immediate disclosure concerns genetically engineering immune effector cells to comprise a mutation, such as a partial reduction of polypeptide function, knock-out, partial inhibition of expression (e.g., inhibition associated with mutation of one or more enhancer and / or TF binding site mutations), or full inhibition of expression of BATF, BATF3, and / or DDIT3. In some embodiments, the mutation of BATF, BATF3, and / or DDIT3 genes may occur by any mechanism, including at least by CRISPR / Cas9 technology, to make innovative and effective cellular therapies for the treatment of diseases of any kind, such as cancers and / or diseases characterized by the presence of TGF-p.

[0251] The present disclosure encompasses immune effector cells of any kind that are modified to have reduced or fully inhibited expression of BATF, BATF3, and / or DDIT3. In specific embodiments, the present disclosure encompasses immune effector cells of any kind that are modified to have reduced or fully inhibited expression of BATF. In specific embodiments, the present disclosure encompasses immune effector cells of any kind that are modified to have reduced or fully inhibited expression oiBATF3. In specific embodiments, the present disclosure encompasses immune effector cells of any kind that are modified to have reduced or fully inhibited expression of DDIT3. In specific embodiments, the reduction or full inhibition of expression of BATF, BATF3, and / or DDIT3 in the cells is a direct or indirect result of deliberate manipulation of the cells by the hand of man. The manipulation of the immune effector cells to have reduced or fully inhibited expression of BATF, BATF3, and / or DDIT3 may be by any mechanism, including by homologous or non-homologous recombination. In specific embodiments, the cells are manipulated to have reduced or fully inhibited expression of BATF, BATF3, and / or DDIT3 as a result of CRISPR technology mediated mutations, for example.

[0252] In some embodiments, immune effector cells have reduced or inhibited expression of BATF, BATF3, and / or DD IT 3 particularly by genetic engineering, as opposed to natural cells having one or more mutations that result in reduced expression of endogenous BATF, BATF 3, and / or DDIT3. Thus, in specific embodiments the immune effector cells are genetically engineered to reduce or inhibit expression of the endogenous BATF, BATF3, and / or DDIT3 in the genome of the immune effector cells. In specific embodiments, the immune effector cells are knocked out for expression of endogenous BATF, BATF3, and / or DDIT3.

[0253] The present disclosure encompasses immune effector cells of any kind, including conventional T cells, gamma-delta T cells, NK cells, NK T cells, invariant NK T cells, regulatory T cells, macrophages, B cells, dendritic cells, tumor-infiltrating lymphocytes, MSCs, or a mixture thereof. The cells may be allogeneic, autologous, or xenogeneic with respect to an individual, including an individual in need of the cells, such as an individual with cancer.

[0254] In particular embodiments, the immune effector cells are modified by the hand of man to express or otherwise produce one or more gene products other than the cell also being modified to have reduced or fully inhibited expression of BATF, BATF3, and / or DDIT3. Such additional modification(s) to the cell are not naturally present in the cell or are of exogenous origin with respect to the cell. The additional modification(s) may be of any kind, such as theimmune effector cells expressing a receptor, a cytokine, a suicide gene, or a chemokine, or a combination thereof, as examples.

[0255] When the immune effector cells having reduced or fully inhibited expression of BATF, BATF3, and / or DDIT3 are also modified additionally to produce or express a gene product that is not naturally present in the cell or is of exogenous origin, the order in which the immune effector cell is modified may be of any kind. For example, immune effector cells having reduced or fully inhibited expression of BATF, BATF3, and / or DDIT3 may be modified to have one or more additional modifications, wherein in other cases immune effector cells are modified to have reduced or fully inhibited expression of BATF, BATF3, and / or DDIT3 after they have been modified to produce or express a gene product that is not naturally present in the cell or is of exogenous origin.

[0256] In particular embodiments, the immune effector cell comprising mutations of BATF, BATF3, and / or DDIT3 is the same cell that is modified to express a receptor, such as an antigen receptor. In some embodiments, any kind of immune effector cell encompassed by the present disclosure may express an antigen receptor that may be of any kind, including a receptor directed towards an antigen that is a cancer antigen that may also be a tumor antigen. In specific embodiments, the receptor is a chimeric antigen receptor (CAR) or a T-cell receptor (TCR), for example. The immune effector cells may be specifically designed to have an engineered mutation of BATF, BATF3, and / or DDIT3 and be designed to express a heterologous antigen receptor that targets an antigen on cancer cells in the individual. That is, the cells may be tailored to include one or more antigen receptors that target antigens known to be present on cancer cells of the individual.

[0257] In particular embodiments, cells of the present disclosure are produced for the purpose of being used as off-the-shelf cells. For example, cells that have engineered mutations of BATF, BATF3, and / or DDIT3 are present in a repository, for example, and they are obtained from the repository and optionally engineered to have a further modification. In other cases, cells that have a modifications other than engineered mutations of BATF, BATF3, and / or DDIT3 are obtained from a repository and are engineered to have one or more engineered mutations of BATF, BATF3, and / or DDIT3. Following such modifications to the cells after obtaining them from a repository, the cells may be stored, or an effective amount of the cells may be provided to an individual in need thereof. Further engineering of BATF, BATF3, and / or DDIT3 KO or knock-down cells may be to engineer them to express an engineered receptor, such as an engineered antigen receptor that targets a tumor antigen suitable for treatment of an individual with a specific cancer expressing antigen.

[0258] In particular embodiments, immune effector cells with engineered mutations of BATF, BATF3, and / or DDIT3 also express one or more engineered antigen-targeting receptors and / or express at least one transfected (as opposed to endogenous to the cell) cytokine and / or express at least one suicide gene. In some cases of cells having engineered mutations of BATF, BATF 3, and / or DDIT3, different vectors encode the antigen-targeting receptor(s) vs. encode the suicide gene(s) and / or transfected cytokine(s). The immune cells, including NK cells, may be derived from cord blood, peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), bone marrow, or a mixture thereof. The NK cells may be derived from a cell line such as, but not limited to, NK-92 cells, for example. The NK cell may be a cord blood mononuclear cell, such as a CD56+ NK cell.

[0259] In some embodiments, the present disclosure describes and / or shows successful knock-out (KO) of the BATF, BATF3, and / or DDIT3 gene using CRISPR / Cas9 from natural killer (NK) cells derived from cord blood stored in cord blood banks. In some embodiments, the present disclosure describes and / or shows how TGF-P signaling pathway disrupted NK cells have enhanced cytotoxicity, secretory function, and / or metabolic fitness relative to WT TGF-P signaling pathway NK cells, following contact of the NK cells with TGF-p. In some embodiments, this enhanced antitumor activity was exemplified using tumor cell lines that have been shown to release TGF-P upon direct cell-cell contact by the NK cells and the tumor cell lines, leading to a microenvironment characterized by the presence of TGF-p. In certain embodiments, a tumor microenvironment is characterized by increased levels of TGF-P relative to non-tumor microenvironments. In some embodiments, a tumor microenvironment characterized by the presence of TGF-P is a solid tumor microenvironment.

[0260] In some cases, the immune effector cells having engineered mutations of BATF, BATF3, and / or DDIT3 have been expanded in the presence of an effective amount of universal antigen presenting cells (UAPCs) or fragments thereof, including in any suitable ratio. The cells may be cultured with the UAPCs at a ratio of 10: 1 to 1 : 10; 9: 1 to 1 :9; 8: 1 to 1 :8; 7: 1 to 1 :7; 6: 1 to 1 :6; 5: 1 to 1 :5; 4: 1 to 1 :4; 3: 1 to 1:3; 2: 1 to 1 :2; or 1 : 1, including at a ratio of 1 :2, for example. In some cases, the NK cells were expanded in the presence of IL-2, such as at a concentration of 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400, or 400-500 U / mL.

[0261] In some embodiments, following genetic modification with any vector(s), the immune effector comprising engineered mutations of BATF, BATF3, and / or DDIT3 may be immediately delivered to an individual or may be stored (or some of the cells are delivered to an individual and the rest of the cells are stored). In certain aspects, following geneticmodification, the cells may be propagated for days, weeks, or months ex vivo as a bulk population within about 1, 2, 3, 4, 5 days or more following gene transfer into cells. In a further aspect, the transfectants are cloned and a clone demonstrating presence of a single integrated or episomally maintained expression cassette or plasmid is expanded ex vivo. In some embodiments, the clone selected for expansion demonstrates reduced expression, or the absence of expression of BATF, BATF3, and / or DDIT3 gene products. In some embodiments, recombinant immune cells may be expanded by stimulation with IL-2, or other cytokines that bind the common gamma-chain (e.g., IL-7, IL-12, IL-15, IL-21, and others). In some embodiments, recombinant immune cells may be expanded by stimulation with artificial antigen presenting cells. In some embodiments, the genetically modified cells may be cryopreserved.

[0262] In some embodiments, cells are treated with one or more deactivating agents (e.g., a kinase inhibitor, e.g., Dasatinib, Nilotinib, Rapamycin, etc.) pre-cryopreservation. In some embodiments, technologies described herein comprise deactivating an NK cell, comprising treating an NK cell with an effective amount of one or more deactivating agents under conditions to produce a deactivated NK cell. In some embodiments, a deactivating agent is a kinase inhibitor. In some embodiments, a deactivating agent is a mechanistic target of rapamycin (mTOR) inhibitor. In some embodiments, the mTOR inhibitor is rapamycin, everolimus, and / or temsirolimus. In some embodiments, the mTOR inhibitor is rapamycin. In some embodiments, the deactivating agent is a tyrosine kinase (TK) inhibitor. In some embodiments, the TK inhibitor is Lorlatinib, Brigatinib, Ceritinib, Alectinib, Crizotinib, Bosutinib, Ponatinib, Nilotinib, Dasatinib, Imatinib, Zanubrutinib, Acalabrutinib, Ibrutinib, Capmatinib, Pexidartinib, Dacomitinib, Osimertinib, Erlotinib, Gefitinib, Lapatinib, Afatinib, Pemigatinib, Erdafitinib, Nintedanib, Gilteritinib, Midostaurin, Tucatinib, Neratinib, Baricitinib, Ruxolitinib, Fedratinib, Tofacitinib, Ripretinib, Selumetinib, Binimetinib, Cobimetinib, Trametinib, Upadacitinib, Avapritinib, Selpercatinib, Cabozantinib, Fostamatinib, Larotrectinib, Entrectinib, Axitinib, Regorafenib, Pazopanib, Sorafenib, Lenvatinib, Vandetanib, and / or Sunitinib. In some embodiments, the TK inhibitor is a BCR- Abl inhibitor. In some embodiments, the TK inhibitor is Bosutinib, Ponatinib, Nilotinib, Dasatinib, and / or Imatinib. In some embodiments, the TK inhibitor is Dasatinib and / or Nilotinib. In some embodiments, the TK inhibitor is Dasatinib.

[0263] In some embodiments, treatment with a deactivating agent is at any point during culturing of the NK cell. In some embodiments, the treatment is for about 24 to about 96 hours, about 36 to about 84 hours, or about 48 to about 72 hours. In some embodiments, the treatmentis for about 24 hours, about 48 hours, or about 72 hours. In some embodiments, the NK cell is treated with the deactivating agent at a concentration of about 1 to about 1000 nM. In some embodiments, the NK cell is treated with the deactivating agent at a concentration of about 5 to about 500 nM. In some embodiments, the NK cell is treated with the deactivating agent at a concentration of about 20 to about 200 nM. In some embodiments, the NK cell is treated with the deactivating agent at a concentration of about 30 to about 100 nM. In some embodiments, the deactivated NK cell has an increased expression of one or more of C-kit, CCR-5, CD62L and / or CXCR4, and / or decreased expression of one or more of NKG2D, DNAM, OX-40, TRAIL, HLA-DR, CD2, CD25, ICOS, and / or CD95 relative to an activated NK cell.

[0264] In some embodiments, technologies described herein comprise methods of maintaining the viability of a population of cells over at least 50% percent following cryopreservation of the population, comprising the step of subjecting the population to an effective amount of one or more deactivating agents (e.g., a tyrosine kinase inhibitor) to deactivate the cells prior to cryopreservation, cryopreserving the cells, and thawing the population, wherein upon thawing the viability of the population is over at least 50%. In some cases, upon thawing of the cells the viability of the population of cells is over at least 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% following cryopreservation of the population.

[0265] Embodiments of the disclosure encompass immune effector cells having engineered mutations (e.g., such as full or partial inhibition of expression, loss of polypeptide function, etc.) of BATF, BATF3, and / or DDIT3 genes, and transgenic expression of one or more engineered receptors, including one or more antigen receptors. In certain embodiments, one or more engineered antigen receptors are generated by the hand of man, for example using recombinant techniques, and are not natural to the immune effector cell. Although the engineered receptor(s) may be of any kind, in specific embodiments the receptor is a chimeric antigen receptor (CAR), T-cell receptor (TCR), homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and so forth.

[0266] In some embodiments, engineered cells described herein comprise an engineered mutation of BATF, BATF3, and / or DDIT3 genes, and one or more transgenic suicide genes. The immune effector cell may have an engineered mutation of BATF, BATF3, and / or DDIT3 genes, and may comprise a recombinant nucleic acid that encodes a suicide gene of any kind. Examples of suicide genes include engineered nonsecretable (including membrane bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see PCT / US2019 / 062009, which is incorporated by reference herein in its entirety), and they may be affected by delivery of anantibody that binds the TNF-alpha mutant. Examples of suicide gene / prodrug combinations that may be used are Herpes Simplex Virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5- fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxy cytidine kinase and cytosine arabinoside. The E.coli purine nucleoside phosphorylase, a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to toxic purine 6- methylpurine, may be utilized. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), Cytochrome p450 enzymes (CYP), Carboxypeptidases (CP), Carboxylesterase (CE), Nitroreductase (NTR), Guanine Ribosyltransferase (XGRTP), Glycosidase enzymes, Methionine-a,y-lyase (MET), and Thymidine phosphorylase (TP), as examples.

[0267] In some embodiments, cells of the disclosure may be obtained from an individual directly or may be obtained from a depository or other storage facility. In some embodiments, cells of the disclosure may be used as part of a cell therapy treatment regimen, and may be autologous or allogeneic with respect to the individual to which the cells are provided as therapy.

[0268] In some embodiments, cells may be from an individual in need of therapy for a medical condition, and following their manipulation to have an engineered mutation of BATF, BATF3, and / or DDIT3 genes, optional suicide gene, optional cytokine(s), and optional receptor(s) (using standard techniques for transduction and expansion for adoptive cell therapy, for example), the cells may be provided back to the individual from which they were originally sourced. In some cases, the cells are stored for later use for the individual or another individual.

[0269] The immune cells may be comprised in a population of cells, and that population may have a majority that have an engineered mutation of BATF, BATF3, and / or DDIT3 genes, and / or one or more suicide genes and / or one or more cytokines. A cell population may comprise 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% of immune cells that have an engineered mutation in BATF, BATF3, and / or DDIT3 genes, and / or one or more suicide genes and / or one or more cytokines and / or one or more engineered receptor; each of these gene products may or may not be produced as separate polypeptides.

[0270] In some embodiments, immune cells may be engineered to have reduced or inhibited BATF, BATF3, and / or DDIT3 gene expression, and engineered to have one or more suicide genes and / or one or more cytokines for the intent of being modular with respect to aspecific purpose. For example, cells may be generated, including for commercial distribution, having reduced or inhibited BATF, BATF3, and / or DDIT3 gene expression, and also expression of one or more suicide genes and / or one or more cytokines (or distributed with a nucleic acid that encodes a suicide gene for subsequent transduction), and a user may modify them to express one or more other genes of interest (including therapeutic genes) dependent upon their intended purpose(s). For instance, an individual interested in treating cancer cells may obtain or generate suicide gene-expressing cells (or heterologous cytokine-expressing cells) and modify them to have reduced or inhibited BATF, BATF3, and / or DDIT3 genes expression, or vice versa.

[0271] In particular embodiments, NK cells are utilized, and the genome of the NK cells comprise an engineered mutation of BATF, BATF3, and / or DDIT3 genes, resulting in reduced expression and / or inhibited function of BATF, BATF3, and / or DDIT3 gene products. In some embodiments, the NK cells may be further modified to include one or more transgenic suicide genes and / or one or more transgenic cytokines. In some embodiments, the genome of a cell may be modified in any manner, but in specific embodiments the genome is modified by CRISPR gene editing, for example. The genome of the cells may be modified to enhance effectiveness of the cells for any purpose.

[0272] Also provided herein, in particular embodiments, are at least methods of making, methods of using, and compositions comprising immune effector cells that have been conditioned (e.g., become accustomed to, adapted to, etc.) to be resistant to TGF-P induced immunosuppression. In some embodiments, cells are conditioned ex vivo through contact with one or more inhibitors of immunosuppressive TGF-P signaling pathways. In some embodiments, cells are conditioned over a period of at least or equal to, exactly or about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29, or 30 days, or greater than 30 days.IV. Methods of Treatment

[0273] Embodiments of the disclosure can include methods of treatment related to treatment of diseases characterized by TGF-P expression and / or microenvironments comprising TGF-P, such as but not limited to cancer immunotherapy or anti-pathogen immunotherapy. In certain embodiments, the methods of treatment comprise administration of one or more compositions comprising immune effector cells having an engineered mutation in BATF, BATF3, and / or DDIT3 genes. In some embodiments, methods include providing to an individual with a disease, such as but not limited to cancer, a pathogenic infection, connectivetissue disorders, tissue fibrosis, and / or chronic inflammatory conditions, an effective amount of immune effector cells having an engineered mutation oi BA IT', BATF3, and / or DDIT3, such that the cells have reduced expression and / or inhibited function of BATF, BATF3, and / or DDIT3 gene products.

[0274] In particular cases, an individual is provided an effective amount of cells having engineered mutations of BATF, BATF3, and / or DDIT3 genes. In specific cases, BATF, BATF3, and / or DDIT3 gene knock-out using CRISPR / Cas9 is utilized to genetically engineer immune cells used in various cellular therapies to increase their effectiveness against a target indication, such as but not limited to AML, MDS, and / or solid tumors, and these cellular therapies are provided to the individual.

[0275] As one example, chimeric antigen receptor (CAR)-T cells, such as those that are FDA-approved for the treatment of leukemia and lymphoma, can be genetically engineered to comprise mutations, such as loss of function mutations, in the BATF, BATF3, and / or DDIT3 genes, for the purpose of increasing their effectiveness in an immunosuppressive microenvironment, such as a microenvironment characterized by the presence of TGF-P, which in particular embodiments, can lead to the expansion of the efficacious use of a previously limited therapy. Moreover, in some embodiments, this genetic engineering strategy can be used in various other forms of cellular therapies, such as CAR-NK cells, TCR-NK cells, engineered TCR-T cells, tumor-infiltrating lymphocytes (TILs), etc. to potentiate them against various types of immunosuppressive microenvironments, such as those found in solid tumors, bone marrow niches, chronically inflamed tissues, acutely inflamed tissues, etc.

[0276] In certain embodiments, cells of the disclosure are provided to an individual for the purpose of improving a medical condition, such as cancer of any kind and / or pathogen infection of any kind. Use of the cells contemplated herein, including pharmaceutical compositions comprising the same, can be used for the prevention, treatment, or amelioration of a cancerous disease, such as a tumorous disease, or a pathogen infection. In particular embodiments, pharmaceutical compositions of the present disclosure may be particularly useful in preventing, ameliorating and / or treating cancer, including cancers that may or may not be solid tumors, for example.

[0277] In particular embodiments, the present disclosure contemplates, in part, the use of cells described herein administered either alone or in any combination with one or more other therapies, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient. In certain embodiments, any nucleic acid molecules or vectors provided herein may be stably integrated into the genome of the cells prior to deliver of the cells to the subject.

[0278] Furthermore, the disclosure relates to a method for the prevention, treatment or amelioration of a tumorous disease comprising the step of administering to a subject in the need thereof an effective amount of any cells that have reduced or inhibited level of expression of BATF, BATF3, and / or DDIT3 gene products, as contemplated herein.

[0279] In some embodiment, isolated cells obtained by any suitable methods or from cell lines and engineered as encompassed herein may be used as a medicament. In some embodiments, medicaments can be used for treating cancer or infections in an individual in need thereof. In some embodiments, medicaments can be used for treating inflammatory conditions in an individual in need thereof. In some embodiments, medicaments can be used for treating connective tissue disorders in an individual in need thereof. In some embodiments, medicaments can be used for treating tissue fibrosis in an individual in need thereof. In some embodiments, isolated cells according to the disclosure can be used in the manufacture of a medicament for treatment of any disease characterized by an immunosuppressive microenvironment comprising TGF-p.

[0280] In some embodiments, the present disclosure provides methods for treating individuals in need thereof, said methods comprising at least one of the following steps: (a) providing immune effector cells; (b) engineering the immune effector cells to have reduced or inhibited expression of at least BATF, BATF3, and / or DDIT3 gene products; (c) engineering the immune effector cells to express one or more engineered receptors (step (c) may come at the same time or before step (b)); (d) engineering the immune effector cells to express one or more cytokines (step (d) may come at the same time or before steps (b) and / or (c)); and (e) administering the engineered cells to an individual in need thereof, including an individual that has been determined to have cancer or is at risk of having cancer (such as at greater risk than an average person of a population).

[0281] In certain embodiments, the engineered cells were engineered for the purpose of producing enhanced expansion, persistence, cytotoxicity, secretory function, and / or metabolic fitness when compared to non-engineered cells. In certain embodiments, the engineered cells were engineered for the purpose of producing enhanced expansion, persistence, cytotoxicity, secretory function, and / or metabolic fitness in engineered cells when the engineered cells are introduced into an immunosuppressive environment, relative to non-engineered cells introduced into an immunosuppressive environment.

[0282] In specific embodiments, the engineered cells are engineered specifically for the purpose of improving cell functionality (e.g., expansion, persistence, cytotoxicity, etc.) relative to non-engineered cells in an microenvironment comprising mature and / or complexed TGF-p.In some embodiments, an immunosuppressive microenvironment characterized by the presence of TGF-P comprises TGF-P (mature and / or complexed) at levels greater than or equal to, exactly or about, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1,11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8,12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5,14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2,16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9,18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6,19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3,21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23,23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7,24.8, 24.9, or 25 ng / ml, or greater than 25 ng / ml, or any range derivable therein. In some embodiments, an immunosuppressive microenvironment characterized by the presence of TGF-P comprises active TGF-P at levels greater than or equal to, exactly or about, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6,11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3,13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15,15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7,16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4,18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1,20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8,21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5,23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, or 25 ng / ml, or greater than 25 ng / ml, or any range derivable therein. In some embodiments, TGF-P comprisesor consists essentially of TGF-pi. In some embodiments, TGF-P comprises or consists essentially of TGF-pi, TGF-P2, and / or TGF-P3. In some embodiments, TGF-P is one of the dominant or is the dominant cytokine in a microenvironment, such as a microenvironment created by a disorder. In some embodiments, TGF-P is one of the most abundant or is the most abundant cytokine in a microenvironment, such as a microenvironment created by a disorder.

[0283] In specific embodiments, engineered cells are engineered specifically for the purpose of improving cell functionality (e.g., expansion, persistence, cytotoxicity, secretion function, metabolic fitness, etc.) relative to non-engineered cells in a microenvironment comprising a levels of TGF-P higher than healthy and / or normal circulating / plasma levels of TGF-p. In specific embodiments, the engineered cells are engineered specifically for the purpose of improving cell functionality (e.g., expansion, persistence, cytotoxicity, secretion function, metabolic fitness, etc.) relative to non-engineered cells in a microenvironment comprising a levels of TGF-P greater than or equal to about 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, or 35 ng / ml, or higher.

[0284] In some embodiments, methods of treatment of the disclosure can be ameliorating, curative, or prophylactic for the individual. Methods may comprise use of autologous immunotherapy or allogeneic immunotherapy treatment. In specific cases, methods are utilized for allogeneic immunotherapy, insofar as it enables the transformation of NK cells, typically obtained from donors, into non-alloreactive cells. In some embodiments, this may be done under standard protocols and reproduced as many times as needed. The resultant engineered immune cells may be pooled and administered to one or several patients, being made available as an "off the shelf therapeutic product. The cells may be stored, such as cryopreserved.

[0285] In some embodiments, administration of the composition(s) of the cells are for cancerous diseases of any kind, including tumorous diseases, including B cell malignancies, multiple myeloma, lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gall bladder, bone, ovary, testes, endometrium, prostate, rectum, anus, or cervix, for example. Exemplary indications for administration of the composition(s) of the cells are cancerous diseases, including any malignancies that express one or more of certain antigens associated with the cancer of an individual. The administration of the composition(s) of the disclosure is useful for all stages (I, II, III, and / or IV) and types of cancer, including for minimal residual disease, early cancer, advanced cancer, and / or metastatic cancer and / or refractory cancer, for example.

[0286] The disclosure further encompasses co-administration protocols with other compounds, e.g., bispecific antibody constructs, targeted toxins, or other compounds, whichact via immune cells. The clinical regimen for co-administration of the inventive compound(s) may encompass co-administration at the same time, before or after the administration of the other component. Particular combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy.

[0287] Embodiments relate to a kit comprising constructs to produce the cells, a nucleic acid sequence as defined herein, a vector as defined herein and / or a host cell (such as an immune effector cell) as defined herein. It is also contemplated that the kit of this disclosure comprises a pharmaceutical composition as described herein above, either alone or in combination with further medicaments to be administered to an individual in need of medical treatment or intervention.V. Genetically Engineered Receptors

[0288] The immune cells of the present disclosure having reduced or inhibited expression of BATF, BATF 3, and / or DDIT3 gene products may be modified further to express one or more non-endogenous gene products. The gene product may or may not be a genetically engineered receptor. The receptor may be of any kind, including a receptor for an antigen, chemokine, or cytokine, for example. In cases wherein the receptor is for an antigen, the antigen may be a cancer antigen, including a solid tumor antigen.

[0289] The immune effector cells having reduced or inhibited expression of BATF, BATF3, and / or DDIT3 may be genetically engineered to express antigen receptors that target specific antigens, and such cells may be specifically designed to target one or more antigens that are present on cancer cells of an individual.

[0290] In specific embodiments, the immune effector cells comprising reduced or inhibited expression cT BA TF, BATF3, and / or DDIT3 may comprise an engineered antigen receptor, such as engineered TCRs or CARs. For example, the immune cells may be NK cells that are modified to express one or more CARs and / or TCRs having antigenic specificity for one or more specific antigens. In some aspects, the immune cells are engineered to express an antigenspecific CAR or antigen-specific TCR by knock-in of the CAR or TCR for example using CRISPR.

[0291] Suitable methods of modification are known in the art. See, for instance, Sambrook and Ausubel, supra. For example, the cells may be transduced to express a TCR having antigenic specificity for a cancer antigen using transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009.

[0292] In some embodiments, the cells comprise one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, ie., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature (e.g., chimeric).

[0293] Exemplary antigen receptors, including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, W02013 / 123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and / or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some aspects, the genetically engineered antigen receptors include a CAR as described in U.S. Patent No. : 7,446, 190, and those described in International Patent Application Publication No. : WO / 2014055668 Al.D. Chimeric Antigen Receptors

[0294] In some embodiments, the antigen-specific CAR comprises: a) one or more intracellular signaling domains, b) a transmembrane domain, and c) an extracellular domain comprising an antigen binding region that targets, including specifically binds, the desired antigen.

[0295] In some embodiments, the engineered antigen receptors include CARs, including activating or stimulatory CARs, costimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see e.g., Fedorov et al., 2013). The CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone.

[0296] Certain embodiments of the present disclosure concern the use of nucleic acids, including nucleic acids encoding an antigen-specific CAR polypeptide, including a CAR that has been humanized to reduce immunogenicity (hCAR), comprising at least one intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the antigen-specific CAR may recognize an epitope comprising the shared space between one or more antigens. In certain embodiments, the binding region can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and / or antigen binding fragments thereof. In another embodiment, that specificity is derived from a peptide (e.g., cytokine) that binds to a receptor.

[0297] It is contemplated that the human antigen targeting CAR component may be encoded by nucleic acids derived from human genes. In some embodiments, such components may be used to enhance cellular immunotherapy for human patients. In a specific embodiment, the disclosure includes a full-length antigen-specific CAR cDNA or coding region. The antigen binding regions or domain can comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Patent 7,109,304, incorporated herein by reference. The fragment can also be any number of different antigen binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells.

[0298] The arrangement could be multimeric, such as a diabody or multimers. The multimers are most likely formed by cross pairing of the variable portion of the light and heavy chains into a diabody. The hinge portion of the construct can have multiple alternatives from being totally deleted, to having the first cysteine maintained, to a proline rather than a serine substitution, to being truncated up to the first cysteine. The Fc portion can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. One could use just one of the Fc domains, e.g., either the CH2 or CH3 domain from human immunoglobulin. One could also use the hinge, CH2 and CH3 region of a human immunoglobulin that has been modified to improve dimerization. One could also use just the hinge portion of an immunoglobulin. One could also use portions of CD8alpha.

[0299] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Other costimulatory receptors include, but are not limited to one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137). In addition to a primarysignal initiated by CD3^, an additional signal provided by a human costimulatory receptor inserted in a human CAR is important for full activation of NK cells and could help improve in vivo persistence and the therapeutic success of the adoptive immunotherapy.

[0300] In some embodiments, an antigen-specific CAR is constructed with specificity for the antigen, such as the antigen being expressed on a normal or non-diseased cell type or on a diseased cell type. Thus, the CAR includes in its extracellular portion one or more antigenbinding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the antigen-specific CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0301] In certain embodiments, the antigen-specific CAR may be co-expressed with a cytokine to improve persistence when there is a low amount of tumor-associated antigen. For example, the CAR may be co-expressed with one or more cytokines, such as IL-7, IL-2, IL- 15, IL- 12, IL- 18, IL-21, or a combination thereof. In some embodiments, the CAR may be coexpressed with IL-15. In some embodiments, the CAR may be co-expressed with IL-2L In some embodiments, the CAR may be co-expressed with IL- 15 and IL-21.

[0302] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR), or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof as it is found that introns stabilize the mRNA. Also, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0303] It is contemplated that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Patent No. 6,410,319. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.

[0304] Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno- associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into immune cells. Suitable vectors for use in accordance with the method of the present disclosure are non-replicating in the immune cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintainthe viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

[0305] In some aspects, the antigen-specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0306] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (z.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD30, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0307] In certain embodiments, the platform technologies disclosed herein to genetically modify immune cells, such as NK cells, comprise (i) non-viral gene transfer using an electroporation device (e.g., a nucleofector), (ii) CARs that signal through endodomains (e.g., CD28 / CD3-(^, CD137 / CD3-(^, or other combinations), (iii) CARs with variable lengths of extracellular domains connecting the recognition domain (e.g., a CD70-recognition domain) to the cell surface, and, in some cases, (iv) artificial antigen presenting cells (aAPC) derived from K562 to be able to robustly and numerically expand CAR+immune cells (see e.g., Singh etal., 2008; Singh et al., 2011).

[0308] In certain embodiments, a CAR recognizes a TROP2 antigen. In certain embodiments, an anti-TROP2 CAR comprises an antigen binding region of a TROP2-specific antibody. In certain embodiments, an anti- TROP2 CAR comprises a scFv of a TROP2-specific antibody. In certain embodiments, a scFv comprises a heavy chain variable region (VH), and a variable region light chain (VL). In certain embodiments, a TROP2-specific antibody ishumanized. In certain embodiments, an anti-TROP2 CAR comprises a scFv of a TROP2- specific humanized antibody. In certain embodiments, an anti-TROP2 CAR comprises a codon optimized polynucleotide encoding a scFv. In certain embodiments, a TROP-2 specific antibody is or comprises an RS7 antibody. In certain embodiments, a RS7 antibody is a murine RS7 (mRS7). In certain embodiments, a RS7 antibody is a humanized RS7 (hRS7). In certain embodiments, an anti-CD70 CAR comprises, or is encoded by, a sequence at least or equal to, exactly or about 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% identity to any one or more of SEQ ID NOs: 46-57. In certain embodiments, an anti-TROP2 CAR and / or an NK cell comprising the same is as described in International Patent Application Publication No. WO 2023 / 283644 A2, published on January 12, 2023, which is incorporated herein by reference in its entirety.SEQ ID NO: 46 - Exemplary anti-TROP2 CAR encoding construct (e.g., iC9mRs7VLVH28H28z!5) nucleic acid sequenceATGCTCGAGGGAGTGCAGGTGGAAACCATCTCCCCAGGCGACGGGCGCACCTTCCCCAAGCG CGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAAGTTGATTCCT CCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGG GAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTA TGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATG TGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTCGACGGATTTGGTGATGTCGGT GCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGG CCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTG GCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAG GTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCAGCAGGA CCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACC TGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTG AACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCA GGCCTGTGGTGGGGAGCAGAAAGATCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACG AGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTC GACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTAC TTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGG ACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCT AATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAA AAAACTTTTCTTTAAAACATCAGCTTCGCGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCG GGGACGTGGAGGAAAATCCCGGGCCCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCT AT T T T AAAAG G T G T C GAG T G C T C T AGAGAG GAG AT T GAG C T GAG C GAG T C T C AC AAAT T C AT GTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTATTG CTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCC TACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTT CACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATATTA CTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGTTGGAAATAAAGGGCTCT ACAAGCGGCTCAGGAAAACCTGGATCAGGCGAAGGGTCTACGGTGAAGCTGCAGGAGTCAGG ACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATATACCT TCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATACTGATGACTTCAAGGGACGGTTTGCCTT CTCTTTGGAAACCTCTGCCACCACTGCCTATTTGCAGATCAACAACCTCAAAAGTGAGGACA TGGCTACATATTTCTGTGCAAGAGGGGGGTTCGGTAGTAGCTACTGGTACTTCGATGTCTGG GGCCAAGGGACCACGGTCACCGTCTCCTCACCGTACGCCATTGAAGTTATGTATCCTCCTCC T T AC C T AGAC AAT GAGAAGAG C AAT G GAAC CATTATCCATGT GAAAG G GAAAC AC C T T T G T C CAAGTCCCCTATTTCCCGGACCTTCTAAGCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAA GAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCC GCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTGAAG TTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCT CAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGA TGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCA CGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGAT GTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCA GTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCA TCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGC GAC C T GAAGAAGAT C GAG GAC C T GAT C C AGAG C AT G C AC AT C GAC G C C AC C C T G TAG AC C GA GAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGG TGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTG GCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGA ACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCA TCAACACCAGCTGA ( SEQ ID NO : 4 6 )SEQ ID NO: 47 - Exemplary anti-TROP2 CAR encoding construct (e.g., iC9mRs7VLVH28H28z!5) amino acid sequenceMLEGVQVET I SPGDGRTFPKRGQTCWHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGW EEGVAQMSVGQRAKLT I SPDYAYGATGHPGI I PPHATLVFDVELLKLESGGGSGVDGFGDVG ALESLRGNADLAYILSMEPCGHCLI INNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVE VKGDLTAKKMVLALLELAQQDHGALDCCVWILSHGCQASHLQFPGAVYGTDGCPVSVEKIV NI FNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTF DQLDAI SSLPTPSDI FVSYSTFPGFVSWRDPKSGSWYVETLDDI FEQWAHSEDLQSLLLRVA NAVSVKGIYKQMPGCFNFLRKKLFFKTSASRAEGRGSLLTCGDVEENPGPMEFGLSWLFLVA ILKGVQCSREDIQLTQSHKFMSTSVGDRVS I TCKASQDVS IAVAWYQQKPGQSPKLLIYSAS YRYTGVPDRFTGSGSGTDFTFT I SSVQAEDLAVYYCQQHYI TPLTFGAGTKLELKRLE IKGS TSGSGKPGSGEGSTVKLQESGPELKKPGETVKI SCKASGYTFTNYGMNWVKQAPGKGLKWMG WINTYTGEPTYTDDFKGRFAFSLETSATTAYLQINNLKSEDMATYFCARGGFGSSYWYFDVW GQGTTVTVSSPYAIEVMYPPPYLDNEKSNGT I IHVKGKHLCPSPLFPGPSKPKFWVLVWGG VLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGD VESNPGPMRI SKPHLRS I S IQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVI S DLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI SLESGDAS IHDTVENLI IL ANNSLSSNGNVTESGCKECEELEEKNIKEFLQS FVHIVQMFINTS ( SEQ ID NO : 47 )SEQ ID NO: 48 - Exemplary anti-TROP2 CAR encoding construct (e.g., iC9mRS7VHVL28H28icz!5) nucleic acid sequenceATGCTCGAGGGAGTGCAGGTGGAAACCATCTCCCCAGGCGACGGGCGCACCTTCCCCAAGCG CGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAAGTTGATTCCT CCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGG GAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTA TGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATG TGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTCGACGGATTTGGTGATGTCGGT GCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGG CCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTG GCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAG GTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCAGCAGGA CCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACC TGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTG AACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCA GGCCTGTGGTGGGGAGCAGAAAGATCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACG AGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTC GACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTAC TTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGG ACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCT AATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAA AAAACTTTTCTTTAAAACATCAGCTTCGCGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCG GGGACGTGGAGGAAAATCCCGGGCCCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCT ATTTTAAAAGGTGTCCAGTGCTCTAGAGAGGTGAAGCTGCAGGAGTCAGGACCTGAGCTGAA GAAGCC T GGAGAGACAGT CAAGAT C T CC T GCAAGGC T T C T GGATATACC T T CACAAAC TAT G GAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACC TACACTGGAGAGCCAACATATACTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAAC CTCTGCCACCACTGCCTATTTGCAGATCAACAACCTCAAAAGTGAGGACATGGCTACATATT TCTGTGCAAGAGGGGGGTTCGGTAGTAGCTACTGGTACTTCGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCATTGGAAATAAAGGGCTCTACAAGCGGCTCAGGAAAACCTGGATC AGGCGAAGGGTCTACGGACATTCAGCTGACCCAGTCTCACAAATTCATGTCCACATCAGTAG GAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTATTGCTGTAGCCTGGTAT CAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGG AGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTG TGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATATTACTCCGCTCACGTTC GGTGCTGGGACCAAGCTGGAGCTGAAACGGCCGTACGCCATTGAAGTTATGTATCCTCCTCC T T AC C T AGAC AAT GAGAAGAG C AAT G GAAC CATTATCCATGT GAAAG G GAAAC AC C T T T G T C CAAGTCCCCTATTTCCCGGACCTTCTAAGCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAA GAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCC GCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTGAAG TTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCT CAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGA TGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCA CGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGAT GTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCA GTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCA TCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGAC C T GAAGAAGAT C GAG GAG C T GAT C C AGAG C AT G GAG AT C GAG G C GAG C C T G TAG AC C GA GAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGG TGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTG GCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGA ACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCA TCAACACCAGCTGA ( SEQ ID NO : 48 )SEQ ID NO: 49 - Exemplary anti-TROP2 CAR encoding construct (e.g., iC9mRS7VHVL28H28icz!5) amino acid sequenceMLEGVQVET I SPGDGRTFPKRGQTCWHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGW EEGVAQMSVGQRAKLT I SPDYAYGATGHPGI I PPHATLVFDVELLKLESGGGSGVDGFGDVG ALESLRGNADLAYILSMEPCGHCLI INNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVE VKGDLTAKKMVLALLELAQQDHGALDCCVWILSHGCQASHLQFPGAVYGTDGCPVSVEKIV NI FNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTF DQLDAI SSLPTPSDI FVSYSTFPGFVSWRDPKSGSWYVETLDDI FEQWAHSEDLQSLLLRVA NAVSVKGIYKQMPGCFNFLRKKLFFKTSASRAEGRGSLLTCGDVEENPGPMEFGLSWLFLVA ILKGVQCSREVKLQESGPELKKPGETVKI SCKASGYTFTNYGMNWVKQAPGKGLKWMGWINT YTGEPTYTDDFKGRFAFSLETSATTAYLQINNLKSEDMATYFCARGGFGSSYWYFDVWGQGT TVTVSSLE IKGSTSGSGKPGSGEGSTDIQLTQSHKFMSTSVGDRVS I TCKASQDVS IAVAWY QQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFT I SSVQAEDLAVYYCQQHYI TPLTF GAGTKLELKRPYAIEVMYPPPYLDNEKSNGT I IHVKGKHLCPSPLFPGPSKPKFWVLVWGG VLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGD VESNPGPMRI SKPHLRS I S IQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVI S DLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI SLESGDAS IHDTVENLI IL ANNSLSSNGNVTESGCKECEELEEKNIKEFLQS FVHIVQMFINTS ( SEQ ID NO : 49 )SEQ ID NO: 50 - Exemplary anti-TROP2 CAR encoding construct (e.g., iC9hRS7VLVH28H28zl5) nucleic acid sequenceATGCTCGAGGGAGTGCAGGTGGAAACCATCTCCCCAGGCGACGGGCGCACCTTCCCCAAGCG CGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAAGTTGATTCCT CCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGG GAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTA TGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATG TGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTCGACGGATTTGGTGATGTCGGT GCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGG CCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTG GCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAG GTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCAGCAGGA CCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACC TGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTG AACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCA GGCCTGTGGTGGGGAGCAGAAAGATCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACG AGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTC GACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTAC TTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGG ACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAA AAAACTTTTCTTTAAAACATCAGCTTCGCGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCG GGGACGTGGAGGAAAATCCCGGGCCCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCT ATTTTAAAAGGTGTCCAGTGCTCTAGAGAGGACATCCAGCTGACCCAGTCTCCATCCTCCCT GTCTGCATCTGTAGGAGACAGAGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTATTG CTGTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACTCGGCATCC TACCGGTACACTGGAGTCCCTGATAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCT GAG CAT GAG GAG T C T G C AAC C T GAAGAT T T T G GAG TTTATTACTGT GAG C AAC AT TAT AT TA CTCCGCTCACGTTCGGTGCTGGGACCAAGGTGGAGATCAAACGTTTGGAAATAAAGGGCTCT ACAAGCGGCTCAGGAAAACCTGGATCAGGCGAAGGGTCTACGCAGGTCCAACTGCAGCAATC TGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACA CCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCCCCTGGACAAGGGCTTAAATGGATG GGCTGGATAAACACCTACACTGGAGAGCCAACATATACTGATGACTTCAAGGGACGGTTTGC CTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTCCAGATCAGCAGCCTAAAGGCTGACG ACACTGCCGTGTATTTCTGTGCAAGAGGGGGGTTCGGTAGTAGCTACTGGTACTTCGATGTC TGGGGCCAAGGGTCCCTGGTCACCGTCTCCTCACCGTACGCCATTGAAGTTATGTATCCTCC T C C T T AC C T AGAC AAT GAGAAGAG C AAT G GAAC CATTATCCATGT GAAAG G GAAAC AC C T T T GTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCAAATTTTGGGTGCTGGTGGTGGTTGGT GGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCA CCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTG AAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGA GCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTG AGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACA TGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGA GATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCAT CCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGT TCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATC AGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACAC CGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGC AGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATC CTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGA GGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGT TCATCAACACCAGCTGA ( SEQ ID NO : 50 )SEQ ID NO: 51 - Exemplary anti-TROP2 CAR encoding construct (e.g., iC9hRS7VLVH28H28zl5) amino acid sequenceMLEGVQVET I SPGDGRTFPKRGQTCWHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGW EEGVAQMSVGQRAKLT I SPDYAYGATGHPGI I PPHATLVFDVELLKLESGGGSGVDGFGDVG ALESLRGNADLAYILSMEPCGHCLI INNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVE VKGDLTAKKMVLALLELAQQDHGALDCCVWILSHGCQASHLQFPGAVYGTDGCPVSVEKIV NI FNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTF DQLDAI SSLPTPSDI FVSYSTFPGFVSWRDPKSGSWYVETLDDI FEQWAHSEDLQSLLLRVA NAVSVKGIYKQMPGCFNFLRKKLFFKTSASRAEGRGSLLTCGDVEENPGPMEFGLSWLFLVA ILKGVQCSREDIQLTQSPSSLSASVGDRVS I TCKASQDVS IAVAWYQQKPGKAPKLLIYSAS YRYTGVPDRFSGSGSGTDFTLT I SSLQPEDFAVYYCQQHYI TPLTFGAGTKVE IKRLE IKGS TSGSGKPGSGEGSTQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQI SSLKADDTAVYFCARGGFGSSYWYFDV WGQGSLVTVSSPYAIEVMYPPPYLDNEKSNGT I IHVKGKHLCPSPLFPGPSKPKFWVLVWG GVLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAG DVESNPGPMRI SKPHLRS I S IQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVI SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI SLESGDAS IHDTVENLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS FVHIVQMFINTS ( SEQ ID NO : 51 )SEQ ID NO: 52 - Exemplary anti-TROP2 CAR encoding construct (e.g., iC9TROP2VLVH28H28z!5) nucleic acid sequenceATGCTCGAGGGAGTGCAGGTGGAAACCATCTCCCCAGGCGACGGGCGCACCTTCCCCAAGCG CGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAAGTTGATTCCT CCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGG GAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTA TGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATG TGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTCGACGGATTTGGTGATGTCGGT GCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTG GCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAG GTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCAGCAGGA CCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACC TGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTG AACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCA GGCCTGTGGTGGGGAGCAGAAAGATCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTC GACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTAC TTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGG ACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCT AATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAA AAAACTTTTCTTTAAAACATCAGCTTCGCGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCG GGGACGTGGAGGAAAATCCCGGGCCCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAGAGAGCAGGTCCAACTGCAGCAATCTGGGTCTGAGTT GAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACAAACT ATGGAATGAACTGGGTGAAGCAGGCCCCTGGACAAGGGCTTAAATGGATGGGCTGGATAAAC ACCTACACTGGAGAGCCAACATATACTGATGACTTCAAGGGACGGTTTGCCTTCTCCTTGGA CACCTCTGTCAGCACGGCATATCTCCAGATCAGCAGCCTAAAGGCTGACGACACTGCCGTGT ATTTCTGTGCAAGAGGGGGGTTCGGTAGTAGCTACTGGTACTTCGATGTCTGGGGCCAAGGG TCCCTGGTCACCGTCTCCTCATTGGAAATAAAGGGCTCTACAAGCGGCTCAGGAAAACCTGGATCAGGCGAAGGGTCTACGGACATCCAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTG TAGGAGACAGAGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTATTGCTGTAGCCTGG TATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACTCGGCATCCTACCGGTACAC TGGAGTCCCTGATAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA G T C T G C AAC C T GAAGAT T T T G GAG TTTATTACTGT GAG C AAC AT TATATTACTCCGCT GAG G TTCGGTGCTGGGACCAAGGTGGAGATCAAACGTCCGTACGCCATTGAAGTTATGTATCCTCC T C C T T AC C T AGAC AAT GAGAAGAG C AAT G GAAC CATTATCCATGT GAAAG G GAAAC AC C T T TGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCAAATTTTGGGTGCTGGTGGTGGTTGGT GGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTG AAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGA GCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTG AGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACA TGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGA GATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCAT CCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGT TCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATC AGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGC AGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATC CTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGA GGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGT TCATCAACACCAGCTGA ( SEQ ID NO : 52 )SEQ ID NO: 53 - Exemplary anti-TROP2 CAR encoding construct (e.g., iC9TROP2VLVH28H28z!5) amino acid sequenceMLEGVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGW EEGVAQMSVGQRAKLTISPDYAYGATGHPGI IPPHATLVFDVELLKLESGGGSGVDGFGDVG ALESLRGNADLAYILSMEPCGHCLI INNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVE VKGDLTAKKMVLALLELAQQDHGALDCCVWILSHGCQASHLQFPGAVYGTDGCPVSVEKIV NI FNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTF DQLDAISSLPTPSDI FVSYSTFPGFVSWRDPKSGSWYVETLDDI FEQWAHSEDLQSLLLRVA NAVSVKGIYKQMPGCFNFLRKKLFFKTSASRAEGRGSLLTCGDVEENPGPMEFGLSWLFLVA ILKGVQCSREQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWIN TYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSLEIKGSTSGSGKPGSGEGSTDIQLTQSPSSLSASVGDRVS ITCKASQDVS IAVAW YQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLT FGAGTKVEIKRPYAIEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSKPKFWVLVWG GVLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAG DVESNPGPMRISKPHLRS IS IQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVI SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAS IHDTVENLI I LANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ( SEQ ID NO : 53 )SEQ ID NO: 54 - Exemplary humanized Rs7 VH amino acid sequenceQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTD DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS ( SEQ ID NO : 54 )SEQ ID NO: 55 - Exemplary humanized Rs7 VL amino acid sequenceDIQLTQSPSSLSASVGDRVS ITCKASQDVS IAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKV ( SEQ ID NO : 55 )SEQ ID NO: 56 - Exemplary murine Rs7 VH amino acid sequenceVKLQESGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYTDD FKGRFAFSLETSATTAYLQINNLKSEDMATYFCARGGFGSSYWYFDVWGQGTTVTVSS ( SEQ ID NO : 56 )SEQ ID NO: 57 - Exemplary murine Rs7 VL amino acid sequenceDIQLTQSHKFMSTSVGDRVS ITCKASQDVS IAVAWYQQKPGQSPKLLIYSASYRYTGVPDRF TGSGSGTDFTFTISSVQAEDLAVYYCQQHYITPLTFGAGTKLELKR ( SEQ ID NO : 57 )

[0309] In certain embodiments, a CAR recognizes a CD70 antigen. In certain embodiments, an anti-CD70 CAR comprises a CD27 derived polypeptide. In certain embodiments, an anti-CD70 CAR comprises, or is encoded by, a sequence at least or equal to, exactly or about 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% identity to any one or more of SEQ ID NOs: 29-37. In certain embodiments, an anti-CD70 CAR and / or an NK cell comprising the same is as described in International Patent Application Publication No. WO 2021 / 142127 Al, published on July 15, 2021, which is incorporated herein by reference in its entirety. In certain embodiments, an anti-CD70 CAR and / or an NK cell comprising the same is as described in International Patent Application Publication No. WO 2022 / 159791 Al, published on July 28, 2022, which is incorporated herein by reference in its entirety. In certain embodiments, an anti-CD70 CAR and / or an NK cell comprising the same is as described in International Patent Application Publication No. WO 2023 / 278520 Al, published on January 5, 2023, which is incorporated herein by reference in its entirety.SEQ ID NO: 29 - Exemplary truncated CD27 with CD28 transmembrane domain amino acid sequenceMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQC DPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLP NPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCS SDFIRFWVLVWGGVLACYSLLVTVAFI I FWV ( SEQ ID NO : 29 )SEQ ID NO: 30 - Exemplary truncated CD27 amino acid sequenceMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQC DPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLP NPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCS SDFIR ( SEQ ID NO : 30 )SEQ ID NO: 31 - Exemplary CD28 TMDFWVLVWGGVLACYSLLVTVAFI I FWV ( SEQ ID NO : 31 )SEQ ID NO: 32 - Exemplary full length WT CD27 amino acid sequenceMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQC DPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLP NPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCS SDFIRILVI FSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQ EDYRKPEPACSP ( SEQ ID NO : 32 )SEQ ID NO: 33 - Exemplary truncated WT CD27 amino acid sequenceATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR ( SEQ ID NO : 33 )SEQ ID NO: 34 - Exemplary CD27 signal peptide (SP) amino acid sequenceMARPHPWWLCVLGTLVGLS ( SEQ ID NO : 34 )SEQ ID NO: 35 - Exemplary CD3z derived Intracellular Signaling Domain (ICD) amino acid sequenceRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG ( SEQ ID NO : 35 )SEQ ID NO: 36 - Exemplary CD28 derived Intracellular Signaling Domain (ICD) amino acid sequenceRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS ( SEQ ID NO : 36 )SEQ ID NO: 37 - Exemplary DAP10 derived Intracellular Signaling Domain (ICD) amino acid sequenceLCARPRRSPAQEDGKVYINMPGRG ( SEQ ID NO : 37 )SEQ ID NO: 58 - Exemplary Homo sapiens CD27 molecule nucleic acid sequenceATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTAC TCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGA TGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGT GATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAG CTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGT GTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCA AACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTT ACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACT TCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGC TCCGATTTTATTCGCATCCTTGTGATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGG GGCCCTGTTCCTCCATCAACGAAGGAAATATAGATCAAACAAAGGAGAAAGTCCTGTGGAGC CTGCAGAGCCTTGTCGTTACAGCTGCCCCAGGGAGGAGGAGGGCAGCACCATCCCCATCCAG GAGGATTACCGAAAACCGGAGCCTGCCTGCTCCCCCTGA ( SEQ ID NO : 58 )SEQ ID NO: 59 - Exemplary codon optimized CD27 nucleic acid sequenceATGGCGAGACCACACCCTTGGTGGCTGTGCGTACTCGGCACACTTGTAGGTCTGTCCGCTAC ACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAA TGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGC GACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTC TTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGT GTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCT AACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACT TTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCT TCCGATTTCATCCGCATACTGGTCATCTTTTCTGGAATGTTCCTTGTGTTCACCCTGGCAGG AGCCCTGTTCCTTCACCAGAGACGCAAGTACAGGTCAAACAAGGGTGAGAGCCCCGTTGAAC CCGCAGAGCCGTGTAGATACTCATGTCCTAGAGAAGAAGAGGGCTCTACTATCCCTATTCAG GAAGATTATAGAAAACCCGAACCCGCGTGCAGCCCC ( SEQ ID NO : 59 )SEQ ID NO: 60 - Exemplary anti-CD70 CAR encoding construct (e.g.,CD27TrCD28tmdicd3zl5) nucleic acid sequenceATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCA GCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAA CCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGG CATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCT CTAGACTGCCATGCTCGAGGATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACC CTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCA GGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGC ATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCAC CATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCA CCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCA CACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCA CATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCAC CCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCC GCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTGAAG TTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCT CAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGA TGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGAT GTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCA TCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGC GAG C T GAAGAAGAT C GAG GAG C T GAT C C AGAG C AT G GAG AT C GAG G C GAG C C T G TAG AC C GA GAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGG TGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA ( SEQ ID NO : 60 )SEQ ID NO: 61 - Exemplary anti-CD70 CAR encoding construct (e.g.,CD27TrCD28tmdicd3zl5) amino acid sequenceMTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPWPHPYRVGDTVWVRRHQTKNLE PRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGT LVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHH TRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSP HPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVWGG VLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGD VESNPGPMRISKPHLRS IS IQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVIS DLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAS IHDTVENLI IL ANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ( SEQ ID NO : 61 )SEQ ID NO: 62 - Exemplary CD27 signal peptide nucleic acid sequenceATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCA ( SEQ ID NO : 62 )SEQ ID NO: 63 - Exemplary truncated wild-type human CD27 nucleic acid sequenceGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTG CCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTC AGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGT GAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGC TGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTC TTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACC CACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGT GCAGCTCCGATTTTATTCGC ( SEQ ID NO : 63 )SEQ ID NO: 64 - Exemplary codon optimized extracellular domain of CD27 nucleic acid sequenceGCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTG TCAAATGTGTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGC AGTGCGACCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGT GAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGC AGAGTGTGCTTGTAGAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCAC TGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCCTCAGCCCGCATCCTCAACCAACA CATCTTCCTTACGTGTCAGAAATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGT GCTCTTCCGATTTCATCCGC ( SEQ ID NO : 64 )

[0310] In certain embodiments, a CAR recognizes a CD5 antigen. In certain embodiments, an anti-CD5 CAR comprises an antigen binding region of a CD5-specific antibody. In certain embodiments, an anti-CD5 CAR comprises a scFv of a CD5-specific antibody. In certainembodiments, a scFv comprises a heavy chain variable region (VH), and a variable region light chain (VL). In certain embodiments, the CD5-specific antibody is humanized (hCD5). In certain embodiments, an anti-CD5 CAR comprises a scFv of a CD5-specific humanized antibody. In certain embodiments, an anti-CD5 CAR comprises a codon optimized polynucleotide encoding a scFv. In certain embodiments, an anti-CD5 CAR comprises, or is encoded by, a sequence at least or equal to, exactly or about 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% identity to any one or more of SEQ ID NOs: 65-79. In certain embodiments, an anti-CD5 CAR and / or an NK cell comprising the same is as described in International Patent Application Publication No. WO 2019 / 023396 Al, published on January 31, 2019, which is incorporated herein by reference in its entirety.SEQ ID NO: 65 - Exemplary hCD5-scFv nucleic acid sequenceATGGAGTTCGGTCTCAGTTGGCTGTTTTTGGTTGCTATCTTGAAGGGCGTCCAATGCAGCCG GGACATCCAGATGACCCAGTCTCCCTCTAGCATGTCAGCGAGTCTTGGTGATCGAGTGACGA TTACCTGCAGAGCCTCTCAAGATATAAACAGCTATCTTTCATGGTTCCAACAGAAGCCGGGG AAGTCCCCAAAAACTCTCATATACAGGGCGAATCGACTCGTAGACGGTGTGCCTTCAAGGTT TTCCGGGAGTGGTAGTGGCACAGATTACACACTTACAATCTCTTCATTGCAGTATGAGGATT TCGGGATCTACTACTGTCAACAGTACGACGAATCCCCATGGACGTTTGGGGGCGGGACCAAA CTTGAGATAAAAGGGAGCACATCTGGAAGTGGTAAACCTGGGTCAGGGGAGGGTTCCACAAA AGGACAAATTCAACTTGTCCAAAGCGGTCCTGGTCTTAAGAAGCCTGGAGGGTCTGTCAGGA TAAGTTGTGCGGCATCCGGCTACACCTTCACCAACTATGGGATGAACTGGGTGAAACAAGCG CCTGGGAAAGGTCTTCGATGGATGGGCTGGATTAATACCCACACTGGAGAGCCCACTTACGC TGATGATTTCAAAGGACGATTTACCTTCTCCTTGGATACTTCCAAGAGTACCGCGTACTTGC AAATCAACAGTCTCCGGGCTGAAGACACGGCCACATACTTCTGTACGCGGAGAGGGTATGAC TGGTATTTTGATGTGTGGGGTCAGGGAACAACCGTGACTGTTTCAAGC ( SEQ ID NO : 65 )SEQ ID NO: 66 - Exemplary hCD5-scFv VL nucleic acid sequenceGACATCCAGATGACCCAGTCTCCCTCTAGCATGTCAGCGAGTCTTGGTGATCGAGTGACGAT TACCTGCAGAGCCTCTCAAGATATAAACAGCTATCTTTCATGGTTCCAACAGAAGCCGGGGA AGTCCCCAAAAACTCTCATATACAGGGCGAATCGACTCGTAGACGGTGTGCCTTCAAGGTTTTCCGGGAGTGGTAGTGGCACAGATTACACACTTACAATCTCTTCATTGCAGTATGAGGATTT CGGGATCTACTACTGTCAACAGTACGACGAATCCCCATGGACGTTTGGGGGCGGGACCAAAC TTGAGATAAAA ( SEQ ID NO : 66 )SEQ ID NO: 67 - Exemplary hCD5-scFv VH nucleic acid sequenceCAAATTCAACTTGTCCAAAGCGGTCCTGGTCTTAAGAAGCCTGGAGGGTCTGTCAGGATAAG TTGTGCGGCATCCGGCTACACCTTCACCAACTATGGGATGAACTGGGTGAAACAAGCGCCTG GGAAAGGTCTTCGATGGATGGGCTGGATTAATACCCACACTGGAGAGCCCACTTACGCTGATGATTTCAAAGGACGATTTACCTTCTCCTTGGATACTTCCAAGAGTACCGCGTACTTGCAAAT CAACAGTCTCCGGGCTGAAGACACGGCCACATACTTCTGTACGCGGAGAGGGTATGACTGGT ATTTTGATGTGTGGGGTCAGGGAACAACCGTGACTGTTTCAAGC ( SEQ ID NO : 67 )SEQ ID NO: 68 - Exemplary hCD5-scFv amino acid sequenceMEFGLSWLFLVAILKGVQCSRDIQMTQSPSSMSASLGDRVT I TCRASQDINSYLSWFQQKPG KSPKTLIYRANRLVDGVPSRFSGSGSGTDYTLT I SSLQYEDFGIYYCQQYDESPWTFGGGTK LE IKGSTSGSGKPGSGEGSTKGQIQLVQSGPGLKKPGGSVRI SCAASGYTFTNYGMNWVKQA PGKGLRWMGWINTHTGEPTYADDFKGRFTFSLDTSKSTAYLQINSLRAEDTATYFCTRRGYDWYFDVWGQGTTVTVSS ( SEQ ID NO : 68 )SEQ ID NO: 69 - Exemplary hCD5-scFv VL amino acid sequenceDIQMTQSPSSMSASLGDRVT I TCRASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRF SGSGSGTDYTLT I SSLQYEDFGIYYCQQYDESPWTFGGGTKLE IK ( SEQ ID NO : 69 )SEQ ID NO: 70 - Exemplary hCD5-scFv VH amino acid sequenceQIQLVQSGPGLKKPGGSVRI SCAASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYAD DFKGRFTFSLDTSKSTAYLQINSLRAEDTATYFCTRRGYDWYFDVWGQGTTVTVSS ( SEQ ID NO : 70 )SEQ ID NO: 71 - Exemplary humanized anti-CD5 CAR encoding construct nucleic acid sequenceAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCAT GGAAAAATACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAAC AGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCT CAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCAT CAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAA TCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCAC AACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCA ATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCT GAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGAC CCCTGCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTC TGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAAC TAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCC TGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCC GATCGTTTAGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGA CGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCC GCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTG TATTTGTCTGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGG TCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTT GGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACC TTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACA CCCAGACCAGGTGGGGTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGG TCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCC CTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCT AGGCGCCCCCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCC CTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTC TACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCG ACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTA AGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGG TGGACCATCCTCTAGACTGCCATGCTCGAGGGAGTGCAGGTGGAAACCATCTCCCCAGGAGA CGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAG ATGGAAAGAAAGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAG CAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAA ACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCAC ATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTC GACGGATTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACAT CCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGT CCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCC TCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTT GCTGGAGCTGGCGCAGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTC ACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCT GTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAA GCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGG CCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTC CAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGA CATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCT CCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAG TCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGG TTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGCTAGCAGAGCCGAGGGCA GGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGAGTTCGGTCTC AGTTGGCTGTTTTTGGTTGCTATCTTGAAGGGCGTCCAATGCAGCCGGGACATCCAGATGAC CCAGTCTCCCTCTAGCATGTCAGCGAGTCTTGGTGATCGAGTGACGATTACCTGCAGAGCCT CTCAAGATATAAACAGCTATCTTTCATGGTTCCAACAGAAGCCGGGGAAGTCCCCAAAAACT CTCATATACAGGGCGAATCGACTCGTAGACGGTGTGCCTTCAAGGTTTTCCGGGAGTGGTAG T GGCACAGAT TACACAC T TACAAT C T C T T CAT T GCAGTAT GAGGAT T T CGGGAT C TAG TAG T GTCAACAGTACGACGAATCCCCATGGACGTTTGGGGGCGGGACCAAACTTGAGATAAAAGGG AGCACATCTGGAAGTGGTAAACCTGGGTCAGGGGAGGGTTCCACAAAAGGACAAATTCAACT TGTCCAAAGCGGTCCTGGTCTTAAGAAGCCTGGAGGGTCTGTCAGGATAAGTTGTGCGGCAT CCGGCTACACCTTCACCAACTATGGGATGAACTGGGTGAAACAAGCGCCTGGGAAAGGTCTT CGATGGATGGGCTGGATTAATACCCACACTGGAGAGCCCACTTACGCTGATGATTTCAAAGG ACGATTTACCTTCTCCTTGGATACTTCCAAGAGTACCGCGTACTTGCAAATCAACAGTCTCC GGGCTGAAGACACGGCCACATACTTCTGTACGCGGAGAGGGTATGACTGGTATTTTGATGTG TGGGGTCAGGGAACAACCGTGACTGTTTCAAGCTACGTCACCGTCTCTTCACAGGATCCCGC CGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACA GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGC CAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG TGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA CGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGC TTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCA GGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCAT TACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGA AAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGC GGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTG CCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAG CAATCCCGGGCCCATGCGGATCAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACC TGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGC TGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAA GAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACG TGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGC CTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAA CAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAG AGAAGAAC AT C AAAGAG T T T C T G C AGAG C T T C G T G GAG AT C G T G C AGAT G T T C AT C AAC AC C AGCTGACGCGTCATCATCGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTC CAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGAT AAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGT TTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATA GAGAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GGGCCAAACAGGATAT C TGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGC CAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCC CAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAA GGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTT CGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGT CCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATC CGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCG GGGGTCTTT C AC AC AT G GAG CAT GT AT CAAAAT TAAT TTGGTTTTTTTTCT TAAGTAT T TAG ATTAAATGGCCATAGTACTTAAAGTTACATTGGCTTCCTTGAAATAAACATGGAGTATTCAG AATGTGTCATAAATATTTCTAATTTTAAGATAGTATCTCCATTGGCTTTCTACTTTTTCTTT TATTTTTTTTTGTCCTCTGTCTTCCATTTGTTGTTGTTGTTGTTTGTTTGTTTGTTTGTTGG TTGGTTGGT TAAT T T T T T T T T AAAGAT C C T ACAC TAT AG T T CAAGC T AGAC TAT T AGC T AC T CTGTAACCCAGGGTGACCTTGAAGTCATGGGTAGCCTGCTGTTTTAGCCTTCCCACATCTAA GATTACAGGTATGAGCTATCATTTTTGGTATATTGATTGATTGATTGATTGATGTGTGTGTG TGTGATTGTGTTTGTGTGTGTGACTGTGAAAATGTGTGTATGGGTGTGTGTGAATGTGTGTA TGTATGTGTGTGTGTGAGTGTGTGTGTGTGTGTGTGCATGTGTGTGTGTGTGACTGTGTCTA TGTGTATGACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTGA AAAAATATTCTATGGTAGTGAGAGCCAACGCTCCGGCTCAGGTGTCAGGTTGGTTTTTGAGA CAGAGTCTTTCACTTAGCTTGGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAA ACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAAT AGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCG CCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTC TCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCT GACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTC CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCC TCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGT GGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAA TATGTATCCGCTCAT GAGAC AAT AAC C C T GAT AAAT G C T T C AAT AAT AT T GAAAAAG GAAGA GTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACG AGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAG AACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATT GACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTG CCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAG GAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACC GGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAA CAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATA GACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGG GGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATG GATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTC AGAC C AAG TTTACTCATATATACTT T AGAT T GAT T T AAAAC TTCATTTTTAATT T AAAAG GA TCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTC CACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCG CGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATC AAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACT GTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATA CCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCG GGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCG TGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGG TCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCT GTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTG CTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAG TGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGC GGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCT GGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAG CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAAT TGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTTGCT C T T AG GAG T T T C C T AAT AC AT C C C AAAC T C AAAT AT AT AAAG C AT T T GAG TTGTTCTATGCC CTAGGGGGCGGGGGGAAGCTAAGCCAGCTTTTTTTAACATTTAAAATGTTAATTCCATTTTA AATGCACAGATGTTTTTATTTCATAAGGGTTTCAATGTGCATGAATGCTGCAATATTCCTGT TAG C AAAG C T AG TAT AAAT AAAAAT AGAT AAAC G T G GAAAT TACT T AGAG TTTCTGTCATTA ACGTTTCCTTCCTCAGTTGACAACATAAATGCGCTGCTGAGCAAGCCAGTTTGCATCTGTCA G GAT C AAT TTCCCATTATGC C AG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GACATATACATGTG ( SEQ ID NO : 71 )SEQ ID NO: 72 - Exemplary anti-CD5 CAR encoding construct (e.g., pSFG4-CD5-Ev3- CAR) nucleic acid sequenceAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCAT GGAAAAATACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAAC AGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCT CAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCAT CAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAA TCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCAC AACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCA ATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCT GAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGAC CCCTGCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCC TGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCC GATCGTTTAGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGA CGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCC GCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTG TATTTGTCTGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGG TCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTT GGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACC TTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACA CCCAGACCAGGTGGGGTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGG TCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCC CTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCT AGGCGCCCCCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCC CTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTC TACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCG ACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTA AGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTC AAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGG TGGACCATCCTCTAGACTGCCATGCTCGAGGGAGTGCAGGTGGAAACCATCTCCCCAGGAGA CGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAG ATGGAAAGAAAGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAG CAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAA ACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCAC ATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTC GACGGATTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACAT CCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGT CCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCC TCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTT GCTGGAGCTGGCGCAGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCT GTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAA GCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGG CCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTC CAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGA CATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCT CCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAG TCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGG TTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGCTAGCAGAGCCGAGGGCA GGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGAGTTCGGCTTG AGTTGGTTGTTCCTTGTGGCGATACTCAAAGGCGTTCAATGTAGCCGAGACATAAAAATGAC CCAGTCTCCGTCATCTATGTATGCAAGCCTCGGCGAGCGAGTGACCATCACGTGCAAGGCGA G T C AAGAT AT AAAC AG CTACTTGTCATGGTTC C AAC AAAAAC GAG G GAAAT GAG C T AAGAC C CTGATCTATAGAGCCAATCGCCTGGTTGACGGTGTCCCCTCCCGCTTTAGCGGCTCCGGAAG CGGTCAAGATTACTCTCTCACAATTTCTTCCTTGGATTATGAAGACATGGGGATCTACTATT GTCAACAGTATGACGAATCCCCGTGGACTTTCGGTGGCGGTACCAAATTGGAAATAAAGGGC TCTACAAGCGGCTCAGGAAAACCTGGATCAGGCGAAGGGTCTACGAAGGGCCAGATACAACT CGTTCAAAGTGGGCCAGAACTCAAAAAACCGGGAGAAACAGTGAAAATTTCTTGTAAGGCAT CAGGATACACATTCACAAACTACGGGATGAATTGGGTCAAACAAGCACCCGGAAAGGGGCTG CGCTGGATGGGGTGGATCAACACACATACTGGGGAACCTACTTACGCAGACGATTTCAAGGGCAGATTCGCCTTTTCTTTGGAGACCTCCGCCTCTACTGCATACTTGCAGATAAACAACCTGAAGAATGAAGATACCGCCACCTACTTCTGTACGCGCAGGGGCTACGATTGGTATTTTGATGTA TGGGGGGCAGGCACCACTGTTACTGTGTCAAGCTACGTCACCGTCTCTTCACAGGATCCCGC CCTGGAAGAGAAGAAAGGCAATTACGTCGTGACCGACCACGGCAGCTGTGTGCGGGCTTGTG GCGCCGATAGCTACGAGATGGAAGAGGACGGCGTGCGGAAGTGCAAGAAGTGCGAGGGCCCC TGCAGAAAAGTGTGCAACGGCATCGGCATCGGAGAGTTCAAGGATAGCCTGAGCATCAACGC CACCAACATCAAGCACTTCAAGAACTGCACCAGCATCAGCGGCGACCTGCACATCCTGCCCG TGGCCTTTAGAGGCGACAGCTTCACCCACACCCCCCCACTGGATCCCCAGGAACTGGACATC CTGAAAACCGTGAAAGAGATCACAGGCTTTCTGCTGATTCAGGCCTGGCCCGAGAACCGGAC AGACCTGCACGCCTTCGAGAACCTGGAAATCATCAGAGGCCGGACCAAGCAGCACGGCCAGT TTTCTCTGGCCGTGGTGTCCCTGAACATCACCAGCCTGGGCCTGCGGAGCCTGAAAGAAATC AGCGACGGCGACGTGATCATCTCCGGCAACAAGAACCTGTGCTACGCCAACACCATCAATTG GAAGAAGCTGTTCGGCACCTCCGGCCAGAAAACAAAGATCATCTCTAACCGGGGCGAGAACA GCTGCAAAGCCACCGGACAAGTGTGCCACGCCCTGTGTAGCCCTGAGGGCTGTTGGGGACCC GAGCCCAGAGATTGCGTGTCCTGCCGGAATGTGTCCAGAGGCCGCGAGTGCGTGGACAAGTG CAACCTGCTGGAAGGCGAGCCCCGCGAGTTTGTGGAAAACAGCGAGTGCATCCAGTGCCACC CCGAGTGTCTGCCCCAGGCCATGAACATTACCTGCACCGGCAGAGGCCCCGACAACTGTATC CAGTGCGCCCACTACATCGACGGCCCCCACTGCGTGAAAACCTGTCCAGCTGGCGTGATGGG AGAGAACAACACCCTCGTGTGGAAGTACGCCGACGCCGGCCATGTGTGCCACCTGTGTCACC CCAATTGCACCTACGGCTGTACCGGCCCTGGCCTGGAAGGCTGTCCTACCAACGGCCCCAAG ATCCCTTCTAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTA TAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCC TGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAG CCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGC AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG AGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGC CTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACC AGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCC CGGGCCCATGCGGATCAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCC TGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTC AGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGAT CGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACC CCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAA AGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCT GAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGA ACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA CGCGTCATCATCGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCT C T AG T T T T GAG T C AAC AAT AT GAG GAG C T GAAG C C T AT AGAG TAG GAG C C AT AGAT AAAAT A AAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCA AGC TAGC T TAAGTAACGCCAT T T T GCAAGGCAT GGAAAAAT AGAT AAC T GAGAATAGAGAAG TTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGT AAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACA GGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATG CGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCT GAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCG CTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCG ATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTT GTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTC T T T CACACAT GCAGCAT G TAT CAAAAT T AAT TTGGTTTTTTTTCT T AAG TAT T T AGAT T AAA TGGCCATAGTACTTAAAGTTACATTGGCTTCCTTGAAATAAACATGGAGTATTCAGAATGTGTCATAAATATTTCTAATTTTAAGATAGTATCTCCATTGGCTTTCTACTTTTTCTTTTATTTT TTTTTGTCCTCTGTCTTCCATTTGTTGTTGTTGTTGTTTGTTTGTTTGTTTGTTGGTTGGTT GGTTAATTTTTTTT T AAAGAT C C TAG AC T AT AG T T C AAG C T AGAC TATTAGCTACTCTGTAA CCCAGGGTGACCTTGAAGTCATGGGTAGCCTGCTGTTTTAGCCTTCCCACATCTAAGATTAC AGGTATGAGCTATCATTTTTGGTATATTGATTGATTGATTGATTGATGTGTGTGTGTGTGAT TGTGTTTGTGTGTGTGACTGTGAAAATGTGTGTATGGGTGTGTGTGAATGTGTGTATGTATG TGTGTGTGTGAGTGTGTGTGTGTGTGTGTGCATGTGTGTGTGTGTGACTGTGTCTATGTGTA TGACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTGAAAAAAT ATTCTATGGTAGTGAGAGCCAACGCTCCGGCTCAGGTGTCAGGTTGGTTTTTGAGACAGAGT CTTTCACTTAGCTTGGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAA GAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGAT GCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTA CAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCG CCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGA TACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT TTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA TCCGCTCAT GAGAC AAT AAG C C T GAT AAAT G C T T C AAT AAT AT T GAAAAAG GAAGAG TAT GA GTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTT GCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGG TTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTT TTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCC GGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACC AGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTA ACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCT GAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGT TGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGG ATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTAT TGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAG ATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAA CGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA AG T T TAG T CAT AT AT AC T T T AGAT T GAT T T AAAAC T T CAT T T T T AAT T T AAAAGGAT C T AGG TGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGA GCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAAT CTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTT CTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGC TCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGG ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGA AAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATG GAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACA TGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCT GATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA GCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACG ACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTTGCTCTTAGG AG T T T C C T AAT AC AT C C C AAAC T C AAAT AT AT AAAG C AT T T GAG TTGTTCTATGCCCTAGGG GGCGGGGGGAAGCTAAGCCAGCTTTTTTTAACATTTAAAATGTTAATTCCATTTTAAATGCA CAGATGTTTTTATTTCATAAGGGTTTCAATGTGCATGAATGCTGCAATATTCCTGTTACCAA AG C TAG TAT AAAT AAAAATAGAT AAAC G T G GAAAT TACT T AGAG TTTCTGTCAT T AAC G T T T CCTTCCTCAGTTGACAACATAAATGCGCTGCTGAGCAAGCCAGTTTGCATCTGTCAGGATCA ATTTCCCATTATGC C AG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GAC AT A TACATGTG ( SEQ ID NO : 72 )SEQ ID NO: 73 - Exemplary anti-CD5 CAR encoding construct (e.g., pSFG4-hCD5-Ev3-CAR) nucleic acid sequenceAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCAT GGAAAAATACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAAC AGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCT CAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCAT CAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAA TCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCAC AACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCA ATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCT GAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGAC CCCTGCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTC TGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAAC TAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCC TGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCC GATCGTTTAGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGA CGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCC GCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTG TATTTGTCTGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGG TCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTT GGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACC TTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACA CCCAGACCAGGTGGGGTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGG TCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCC CTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCT AGGCGCCCCCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCC CTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTC TACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCG ACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTA AGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTC AAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGG TGGACCATCCTCTAGACTGCCATGCTCGAGGGAGTGCAGGTGGAAACCATCTCCCCAGGAGA CGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAG ATGGAAAGAAAGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAG CAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAA ACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCAC ATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTC GACGGATTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACAT CCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCC TCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTT GCTGGAGCTGGCGCAGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTC ACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCT GTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAA GCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGG CCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTC CAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGA CATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCT CCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAG TCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGG TTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGCTAGCAGAGCCGAGGGCA GGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGAGTTCGGTCTC AGTTGGCTGTTTTTGGTTGCTATCTTGAAGGGCGTCCAATGCAGCCGGGACATCCAGATGAC CCAGTCTCCCTCTAGCATGTCAGCGAGTCTTGGTGATCGAGTGACGATTACCTGCAGAGCCT CTCAAGATATAAACAGCTATCTTTCATGGTTCCAACAGAAGCCGGGGAAGTCCCCAAAAACT CTCATATACAGGGCGAATCGACTCGTAGACGGTGTGCCTTCAAGGTTTTCCGGGAGTGGTAG T GGCACAGAT TACACAC T TACAAT C T C T T CAT T GCAGTAT GAGGAT T T CGGGAT C TAG TAG T GTCAACAGTACGACGAATCCCCATGGACGTTTGGGGGCGGGACCAAACTTGAGATAAAAGGG AGCACATCTGGAAGTGGTAAACCTGGGTCAGGGGAGGGTTCCACAAAAGGACAAATTCAACT TGTCCAAAGCGGTCCTGGTCTTAAGAAGCCTGGAGGGTCTGTCAGGATAAGTTGTGCGGCAT CCGGCTACACCTTCACCAACTATGGGATGAACTGGGTGAAACAAGCGCCTGGGAAAGGTCTT CGATGGATGGGCTGGATTAATACCCACACTGGAGAGCCCACTTACGCTGATGATTTCAAAGG ACGATTTACCTTCTCCTTGGATACTTCCAAGAGTACCGCGTACTTGCAAATCAACAGTCTCC GGGCTGAAGACACGGCCACATACTTCTGTACGCGGAGAGGGTATGACTGGTATTTTGATGTG TGGGGTCAGGGAACAACCGTGACTGTTTCAAGCTACGTCACCGTCTCTTCACAGGATCCCGC CCTGGAAGAGAAGAAAGGCAATTACGTCGTGACCGACCACGGCAGCTGTGTGCGGGCTTGTG GCGCCGATAGCTACGAGATGGAAGAGGACGGCGTGCGGAAGTGCAAGAAGTGCGAGGGCCCC TGCAGAAAAGTGTGCAACGGCATCGGCATCGGAGAGTTCAAGGATAGCCTGAGCATCAACGC CACCAACATCAAGCACTTCAAGAACTGCACCAGCATCAGCGGCGACCTGCACATCCTGCCCG TGGCCTTTAGAGGCGACAGCTTCACCCACACCCCCCCACTGGATCCCCAGGAACTGGACATC CTGAAAACCGTGAAAGAGATCACAGGCTTTCTGCTGATTCAGGCCTGGCCCGAGAACCGGAC AGACCTGCACGCCTTCGAGAACCTGGAAATCATCAGAGGCCGGACCAAGCAGCACGGCCAGT TTTCTCTGGCCGTGGTGTCCCTGAACATCACCAGCCTGGGCCTGCGGAGCCTGAAAGAAATC AGCGACGGCGACGTGATCATCTCCGGCAACAAGAACCTGTGCTACGCCAACACCATCAATTG GAAGAAGCTGTTCGGCACCTCCGGCCAGAAAACAAAGATCATCTCTAACCGGGGCGAGAACA GCTGCAAAGCCACCGGACAAGTGTGCCACGCCCTGTGTAGCCCTGAGGGCTGTTGGGGACCC GAGCCCAGAGATTGCGTGTCCTGCCGGAATGTGTCCAGAGGCCGCGAGTGCGTGGACAAGTG CAACCTGCTGGAAGGCGAGCCCCGCGAGTTTGTGGAAAACAGCGAGTGCATCCAGTGCCACC CCGAGTGTCTGCCCCAGGCCATGAACATTACCTGCACCGGCAGAGGCCCCGACAACTGTATC CAGTGCGCCCACTACATCGACGGCCCCCACTGCGTGAAAACCTGTCCAGCTGGCGTGATGGG AGAGAACAACACCCTCGTGTGGAAGTACGCCGACGCCGGCCATGTGTGCCACCTGTGTCACC CCAATTGCACCTACGGCTGTACCGGCCCTGGCCTGGAAGGCTGTCCTACCAACGGCCCCAAG ATCCCTTCTAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTA TAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCC TGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAG CCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGC AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG AGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGC CTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCC CGGGCCCATGCGGATCAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCC TGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTC AGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGAT CGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACC CCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAA AGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCT GAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGA ACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA CGCGTCATCATCGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCT C T AG T T T T GAG T C AAC AAT AT GAG GAG C T GAAG C C T AT AGAG TAG GAG C C AT AGAT AAAAT A AAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCA AGC TAGC T TAAGTAACGCCAT T T T GCAAGGCAT GGAAAAAT AGAT AAC T GAGAATAGAGAAG TTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGT AAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACA GGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATG CGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCT GAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCG CTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCG ATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTT GTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTC T T T CACACAT GCAGCAT G TAT CAAAAT T AAT TTGGTTTTTTTTCT T AAG TAT T T AGAT T AAA TGGCCATAGTACTTAAAGTTACATTGGCTTCCTTGAAATAAACATGGAGTATTCAGAATGTG TCATAAATATTTCTAATTTTAAGATAGTATCTCCATTGGCTTTCTACTTTTTCTTTTATTTT TTTTTGTCCTCTGTCTTCCATTTGTTGTTGTTGTTGTTTGTTTGTTTGTTTGTTGGTTGGTT GGTTAATTTTTTTT T AAAGAT C C TAG AC T AT AG T T C AAG C T AGAC TATTAGCTACTCTGTAA CCCAGGGTGACCTTGAAGTCATGGGTAGCCTGCTGTTTTAGCCTTCCCACATCTAAGATTAC AGGTATGAGCTATCATTTTTGGTATATTGATTGATTGATTGATTGATGTGTGTGTGTGTGAT TGTGTTTGTGTGTGTGACTGTGAAAATGTGTGTATGGGTGTGTGTGAATGTGTGTATGTATG TGTGTGTGTGAGTGTGTGTGTGTGTGTGTGCATGTGTGTGTGTGTGACTGTGTCTATGTGTA TGACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTGAAAAAAT ATTCTATGGTAGTGAGAGCCAACGCTCCGGCTCAGGTGTCAGGTTGGTTTTTGAGACAGAGT CTTTCACTTAGCTTGGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAA GAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGAT GCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTA CAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCG CCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAG CTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGA TACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT TTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA TCCGCTCAT GAGAC AAT AAC C C T GAT AAAT G C T T C AAT AAT AT T GAAAAAG GAAGAG TAT GA GTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTT GCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGG TTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTT TTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCC GGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACC AGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTA ACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGT TGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGG ATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTAT TGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAG ATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAA CGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA AG T T TAG T CAT AT AT AC T T T AGAT T GAT T T AAAAC T T CAT T T T T AAT T T AAAAGGAT C T AGG TGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGA GCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAAT CTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTT CTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGC TCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGG ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGA AAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATG GAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACA TGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCT GATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA GCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACG ACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAG CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTTGCTCTTAGG AG T T T C C T AAT AC AT C C C AAAC T C AAAT AT AT AAAG C AT T T GAC TTGTTCTATGCCCTAGGG GGCGGGGGGAAGCTAAGCCAGCTTTTTTTAACATTTAAAATGTTAATTCCATTTTAAATGCA CAGATGTTTTTATTTCATAAGGGTTTCAATGTGCATGAATGCTGCAATATTCCTGTTACCAA AG C TAG TAT AAAT AAAAAT AGAT AAAC G T G GAAAT TACT T AGAG TTTCTGTCAT T AAC G T T T CCTTCCTCAGTTGACAACATAAATGCGCTGCTGAGCAAGCCAGTTTGCATCTGTCAGGATCA ATTTCCCATTATGC C AG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GAC AT A TACATGTG ( SEQ ID NO : 73 )SEQ ID NO: 74 - Exemplary anti-CD5 scFv (humanized) nucleic acid sequenceATGGAGTTCGGTCTCAGTTGGCTGTTTTTGGTTGCTATCTTGAAGGGCGTCCAATGCAGCCG GGACATCCAGATGACCCAGTCTCCCTCTAGCATGTCAGCGAGTCTTGGTGATCGAGTGACGA TTACCTGCAGAGCCTCTCAAGATATAAACAGCTATCTTTCATGGTTCCAACAGAAGCCGGGG AAGTCCCCAAAAACTCTCATATACAGGGCGAATCGACTCGTAGACGGTGTGCCTTCAAGGTT TTCCGGGAGTGGTAGTGGCACAGATTACACACTTACAATCTCTTCATTGCAGTATGAGGATT TCGGGATCTACTACTGTCAACAGTACGACGAATCCCCATGGACGTTTGGGGGCGGGACCAAA CTTGAGATAAAAGGGAGCACATCTGGAAGTGGTAAACCTGGGTCAGGGGAGGGTTCCACAAA AGGACAAATTCAACTTGTCCAAAGCGGTCCTGGTCTTAAGAAGCCTGGAGGGTCTGTCAGGA TAAGTTGTGCGGCATCCGGCTACACCTTCACCAACTATGGGATGAACTGGGTGAAACAAGCG CCTGGGAAAGGTCTTCGATGGATGGGCTGGATTAATACCCACACTGGAGAGCCCACTTACGC TGATGATTTCAAAGGACGATTTACCTTCTCCTTGGATACTTCCAAGAGTACCGCGTACTTGC AAATCAACAGTCTCCGGGCTGAAGACACGGCCACATACTTCTGTACGCGGAGAGGGTATGAC TGGTATTTTGATGTGTGGGGTCAGGGAACAACCGTGACTGTTTCAAGC ( SEQ ID NO : 74 )SEQ ID NO: 75 - Exemplary anti-CD5 scFv (humanized) amino acid sequenceMEFGLSWLFLVAILKGVQCSRDIQMTQSPSSMSASLGDRVTITCRASQDINSYLSWFQQKPG KSPKTLIYRANRLVDGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTK LEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPGLKKPGGSVRISCAASGYTFTNYGMNWVKQA PGKGLRWMGWINTHTGEPTYADDFKGRFTFSLDTSKSTAYLQINSLRAEDTATYFCTRRGYD WYFDVWGQGTTVTVSS ( SEQ ID NO : 75 )SEQ ID NO: 76 - Exemplary anti-CD5 scFv (murine) nucleic acid sequenceATGGAGTTCGGCTTGAGTTGGTTGTTCCTTGTGGCGATACTCAAAGGCGTTCAATGTAGCCG AGACATAAAAATGACCCAGTCTCCGTCATCTATGTATGCAAGCCTCGGCGAGCGAGTGACCA TCACGTGCAAGGCGAGTCAAGATATAAACAGCTACTTGTCATGGTTCCAACAAAAACCAGGG AAATCACCTAAGACCCTGATCTATAGAGCCAATCGCCTGGTTGACGGTGTCCCCTCCCGCTTTAGCGGCTCCGGAAGCGGTCAAGATTACTCTCTCACAATTTCTTCCTTGGATTATGAAGACA TGGGGATCTACTATTGTCAACAGTATGACGAATCCCCGTGGACTTTCGGTGGCGGTACCAAA TTGGAAATAAAGGGCTCTACAAGCGGCTCAGGAAAACCTGGATCAGGCGAAGGGTCTACGAA GGGCCAGATACAACTCGTTCAAAGTGGGCCAGAACTCAAAAAACCGGGAGAAACAGTGAAAATTTCTTGTAAGGCATCAGGATACACATTCACAAACTACGGGATGAATTGGGTCAAACAAGCA CCCGGAAAGGGGCTGCGCTGGATGGGGTGGATCAACACACATACTGGGGAACCTACTTACGC AGACGATTTCAAGGGCAGATTCGCCTTTTCTTTGGAGACCTCCGCCTCTACTGCATACTTGC AGATAAACAACCTGAAGAATGAAGATACCGCCACCTACTTCTGTACGCGCAGGGGCTACGATTGGTATTTTGATGTATGGGGGGCAGGCACCACTGTTACTGTGTCAAGC ( SEQ ID NO : 76 )SEQ ID NO: 77 - Exemplary anti-CD5 scFv (murine) amino acid sequenceMEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCQQYDESPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVSS ( SEQ ID NO : 77 )SEQ ID NO: 78 - Exemplary anti-CD5 CAR encoding construct (e.g.,CD5CARCD28hinge.28tmd3z.IL15) nucleic acid sequenceATGGAGTTCGGCTTGAGTTGGTTGTTCCTTGTGGCGATACTCAAAGGCGTTCAATGTAGCCGAGACATAAAAATGACCCAGTCTCCGTCATCTATGTATGCAAGCCTCGGCGAGCGAGTGACCATCACGTGCAAGGCGAGTCAAGATATAAACAGCTACTTGTCATGGTTCCAACAAAAACCAGGGAAATCACCTAAGACCCTGATCTATAGAGCCAATCGCCTGGTTGACGGTGTCCCCTCCCGCTTTAGCGGCTCCGGAAGCGGTCAAGATTACTCTCTCACAATTTCTTCCTTGGATTATGAAGACATGGGGATCTACTATTGTCAACAGTATGACGAATCCCCGTGGACTTTCGGTGGCGGTACCAAATTGGAAATAAAGGGCTCTACAAGCGGCTCAGGAAAACCTGGATCAGGCGAAGGGTCTACGAAGGGCCAGATACAACTCGTTCAAAGTGGGCCAGAACTCAAAAAACCGGGAGAAACAGTGAAAATTTCTTGTAAGGCATCAGGATACACATTCACAAACTACGGGATGAATTGGGTCAAACAAGCACCCGGAAAGGGGCTGCGCTGGATGGGGTGGATCAACACACATACTGGGGAACCTACTTACGCAGACGATTTCAAGGGCAGATTCGCCTTTTCTTTGGAGACCTCCGCCTCTACTGCATACTTGCAGATAAACAACCTGAAGAATGAAGATACCGCCACCTACTTCTGTACGCGCAGGGGCTACGATTGGTATTTTGATGTATGGGGGGCAGGCACCACTGTTACTGTGTCAAGCCGTACGATTGAAGT TATGTATCCTCCTCCTTACC T AGAC AAT GAGAAGAG C AAT G GAAC CATTATCCATGT GAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTG GGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCC CCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGC TCAACGCGTGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCA GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTG GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAAT GAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCG GAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTG AAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCG GAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCG GCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGG GTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGC CACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTC TGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAG AACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTG CAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACA TCGTGCAGATGTTCATCAACACCAGCTGA ( SEQ ID NO : 78 )SEQ ID NO: 79 - Exemplary anti-CD5 CAR encoding construct (e.g.,CD5CARCD28hinge.28tmd3z.IL15) amino acid sequenceMEFGLSWLFLVAILKGVQCSRDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPG KSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCQQYDESPWTFGGGTK LEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQA PGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYD WYFDVWGAGTTVTVSSRTIEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSKPFWVLV WGGVLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR STRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALL KLAGDVESNPGPMRISKPHLRS IS IQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANW VNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAS IHDTVE NLI ILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS ( SEQ ID NO : 79 )E. T Cell Receptors (TCR)

[0311] In some embodiments, the genetically engineered antigen receptors include recombinant TCRs and / or TCRs cloned from naturally occurring T cells. A "T cell receptor" or "TCR" refers to a molecule that contains a variable a and P chains (also known as TCRa and TCRP, respectively) or a variable y and 5 chains (also known as TCRy and TCRS, respectively) and that is capable of specifically binding to an antigen peptide bound to a major histocompatibility complex (MHC) receptor. In some embodiments, the TCR is in the aP form.

[0312] In particular embodiments, provided herein are NK cells that have been modified by the hand of man to express part or all of a CD3 / TCR receptor complex. In specific embodiments, the NK cells are modified to include all components of the CD3 complex,including CD3^, CD3s, CD3y and CD36. In particular cases the full lengths of CD3^, CD3s, CD3y and CD36 are utilized, including their extracellular domain, transmembrane domain, and intracellular domain, however in alternative embodiments only part of one or more of CD3^, CD3s, CD3y and CD36 are utilized each of which that may or may not be combined with one or more intracellular signaling domains such as CD 16, NKG2D, DAP 10, DAP 12, CD28, 4 IBB, 2B4, CD27, 0X40, or any combination thereof.

[0313] In certain embodiments, an amino acid sequence (e.g., a polypeptide) may comprise an amino acid represented by a single letter “X” or a three letter code “Xaa”. In some embodiments, the amino acid represented by “X” or “Xaa” is any naturally occurring amino acid, such as but not limited to, Arginine (Arg, R), Histidine (His, H), Lysine (Lys, K), Aspartic Acid (Asp, D), Glutamic Acid (Glu, E), Serine (Ser, S), Threonine (Thr, T), Asparagine (Asn, N), Glutamine (Gin, Q), Glycine (Gly, G), Proline (Pro, P), Cysteine (Cys, C), Alanine (Ala, A), Valine (Val, V), Isoleucine (He, I), Leucine (Leu, L), Methionine (Met, M), Phenylalanine (Phe, F), Tyrosine (Tyr, Y), or Tryptophan (Trp, W).

[0314] In certain embodiments, particular sequences for any of the CD3 receptor components are utilized, including wildtype or mutants of the components so long as the CD3 receptor having the mutant is able to allow signaling through the CD3 complex leading to activation and killing of targets. In some cases, CD3 / TCR complex associated polypeptides, polynucleotides encoding the same, and / or constructs comprising said polynucleotides are described in the Inventors international patent application publication W02023004425A2 (PCT / US2022 / 074062), published on January 26, 2023, which is incorporated herein by reference in its entirety for the purposes described herein.

[0315] Typically, TCRs that exist in aP and y5 forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to MHC molecules. In some embodiments, a TCR also can contain a constant domain, a transmembrane domain and / or a short cytoplasmic tail (see, e.g., Janeway et al, 1997). For example, in some aspects, each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass functional TCRfragments thereof. The term also encompasses intact or full-length TCRs, including TCRs in the aP form or y5 form.

[0316] Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR that binds to a specific antigenic target bound in an MHC molecule, i.e. MHC-target complex. An "antigen-binding portion" or “antigen-binding fragment" of a TCR, which can be used interchangeably, refers to a molecule that contains a portion of the structural domains of a TCR, but that binds the antigen (e.g. MHC- target complex) to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable P chain of a TCR, sufficient to form a binding site for binding to a specific MHC-target complex, such as generally where each chain contains three complementarity determining regions.

[0317] In some embodiments, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) analogous to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule and determine peptide specificity. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., lores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC molecule. In some embodiments, the variable region of the P-chain can contain a further hypervariability (HV4) region.

[0318] In some embodiments, the TCR chains contain a constant domain. For example, like immunoglobulins, the extracellular portion of TCR chains (e.g., a-chain, P-chain) can contain two immunoglobulin domains, a variable domain (see e.g., Va or Vp; typically amino acids 1 to 116 based on Kabat numbering Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5thed.) at the N-terminus, and one constant domain (e.g., a-chain constant domain or Ca, typically amino acids 117 to 259 based on Kabat, P-chain constant domain or Cp, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain contains short connectingsequences in which a cysteine residue forms a disulfide bond, making a link between the two chains. In some embodiments, a TCR may have an additional cysteine residue in each of the a and P chains such that the TCR contains two disulfide bonds in the constant domains.

[0319] In some embodiments, the TCR chains can contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chains contain a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules like CD3. For example, a TCR containing constant domains with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.

[0320] Generally, CD3 is a multi-protein complex that can possess three distinct chains (y, 5, and a) in mammals and the ^-chain. For example, in mammals the complex can contain a CD3y chain, a CD36 chain, two CD3s chains, and a homodimer of CD3(^ chains. The CD3y, CD36, and CD3s chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD36, and CD3s chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3y, CD36, and CD3s chains each contain a single conserved motif known as an immunoreceptor tyrosine -based activation motif or ITAM, whereas each CD3(^ chain has three. Generally, IT AMs are involved in the signaling capacity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating the signal from the TCR into the cell. The CD3- and (^-chains, together with the TCR, form what is known as the T cell receptor complex. In some embodiments, immune cells are engineered to express a CD3 multi-protein complex.

[0321] In some embodiments, the TCR may be a heterodimer of two chains a and P (or optionally y and 5) or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (a and P chains or y and 5 chains) that are linked, such as by a disulfide bond or disulfide bonds. In some embodiments, a TCR for a target antigen (e.g., a cancer antigen) is identified and introduced into the cells. In some embodiments, nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, such as from cells such as from a T cell (e.g. cytotoxic T cell), T cell hybridomas or other publicly available sources. In some embodiments, the T cells can be obtained from in vivo isolated cells. In someembodiments, a high-affinity T cell clone can be isolated from a patient, and the TCR isolated. In some embodiments, the T cells can be a cultured T cell hybridoma or clone. In some embodiments, the TCR clone for a target antigen has been generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system, or HL A). See, e.g., tumor antigens (see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR or antigen-binding portion thereof can be synthetically generated from knowledge of the sequence of the TCR.

[0322] In certain embodiments, a TCR recognizes an NY-ESO-1 antigen. In certain embodiments, a NY-ESO-1 TCR comprises, or is encoded by, a sequence at least or equal to, exactly or about 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% identity to any one or more of SEQ ID NOs: 80-81 and 104-109.SEQ ID NO: 80 - Exemplary NY-ESO-1 TCR alpha chain amino acid sequenceXQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRL NASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVY QLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFA CANAFNNS I IPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLM TLRLWSS ( SEQ ID NO : 80 )SEQ ID NO: 81 - Exemplary NY-ESO-1 TCR beta chain amino acid sequenceGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGY NVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPKVAV EE P S E AE I S H T QKAT L VC LAT G F Y P DHVE L S WWVNGKE VH SGVSTDPQPLKEQ PALND S R YC LSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSES YQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG ( SEQ ID NO : 81 )SEQ ID NO: 104 - Exemplary NY-ESO-1 TCR alpha chain with signal peptide amino acid sequenceMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKG LTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFG RGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMR SMDFKSNSAVAWSNKSDFACANAFNNS I IPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL SVIGFRILLLKVAGFNLLMTLRLWSS ( SEQ ID NO : 104 )SEQ ID NO: 105 - Exemplary NY-ESO-1 TCR beta chain with signal peptide amino acid sequenceMS IGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHS GVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP VTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSR G ( SEQ ID NO : 105 )SEQ ID NO: 106 - Exemplary NY-ESO-1 construct open reading frameMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKG LTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFG RGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMR SMDFKSNSAVAWSNKSDFACANAFNNS I IPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL SVIGFRILLLKVAGFNLLMTLRLWSSGSGATNFSLLKQAGDVEENPGPMS IGLLCCAALSLL WAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQ GEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNV FPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPA LNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD CGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG ( SEQ ID NO : 106 )SEQ ID NO: 107 - Exemplary NY-ESO-1 TCR alpha chain with signal peptide nucleic acid sequence atggagaccctcttgggcctgcttatcctttggctgcagctgcaatgggtgagcagcaaaca ggaggtgacacagattcctgcagctctgagtgtcccagaaggagaaaacttggttctcaact gcagtttcactgatagcgctatttacaacctccagtggtttaggcaggaccctgggaaaggt ctcacatctctgttgcttattcagtcaagtcagagagagcaaacaagtggaagacttaatgc ctcgctggataaatcatcaggacgtagtactttatacattgcagcttctcagcctggtgact cagccacct acct ctgt get gtgaggcccCTTTATggaggaagctacat acct acatttgga agaggaaccagccttattgttcatccgtatatccagaaccctgaccctgccgtgtaccagct gagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaa atgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgagg tctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgc aaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcct gtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctg tcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgct gcggctgtggtccagc ( SEQ ID NO : 107 )SEQ ID NO: 108 - Exemplary NY-ESO-1 TCR beta chain with signal peptide nucleic acid sequence at gagcat cggcctcctgt get gtgcagccttgtctctcctgtgggcaggtccagtgaatgc tggtgtcactcagaccccaaaattccaggtcctgaagacaggacagagcatgacactTcagt gtgcccaggatatgaaccatgaatacatgtcctggtatcgacaagacccaggcatggggctg aggctgattcattactcagttggtgctggtatcactgaccaaggagaagtccccaatggcta caatgtctccagatcaaccacagaggatttcccgctcaggctgctgtcggctgctccctccc agacatctgtgtacttctgtgccagcagttacgtcgggaacaccggggagctgttttttgga gaaggctctaggctgaccgtactggaggacctgaaaaacgtgttcccacccAaggtcgctgt gtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtatgcctggcca caggcttctaccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagt ggggtcagcacagacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgct gtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaaccc gtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggcttcacctccgagtc ttaccagcaaggggtcctgtctgccaccatcctctatgagatcttgctagggaaggccacct tgtatgccgtgctggtcagtgccctcgtgctgatggccatggtcaagagaaaggattccaga ggc ( SEQ ID NO : 108 )SEQ ID NO: 109 - Exemplary NY-ESO-1 construct open reading frame atggagaccctcttgggcctgcttatcctttggctgcagctgcaatgggtgagcagcaaaca ggaggtgacacagattcctgcagctctgagtgtcccagaaggagaaaacttggttctcaact gcagtttcactgatagcgctatttacaacctccagtggtttaggcaggaccctgggaaaggt ctcacatctctgttgcttattcagtcaagtcagagagagcaaacaagtggaagacttaatgc ctcgctggataaatcatcaggacgtagtactttatacattgcagcttctcagcctggtgact cagccacct acct ctgt get gtgaggcccCTTTATggaggaagctacat acct acatttgga agaggaaccagccttattgttcatccgtatatccagaaccctgaccctgccgtgtaccagct gagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaa atgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgagg tctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgc aaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcct gtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctg tcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgct gcggctgtggtccagcggaagcggagctactaactttagcctgctgaagcaggctggagatg tggaggagaaccctggacct at gagcat cggcctcctgt get gtgcagccttgtctctcctg tgggcaggtccagtgaatgctggtgtcactcagaccccaaaattccaggtcctgaagacagg acagagcatgacactTcagtgtgcccaggatatgaaccatgaatacatgtcctggtatcgac aagacccaggcatggggctgaggctgattcattactcagttggtgctggtatcactgaccaa ggagaagtccccaatggctacaatgtctccagatcaaccacagaggatttcccgctcaggct gctgtcggctgctccctcccagacatctgtgtacttctgtgccagcagttacgtcgggaaca ccggggagctgttttttggagaaggctctaggctgaccgtactggaggacctgaaaaacgtg ttcccacccAaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggc cacactggtatgcctggccacaggcttctaccccgaccacgtggagctgagctggtgggtga atgggaaggaggtgcacagtggggtcagcacagacccgcagcccctcaaggagcagcccgcc ctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaa cccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtgga cccaggatagggccaaacccgtcacccagatcgtcagcgccgaggcctggggtagagcagac tgtggcttcacctccgagtcttaccagcaaggggtcctgtctgccaccatcctctatgagat cttgctagggaaggccaccttgtatgccgtgctggtcagtgccctcgtgctgatggccatgg tcaagagaaaggattccagaggc ( SEQ ID NO : 109 )

[0323] In certain embodiments, a TCR recognizes a PRAME antigen. In certain embodiments, an anti-PRAME TCR comprises, or is encoded by, a sequence at least or equal to, exactly or about 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% identity to any one or more of SEQ ID NOs: 82-87 and 110-112.SEQ ID NO: 82 - Exemplary PRAME TCR clone 46 alpha chain amino acid sequenceMLLEHLLI ILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSS I FNTWLWYKQDPGEGPV LLIALYKAGELTSNGRLTAQFGITRKDSFLNISAS IPSDVGIYFCAGIPRDNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSM DFKSNSAVAWSNKSDFACANAFNNS I IPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSV IGFRILLLKVAGFNLLMTLRLWSS ( SEQ ID NO : 82 )SEQ ID NO: 83 - Exemplary PRAME TCR clone 46 beta chain amino acid sequenceMGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASTPWLAGGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG ( SEQ ID NO : 83 )SEQ ID NO: 84 - Exemplary PRAME TCR clone 54 alpha chain amino acid sequenceMLLLLVPVLEVI FTLGGTRAQSVTQLGSHVSVSERALVLLRCNYSSSVPPYLFWYVQYPNQGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVSGQTGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS I IPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS ( SEQ ID NO : 84 )...

Claims

CLAIMSWhat is claimed is:

1. An engineered Natural Killer (NK) cell, wherein the NK cell comprises one or more engineered mutations in an endogenous basic leucine zipper ATF-like transcription factor (BATF basic leucine zipper ATF-like transcription factor 3 (BATF3), and / or DNA damage inducible transcript 3 (DDITS) gene of the cell.

2. An engineered immune cell, wherein the immune cell comprises one or more engineered mutations in endogenous BATF and BATF3, BATF and DDIT3, or BATF3 and DDIT3, genes of the cell.

3. An engineered immune cell, wherein the immune cell comprises one or more engineered mutations in endogenous BATF, BA TF3, and DDIT3 genes of the cell.

4. The engineered cell of any one of claims 1-3, wherein the one or more mutations comprise a partial or complete loss of function, and / or knock-out (KO) mutation.

5. The engineered cell of any one of claims 1-3 wherein the one or more mutations reduces or inhibits transcription or post-transcriptional processing of one or more mRNA isoforms encoded by the one or more mutated endogenous genes relative to a non-mutated locus encoding the same endogenous gene.

6. The engineered cell of any one of claims 1-3 wherein the one or more mutations is in a coding region of the one or more genes.

7. The engineered cell of any one of claims 1-3 wherein the one or more mutations is in an intron of the one or more genes.

8. The engineered cell of any one of claims 1-3 wherein the one or more mutations is in an enhancer and / or promoter region of the one or more genes.

9. The engineered cell of any one of claims 1-3 wherein the one or more mutations is in a phosphorylated mothers against decapentaplegic homolog 2 (p-SMAD2) and / or phosphorylated mothers against decapentaplegic homolog 3 (p-SMAD3) binding site.

10. The engineered cell of any one of claims 1-3 wherein at least one of the one or more mutations result in substantially no polypeptide gene products from at least one of the one or more genes.

11. The engineered cell of any one of claims 1-3 wherein the one or more mutations comprise a homozygous mutation in at least one of the BATF, BATF3, and / or DDIT3 genes.

12. The engineered cell of any one of claims 1-3 wherein the one or more mutations comprise a heterozygous mutation in one or more genes.

13. The engineered cell of any one of claims 1-3, wherein relative to a control nonengineered cell, the one or more mutations result in decreased BATF regulon transcriptional activity, increased metabolic capacity, increased cytotoxicity, increased persistence, and / or increased secretory function of the engineered cell in a microenvironment and / or a tumor microenvironment (TME) characterized by presence of transforming growth factor beta (TGF- P).

14. The engineered cell of claim 13, wherein following exposure to TGF-P, the one or more mutations provides the engineered cell with improved cytotoxicity capacity relative to a nonengineered cell exposed to TGF-p.

15. The engineered cell of claim 13, wherein following exposure to TGF-P, the one or more mutations provides the engineered cell with improved metabolic function relative to a nonengineered cell following exposure to TGF-p.

16. The engineered cell of claim 15, wherein the improved metabolic function comprises increased glycolytic capacity, increased oxidative phosphorylation capacity, and / or increased oxygen consumption rates.

17. The engineered cell of claim 13, wherein following exposure to TGF-P, the one or more mutations provides the engineered cell with reduced levels of exhaustion markers and / or exhaustion score relative to a non-engineered cell following exposure to TGF-p.

18. The engineered cell of claim 17, wherein the reduced exhaustion markers comprise LAG3, TIM3, TIGIT, KLRG1, KLRC1, and / or CISH.

19. The engineered cell of claim 13, wherein following exposure to TGF-P, the one or more mutations provides for reduced levels of p-SMAD2 and / or p-SMAD3 in the engineered cell relative to a non-engineered cell following exposure to TGF-p.

20. The engineered cell of claim 13, wherein following exposure to TGF-P, the one or more mutations provides for reduced levels of transcripts and / or polypeptides encoded by genesTBX21, HP1BP3, TIPI, BATF, ASCL2, HOMEZ, GATA3, ZNF444, CEBPD, LEF1, ZNF71, and / or ZNF319 relative to a non-engineered cell following exposure to TGF-p.

21. The engineered cell of claim 13, wherein following exposure to TGF-P, the one or more mutations provides for reduced levels of BATF regulon activity relative to a non-engineered cell following exposure to TGF-p.

22. The engineered cell of claim 13, wherein following exposure to TGF-P, the one or more mutations provides for reduced levels of expression of one or more, five or more, or ten or more genes identified in Table 3 relative to a non-engineered cell following exposure to TGF- P-23. The engineered cell of claim 13, wherein following exposure of the cell to TGF-P, the one or more mutations provides for reduced levels of expression of genes LAG3, HAVCR2, TIGIT, BATF, CTLA4, TGFBR2, ENTPD1, DNAJB1, and / or HSPA1A relative to a nonengineered cell following exposure to TGF-p.

24. The engineered cell of claim 13, wherein the TGF-P comprises TGF-pi, TGF-P2, and / or TGF-P 3.

25. The engineered cell of claim 13, wherein the TGF-P comprises active TGF-P, TGF-P complexed with TGF-P 1 -latency-associated peptide (LAP), and / or TGF-P complexed with latent transforming growth factor beta binding protein 1 (LTBP1).

26. The engineered cell of claim 13, wherein the TGF-P comprises, consists essentially of, or consists of active and / or soluble TGF-p.

27. The engineered cell of any one of claims 1-3, wherein the one or more mutations results in enhanced polyfunctionality of the engineered cell relative to a control non-engineered cell in response to stimulation by and / or immunosuppressive signaling from a tumor cell.

28. The engineered cell of claim 27, wherein the enhanced polyfunctionality is evidenced by an increase in cytokine release in response to stimulation by tumor cells.

29. The engineered cell of claim 28, wherein the increase in cytokine release comprises an increase in interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), and / or the degranulation marker CD 107a, in response to stimulation by tumor cells.

30. The engineered cell of claim 27, wherein the increase in cytokine release comprises an increase in granulocyte-macrophage colony-stimulating factor (GMCSF), soluble CD137(sCD137), INF-g, Granzyme A, interleukin 13 (IL-13), Granzyme B, soluble FAS cell surface death receptor (sFas), interleukin 6 (IL-6), soluble FAS cell surface death receptor ligand (sFasL), macrophage inflammatory protein- 1 alpha (MIP-la), macrophage inflammatory protein-1 beta (MIP-lb), TNF-a, and / or Perforin, in response to stimulation by tumor cells.

31. The engineered cell of any one of claims 1-3, wherein the one or more mutations provides an enhanced activated and / or cytotoxic phenotype to the engineered cell relative to a control non-engineered cell.

32. The engineered cell of any one of claims 1-3, wherein the one or more mutations provides an enhanced activated and / or cytotoxic phenotype for the engineered cell relative to a control non-engineered cell, wherein the enhanced activated and / or cytotoxic phenotype is associated with one or more of GSEA identified pathways: Fc Epsilon signaling, Regulation of Actin Cytoskeleton, T cell Receptor Signaling Pathway, TNF- Alpha Signaling via NF-kB, MAPK Signaling Pathway, and / or Rho GTPase Cycle.

33. The engineered cell of any one of claims 1-3, wherein the one or more mutations provides for enhanced upregulation of G2M, E2F, MYC, MT0RC1, oxidative phosphorylation, and / or TNFa signaling in the engineered cell relative to a control cell.

34. The engineered cell of any one of claims 1-3, wherein the one or more mutations provides for an increase in open chromatin peaks associated with transcription factors ELF3, CTCFL, RUNX1, NKX2-2, and / or IRF8 in the engineered cell relative to a control cell.

35. The engineered cell of any one of claims 1-3, wherein the one or more mutations provides for an increase in closed chromatin peaks associated with transcription factors BATF, FRA1, JUNB, ATF3, and / or FOS.

36. The engineered cell of any one of claims 1-3, wherein at least one of the one or more mutations is in endogenous gene BATF.

37. The engineered cell of claim 36, wherein the BATF mutation is a result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NOs: 10, 11, and / or 12.

38. The engineered cell of claim 36, wherein the BATF mutation results in a decrease in BATF polypeptide levels of greater than 60%, greater than 80%, or greater than 90% relative to a non-engineered cell.

39. The engineered cell of any one of claims 1-3, wherein at least one of the one or more mutations is in endogenous gene BATF3.

40. The engineered cell of claim 39, wherein the BATF3 mutation is a result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NO: 27.

41. The engineered cell of claim 39, wherein the BATF3 mutation results in a decrease in BATF3 polypeptide levels of greater than 60%, greater than 80%, or greater than 90% relative to a non-engineered cell.

42. The engineered cell of any one of claims 1-3, wherein at least one of the one or more mutations is in endogenous gene DDIT3.

43. The engineered cell of claim 42, wherein the DDIT3 mutation is a result of exposure of the cell to a polynucleotide comprising the sequence of SEQ ID NOs: 27 and / or 28.

44. The engineered cell of claim 42, wherein the DDIT3 mutation results in a decrease in one or more DDIT3 polypeptide isoform levels of greater than 60%, greater than 80%, or greater than 90% relative to a non-engineered cell.

45. The engineered cell of any one of claims 1-44, wherein the cell is a T cell, natural killer (NK) cell, NK T cell, macrophage, B cell, invariant NKT cells, gamma delta T cells, MSCs, tumor-infiltrating lymphocyte, or dendritic cell.

46. The engineered cell of any one of claims 1-45, wherein the cell is an NK cell.

47. The engineered cell of any one of claims 1-46, wherein the cell is an NK cell derived from cord blood (CB), peripheral blood (PB), an NK cell line, bone marrow, a stem cell, or a mixture thereof.

48. The engineered NK cell of any one of claims 45-47, wherein the NK cell is derived from cord blood.

49. The engineered cell of any one of claims 1-3, wherein the cell comprises one or more transgenic engineered receptors.

50. The engineered cell of claim 49, wherein the one or more engineered transgenic receptors comprises an engineered antigen receptor that specifically targets an antigen.

51. The engineered cell of claim 50, wherein the one or more engineered transgenic antigen receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

52. The engineered cell of claim 51, wherein the engineered transgenic antigen receptor is a CAR.

53. The engineered cell of claim 51, wherein the engineered transgenic antigen receptor is a TCR.

54. The engineered cell of claim 53, wherein the TCR is an invariant TCR (iTCR).

55. The engineered cell of claim 53, wherein the TCR comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 95-98.

56. The engineered cell of claim 51, wherein the engineered cell transgenically expresses a CD3 complex.

57. The engineered cell of claim 56, wherein the CD3 complex comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 99-100.

58. The engineered cell of claim 51, wherein the engineered cell transgenically expresses a Fc Receptor (FcR) extracellular binding domain.

59. The engineered cell of claim 58, wherein the FcR comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 101-103.

60. The engineered cell of any one of claims 1-3, wherein the engineered cell is complexed with one or more antibodies targeting antigens BCMA, CD20, CD19, EGFR, CD30, HER2, GPRC5D, CD16, CD3, CD28, c-MET, PSMA, MUC17, CD33, FLT3, STEAP1, CLDN18.2, CD 123, EpCAM, CEA, GPC3, CD38, CD33, CD22, GPA33, GD2, MUC16, DLL-3, CLEC12A, FcRH5, BlyS, and / or SSTR.

61. The engineered cell of claim 60, wherein the one or more antibodies comprise Elranatamab, Glofitamab, Tafasitamab, Cetuximab, Imgatuzumab, Margetuximab, Amivantamab, Blinatumomab, Obinutuzumab, IPH61 (also known as IPH6101 or SAR443579), Teclistamab, Talquetamab, Pertuzumab, Trastuzumab, Brentuximab, Mosunetuzumab, Epcoritamab, GEN3017, Loncastuximab tesirine, Belimumab, and / or Rituximab.

62. The engineered cell of claim 50, wherein the antigen is a cancer antigen.

63. The engineered cell of claim 50, wherein the antigen is a solid tumor antigen.

64. The engineered cell of claim 50, wherein the antigen is a blood cancer antigen.

65. The engineered cell of claim 50, wherein the antigen is selected from the group consisting of CD70, 5T4, 8H9, avp6integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD47, CD 123, CD 138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, FBP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA- Al+NY-ESO-1, IL-l lRa, IL-13Ra2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, R0R1, SP17, Survivin, TAG72, TIM3, TIGIT, TROP2, TEMs, HMW-MAA, VEGFR2, and any combination thereof.

66. The engineered cell of claim 50, wherein the antigen comprises CD70, CD47, CD 19, CD20, BCMA, CD5, TROP2, CD123, CD33, PRAME, NY-ESO-1, EGFRvIII, IL-13Ra2, and / or TIM3.

67. The engineered cell of claim 66, wherein the target antigen comprises CD70.

68. The engineered cell of claim 67, wherein the engineered transgenic antigen receptor comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 29-37, or 58-64.

69. The engineered cell of claim 66, wherein the target antigen comprises CD5.

70. The engineered cell of claim 69, wherein the engineered transgenic antigen receptor comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 65-79.

71. The engineered cell of claim 66, wherein the target antigen comprises TROP2.

72. The engineered cell of claim 71, wherein the engineered transgenic antigen receptor comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 46-57.

73. The engineered cell of claim 66, wherein the target antigen comprises PRAME.

74. The engineered cell of claim 73, wherein the engineered transgenic antigen receptor comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 82-87 and / or 110- 112.

75. The engineered cell of claim 66, wherein the target antigen comprises NY-ESO-1.

76. The engineered cell of claim 75, wherein the engineered transgenic antigen receptor comprises a polypeptide sequence and / or is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 80-81 and / or 104- 109.

77. The engineered cell of claim 66, wherein the target antigen comprises EGFRvIII and IL-13Ra2.

78. The engineered cell of claim 50, wherein the one or more engineered receptors comprises a cytokine receptor, chemokine receptor, homing receptor, or a combination thereof.

79. The engineered cell of any one of claims 1-3, wherein the cell is engineered to transgenically express or overexpress one or more chemokines and / or one or more cytokines.

80. The engineered cell of claim 79, wherein the cytokine is IL-15, IL-12, IL-21, IL-2, IL- 18, IL-7, or a combination thereof.

81. The engineered cell of claim 80, wherein the cytokine is IL- 15 and / or IL-21.

82. The engineered cell of any one of claims 1-3, wherein the cell is engineered to comprise a transgenic suicide gene.

83. The engineered cell of any one of claims 1-3, wherein the one or more mutations in an endogenous are the result of homologous recombination and / or non-homologous recombination.

84. The engineered cell of any one of claims 1-3, wherein the one or more mutations is mediated by contact of the cell with an endonuclease.

85. The engineered cell of claim 84, wherein the endonuclease is an RNA guided endonuclease.

86. The engineered cell of claim 85, wherein the RNA guided endonuclease is CRISPR- Cas9.

87. The engineered cell of any one of claims 1-3, wherein the cell comprises one or more additional mutations in one or more genes, wherein the one or more additional gene is selected from the group consisting of TGFBR2, GPR4, GPR31, GPR68, GPR81, GPR132, GPR151, CREM, ICER, CREB1, NKG2A, SIGLEC-7, LAG3, TIM3, GISH, F0X01, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD38, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, GR, and CD7.

88. A method of making the engineered cell according to any one of claims 1-87.

89. A population of cells comprising the engineered cells of any one of claims 1-87.

90. A composition comprising a pharmaceutically acceptable excipient and the population of engineered cells of claim 89.

91. A method of treating a disorder in a patient in need thereof, the method comprising a step of administering a therapeutically effective amount of the engineered cell, population of engineered cells, or composition of claims 1-3, 89, or 90 to the individual.

92. A method of treating a disorder in a patient in need thereof, the method comprising a step of administering a therapeutically effective amount of the engineered cell of claims 1-3 to the individual.

93. The method of claim 92, wherein the disorder is characterized by the presence of extracellular TGF-P, optionally soluble extracellular TGF-p.

94. The method of claim 93, wherein the extracellular TGF-P is one the dominant or is the dominant cytokine in a microenvironment created by the disorder.

95. The method of claim 93, wherein the extracellular TGF-P is one of the most abundant, or is the most abundant cytokine in a microenvironment created by the disorder.

96. The method of claim 92, wherein the disorder comprises cancer, a pathogenic infection, a connective tissue disorders, tissue fibrosis, and / or a chronic inflammatory condition.

97. The method of claim 92, wherein the disorder comprises cancer.

98. The method of claim 92, wherein the cells are autologous, allogeneic, or xenogeneic with respect to the individual.

99. The method of claim 98, wherein the cells are allogeneic with respect to the individual.

100. The method of claim 96, wherein the cancer comprises a solid tumor.

101. The method of claim 96, wherein the cancer does not comprise a solid tumor.

102. The method of claims 96, wherein the cancer is of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, and / or cervix.

103. The method of claim 96, wherein the cancer comprises AML and / or MDS.

104. The method of claim 96, wherein the cancer comprises cells expressing one or more of antigens CD70, 5T4, 8H9, avp6integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD47, CD 123, CD 138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, FBP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA- Al+NY-ESO-1, IL-l lRa, IL-13Ra2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TIM3, TIGIT, TROP2, TEMs, HMW-MAA, VEGFR2, or any combination thereof.

105. The method of claim 96, wherein the cancer comprises cells expressing one or more of antigens CD70, CD19, CD20, BCMA, CD47, CD5, TROP2, CD123, CD33, PRAME, NY- ESO-1, EGFRvIII, IL-13Ra2, and / or TIM3.

106. The method of claim 96, wherein the cancer comprises cells expressing CD70.

107. The method of claim 96, wherein the cancer comprises cells expressing CD5.

108. The method of claim 96, wherein the cancer comprises cells expressing TROP2.

109. The method of claim 96, wherein the cancer comprises cells expressing PRAME.

110. The method of claim 96, wherein the cancer comprises cells expressing NY-ESO-1.

111. The method of claim 96, wherein the cancer comprises cells expressing EGFRvIII and IL-13Ra2.

112. The method of claim 92, wherein the individual is a mammal.

113. The method of claim 112, wherein the individual is a human, dog, cat, horse, cow, sheep, pig, or rodent.

114. The method of claim 112, wherein the individual is a human.

115. The method of claim 92, wherein the individual is administered at least two doses of the cells.

116. The method of claim 92, wherein the individual is administered one or more additional cancer therapy.

117. The method of claim 116, wherein the additional cancer therapy comprises surgery, radiation, chemotherapy, hormone therapy, immunotherapy, or a combination thereof.

118. The method of claim 96, further comprising the step of diagnosing cancer in the individual.

119. A polynucleotide comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 10-12, 17, or 27-28.

120. A method of treating a disease or disorder in an individual in need thereof, wherein the method comprises administering a cell or population of cells comprising one or more mutations in the BA IT'. BA TT' 3. and / or DDIT3 genes, wherein the disorder is characterized by the production of TGF-P and / or presence of extracellular TGF-P in a microenvironment generated by the disorder.

121. A method of shielding an immune cell from the immunosuppressive effects of TGF-P, the method comprising creating one or more mutations in BA TT'. BA TT' 3. and / or DDIT3 genes in the cell.

122. The method of claim 121, wherein the immune cell is an NK cell.

123. A kit comprising the engineered cell, population of engineered cells, composition, and / or means to perform the methods of any one of claims 1-122.

124. Use of the engineered cell of any one of claims 1-87 in the manufacture of a medicament.

125. The engineered cell of any one of claims 1-87 for use in the treatment of cancer.

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